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Genetic basis of hypertrophic cardiomyopathy

Bos, J.M.

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Download date:01 Sep 2021

GENETIC BASIS OF HYPERTROPHIC CARDIOMYOPATHY

© 2010 by Johan Martijn Bos

Genetic basis of hypertrophic cardiomyopathy Johan Martijn Bos / University of Amsterdam, 2010. Thesis

Printed by Ipskamp Drukkers B.V.

ISBN: 978-90-9024905-6

No parts of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means without permission of the author

GENETIC BASIS OF HYPERTROPHIC CARDIOMYOPATHY

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het college voor promoties ingestelde

commissie, in het openbaar te verdedigen in de Agnietenkapel

op vrijdag 15 januari 2010, te 12.00 uur

doorJohan Martijn Bos geboren te Vorden

Promotiecommissie

Promotor: Prof. dr. A.A.M. Wilde

Co-promotor: Prof. dr. M.J. Ackerman

Leden: Prof. dr. R.J.G Peters Prof. dr. Y.M. Pinto Prof. dr. R.J.A. Wanders Prof. dr. N.A. Blom Prof. dr. P.A.F.M Doevendans Dr. L. Kapusta

Faculteit der Geneeskunde

The research described in this thesis was carried out in the Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory in Rochester, MN (USA) in collaboration with the Heart Failure Research Center of the Academic Medical Center, Amsterdam (The Netherlands).

Financial support by the Netherlands Heart Foundation for the publication of this thesis is gratefully acknowledged.

Additional support was generously provided by the Medtronic Bakken Research Center, Maastricht; AstraZeneca, Zoetermeer; St. Jude Medical, Veenendaal and the University of Amsterdam.

Voor mijn ouders

Table of contents Page

Chapter 1 Introduction Genetics of hypertrophic cardiomyopathy: one, two, or more diseases? 9

Curr Opin Cardiol 2007; 22(3): 193 – 199 [Review]

Chapter 2 Genotype-phenotype relationships involving hypertrophic cardiomyopathy - associated mutations in titin, muscle LIM protein, and telethonin. 27

Mol Genet Metab 2006; 88(1): 78 – 85

Chapter 3 Cardiac ankyrin repeat protein gene (ANKRD1) mutations in hypertrophic cardiomyopathy. 49

J Am Coll Cardiol 2009; 54(4): 334 – 42

Chapter 4 Echocardiographic-determined septal morphology in Z-disc hypertrophic cardiomyopathy. 71

Biochem Biophys Res Commun 2006; 351(4): 896 – 902

Chapter 5 Relationship between sex, shape and substrate in hypertrophic cardiomyopathy. 89 Am Heart J 2008; 155: 1128 – 1134

Chapter 6 TGFß-inducible early gene-1 (TIEG1): a novel hypertrophic cardiomyopathy susceptibility gene. 107

Manuscript in preparation

Chapter 7 Diagnostic, prognostic and therapeutic implications of genetic testing for hypertrophic cardiomyopathy. 129

J Am Coll Cardiol 2009; 54(3): 201 – 211 [Review]

The growing field of genetic contributors to the pathogenesis of HCM or,About ‘lumpers’ and ‘splitters’: McKusick revisited. 161

Summary 169

Samenvatting 173

Acknowledgements 177

List of publications 179

Chapter 1

Genetics of Hypertrophic Cardiomyopathy: One, Two, or More Diseases?

J. Martijn Bos, Steve R. Ommen, Michael J. Ackerman

Curr Opin Cardiol 2007; 22(3): 193 – 9 [Review]

Abstract

Purpose of review. Hypertrophic cardiomyopathy (HCM), affecting 1 in 500 persons, is the most common identifiable cause of sudden death in the young. This review details the history of HCM, recent discoveries in its genetic underpinnings and important genotype-phenotype relationships described in recent studies. Recent findings. Since the discovery of the genetic underpinnings of hypertrophic cardiomyopathy in 1989 hundreds of mutations scattered amongst at least 10 sarcomeric genes confer the pathogenetic substrate for this “disease of the sarcomere/myofilament”. More recently, the genetic spectrum of HCM has expanded to encompass mutations in Z-disc associated genes (Z-disc hypertrophic cardiomyopathy) and glycogen storage diseases mimicking HCM (metabolic hypertrophic cardiomyopathy). Recent genotype-phenotype studies have discovered an important relationship between morphology of the left ventricle, its underlying genetic substrate and long-term outcome of this disease. Summary. Genomic medicine has entered the clinical practice and the diagnostic utility of genetic testing for HCM diseases is clearly evident, but with the growing number of hypertrophic cardiomyopathy-associated genes strategic choices have to be made. With recent discoveries in genotype-phenotype relationships, especially pertaining to the echocardiographic septal shape and the underlying pathogenetic mutation, time has come to subdivide the one disease we call HCM.

Keywords

Genetic testing, hypertrophic cardiomyopathy, myofilament, septal morphology

10

Introduction

Hypertrophic cardiomyopathy (HCM) is a disease of enormous phenotypic and genotypic heterogeneity. Affecting 1 in 500 people, it is the most prevalent genetic cardiovascular disease, and more importantly the most common cause of sudden cardiac death in young athletes[1]. Anatomically/physiologically, HCM can manifest with negligible to extreme hypertrophy, minimal to extensive fibrosis and myocyte disarray, absent to severe left ventricular outflow tract obstruction, and distinct septal contours/morphologies such as reverse curve-, sigmoidal-, and apical variant-HCM. The clinical course varies extremely, ranging from an asymptomatic lifelong course to dyspnea/angina refractory to pharmacotherapy to sudden death as the sentinel event.

HCM was fully described for the first time by Teare in 1958 as ‘asymmetrical hypertrophy of the heart in young adults’[2]. It has since been known by a confusing array of names, reflecting its clinical heterogeneity and its uncommon occurrence in daily cardiologic practice. In 1968, the World Health Organization (WHO) defined cardiomyopathies as ‘diseases of different and often unknown etiology in which the dominant feature is cardiomegaly and heart failure’[3]. This statement was updated in 1980 and defined cardiomyopathies as ‘heart muscles diseases of unknown cause’, thereby differentiating it from specific identified heart muscle diseases of known cause, like myocarditis[4].

Throughout the years, names such as idiopathic hypertrophic subaortic stenosis[5], muscular subaortic stenosis[6] and hypertrophic obstructive cardiomyopathy[7] have been widely and interchangeably used to define the same disease. In 1995, a WHO/International Society and Federation of Cardiology Task Force on cardiomyopathies classified the different cardiomyopathies by dominant pathophysiology , or if possible, by etiological/pathogenetic factors[8]. The four most important cardiomyopathies - dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), arrhythmogenic right ventricular cardiomyopathy (ARVC) and HCM - were recognized, next to a number of specific and mostly acquired cardiomyopathies, like ischemic- or inflammatory cardiomyopathy[8].

11

Accordingly, HCM is described as ‘left and/or right ventricular hypertrophy, usually asymmetric and involving the interventricular septum with predominant autosomal dominant inheritance involving sarcomeric contractile proteins’[8]. This nomenclature has been upheld in the most recent ACC/ESC expert consensus document of 2003[9], although with expanding knowledge on the genetic background of these diseases voices have recently been subclassified into primary cardiomyopathies into genetic - , mixed - and acquired cardiomyopathies[10]. Under this approach, the genetic subgroup entails HCM, ARVC and glycogen storage diseases presenting as HCM, but also includes ion channel disorders such as long QT syndrome (LQTS)[10].

Genetic background of HCM

Since the sentinel discovery of the first locus for familial HCM (1989) and the first mutations involving the MYH7-encoded beta myosin heavy chain (1990) as the pathogenic basis for HCM[11, 12], over 300 mutations scattered among at least 24 genes encoding various sarcomeric, calcium handling and mitochondrial proteins have been identified (Table 1). The most common genetically-mediated form of HCM is myofilament-HCM, with hundreds of disease-associated mutations in 8 genes

encoding proteins critical to the sarcomere’s thick myofilament [ -myosin heavy chain (MYH7)[12], regulatory myosin light chain (MYL2) and essential myosin light chain (MYL3)][13], intermediate myofilament [myosin binding protein C (MYBPC3)][14], and thin myofilament [cardiac troponin T (TNNT2), -tropomyosin (TPM1)[15], cardiac troponin I (TNNI3)[16], and actin (ACTC)[17, 18]. Targeted screening of giant sarcomeric TTN-encoded titin, which extends throughout half of the sarcomere, has thus far revealed only one mutation[19]. More recently, mutations have been described in the myofilament protein alpha-myosin heavy chain encoded by MYH6[20]. Although up until 2001 it was thought that specific mutations in these myofilament genes were inherently ‘benign’ or ‘malignant’ [21, 22, 23, 24, 25, 26, 27, 28], genotype-phenotype studies involving a large cohort of unrelated patients have indicated that great caution must be exercised with assigning particular prognostic significance to any particular mutation[29, 30, 31].

12

Furthermore, those studies have demonstrated that the two most common forms of genetically mediated HCM – MYH7-HCM and MYBPC3-HCM – are phenotypically indistinguishable[32]. The prevalence of mutations in the 8 most common myofilament associated genes, currently comprising the commercially available HCM genetic test (www.hpcgg.org) in different international cohorts ranges from 30 to 61%, leaving still a large number of patients with genetically unexplained disease[33].

Over the last few years, the spectrum of HCM-associated genes expanded outside the myofilament to encompass additional subgroups that could be classified as ‘Z-disc-HCM’, ‘calcium-handling HCM’, and ‘metabolic HCM’; all genes currently implicated in the pathogenesis of HCM are shown in Table 1. As a result of its close proximity to the contractile apparatus of the myofilament and its specific structure-function relationship with regards to cyto-architecture, as well as its role in the stretch-sensor mechanism of the sarcomere, recent attention has been focused on the cardiac Z-disc. Initial mutations were described in muscle LIM protein encoded by CSRP3[34] and telethonin encoded by TCAP[35], an observation replicated in our large cohort of unrelated patients with HCM[36]. LDB3-encoded LIM domain binding 3, ACTN2-encoded alpha actinin 2 and VCL-encoded vinculin/metavinculin have been added to that list[37]. Interestingly, although the first HCM-associated mutation in vinculin was found in the cardiac-specific insert of the gene, yielding the protein called metavinculin[38], the follow up study also identified a mutation in the ubiquitously expressed protein vinculin[39].

As the critical ion in the excitation-contraction coupling of the cardiomyocyte, calcium and proteins involved in calcium induced calcium release (CICR) have always been of high interest in the pathogenesis of HCM. Although with very low frequency, mutations have been described in the promoter – and coding region of PLN-encoded phospholamban, an important inhibitor of cardiac muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA)[40, 41] as well as in the RyR2-encoded cardiac ryanodine receptor[42]. Recently, our HCM genetic research program discovered three novel mutations in JPH2-encoded junctophilin 2 in three, previously genotype negative, patients with HCM. This is the first time that JPH2, which is thought to play a role in approximating the sarcoplasmic reticulum calcium release channels and plasmalemmal L-type calcium channels, has been implicated in the pathogenesis of HCM[43].

13

Tabl

e 1:

Sum

mar

y of

hyp

ertro

phic

car

diom

yopa

thy

(HC

M)-

susc

eptib

ility

gen

es a

nd t

he e

stim

ated

/ext

rapo

late

d fre

quen

cy (

%)

of s

peci

fic

mut

atio

ns b

y m

orph

olog

ic s

ubgr

oup.

Gen

eLo

cus

Prot

ein

Rev

erse

Cur

ve H

CM

Si

gmoi

dal

HC

MA

pica

l HC

M

Myo

filam

ent H

CM

70 –

85

10 -1

5 30

– 4

0

Gia

ntfil

amen

t T

TN

2q24

.3

Titin

-

- -

Thi

ckfil

amen

t M

YH

714

q11.

2-q1

2 -m

yosi

n he

avy

chai

n 30

- 40

<

5 10

- 15

MY

H6

14q1

1.2-

q12

-myo

sin

heav

y ch

ain

- -

-

MY

L212

q23-

q24.

3 V

entri

cula

r reg

ulat

ory

myo

sin

lig

ht c

hain

<

5 0

2 - 4

MY

L33p

21.2

-p21

.3

Ven

tricu

lar e

ssen

tial m

yosi

n

light

cha

in

- -

-

Inte

rmed

iate

fil

amen

t M

YB

PC

3 11

p11.

2 C

ardi

ac m

yosi

n-bi

ndin

g pr

otei

n C

30

- 40

5

10 -

15

Thi

n fil

amen

t T

NN

T2

1q32

C

ardi

ac tr

opon

in T

5

- 10

<1

< 5

T

NN

I319

p13.

4 C

ardi

ac tr

opon

in I

1-2

<1

0

T

PM

115

q22.

1 -tr

opom

yosi

n 1-

2 0

0

AC

TC

15q1

4 -c

ardi

ac a

ctin

<1

0

0

Z-di

sc H

CM

0

5 - 1

0 <

5

LB

D3

10q2

2.2-

q23.

3 LI

M b

indi

ng d

omai

n 3

(A

lias:

ZA

SP

) 0

3 3

C

SR

P3

11p1

5.1

Mus

cle

LIM

pro

tein

0

<1

0

TC

AP

17q1

2-q2

1.1

Tele

thon

in

0 <1

0

V

CL

10q2

2.1-

q23

Vin

culin

/met

avin

culin

0

<1

<1

A

CT

N2

1q42

-q43

A

lpha

-act

inin

2

0 1

0

Cal

cium

han

dlin

g H

CM

-

--

R

yR2

1q42

.1-q

43

Car

diac

ryan

odin

e re

cept

or

- -

-

JP

H2

20q1

2 Ju

ncto

phili

n-2

<1

<1

0

P

LN6q

22.1

P

hosp

hola

mba

n -

- -

Met

abol

ic H

CM

P

RK

AG

2 7q

35- q

36.3

6 A

MP

-act

ivat

ed p

rote

in k

inas

e -

- -

LA

MP

2Xq

24

Lyso

som

e-as

soci

ated

m

embr

ane

prot

ein

2 -

- -

G

LAXq

22

Alp

ha-g

alac

tosi

dase

A

- -

-

FX

N9q

13

Frat

axin

-

- -

- in

dica

tes

that

no

geno

type

-phe

noty

pe s

tudi

es in

volv

ing

a la

rge

coho

rt of

unr

elat

ed p

atie

nts

have

bee

n pe

rform

ed to

est

imat

e th

e fre

quen

cy o

f tha

t gen

e’s

parti

cula

r inv

olve

men

t in

that

par

ticul

ar m

orph

olog

ical

sub

type

of H

CM

.

The last important genetic subgroup of HCM is that of the metabolic HCM, involving mitochondrial and lysosomal proteins. In 2005, Arad et al. first described mutations in lysosome-associated membrane protein-2 encoded by LAMP2 and protein kinase gamma-2 encoded by PRKAG2 in glycogen storage disease-associated genes mimicking the clinical phenotype of HCM[44, 45, 46, 47]. In 2005, a mutation in FXN-encoded frataxin was described in a patient with HCM. Although this patient also harbored a myofilament mutation in MYBPC3-encoded myosin binding protein C, functional characterization showed significant influence of the FXN-mutant on the phenotype, suggesting that the observed alterations in energetics may act in synergy with the present myofilament mutation[48]. Similar to PRKAG2 and LAMP2, Fabry’s disease can express predominant cardiac features of left ventricular hypertrophy. Over the years, mutations in GLA-encoded alpha-galactosidose A have been found in patients with this multi-system disorder [49, 50, 51].

Although up to 24 HCM-susceptibility genes involving different pathways have been identified, the search for novel mutations in new genes continues. Recently, a genome wide-linkage study identified a new locus for HCM in a large family with left ventricular hypertrophy located to chromosome 7. Subsequent studies of genes located to this region however have thus far not yielded the causative gene[52]. As a result of the increasing genetic heterogeneity of HCM, a classification based on functional genetics might seem very helpful, but in light of the low yield of mutations in a large number of these genes as well as the commercial availability of just a small number of these genes, a phenotypic classification might be a more useful tool in looking at this disease from a clinical practice vantage point.

16

Genotype-phenotype analyses in HCM

Numerous studies have tried to identify phenotypic characteristics most indicative of myofilament/sarcomeric-HCM to facilitate genetic counseling and strategically direct clinical genetic testing[29, 31, 32, 53, 54, 55, 56]. Although several phenotype-genotype relationships have emerged to enrich the yield of genetic testing, these patient profiles have not been particularly clinically informative. An important discovery, linking the echocardiographically determined septal morphology to the underlying genetic substrate, was recently made.

The first link to be drawn between septal morphologies was a result of HCM study by Lever and colleagues in the 1980s, in which septal contour – classified as reverse septal contour, sigmoidal septal contour, apical - and neutral contour - was found to be age-dependent with a predominance of sigmoidal-HCM being present in the elderly[57]. In the early 90’s Seidman et al described an early genotype-phenotype observation involving a small number of patients and family members and discovered that patients with mutations in the beta myosin heavy chain (MYH7-HCM) generally had reversed curvature septal contours (reverse curve-HCM)[58].

Inspired by these two initial observations, we recently finished a large genotype-phenotype analysis correlating the septal morphology with the underlying genotype. After extensive analysis of the echocardiograms of 382 previously genotyped and published patients[32, 53, 56], we observed that sigmoidal-HCM (47% of cohort) and reverse curve-HCM (35% of cohort) were the two most prevalent anatomical subtypes of HCM, and discovered that the septal contour was the strongest predictor for the presence of a myofilament mutation, regardless of age [59]. Multivariate analysis in this cohort demonstrated septal morphology was the only independent predictor of myofilament HCM with an odds ratio of 21 (p<0.001), when reverse curve morphology was present[59]. Apical HCM, in which the hypertrophy is mostly concentrated around the apex of the heart, was found in 10% (n=37) of our cohort. The yield of the commercially available HCM genetic test for myofilament-HCM was 79% in reverse curve-HCM but only 8% in patients with sigmoidal-HCM. Of the smaller subgroup of patients with apical HCM, 32% had a mutation in one of the myofilaments [56].

17

These observations may facilitate echo-guided genetic testing by enabling informed genetic counseling about the a priori probability of a positive genetic test based upon the patient’s expressed anatomical phenotype (Figure 1). In addition, the paucity of myofilament mutations in sigmoidal-HCM opens the door for research to elucidate the molecular/genetic determinants of sigmoidal HCM.

Figure 1: Functional subgroups of genetic hypertrophic cardiomyopathy (HCM) and the yield of genetic testing for the two most common septal morphologies with their respective subgroup. Shown are the most important functional subgroups of genetically mediated HCM and the yield of mutations over various cohorts. Blue arrows indicate the functional relationship between the different elements. The black arrows show the yield of genetic testing for the subgroups of myofilament HCM and Z-disc HCM and their morphologic subgroups. LAMP2, lysosome-associated membrane protein 2; PLN, phospholamban; PRKAG2, AMP-activated protein kinase; SR, sarcoplasmic reticulum;RyR2, cardiac ryanodine receptor.

18

With the majority of known myofilament proteins studied, except for a complete analysis of the giant protein TTN-encoded titin, recent research has been focused proteins beyond the cardiac myofilaments, especially proteins involved in the cyto-architecture and cardiac stretch sensor mechanism of the cardiomyocyte localized to the cardiac Z-disc (Figure 1). The Z-disc is an intricate assembly of proteins at the Z-line of the cardiomyocyte sarcomere. Extensively reviewed, proteins of the Z-disc are important in the structural and mechanical stability of the sarcomere as they appear to serve as a docking station for transcription factors, calcium signaling proteins, kinases and phosphatases [60, 61]. In addition, this assembly of proteins seems to serve as a way station for proteins that regulate transcription by aiding in their controlled translocation between the nucleus and the Z-disc[60, 61].

With all of these roles, a main implication for the Z-disc is its involvement in the cardiomyocyte stretch sensing and response systems[62]. Mutations in three such proteins localized to the cardiac Z-disc, CSRP3-encoded muscle LIM protein (MLP), TCAP-encoded telethonin and VCL-encoded vinculin, including its cardiac specific insert of exon 19 that yields metavinculin, have previously been established as both HCM[34, 35, 36, 38, 39] and dilated cardiomyopathy (DCM)-susceptibility genes[34, 35, 36, 38, 63, 64]. Additionally, it is now fully appreciated that these divergent cardiomyopathic phenotypes of HCM and DCM are partially allelic disorders with ACTC, MYH7, TNNT2, TPM1, MYBPC3, TTN, MLP, TCAP, and VCL established as both HCM- and DCM-susceptibility genes[34, 35, 38, 63, 65, 66, 67, 68, 69].

Mutations in ACTN2-encoded alpha-actinin-2 (ACTN2) and LDB3-encoded LIM domain binding 3 (LDB3) as novel HCM-susceptibility genes[37] were described. Building on our discovery linking reverse-curve HCM to the presence of myofilament mutation, and recognizing that the Z-disc may transduce multiple signaling pathways during stress, translating into hypertrophic responses, cell growth and remodeling [70], we have observed that Z-disc HCM, in contrast to myofilament HCM, is preferentially sigmoidal. Eleven out of 13 patients with Z-disc HCM had a sigmoidal septal contour and no reverse septal curvatures were seen [37]. We speculate that Z-disc HCM leads to a hypertrophic response that is expressed in the areas of highest stress (i.e. LVOT) and therefore predisposes to a sigmoidal septal contour.

19

Intriguing conclusions can be drawn from these observations. Whereas in initial morphologic studies, sigmoidal-HCM seemed to be associated with older age [57], the underlying genotype rather than age appears to be the predominant determinant of septal morphology[59]. Furthermore, Z-disc HCM seems to have a predilection for sigmoidal contour status. Given that the vast majority of our patients with sigmoidal HCM still lack a putative disease-causing mutation, the molecular underpinnings responsible for a sigmoidal morphology remain to be elucidated. Alternatively, it seems plausible that a HCM-predisposing mutation might not be the principle determinant for many patients with sigmoidal-HCM. Instead, a multi-factorial model may be responsible for this subtype of clinically diagnosed HCM.

In this model, the sum of all contributors – the presence or absence of a mutation or LVH promoting polymorphisms[71], an unidentified genetic substrate, environmental factors and hypertension, culminates in what is clinically labeled as HCM. This multi-factorial model for sigmoidal-HCM is supported by the significantly older age at diagnosis of patients with sigmoidal-HCM (49 years) compared to those with reverse curve-HCM (32 years)[59] and the fact that nearly 20% of patients classified with sigmoidal-HCM were noted to have mild hypertension[59]. Diagnosed with HCM by experienced physicians, a subset of this group may have a basal septum more sensitive to the pro-hypertrophy trigger of increased afterload, precipitating basal septal hypertrophy (sigmoidal disease).

20

Conclusions

Genomic medicine has entered the clinical practice as it pertains to the evaluation and management of HCM. The diagnostic utility of genetic testing for HCM diseases is clearly evident, but strategic choices have to be made with the growing number of genes implicated in this disease. With recent discoveries in genotype-phenotype relationships, especially pertaining the echocardiographic septal shape and the underlying pathogenetic mutation, time has come to further subdivide the one disease we call HCM.

Clinical HCM specialists are accustomed already to prefacing the HCM label with physiological descriptors of obstructive- and non-obstructive-HCM and anatomical/morphological descriptors: reverse curve- , sigmoidal- , and apical-HCM. Accordingly, a pathogenetic subdivision seems warranted. Just as there is no prerequisite for clinically diagnosed HCM to necessarily be obstructive or reverse curve in nature, it should not be mandated that clinically diagnosed HCM requires a genetic perturbation in one of the sarcomeric myofilaments. Instead, what is emerging is a clear picture that the two most common anatomical/morphological subtypes of HCM (reverse curve- and sigmoidal-HCM) largely emanate from fundamentally distinct pathogenetic mechanisms. Herein, most (but not all) of reverse curve-HCM is indeed a “disease of the sarcomere” and most (but not all) sigmoidal-HCM is in search of its etiology.

21

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23

31. Van Driest SV, Ackerman MJ, Ommen SR, Shakur R, et al. Prevalence and severity of "benign" mutations in the beta myosin heavy chain, cardiac troponin-T, and alpha tropomyosin genes in hypertrophic cardiomyopathy. Circulation 2002; 106: 3085-3090.

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35. Hayashi T, Arimura T, Itoh-Satoh M, Ueda K, et al. Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy. J Am Coll Cardiol 2004; 44(11):2192-2201.

36. Bos JM, Poley RN, Ny M, Tester DJ, et al. Genotype-phenotype relationships involving hypertrophic cardiomyopathy-associated mutations in titin, muscle LIM protein, and telethonin. Mol Genet Metab 2006; 88(1): 78-85.

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40. Minamisawa S, Sato Y, Tatsuguchi Y, Fujino T, et al. Mutation of the phospholamban promoter associated with hypertrophic cardiomyopathy. Biochem Biophys Res Commun 2003; 304(1): 1-4.

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42. Fujino N, Ino H, Hayashi K, Uchiyama K, et al. A novel missense mutation in cardiac ryanodine receptor gene as a possible cause of hypertrophic cardiomyopathy: evidence from familial analysis. Circulation 2006; 114(18): II-165: 915.

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24

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48. Van Driest SL, Gakh O, Ommen SR, Isaya G, et al. Molecular and functional characterization of a human frataxin mutation found in hypertrophic cardiomyopathy. Mol Genet Metab 2005; 85(4):280-5

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50. Sachdev B, Takenaka T, Teraguchi H, Tei C, et al. Prevalence of Anderson-Fabry disease in male patients with late onset hypertrophic cardiomyopathy. Circulation 2002; 105(12): 1407-1411.

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52. Song L, DePalma SR, Kharlap M, Zenovich AG, et al. Novel locus for an inherited cardiomyopathy maps to chromosome 7. Circulation 2006; 113(18): 2186-2192.

53. Van Driest SL, Jaeger MA, Ommen SR, Will ML, et al. Comprehensive analysis of the beta-myosin heavy chain gene in 389 unrelated patients with hypertrophic cardiomyopathy. JAm Coll Cardiol 2004; 44(3): 602-610.

54. Woo A, Rakowski H, Liew JC, Zhao MS, et al. Mutations of the beta myosin heavy chain gene in hypertrophic cardiomyopathy: critical functional sites determine prognosis. Heart 2003; 89(10): 1179-1185.

55. Richard P, Charron P, Carrier L, Ledeuil C, et al. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation 2003; 107(17): 2227-2232.

56. Van Driest SL, Ellsworth EG, Ommen SR, Tajik AJ, et al. Prevalence and spectrum of thin filament mutations in an outpatient referral population with hypertrophic cardiomyopathy. Circulation 2003; 108: 445-451.

57. Lever HM, Karam RF, Currie PJ, Healy BP. Hypertrophic cardiomyopathy in the elderly. Distinctions from the young based on cardiac shape. Circulation 1989; 79(3): 580-589.

58. Solomon SD, Wolff S, Watkins H, Ridker PM, et al. Left ventricular hypertrophy and morphology in familial hypertrophic cardiomyopathy associated with mutations of the beta-myosin heavy chain gene. J Am Coll Cardiol 1993; 22(2): 498-505.

59. Binder J, Ommen SR, Gersh BJ, Van Driest SL, et al. Echocardiography-guided genetic testing in hypertrophic cardiomyopathy: septal morphological features predict the presence of myofilament mutations. Mayo Clin Proc 2006; 81(4): 459-467.

60. Frank D, Kuhn C, Katus HA, Frey N. The sarcomeric Z-disc: a nodal point in signalling and disease. J Mol Med 2006; 84(6):446-68

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25

62. Knoll R, Hoshijima M, Hoffman HM, Person V, et al. The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell 2002; 111(7): 943-955.

63. Mohapatra B, Jimenez S, Lin JH, Bowles KR, et al. Mutations in the muscle LIM protein and alpha-actinin-2 genes in dilated cardiomyopathy and endocardial fibroelastosis. Mol Genet Metab 2003; 80(1-2): 207-215.

64. Olson TM, Illenberger S, Kishimoto NY, Huttelmaier S, et al. Metavinculin mutations alter actin interaction in dilated cardiomyopathy. Circulation 2002; 105(4): 431-437.

65. Kamisago M, Sharma SD, DePalma SR, Solomon S, et al. Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N Engl J Med 2000; 343(23): 1688-1696.

66. Olson TM, Doan TP, Kishimoto NY, Whitby FG, et al. Inherited and de novo mutations in the cardiac actin gene cause hypertrophic cardiomyopathy. J Mol Cell Cardiol 2000; 32:1687-1694.

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68. Gerull B, Gramlich M, Atherton J, McNabb M, et al. Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy. Nat Genet 2002; 30(2): 201-204.

69. Daehmlow S, Erdmann J, Knueppel T, Gille C, et al. Novel mutations in sarcomeric protein genes in dilated cardiomyopathy. Biochem Biophys Res Commun 2002; 298(1): 116-120.

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71. Perkins MJ, Van Driest SL, Ellsworth EG, Will ML, et al. Gene-specific modifying effects of pro-LVH polymorphisms involving the renin-angiotensin-aldosterone system among 389 unrelated patients with hypertrophic cardiomyopathy. Eur Heart J 2005; 26(22): 2457-2462.

26

Chapter 2

Genotype-Phenotype Relationships Involving Hypertrophic Cardiomyopathy-Associated Mutations in

Titin, Muscle LIM Protein and Telethonin

J. Martijn Bos, Rainer N. Poley, Melissa Ny, David J. Tester, Xiaolei Xu, Matteo Vatta, Jeffrey A. Towbin, Bernard J. Gersh, Steve R. Ommen, Michael J. Ackerman

Mol Genet Metab 2006; 88(1): 78 – 85

Letter to editor:

Mol Genet Metab 2006; 88(2): 199 – 200

Mol Genet Metab 2006; 89(3): 286 – 287

Abstract Background: TTN-encoded titin, CSRP3-encoded muscle LIM protein, and TCAP-

encoded telethonin are Z-disc proteins essential for the structural organization of the

cardiac sarcomere and the cardiomyocyte’s stretch sensor. All 3 genes have been

established as cardiomyopathy-associated genes for both dilated cardiomyopathy

(DCM) and hypertrophic cardiomyopathy (HCM). Here, we sought to characterize the

frequency, spectrum, and phenotype associated with HCM-associated mutations in

these 3 genes in a large cohort of unrelated patients evaluated at a single tertiary

outpatient center.

Methods: DNA was obtained from 389 patients with HCM (215 male, left ventricular

wall thickness of 21.6 ± 6 mm) and analyzed for mutations involving all translated

exons of CSRP3 and TCAP and targeted HCM-associated exons (2, 3, 4 and 14) of

TTN using polymerase chain reaction (PCR), denaturing high performance liquid

chromatography (DHPLC), and direct DNA sequencing. Clinical data was extracted

from patient records and maintained independent of the genotype.

Results: Overall, 16 patients (4.1%) harbored a Z-disc mutation: 12 had a MLP

mutation and 4 patients a TCAP mutation. No TTN mutations were detected. Seven

patients were also found to have a concomitant myofilament mutation. Seven patients

with a MLP-mutation were found to harbor the DCM-associated, functionally

characterized W4R mutation. W4R-MLP was also noted in a single white control

subject. Patients with MLP/TCAP-associated HCM clinically mimicked myofilament-

HCM.

Conclusions: Approximately 4.1% of unrelated patients had HCM-associated MLP or

TCAP mutations. MLP/TCAP-HCM phenotypically mirrors myofilament-HCM and is

more severe than the subset of patients who still remain without a disease-causing

mutation. The precise role of W4R-MLP in the pathogenesis of either DCM or HCM

warrants further investigation.

Keywords

Genetics, genes, hypertrophy, cardiomyopathy, Z-disc, muscle LIM protein, telethonin,

TCAP, titin

28

Introduction Affecting one in 500 persons, hypertrophic cardiomyopathy (HCM) is a disease

associated with remarkable genotypic and phenotypic heterogeneity[1, 2]. Clinical

outcomes range from an entirely asymptomatic course with normal longevity to chronic

progressive heart failure or sudden cardiac death (SCD). Indeed, HCM is one of the

leading causes of SCD in young persons [1].

The most common genetically mediated form of HCM is myofilament-HCM with

hundreds of disease-associated mutations in 8 genes encoding proteins critical to the

sarcomere’s thick - [ -myosin heavy chain (MYH7)[3], regulatory myosin light chain

(MYL2) and essential myosin light chain (MYL3)][4], intermediate - [myosin binding

protein C (MYBPC3)][5], and thin myofilament [cardiac troponin T (TNNT2), -

tropomyosin (TPM1)[6], cardiac troponin I (TNNI3)[7], and actin (ACTC)[8, 9]].

Myofilament-HCM accounts for approximately 40-65% of HCM among cohorts of

unrelated patients[10]. In general, patients with myofilament-HCM have greater

hypertrophy and present at a younger age than those who remain without an

established disease-causing mutation[11]. The 2 most common genotypes of

myofilament-HCM, MYBPC3- and MYH7-HCM, are phenotypically indistinguishable

from each other[12, 13, 14, 15, 16, 17, 18, 19, 20].

Besides perturbations involving the sarcomere’s myofilaments, the Z-disc,

which comprises a cadre of proteins involved in cardiomyocyte cytoarchitecture and

mechano-sensor- signaling, has emerged recently as host to several HCM-associated

mutations extending the spectrum of “sarcomeric”-HCM. To date, 3 genes encoding

critical Z-disc proteins: TTN-encoded titin, CSRP3-encoded muscle LIM protein (MLP),

and the TCAP-encoded telethonin, have been implicated in the pathogenesis of both

dilated cardiomyopathy (DCM) and HCM[21, 22, 23, 24].

29

As part of the cardiomyocyte stretch response machinery, TTN-encoded titin,

which extends throughout half of the sarcomere from the M-line to the Z-disc is the

largest of the three proteins; mapped on chromosome 2q31, TTN encodes for a giant

26,926 amino-acid protein with a molecular weight of 2,993 kD[25]. CSRP3-encoded

MLP and TCAP-encoded telethonin are mapped to 11p15.1 and 17q12 respectively

and contain 194 and 167 amino acids respectively [26, 27]. Prior to this study, 1 HCM-

associated mutation in TTN (R740L-TTN)[28], 3 HCM-associated mutations in MLP

(L44P-MLP, C58G-MLP and S54R/E55G-MLP)[22], and 2 HCM-associated mutations

in TCAP (T137I-TCAP and R153H-TCAP) have been reported[21].

Having completed a comprehensive mutational analysis involving all translated

exons of the 8 genes responsible for myofilament-HCM[14, 15, 29, 30], we sought to

determine the frequency, spectrum, and phenotype associated with these 3 genes that

encode essential Z-disc proteins among a large cohort of unrelated patients diagnosed

clinically with HCM.

30

Methods Study population

Following a written informed consent for this IRB-approved research protocol, blood

samples were obtained from 389 unrelated patients with HCM (215 male, left

ventricular wall thickness of 21.6 6 mm) evaluated at the Mayo Clinic’s HCM clinic

between April 1997 and December 2001. Subsequently DNA was extracted from the

blood samples using Purgene DNA extraction kits (Gentra, Minneapolis, Minnesota).

HCM-associated mutational analysis of TTN, CSRP3, and TCAP

Using polymerase chain reaction (PCR) and denaturing high performance liquid

chromatography (DHPLC) (WAVE, Transgenomic, Omaha, Nebraska), the 3 genes

implicated in Z-disc-HCM: TTN-encoded titin, CSRP3-encoded muscle LIM protein,

and TCAP-encoded telethonin, were analyzed. Abnormal elution profiles were further

characterized by direct DNA sequencing (ABI Prism 377; Applied Biosystem, Foster

City, California).

For TTN, only a targeted analysis of the exons (2, 3, 4 and 14) hosting

cardiomyopathy-associated mutations was performed while a comprehensive open

reading frame/splice-site analysis was conducted for all translated exons of CSRP3 (5

exons) and TCAP (2 exons). A topological schematic of both MLP and telethonin

including key functional domains is depicted in Figure 1. Primers, annealing

temperatures and optimized WAVE conditions are available upon request. Four

hundred reference alleles, derived from 100 white and 100 black healthy controls

(Coriell Cell Repositories), were also examined to determine whether an identified

amino acid variant was a common polymorphism. The non-synonymous mutations

were annotated using the single letter convention as in L44P whereby the wild type

leucine (L) at residue 44 has been replaced by proline (P).

Statistical analysis

Analysis of variance tests were used to assess differences between continuous

variables; contingency tables or z-tests were used as appropriate to analyze nominal

variables independency of the different variables. Student’s T-tests were performed to

elucidate differences between the different subgroups. A p-value less than 0.05 was

considered statistically significant.

31

Figure 1: Topological schematic of muscle LIM protein and telethonin. Shown are the important domains of the protein. For MLP, the TCAP-binding domain, both LIM-domains and its nuclear localization signal (NLS) are shown. For telethonin, the MLP -, titin- and minK-binding domains are shown. Amino-acid localization of the specific domains between parentheses

32

Results

Table 1 summarizes the phenotype of the entire HCM cohort including those with

perturbations involving either MLP or telethonin. The mean age at diagnosis for our

total cohort was approximately 41 ± 19 years with 216 patients (55%) having cardiac

symptoms at presentation and 60 (15%) having received an implantable cardioverter-

defibrillator (ICD). The mean maximum left ventricular wall thickness (LVWT) was 21.6

± 6 mm. Of the 389, 161 (41%) were treated in part by a surgical myectomy, reflecting

the surgical referral bias and subsequent over-representation of obstructive HCM in

this cohort. Approximately one-third had a family history of HCM whereas one-seventh

was found to have a family history of sudden cardiac death. Myofilament-HCM was

demonstrated previously for 147 of the 389 subjects (38%)[14, 15, 30].

Overall, 16 (4.1%) individuals with HCM hosted possible mutations in the genes

underlying Z-disc-HCM: TTN (0), CSRP3 (12), and TCAP (4). The clinical phenotypes

of these patients are described in Table 2. The average at diagnosis for MLP (CSRP3)

- and TCAP-associated HCM was 48.5 ± 17 and 38.8 ± 9 years, respectively, while the

mean maximal left ventricular wall thickness (MLVWT) was 20.1 ± 3 mm and 29.5 ± 12

mm, respectively. Three patients (25%) with a MLP-mutation and 1 patient (25%) with

a TCAP-mutation reported a family history of HCM, while 2 and 1 patient (17 and 25%)

respectively had a family history of SCD. A total of 8 patients underwent a surgical

myectomy due to refractory symptoms despite optimal medical treatment.

33

Table 1: Clinical characteristics of HCM cohort

HCM-cohort

Genotype negative

Singlemyofilament

mutationMLP TCAP

No. of

individuals 389 233 140 12 4

Sex,

male/female 215/174 127/106 79/61 7/5 2/2

Age at Dx 41.2 ±19 45.1 ± 19 34.5 ± 17 48.5 ± 17 38.8 ± 9

Cardiac

symptoms 216 (56%) 128 (55%) 74 (53%) 9 (75%) 4 (100%)

Max LVWT

(mm) 21.6 ± 6 20.6 ± 6 23.0 ± 7 20.1 ± 3 29.5 ± 12

LVWT 25 mm 78 (20%) 38 (16%) 38 (28%) 0 2 (50%)

Resting LVOTO

(mmHg) 46.6 ± 42 46.6 ± 42 42.8 ± 42 80 ± 43 75 ± 38

Pos. FH for

HCM 121 (31%) 54 (23%) 61 (44%) 3 (25%) 1 (25%)

Pos. FH for

SCD 54 (14%) 26 (11%) 27 (19%) 2 (17%) 1 (25%)

Myectomy 160 (41%) 92 (39%) 62 (44%) 5 (42%) 3 (75%)

Pacemaker 67 (17%) 35 (15%) 26 (19%) 5 (42%) 2 (50%)

ICD 60 (15%) 23 (10%) 36 (26%) 1 (8%) 0

Multiple or

concomitant

myofilament

mutation

147 - 10/140 6/12 1/4

Values are mean ± SD or % (n). Dx indicates diagnosis; FH, family history; HCM, Hypertrophic cardiomyopathy; ICD, implantable cardioverter-defibrillator; LVOTO, left ventricular outflow tract obstruction; LVWT, left ventricular wall thickness; SCD, sudden cardiac death

34

HCM-associated MLP mutations

Figure 2 depicts the mutations found in the CSRP3-encoded MLP; novel mutations

are indicated by an asterisk. Five CSRP3 variants were identified in 12 patients,

including 4 missense mutations and 1 frame-shift mutation, involving residues highly

conserved across species (data not shown) and not seen in 400 reference alleles.

Figure 2: Schematic representation of mutations in muscle LIM protein and telethonin. Representation of mutations found in our cohort of 389 patients with HCM. The L44P-MLP has been previously published as a HCM-associated mutation. The W4R-MLP mutation has been previously published and functionally characterized in patients with DCM. Novel mutations are indicated with an asterisk.

35

Clinical phenotypes are described in Table 2. K42fs/165 and Q91L were

detected in patients having no HCM-associated myofilament mutations (cases 8 and 12). The previously published HCM-causing mutation (L44P, case 9) localized to the

LIM1 -actinin binding domain while the R64C and Y66C mutations (cases 10 and 11)

localized to the 6 amino acid nuclear localization signal (NLS). These 3 mutations

(cases 9-11) were detected in patients also hosting HCM-associated myofilament

mutations.

The missense mutation, W4R-MLP, which localizes to telethonin’s binding domain,

was noted in 7 patients (cases 1-7). Three of these patients (cases 5-7) also had a

mutation involving either the beta myosin heavy chain or myosin binding protein C.

W4R was also observed in one of the 400 reference alleles examined (a healthy

Caucasian control).

36

HCM-associated TCAP mutations

Three different, novel TCAP mutations were identified in 4 patients with HCM (Table 2,

cases 13 – 16). Two patients (cases 13 and 14) had an in-frame deletion involving

glutamic acid at position 13 (E13del). The R70W mutation was located in the reciprocal

MLP-binding domain of telethonin in a patient (case 15) with a MLVWT of 46 mm and

a positive family history for HCM. The titin-binding domain of telethonin was host to a

missense mutation, P90L, for one patient (case 16) who also had a missense mutation

involving myosin binding protein C.

Genotype-phenotype relationships in MLP/TCAP-HCM

Compared to patients still lacking a mutation (genotype negative) and patients with

myofilament-HCM, patients with mutations involving either MLP or TCAP more closely

resembled the subset with myofilament-HCM (Figure 3 a-c). The subset with MLP-

HCM were, however, more obstructive (80 ± 43 mmHg) than both myofilament-HCM

(42.8 ± 42 mmHg; p = 0.01) and genotype negative-HCM (46.6 ± 42 mmHg; p =

0.007). Despite the small sample size, patients with TCAP-HCM had significantly

greater MLVWT (29.5 ± 12 mm) compared with either genotype negative- (20.6 ± 6

mm; p = 0.006), myofilament- (23.0 ± 7.0 mm; p = 0.04), or MLP-HCM (20.1 ± 3 mm; p

= 0.01) and a similar age at diagnosis as myofilament positive-HCM (38.8 ± 9 vs. 34.5

± 17 years old; p = 0.6). When a subset analysis of patients with either Z-disc only

(n=9) mutations or Z-disc mutation plus a concomitant myofilament (n=7) mutation was

performed, the phenotypes of these two subgroups did not differ from each other on

MLVWT (23.9 ± 9 mm vs. 20.6 ± 3 mm; p = 0.3), MLVOTO (67.4 ± 49 mmHg vs. 93.5

± 20 mmHg; p = 0.2) or age at diagnosis (46.3 ± 6 yrs vs. 45.8 ± 6.8 yrs; p = 0.9),

supporting the role of MLP/TCAP mutations in pathogenesis of HCM.

37

Tabl

e 2.

Clin

ical

pro

files

of P

atie

nts

with

a H

CM

-ass

ocia

ted

CS

PR

3 (M

LP) o

r TC

AP

Mut

atio

n

C a s eG

ene

Mut

atio

n(e

xon)

M

yofil

amen

t M

utat

ion

Age/

Se

x

Age

at DxRa

ce*

Sym

ptom

s a

tPr

esen

tatio

n Su

bseq

uent

sy

mpt

oms

A F

Max

.LV

WT

(mm

)

Res

ting

LVO

TO

(mm

Hg)

FH of HC

M

FH o

f SC

D(A

ge a

t SC

D) †

Tr

eatm

ent

1C

SR

P3

W4R

(1)

80/F

69

1An

gina

, dys

pnea

An

gina

, dy

spne

a Y

1521

Yes

No

PM

2C

SR

P3

W4R

(1)

29/M

161

Asym

ptom

atic

D

yspn

ea

N25

0N

oN

o…

3C

SR

P3

W4R

(1)

56/M

411

Asym

ptom

atic

An

gina

, dy

spne

a,

(pre

)syn

cope

N17

32No

NoPM

4C

SR

P3

W4R

(1)

78/F

68

1n/

aD

yspn

ea

N20

0N

oN

oM

yect

omy

5C

SR

P3

W4R

(1)

F111

3I-

MYB

PC3

59/M

501

Asym

ptom

atic

D

yspn

ea,

(pre

)syn

cope

N

2311

7No

NoM

yect

omy,

PM

, IC

D

6C

SR

P3

W4R

(1)

T137

7M-

MYH

750

/F

431

n/a

Angi

na,

dysp

nea,

(p

re)s

ynco

pe

N18

86Ye

sN

oM

yect

omy

7C

SR

P3

W4R

(1)

I511

T-M

YH7

60/F

53

1D

yspn

ea

Dys

pnea

, (p

re)s

ynco

pe

N16

0Ye

sN

o…

8C

SR

P3

K42

fs/1

65

(2)

53/M

462

Angi

na, d

yspn

ea

Angi

na,

dysp

nea,

(p

re)s

ynco

pe

N18

112

NoNo

…

9C

SR

P3

L44P

(2)

G10

41 fs

/5-

MYB

PC3

71/F

62

n/a

Pres

ynco

pe

Angi

na,

dysp

nea,

(p

re)s

ynco

pe

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100

Yes

Yes

(40,

32,

39)

Mye

ctom

y,

PM

10C

SR

P3

R64

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) I1

131T

-M

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372

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n/a

Dys

pnea

, (p

re)s

ynco

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na,

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58No

No…

11C

SR

P3

Y66C

(2)

R16

2Q-

TNN

I3

36/M

281

n/a

Asym

ptom

atic

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1910

0N

oYe

s(4

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omy

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) 57

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gina

An

gina

, dy

spne

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(pre

)syn

cope

Y22

18No

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13T

CA

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3del

(1)

53/M

471

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pnea

, (p

re)s

ynco

pe

Dys

pnea

, (p

re)s

ynco

pe

N22

100

NoNo

….

14T

CA

PE1

3del

(1)

42/M

371

Angi

na, d

yspn

ea

Angi

na,

dysp

nea

N30

81Ye

sYe

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4)M

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omy,

IC

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15T

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44

1As

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Mye

ctom

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PM

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PP9

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2)

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26

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na,

dysp

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pr

esyn

cope

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100

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Mye

ctom

y,

PM

AF, a

trial

fibr

illatio

n; D

x, d

iagn

osis;

FH

, fam

ily h

isto

ry; H

CM

, Hyp

ertro

phic

card

iom

yopa

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ICD

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plan

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e ca

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t ven

tricu

lar

outfl

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act o

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n; L

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left

vent

ricul

ar w

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ness

; n/a

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ava

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e; P

M, p

acem

aker

;SC

D, s

udde

n ca

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c de

ath;

* 1 =

Cau

casia

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= H

ispan

ic; †

, in

a fir

st d

egre

e re

lativ

e

Figure 3a

Figure 3b

40

Figure 3c

Figure 3a – c: Degree of hypertrophy (a), degree of left ventricular outflow tract obstruction (b) and age at diagnosis (c) for genotyped subjects. Genotyped patients with hypertrophic cardiomyopathy are grouped on the X-axis as hosting as hosting no putative mutation (genotype negative), hosting a myofilament mutation (myofilament-HCM), a MLP-mutation or a TCAP-mutation. Unless otherwise noted, all pair wise comparisons are not statistically significant. *, p<0.05 compared to all other groups

41

Discussion

As critical components of the dynamic protein scaffolding between the sarcomere and

cytoskeleton at the Z-line, the titin-muscle LIM protein-telethonin complex is involved in

both cyto-architecture and mechano-signaling, thus serving as a potential link between

myofilament-HCM and Z-disc-HCM. 1 Prior to this study, 1 HCM-associated mutation

in titin[28], 4 HCM-associated mutations in MLP[22, 31] and 2 HCM-associated

mutations in telethonin have been reported[21]. In addition, consistent with the notion

that HCM and DCM are often allelic disorders, several DCM-associated mutation in

these 3 Z-disc proteins have been discovered as well[23, 24, 32, 33, 34]. Based upon

our observations in this study, the genes encoding Z-disc proteins currently implicated

so far as only DCM-susceptibility genes constitute rational candidate genes to explore

in HCM.

This study represents the largest series of patients examined for the 3 known

subtypes of Z-disc-HCM whereby approximately 4% of unrelated patients harbored a

mutation in either MLP (CSRP3) or TCAP. We did not observe any mutations in the

giant protein, titin, which extends across half of the entire sarcomere. However, only

those regions implicated previously in either HCM or DCM were examined. Among the

12 patients with a non-synonymous, amino-acid altering variant in the CSRP3-encoded

MLP, a compelling case for disease-association exists at the present time for 5

patients (cases 8-12). Besides the L44P-MLP, R64C-MLP, and Y66C-MLP missense

mutations, 3 patients (cases 9-11) also possessed a concomitant myofilament

mutation: G1041fs/5-MYBPC3, I1131T-MYBPC3 and R162Q-TNNI3 respectively. The

L44P-MLP variant along with the K42fs/165-MLP frameshift mutation localize to the

LIM1-domain which is responsible for binding to -actinin. In a yeast 2-hybrid assay,

Geier et al. recently showed a significantly impaired binding affinity for -actinin due to

C58G-MLP[22].

42

The pathogenic mechanism for HCM in these patients hosting both MLP

variants and myofilament mutations may be due to synergistic heterozygosity (two-hit

hypothesis) as we have previously demonstrated in a patient hosting a known myosin

binding protein C missense mutation and a functionally-compromised frataxin

mutation[35]. Previously, we demonstrated that among the 140 patients in our cohort

previously established to have solely myofilament-HCM, 10 patients (7%) hosted 2

myofilament mutations with one of the variants usually involving myosin binding protein

C[14]. Supporting the notion that both variants contributed to the expressed

phenotype, these patients with multiple myofilament-HCM were younger at diagnosis

and had greater hypertrophy than those having a single myofilament mutation. Herein,

proportionately more patients with putative Z-disc-HCM also had a myofilament

mutation raising the possibility that some of these variants may represent false

positives. Future studies of the families represented by these HCM cases may shed

light on the relative contributions of both the myofilament and the Z-disc mutation in the

expressed phenotype.

The precise contribution of W4R-MLP (seen in 7 patients, cases 1-7) in the

pathogenesis of HCM remains an enigma. Four of the 7 patients with W4R-MLP in the

present study also have a published HCM-associated myofilament mutation. Initially,

W4R was discovered as a DCM-associated mutation and was reportedly absent in 640

normal reference alleles[34]. Localizing to the telethonin-binding domain of MLP, it was

not surprising to see in vitro assays demonstrating markedly reduced

interaction/localization with telethonin[34]. Transgenic mouse models of W4R-MLP

yield mice with a rather pronounced cardiomyopathy characterized by significant

ventricular dilation and systolic dysfunction[36].

Recently, W4R-MLP was observed in 1 of 137 unrelated patients with

HCM[31]. This variant was found in a patient with predominant apical HCM in which no

myofilament mutations were identified. However, these investigators also observed

W4R in 3 of 500 reference alleles (0.6% allelic frequency). We have now observed

W4R in 1/400 reference alleles. While clearly a phenotype producing mutation in an

overexpression transgenic mouse model, further studies are necessary to elucidate the

precise role of W4R-MLP in the pathogenesis of cardiomyopathies in humans.

43

Finally, 4 patients hosted mutations in telethonin with 1 patient also having a

myofilament Q998R-MYBPC3 genotype. These patients had severe hypertrophy

(mean MLVWT = 29.5 mm) whereas previously published TCAP probands had a mean

MLVWT of 20 mm. In particular, the patient in our study with R70W-TCAP had

massive hypertrophy with a septal wall thickness of 46 mm. No other mutations in

known HCM genes have been found in this individual. R70W-TCAP localizes to the

functional domain essential for binding MLP.

Most of the HCM- and DCM-associated mutations reported in these 3 Z-disc

proteins have not been characterized functionally. It remains to be determined whether

or not the various mutations selectively perturb force generating (HCM-predisposing)

or force transmitting (DCM-predisposing) functions.

44

Conclusions

In this study, HCM-susceptibility mutations in CSRP3 and TCAP represent uncommon

causes of HCM, with a prevalence similar to troponin I- and actin-HCM. The combined

clinical phenotype of MLP/TCAP-HCM resembles that of myofilament-HCM. Co-

segregation and functional studies are now needed to dissect the relative contributions

of the various Z-disc mutations to the pathogenesis and phenotypic expression of

HCM.

Acknowledgements

We are grateful to the patients seen at the HCM Clinic for their participation in this

study and to Mr. Doug Kocer, the nurse coordinator of the HCM Clinic.

45

References

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46

16. Erdmann J, Raible J, Maki-Abadi J, Hummel M, et al. Spectrum of clinical phenotypes and gene variants in cardiac myosin-binding protein C mutation carriers with hypertrophic cardiomyopathy. J Am Coll Cardiol 2001; 38(2): 322-330. 17. Morner S, Richard P, Kazzam E, Hellman U, et al. Identification of the genotypes causing hypertrophic cardiomyopathy in northern Sweden. J Mol Cell Cardiol 2003; 35(7): 841-849. 18. Garcia-Castro M, Reguero JR, Batalla A, Diaz-Molina B, et al. Hypertrophic cardiomyopathy: low frequency of mutations in the beta-myosin heavy chain (MYH7) and cardiac troponin T (TNNT2) genes among Spanish patients. Clinical Chemistry 2003; 49(8): 1279-1285. 19. Jaaskelainen P, Soranta M, Miettinen R, Saarinen L, et al. The cardiac beta-myosin heavy chain gene is not the predominant gene for hypertrophic cardiomyopathy in the Finnish population. J Am Coll Cardiol 1998; 32(6): 1709-1716. 20. Jaaskelainen P, Kuusisto J, Miettinen R, Karkkainen P, et al. Mutations in the cardiac myosin-binding protein C gene are the predominant cause of familial hypertrophic cardiomyopathy in eastern Finland. J Mol Med 2002; 80: 412-422. 21. Hayashi T, Arimura T, Itoh-Satoh M, Ueda K, et al. Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy. J Am Coll Cardiol 2004; 44(11): 2192-2201. 22. Geier C, Perrot A, Ozcelik C, Binner P, et al. Mutations in the human muscle LIM protein gene in families with hypertrophic cardiomyopathy. Circulation 2003; 107(10): 1390-1395. 23. Gerull B, Gramlich M, Atherton J, McNabb M, et al. Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy. Nat Genet 2002; 30(2): 201-204. 24. Itoh-Satoh M, Hayashi T, Nishi H, Koga Y, et al. Titin mutations as the molecular basis for dilated cardiomyopathy. Biochem Biophys Res Commun 2002; 291(2): 385-393. 25. Labeit S, Kolmerer B. Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science 1995; 270(5234): 293-296. 26. Valle G, Faulkner G, De Antoni A, Pacchioni B, et al. Telethonin, a novel sarcomeric protein of heart and skeletal muscle. FEBS Lett 1997; 415(2): 163-168. 27. Fung YW, Wang RX, Heng HH, Liew CC. Mapping of a human LIM protein (CLP) to human chromosome 11p15.1 by fluorescence in situ hybridization. Genomics 1995; 28(3): 602-603. 28. Satoh M, Takahashi M, Sakamoto T, Hiroe M, et al. Structural analysis of the titin gene in hypertrophic cardiomyopathy: Identification of a novel disease gene. Biochem Biophys Res Commun 1999; 262: 411-417. 29. Ackerman MJ, Van Driest SV, Ommen SR, Will ML, et al. Prevalence and age-dependence of malignant mutations in the beta-myosin heavy chain and troponin T gene in hypertrophic cardiomyopathy: a comprehensive outpatient perspective. J Am Coll Cardiol 2002; 39(12): 2042-2048. 30. Van Driest SL, Ellsworth EG, Ommen SR, Tajik AJ, et al. Prevalence and spectrum of thin filament mutations in an outpatient referral population with hypertrophic cardiomyopathy. Circulation 2003; 108: 445-451.

47

31. Newman B, Cescon D, Woo A, Rakowski H, et al. W4R variant in CSRP3 encoding muscle LIM protein in a patient with hypertrophic cardiomyopathy. Mol Genet Metab 2005; 84(4): 374-375. 32. Hayashi T, Arimura T, Ueda K, Shibata H, et al. Identification and functional analysis of a caveolin-3 mutation associated with familial hypertrophic cardiomyopathy. Biochem Biophys Res Commun 2004; 313(1): 178-184. 33. Mohapatra B, Jimenez S, Lin JH, Bowles KR, et al. Mutations in the muscle LIM protein and alpha-actinin-2 genes in dilated cardiomyopathy and endocardial fibroelastosis. Mol Genet Metab 2003; 80(1-2): 207-215. 34. Knoll R, Hoshijima M, Hoffman HM, Person V, et al. The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell 2002; 111(7): 943-955. 35. Van Driest SL, Gakh O, Ommen SR, Isaya G, et al. Molecular and functional characterization of a human frataxin mutation found in hypertrophic cardiomyopathy. Mol Genet Metab 2005; 85(4): 280-5 36. Arber S, Hunter JJ, Ross J, Jr., Hongo M, et al. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell 1997; 88(3): 393-403.

48

Chapter 3

Cardiac Ankyrin Repeat Protein Gene (ANKRD1)

Mutations in Hypertrophic Cardiomyopathy

Takuro Arimura*, J. Martijn Bos*, Akinori Sato, Toru Kubo, Hiroshi Okamoto, Hirofumi Nishi, Haruhito Harada, Yoshinori Koga, Mousumi Moulik, Yoshinori L. Doi, Jeffrey A. Towbin, Michael J. Ackerman, Akinori Kimura* These authors equally contributed to the work

J Am Coll Cardiol 2009; 54(4): 334 – 42

Editorial comment by:

L. Mestroni. Phenotypic Heterogeneity of Sarcomeric

Gene Mutations: a Matter of Gain and Loss?

J Am Coll Cardiol 2009; 54(4): 343 – 5

Abstract

Objectives: The purpose of this study was to explore a novel disease gene for

hypertrophic cardiomyopathy (HCM) and evaluate functional alteration(s) caused by

mutations.

Background: Mutations in genes encoding myofilaments or Z-disc proteins of the

cardiac sarcomere cause HCM, but the disease-causing mutations can be found in half

of the patients, indicating that novel HCM-susceptibility genes await discovery. We

studied a candidate gene ANKRD1 encoding for cardiac ankyrin repeat protein (CARP)

that is a Z-disc component interacting with N2A domain of titin/connectin and N-terminal

domain of myopalladin.

Methods: We analyzed 384 HCM patients for mutations in ANKRD1 and in the N2A

domain of titin/connectin gene (TTN). Interaction of CARP with titin/connectin or

myopalladin was investigated using co-immunoprecipitation assay to demonstrate the

functional alteration caused by ANKRD1 or TTN mutations. Functional abnormalities

caused by the ANKRD1 mutations were also examined at the cellular level in neonatal

rat cardiomyocytes.

Results: Three ANKRD1 missense mutations, Pro52Ala, Thr123Met and Ile280Val,

were found in 3 patients. All mutations increased binding of CARP to both titin/connectin

and myopalladin. In addition, TTN mutations, Arg8500His and Arg8604Gln, in the N2A

domain were found in 2 patients and these mutations increased binding of

titin/connectin to CARP. Myc-tagged CARP showed that the mutations resulted in

abnormal localization of CARP in cardiomyocytes.

Conclusion: CARP abnormalities may be involved in the pathogenesis of HCM.

Keywords

Hypertrophic cardiomyopathy, mutation, Z-disc, CARP, titin/connectin

50

Abbreviations Ab Antibody

ANKRD1 Ankyrin repeat domain 1

CARP Cardiac ankyrin repeat protein

cDNA Complementary deoxyribonucleic acid

Co-IP Co-immunoprecipitation

DAPI 4’6-diamidino-2-phenylindole

DCM Dilated cardiomyopathy

HCM Hypertrophic cardiomyopathy

PCR Polymerase chain reaction

WT Wild-type

51

Introduction

Cardiomyopathy is a primary heart muscle disorder caused by functional abnormalities

of cardiomyocytes. There are several clinical subtypes of cardiomyopathy and the most

prevalent subtype is hypertrophic cardiomyopathy (HCM)[1,2]. HCM is characterized by

hypertrophy and diastolic dysfunction of cardiac ventricles accompanied by

cardiomyocyte hypertrophy, fibrosis and myofibrillar disarray[1]. Although the etiology of

HCM has not been fully elucidated, 50-70% of the patients with HCM have apparent

family histories consistent with autosomal dominant genetic trait[2], and recent genetic

analyses have revealed that a significant percentage of HCM is caused by mutations in

the genes encoding for myofilaments and Z-disc proteins of the cardiac sarcomere with

the majority of mutations identified in MYH7-encoded beta myosin heavy chain and

MYBPC3-encoded myosin binding protein C[2].

ANKRD1 (ankyrin repeat domain 1)-encoded “cardiac adriamycin responsive

protein” [3] or “cardiac ankyrin repeat protein”(CARP)[4], is a transcription co-factor and

an early differentiation marker of cardiac myogenesis, expressed in the heart during

embryonic and fetal development. CARP expression is up-regulated in the adult hearts

at end-stage heart failure [5]. In addition, increased CARP expression was found in

hypertrophied hearts from experimental murine models [6, 7]. These observations

suggest a pivotal role of CARP in cardiac muscle function in both physiological and

pathological conditions. Although CARP is known to be involved in the regulation of

gene expression in the heart, Bang et al. demonstrated that CARP located to both the

sarcoplasm and nucleus, suggesting a shuttling of CARP in cellular components [8].

Within the I-band region of sarcomere, CARP bound to both N2A domain of

titin/connectin encoded by TTN and the N-terminal domain of myopalladin encoded by

MYPN. Hence, titin/connectin and myopalladin function in part as anchoring proteins of

“sarcomeric CARP” [8, 9].

52

Titin/connectin is the most giant protein expressed in the striated muscles,

which is involved in sarcomere assembly, force transmission at the Z-disc, and

maintenance of resting tension in the I-band region[10, 11]. In cardiac muscle, there are

two titin isoforms, N2B and N2BA. The N2B isoform contains a cardiac specific N2B

domain, and the N2BA isoform contains both N2B and N2A domains. Both N2A and

N2B domains, within the extensible I-band region, function as a molecular spring that

develops passive tension; the expression of N2B isoform results in a higher passive

stiffness than that of N2AB isoform. We previously reported an HCM-associated

mutation localizing to the N2B domain[12], and Gerull et al.[13] reported other TTN

mutations in the Z/I transition domain. These observations suggest that the I-band

region of titin/connectin contains elastic components extending with stretch to generate

passive force, which plays an important role in the maintenance of cardiac function.

Another protein that anchors CARP at the Z/I band is myopalladin, a

cytoskeletal protein containing 3 proline-rich motifs and 5 Ig domains. The proline-rich

motifs in the central part is required for binding to nebulin/nebulette, and the Ig domains

at the N-terminus and C-terminus are involved in the binding to CARP and sarcomeric

-actinin, respectively[8]. It was suggested that myopalladin played key roles in

sarcomere/Z-disc assembly, myofibrillogenesis, recruitment of the other Z/I-band

elements, and signaling in the Z/I-band[8].

In this study, we analyzed unrelated patients with heretofore

genotype-negative HCM for mutations in ANKRD1 and found 3 mutations that showed

abnormal binding to myopalladin and titin/connectin. In addition, we searched for

mutations in the recipcrocal CARP-binding N2A domain of titin/connectin and identified

2 HCM-associated mutations in TTN causing abnormal binding to CARP. We report

here that abnormal CARP assembly in the cardiac muscles may be involved in the

pathogenesis of HCM.

53

Methods Subjects

A total of 384 unrelated patients with HCM were included in this study. The patients

were diagnosed based on medical history, physical examination, 12-lead

electrocardiogram, echocardiography, and other special tests if necessary. The

diagnostic criteria for HCM included LV wall thickness >13mm on echocardiography, in

the absence of coronary artery disease, myocarditis, and hypertension. The patients

had been analyzed previously for mutations in previously published myofilament- and

Z-disc associated genes and no mutation was found in any of the known

HCM-susceptibility genes (15-18). Ethnically-matched healthy individuals (400 and 300

from Japan and USA, respectively) were used as controls. Blood samples were

obtained from the subjects after given informed consent. The protocol for research was

approved by the Ethics Reviewing Committee of Medical Research Institute, Tokyo

Medical and Dental University (Japan) and by the Mayo Foundation Institutional Review

Board (US).

Mutational analysis

Using intronic primers, each translated ANKRD1 exon was amplified by polymerase

chain reaction (PCR) from genomic DNA samples. TTN exons 99 to 104 corresponding

to the N2A domain including binding domains to CARP and p94/calpain were amplified

by PCR in exon-by-exon manner. Sequence of primers and PCR conditions used in this

study are available upon request. PCR products were analyzed by direct sequencing or

by denaturing high performance liquid chromatography (DHPLC) followed by

sequencing analysis. Sequencing was performed using Big Dye Terminator chemistry

(version 3.1) and ABI3100 DNA Analyzer (Applied Biosystems, CA, USA).

54

Co-immunoprecipitation (co-IP) assay

We obtained cDNA fragments of human ANKRD1 and TTN by RT-PCR from adult heart

mRNA. A wild-type (WT) full-length CARP cDNA fragment spanned from bp249 to

bp1208 of GenBank Accession No. NM_014391 (corresponding to aa1-aa319). Three

equivalent mutant cDNA fragments containing C to G (Pro52Ala mutation), C to T

(Thr123Met mutation) or A to G (Ile280Val mutation) substitutions were obtained by

primer-directed mutagenesis method. A WT TTN cDNA fragment encoding N2A

domains (from bp25535 to bp26465 of NM_133378 corresponding aa8437-aa8747)

was obtained and 3 TTN mutants carrying T to C (non disease-associated Ile8474Thr

polymorphism), G to A (HCM-associated Arg8500His mutation) or G to A

(HCM-associated Arg8604Gln mutation) substitutions were created by the

primer-mediated mutagenesis method. The cDNA fragments of ANKRD1 were cloned

into myc-tagged pCMV-Tag3 (Stratagene, CA, USA), while TTN and MYPN cDNA

fragments were cloned into pEGFP-C1 (Clontech, CA, USA). These constructs were

sequenced to ensure that no errors were introduced.

Cellular transfection and protein extractions were performed as described

previously [14], and co-IP assays were performed using the Catch and Release v2.0

Reversible Immunoprecipitation System according to the manufacturer‘s instructions

(Millipore, Billerica, MA). Immunoprecipitates were separated on SDS-PAGE gels and

transferred to a nitrocellulose membrane. After a pre-incubation with 3% skim milk in

PBS, the membrane was incubated with primary rabbit anti-myc polyclonal or mouse

anti-GFP monoclonal Ab (1:100, Santa Cruz Biotechnology, CA, USA), and with

secondary goat anti-rabbit (for polyclonal Ab) or rabbit anti-mouse (for monoclonal Ab)

IgG HRP-conjugated Ab (1:2000, Dako A/S, Grostrup, Denmark). Signals were

visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA,

USA) and Luminescent Image Analyzer LAS-3000mini (Fujifilm, Tokyo, Japan), and

their densities were quantified by using Multi Gauge ver3.0 (Fujifilm, Tokyo, Japan).

Numerical data were expressed as means ± SEM. Statistical differences were

analyzed using one-way ANOVA and Student’s t test for paired values. Means were

compared by independent samples t-tests without correction for multiple comparisons.

A p-value<0.05 was considered to be statistically significant.

55

Indirect immunofluorescence microscopy

All care and treatment of animals were in accordance with “Guidelines for the Care and

Use of Laboratory Animals” published by the National Institute of Health (NIH

Publication 85-23, revised 1985) and subjected to prior approval by the local animal

protection authority. Neonatal rat cardiomyocytes were prepared as described

previously[14]. Eighteen and 48 hours after the transfection, cardiomyocytes were

washed with PBS, fixed for 15 min in 100% ethanol at -20°C. Transfected cells were

incubated in blocking solution, and stained by primary rabbit anti-myc polyclonal Ab

(1:100, Santa Cruz Biotechnology) and mouse anti- -actinin monoclonal Ab (1:800,

Sigma-Aldrich), followed by secondary sheep anti-rabbit IgG FITC-conjugated Ab

(1:500, Chemicon, Victoria, Australia) and Alexa fluor 568 goat anti-mouse IgG (1:500,

Molecular Probes, OR, USA). All cells were mounted on cover-glass using Mowiol 4-88

Reagent (Calbiochem, Darmstadt, Germany) with 4’6-diamidino-2-phenylindole (DAPI,

Sigma-Aldrich) and images from at least 200 transfected cells were analyzed with an

LSM510 laser-scanning microscope (Carl Zeiss Microscopy, Jena, Germany).

56

Results Identification of ANKRD1 (CARP) and TTN mutations in HCM

Eleven distinct sequence variations in ANKRD1 were identified among the 384 patients

with HCM (Figure 1A). Four intronic variants, 2 non-synonymous substitutions and 1

synonymous variation were polymorphisms, because they were also found in the

controls. A nonsense mutation (c.423C>T in exon 2 yielding Gln59ter) was found in

2patients with familial HCM and was absent in the controls, but was not co-segregated

with the disease in both families, suggesting that they were not associated with HCM. In

contrast, 3 missense mutations, Pro52Ala (c.402C>G in exon 2), Thr123Met (c.616C>T

in exon 4) and Ile280Val (c.1086A>G in exon 8), identified in three unrelated HCM

patients, were not found in the controls.

Sequence variations in TTN at the N2A domain containing binding region to

CARP and p94/calpain were searched for in the patients and 8 variations were

identified (Figure 1B). An intronic variation and 3 synonymous variations were

polymorphisms observed in the controls. Two non-synonymous variations, Ile8474Thr

(c.25645T>C in exon 99) and Asp8672Val (c.26239A>T in exon 102), were not

associated with HCM, because Ile8474Thr was found in the controls and Asp8672Val

did not co-segregate with the disease in a multiplex family. On the other hand, 2

missense mutations, Arg8500His (c.25723G>A in exon 99) and Arg8604Gln

(c.26035G>A in exon 100), identified in familial HCM patients, were not found in the

controls.

Clinical phenotypes

Clinical findings of the patients carrying the ANKRD1 or TTN mutations are summarized

in Table 1. All patients manifested with HCM except CM1288 II-2 who had mild cardiac

hypertrophy. Her father had died suddenly of unknown etiology at the age of 30. Two

unaffected brothers of the patient did not harbor the mutation (Figure 1C). The proband

patient with the TTN Arg8606Gln mutation (CM1480, Table 1) showed asymmetric

septum hypertrophy. A family study revealed that his father had unexplained sudden

cardiac death. His son (CM1481, Table 1) was affected and carried the same mutation

(Figure 1D).

57

58

Figure 1: Mutational analyses of ANKRD1 and TTN in HCM. (A) Sequence variations found in ANKRD1. Single letter code was used to indicate the amino acid residue. Solid boxes represent protein coding region corresponding to exons 1-9. Dotted boxes indicate ankyrin repeat domains encoded by exons 5-8. (B) Sequence variations found in TTN. Solid boxes represent Ig domains corresponding to exons 98, 99 and 102-104. Dotted boxes indicate tyrosine- rich motif encoded by exons 99-101. (C and D) Pedigrees of HCM families with the ANKRD1 T123M (C, CM 1288 family) and the TTN R8604Q (D, CM 1480 family). Filled square and filled circle indicate affected male and female, respectively. Open square and open circle represent unaffected or unexamined male and female with HCM, respectively. An arrow indicates the proband patient. Presence (+) or absence (-) of the mutations is noted.

Tabl

e 1:

Clin

ical

cha

ract

eris

tics

of in

divi

dual

s ca

rryin

g A

NK

RD

1 or

TT

N m

utat

ions

IDM

utat

ion

Age

,ge

nder

Age at

onse

tC

linic

alD

xA

ge a

t cl

inic

alex

amFH

of

HC

MNY

HALV

Dd

(mm

) LV

Ds

(mm

) IV

S(m

m)

PW (mm

)%

FS%

EFO

ther

rem

arks

May

o I

AN

KR

D1

P52A

44

,

mal

e 30

H

CM

32

N

o II

- -

22

- -

70

LVH

on

ECG

; pro

voca

ble

grad

ient

100

mm

HG

, but

as

ympt

omat

ic

May

o II

AN

KR

D1

P52A

65

,

mal

e 41

H

CM

54

N

o III

38

16

14

14

-

84

Mid

-ven

tricu

lar-

apic

al

hype

rtrop

hy w

ith m

idve

ntric

ular

w

all t

hick

ness

up

to 3

5mm

CM

1288

II-

2 A

NK

RD

1 T1

23M

62

, fe

mal

e 40

H

CM

40

N

o I

41

22

13

13

46

78

Late

ral L

V h

yper

troph

y (1

5mm

), LA

D=3

7 m

m, E

CG

; abn

orm

al

Q-w

ave

in II

, III

,aV

f, V

4-6

May

o III

A

NK

RD

1 I2

80V

82

, fe

mal

e 61

H

CM

73

N

o III

52

30

20

14

-

70

Sep

tal a

blat

ion

(relie

ved

obst

ruct

ion

73m

mH

g ->

22

mm

Hg)

CM

89

TT

N

R85

00H

59

,

mal

e 53

H

CM

59

N

o I

42

25

28

8 40

79

LV

H (A

SH)

CM

1480

II-

4 T

TN

R

8604

Q

52,

m

ale

43

HC

M

43

Yes

I 41

24

18

10

41

80

LV

H (A

SH

), A

trial

fibr

illatio

n E

CG

; Inv

erte

d T-

wav

e in

V4-

V6

CM

1481

III

-1

TT

N

R86

04Q

25

,

mal

e 16

H

CM

16

Ye

s I

45

27

22

9 40

66

LV

H (A

SH

),

EC

G; I

nver

ted

T-w

ave

in V

1-V

3

Dx,

dia

gnos

is; E

F, e

ject

ion

fract

ion;

FH

, fam

ily h

isto

ry; L

AD

, lef

t atri

al d

imen

sion

; LVH

, lef

t ven

tricu

lar

hype

rtrop

hy; L

VDd,

left

vent

ricul

ar d

imen

sion

dia

stol

e,

LVD

s, le

ft ve

ntric

ular

dim

ensi

on s

ysto

le, I

VS

, int

rave

ntric

ular

sep

tum

; FS

, fra

ctio

nal s

horte

ning

; NY

HA

, New

Yor

k H

eart

Ass

ciat

ion

Altered interaction between titin/connectin and CARP caused by the TTN

or CARP mutations

To investigate the functional alterations caused by the CARP mutations in the binding to

titin/connectin N2A domain, WT-, Pro52Ala-, Thr123Met-, or Ile280Val-CARP construct

was co-transfected with the WT TTN-N2A construct into COS-7 cells. Western blot

analyses of immunoprecipitates from the transfected cells demonstrated that

HCM-associated CARP mutations significantly increased binding to TTN-N2A

(2.22±0.76 AU, p<0.05, 1.98±0.52 AU, p<0.01 or 2.16±0.64 AU, p<0.05, respectively)

(Figure 2A and B). Reciprocally, the effect of titin/connectin mutations in binding to

CARP was assessed. TTN-N2A constructs, WT-, HCM-associated mutants

(Arg8500His- and Arg8604Gln-TTN), or non-disease-related variant (Ile8474Thr)

TTN-N2A were co-transfected with WT CARP. Western blot analyses showed that

Arg8500His and Arg8604Gln significantly increased the binding to CARP (2.78±0.40 or

3.16±0.40 AU, respectively, p<0.001 in each case) (Figure 2A and B), while the

non-disease related variant (Ile8474Thr) did not alter the binding (1.18±0.11 AU),

despite equal expression of proteins.

Altered interaction between myopalladin and CARP caused by the CARP

mutations

Because CARP bound also to myopalladin, we investigated the effects of CARP

mutations in binding to myopalladin. WT or mutant CARP construct was co-transfected

with a MYPN construct. Western blot analysis revealed that binding of mutant CARPs,

Pro52Ala, Thr123Met or Ile280Val, to myopalladin was significantly increased

(3.60+/-0.67 AU, p<0.001, 1.87+/-0.47 AU, p<0.01 or 2.48+/-0.45 AU, p<0.001,

respectively) (Fig. 2C and D).

60

Figure 2: Binding of CARP to titin/connectin and myopalladin. Binding of CARP to titin/connectin (TTN) or myopalladin (MYPN) was analyzed by co-IP assays. (A) Myc-tagged CARPs co-precipitated with GFP-tagged TTN-N2A domain were shown (top panel). Expressions of GFP-tagged TTN- N2A (middle panel) and myc-tagged CARP (lower panel) were confirmed by immunobloting of whole cell supernatants. Binding pairs were WT CARP in combination with WT, I8474T, R8500H or R8604Q mutant TTN-N2A , or WT TTN-N2A with WT, P52A, T123M or I280V mutant CARP. Dashes indicate no GFP- or myc-tagged proteins (transfected only with pEGFP-C1 or pCMV-Tag3 vectors, respectively). (B) Densitometric data obtained in the co-IP assay. Data for WT CARP with WT TTN-N2A were arbitrarily defined as 1.00 arbitrary unit (AU). Data are represented as mean ± SEM. (n= 6 for each case). *** p<0.001 vs WT; ** p<0.01 vs WT; * p<0.05 vs WT.

61

Figure 2 cont’d. (C): Myc-tagged CARP co-precipitated with GFP-tagged full-length MYPN was detected by immunobloting using anti-myc antibody (top panel). Expressed amounts of GFP-tagged MYPN (middle panel) and myc-tagged CARP (lower panel) were confirmed as in(A). Binding pairs were full-length WT-MYPN with WT, P52A, T123M or I280V mutant CARP. (D) Densitometric analysis of myc-blotting data in (C). Data were arbitrarily represented as intensities and that for WT CARP with full length or N-terminal half WT MYPN was defined as 1.00 AU. Data are expressed as mean± SEM. (n = 9 for each case). *** p<0.001 vs WT; ** p<0.01 vs WT.

62

Altered localization of CARP caused by the mutations

To further investigate the functional consequence of the CARP mutations, we examined

cellular distribution of the mutant CARP proteins expressed in neonatal rat primary

cardiomyocytes. Cells were transfected with myc-tagged WT or mutant CARP

constructs, co-immunostained for myc (a marker for CARP) and -actinin (a marker for

Z-disc). WT and mutant myc-CARP proteins were expressed at a similar level in the

transfected cells as assessed by Western-blot analyses, suggesting that the mutations

did not affect the expression level and stability of CARP proteins (data not shown).

Control cells expressing myc-tag alone showed negative staining for myc-tag with

striated staining pattern of sarcomeric -actinin at the Z-disc (data not shown). In

premature cardiomyocytes containing Z-bodies (Z-disc precursors), myc-tagged WT

CARP was mainly targeted to nucleus and colocalization of CARP with -actinin, which

formed patchy dense bodies in the cytoplasm, was observed (Figure 3A-C). No

apparent changes in localization of mutant CARP proteins were observed in the

nascent and immature cardiomyocytes (Figure 3D-F, G-I and J-L).

In the mature cardiomyocytes where Z-discs were well organized, myc-tagged

WT CARP was assembled in the striated pattern at the Z-I bands and co-localized with

-actinin (Figure 4A-C). It was found that most ( 90%) of mature cardiomyocytes did

not contain nuclear CARP (Figure 4A-C). On the other hand, higher intensity of

CARP-related fluorescence at the Z-I bands and diffused localization in the cytoplasm

was observed in the most ( 80%) of mature cardiomyocytes expressing myc-tagged

mutant CARPs, albeit that the Z-disc assembly was not impaired (Figure 4D-F, G-I and J-L). Quite interestingly, myc-tagged mutant CARP proteins displayed localization

within the nuclear and/or at nuclear membrane in 60% of mature cardiomyocytes

(Figure 4D-F, G-I and J-L).

63

Discussion

CARP encoded by ANKRD1 is a nuclear transcription co-factor expressing in the

embryonic hearts. Its expression progressively decreases in adult hearts [3, 4] and

reappears in the hypertrophied or failing adult heart [5, 15], suggesting that CARP may

be involved in the regulation of muscle gene expression. CARP also localizes in cardiac

sarcomere although the roles of “sarcomeric CARP” are not fully elucidated. Several

reports have demonstrated that CARP binds titin/connectin [9] , myopalladin [8] and

desmin [16] at the Z/I-region of sarcomere. In this study, we found that the

HCM-associated ANKRD1 mutations increased the binding of CARP to titin/connectin

and myopalladin, and HCM-associated TTN mutations in its reciprocal CARP

N2A-binding domain increased the binding of titin/connectin to CARP. These

observations in association with HCM suggested that the assembly or binding of

sarcomeric CARP with titin/connectin and/or myopalladin would be required for the

maintenance of cardiac function.

In the nascent myofibrils, myc-tagged CARP proteins were detected within the

nucleus irrespective of mutations. Because CARP is an early differentiation marker

during heart development, recruitment of CARP into nuclei may be important in the

embryonic gene expression. Interestingly, abnormal intra-nuclear accumulation of

myc-tagged mutant CARP proteins was observed in mature myofibrils. It is well known

that the embryonic and fetal gene program of cardiac cytoskeletal proteins is initiated

during the cardiac remodeling [17, 18]. Hence, one could hypothesize that nuclear

CARP may cause embryonic/fetal gene expression in mature myofibrils and this

abnormal gene expression is a possible mechanism leading to the pathogenesis of

HCM. It was reported that CARP negatively regulated expression of cardiac genes

including MYL2, TNNC1 and ANP [3, 4].

64

Conversely, another report suggested that different expression level of CARP

did not correlate with the altered expression of cardiac genes such as MYL2, MYH7,

ACTC, CACTN, TPM1, ACTN2 and DES [19]. Thus, the role of CARP as a regulator of

cardiac gene expression remains to be resolved. During the preparation of this paper,

Cinquetti et al. [20] reported other CARP mutations, rearrangements or Thr116Met, in

association with the cyanotic congenital heart anomaly known as total anomalous

pulmonary venous return (TAPVR). These mutations were demonstrated to be

associated with increased expression or stability of CARP. It is not clear whether the

mutations associated with HCM altered expression or stability of CARP, though our data

suggested that HCM-associated CARP mutations did not alter the stability. The

molecular mechanisms underlying the CARP-related pathogenesis should be different

between TAPVR and HCM.

65

Figure 3: Distribution myc-tagged CARP in immature rat cardiomyocytes. Neonatal rat cardiomyocytes transfected with myc-tagged WT (A-C) or mutant (P52A, T123M or I280V) (D-F, G-I or J-L, respectively) CARP constructs were fixed 18 h after the transfection, and stained with DAPI and anti- -actinin antibody followed by secondary antibody (B, E, H, and K). Merged images were shown in C, F, I, and L. In the immature cardiomyocytes showing nascent myofibrils with Z bodies (Z-disc precursors), myc-tagged CARPs were preferentially localized to the nucleus and mutant CARP showed relatively low expression in the cytoplasm. Scale bars=10 m.

66

Figure 4: Distribution of myc-tagged CARP in mature rat cardiomyocytes. Neonatal rat cardiomyocytes transfected with myc-tagged WT (A-C) or mutant (P52A, T123M or I280V) (D-F, G-I or J-L, respectively) CARP constructs were fixed 48 h after the transfection, and stained with DAPI and anti- -actinin antibody followed by secondary antibody (B, E, H, and K). Merged images were shown in C, F, I, and L. In the mature cardiomyocytes showing myofibrils with Z-discs, normal localization of myc-tagged WT CARP at the Z-discs was observed (A-C). In contrast, myc-tagged mutant CARP proteins showed intense localization at the I-discs (colocalization with -actinin) and diffused localization in the cytoplasm (D-F, G-I and J-L). In addition, myc-tagged mutant CARPs expressed at high levels around the nuclear membrane (white arrow) and/or in the nucleus (white arrowhead).

67

Conclusions

We identified 3 missense CARP mutations in < 1% of unrelated patients with HCM,

which not only increased the binding of sarcomeric CARP to I-band components but

also resulted in the mis-localization of CARP to the nucleus. Although the molecular

mechanisms of HCM due to the CARP mutations remain to be elucidated, our findings

imply that HCM may be associated with the abnormal recruitment of CARP in

cardiomyocytes leading to pathological hypertrophy.

Acknowledgements

We thank Drs. H. Toshima, C. Kawai, K. Kawamura, M. Nagano, T. Sugimoto, S.

Ogawa, A. Matsumori, S. Sasayama, R. Nagai, and Y. Yazaki for their contributions in

clinical evaluation and blood sampling from patients with cardiomyopathy, and Ms. M.

Yanokura, M. Emura and A. Nishimura for their technical assistance.

68

References

1. Richardson P, McKenna W, Bristow M, Maisch B, et al. Report of the 1995 World Health Organization/International Society and Federation of Cardiology task force on the definition and classification of cardiomyopathies. Circulation 1996; 93(5): 841-842. 2. Bos JM, Ommen SR, Ackerman MJ. Genetics of hypertrophic cardiomyopathy: One, two, or more diseases? Curr Opin Cardiol 2007; 22(3): 193-199. 3. Jeyaseelan R, Poizat C, Baker RK, Abdishoo S, et al. A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes. J Biol Chem 1997; 272(36): 22800-22808. 4. Zou Y, Evans S, Chen J, Kuo HC, et al. CARP, a cardiac ankyrin repeat protein, is downstream in the nkx2-5 homeobox gene pathway. Development 1997; 124(4): 793-804. 5. Zolk O, Frohme M, Maurer A, Kluxen FW, et al. Cardiac ankyrin repeat protein, a negative regulator of cardiac gene expression, is augmented in human heart failure. Biochem Biophys Res Commun 2002; 293(5): 1377-1382. 6. Ihara Y, Suzuki YJ, Kitta K, Jones LR, et al. Modulation of gene expression in transgenic mouse hearts overexpressing calsequestrin. Cell Calcium 2002; 32(1): 21-29. 7. Baudet S. Another activity for the cardiac biologist: CARP fishing. Cardiovasc Res 2003; 59(3): 529-531. 8. Bang ML, Mudry RE, McElhinny AS, Trombitas K, et al. Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies. JCell Biol 2001; 153(2): 413-427. 9. Miller MK, Bang ML, Witt CC, Labeit D, et al. The muscle ankyrin repeat proteins: CARP, Ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules. JMol Biol 2003; 333(5): 951-964. 10. Granzier HL, Labeit S. The giant protein titin: A major player in myocardial mechanics, signaling, and disease. Circ Res 2004; 94(3): 284-295. 11. LeWinter MM, Wu Y, Labeit S, Granzier H. Cardiac Titin: Structure, functions and role in disease. Clin Chim Acta 2007; 375(1-2): 1-9. 12. Itoh-Satoh M, Hayashi T, Nishi H, Koga Y, et al. Titin mutations as the molecular basis for dilated cardiomyopathy. Biochem Biophys Res Commun 2002; 291(2): 385-393. 13. Gerull B, Gramlich M, Atherton J, McNabb M, et al. Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy. Nat Genet 2002; 30(2): 201-204. 14. Arimura T, Matsumoto Y, Okazaki O, Hayashi T, et al. Structural analysis of Obscurin gene in hypertrophic cardiomyopathy. Biochem Biophys Res Commun 2007; 362(2): 281-287. 15. Aihara Y, Kurabayashi M, Saito Y, Ohyama Y, et al. Cardiac ankyrin repeat protein is a novel marker of cardiac hypertrophy: Role of m-cat element within the promoter. Hypertension 2000; 36(1): 48-53. 16. Witt SH, Labeit D, Granzier H, Labeit S, et al. Dimerization of the cardiac ankyrin protein CARP: Implications for MARP titin-based signaling. J Muscle Res Cell Motil 2006; 26(6-8): 401-408.

69

17. Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev 1999; 79(1): 215-262. 18. Swynghedauw B, Baillard C. Biology of hypertensive cardiopathy. Curr Opin Cardiol 2000; 15(4): 247-253. 19. Torrado M, Lopez E, Centeno A, Castro-Beiras A, et al. Left-right asymmetric ventricular expression of CARP in the piglet heart: Regional response to experimental heart failure. Eur J Heart Fail 2004; 6(2): 161-172. 20. Cinquetti R, Badi I, Campione M, Bortoletto E, et al. Transcriptional deregulation and a missense mutation define ANKRD1 as a candidate gene for total anomalous pulmonary venous return. Hum Mutat 2008; 29(4): 468-474.

70

Chapter 4

Echocardiographic-Determined Septal Morphology in Z-Disc Hypertrophic Cardiomyopathy

Jeanne L. Theis*, J. Martijn Bos *, Virginia B. Bartleson, Melissa L. Will, Josepha Binder, Matteo Vatta, Jeffrey A. Towbin, Bernard J. Gersh, Steve R. Ommen, Michael J. Ackerman * These authors contributed equally to this study

Biochem Biophys Res Commun 2006; 351(4): 896 – 902

Abstract Hypertrophic cardiomyopathy (HCM) can be classified into at least 4 major anatomic

subsets based upon the septal contour, and the location and extent of hypertrophy:

reverse curvature-, sigmoidal-, apical-, and neutral contour-HCM. Here, we sought to

identify genetic determinants for sigmoidal-HCM and hypothesized that Z-disc HCM

may be associated preferentially with a sigmoidal phenotype. Utilizing PCR, DHPLC,

and direct DNA sequencing, we performed mutational analysis of five genes encoding

cardiomyopathy associated Z-disc proteins. The study cohort consisted of 239

unrelated patients with HCM previously determined to be negative for mutations in the

8 genes associated with myofilament-HCM. Blinded to the Z-disc genotype status, the

septal contour was graded qualitatively using standard transthoracic

echocardiography. Thirteen of the 239 patients (5.4%) had one of 13 distinct HCM-

associated Z-disc mutations involving residues highly conserved across species and

absent in 600 reference alleles: LDB3 (6), ACTN2 (3), TCAP (1), CSRP3 (1) and VCL

(2). For this subset with Z-disc-associated HCM, the septal contour was sigmoidal in

11 (85%) and apical in 2 (15%). While Z-disc-HCM is uncommon, it is equal in

prevalence to thin filament-HCM. In contrast to myofilament HCM, Z-disc HCM is

associated preferentially with sigmoidal morphology.

Keywords Hypertrophy, cardiomyopathy, septum, echocardiography, genes, Z-disc

72

Introduction Affecting 1 in 500 persons, hypertrophic cardiomyopathy (HCM) is the most common

identifiable cause of sudden death in young athletes and is the most common heritable

cardiovascular disease[1]. Characterized by unexplained myocardial hypertrophy in the

absence of precipitating factors such as hypertension or aortic stenosis, HCM is

underscored by profound genetic and phenotypic heterogeneity. Since the sentinel

discovery of mutations involving the MYH7-encoded -myosin heavy chain as the

pathogenetic basis for HCM in 1990[2], more than 300 mutations scattered among at

least 12 HCM-susceptibility genes encoding sarcomeric proteins have been identified.

The first link to be drawn between septal morphologies was a result of a pre-

genomics era HCM study by Lever and colleagues where septal contour was found to

be age-dependent with a predominance of the sigmoid septum with normal curvature

being present in the elderly[3]. This was followed up by an early genotype-phenotype

observation by Seidman and colleagues involving a small number of patients and

ultimately revealed that patients with mutations in the beta myosin heavy chain (MYH7-

HCM) generally had reversed curvature septal contours[4]. Most recently, we

discovered that myofilament-HCM may have a predilection for a reverse curvature

septal phenotype regardless of age[5]. After analyzing all echocardiograms of 382

previously genotyped and published patients[6, 7, 8], multivariate analysis

demonstrated that reverse septal curvature was the only, independent predictor of

myofilament HCM with an odds ratio of 21[5]. Moreover, the yield of the commercially

available HCM genetic test (panel A and panel B) which examines 8 genes responsible

for myofilament-HCM was 79% in reverse curve-HCM but only 8% in sigmoidal-HCM.

These observations provide the rationale for seeking novel genetic

determinants that confer susceptibility for sigmoidal-HCM. Recent attention has been

focused on proteins outside the cardiac myofilament, involved in the cyto-architecture

and cardiac stretch sensor mechanism of the cardiomyocyte. Mutations in three such

proteins localized to the cardiac Z-disc, CSRP3-encoded muscle LIM protein (MLP),

TCAP-encoded telethonin and VCL-encoded vinculin, including its cardiac specific

insert of exon 19 that yields metavinculin, have previously been established as both

HCM[9, 10, 11, 12, 13] and dilated cardiomyopathy (DCM)-susceptibility genes[9, 10,

11, 12, 14, 15].

73

Additionally, it has been recognized that these divergent cardiomyopathic

phenotypes of HCM and DCM are partially allelic disorders with ACTC, MYH7, TNNT2,

TPM1, MYBPC3, TTN, MLP, TCAP, and VCL established as both HCM- and DCM-

susceptibility genes[10, 11, 12, 14, 16, 17, 18, 19, 20].

These observations prompted us to consider perturbations in the cardiac Z-disc

as another pathway for hypertrophic or dilated cardiomyopathy. Besides the three

aforementioned Z-disc proteins implicated in HCM, we considered two additional

genes: ACTN2-encoded alpha-actinin 2 and LDB3-encoded LIM domain binding 3

(official HUGO nomenclature; also known as ZASP-encoded Z-band associated

alternatively spliced PDZ-motif protein), as candidates for HCM. Both genes have been

implicated in the pathogenesis of DCM and encode proteins that are key binding

partners of the previously mentioned HCM-associated, Z-disc proteins[14, 21]. The

published Q9R-ACTN2 missense mutation inhibited cellular function and was

associated with extra-nuclear localization in cultured cells with co-immunoprecipitation

studies showing its failure to bind to MLP[14]. LDB3-associated animal models reveal

that null mice completely devoid of this protein lose their ability to maintain structural

integrity of the Z-disc, leading to impaired contraction and perinatal death[22].

Because of the specific structure-function relationship of the proteins in the

cardiac Z-disc[23, 24] and the specific cardiomyocyte stretch response mechanism of

these proteins[25], we hypothesized that Z-disc HCM might be preferentially sigmoidal.

We speculate that in the presence of Z-disc mutations, the compensatory hypertrophic

response may be greatest in areas of highest stress (i.e. LVOT), thereby resulting in

the basal septal bulge and sigmoidal shaped contour.

74

Methods

Between April 1997 and December 2001, a total of 382 unrelated patients (210 male,

mean maximum left ventricular wall thickness (MLVWT) 21.5 ± 6mm) had both

comprehensive echocardiographic examination and evaluation in Mayo Clinic’s HCM

clinic and genetic testing. HCM was diagnosed according to WHO criteria as

unexplained cardiac hypertrophy (>13 mm) in the absence of hypertrophy inciting

factors such as aortic stenosis. Following written informed consent for this IRB-

approved research protocol, DNA was extracted from the blood samples using

Purgene DNA extraction kits (Gentra, Inc., Minneapolis, Minnesota). After a

comprehensive analysis of the eight most common myofilament HCM-associated

genes, 239 patients (131 male, mean MLVWT 20.7 ± 6mm) remained without a

pathogenetic explanation and are in this study referred to as “myofilament genotype

negative” [6, 7, 8, 26].

This subset was analyzed for mutations in all translated exons of all published,

cardiomyopathy-associated Z-disc genes: CSRP3-encoded muscle LIM protein (MLP),

TCAP-encoded telethonin (TCAP), VCL-encoded vinculin, ACTN2-encoded alpha-

actinin 2 (ACTN2) and LDB3-encoded LIM domain binding protein 3 (LDB3), using

polymerase chain reaction (PCR), denaturing high performance liquid chromatography

(DHPLC) (Transgenomic, Omaha NE) and direct DNA sequencing(ABI Prism 377;

Applied Biosystem, Foster City, California). Primer sequences and DHPLC-methods

are available upon request. To exclude common non-synonymous polymorphisms, we

examined 600 ethnically matched reference alleles.

Echocardiography

Septal curvature and cavity contour were evaluated in the long axis view at end-

diastole. Sigmoid septal morphology was defined as a generally ovoid left ventricular

(LV) cavity with the septum being concave toward the LV with a pronounced basal

septal bulge. Reverse curve septal morphology was defined as a predominant mid-

septal convexity toward the left ventricular cavity with the cavity itself having an overall

crescent shape. Apical variant HCM was defined as a predominant apical distribution

of hypertrophy. Neutral septal contour was defined by an overall straight or variable

convexity that was neither predominantly convex nor concave toward the LV cavity.

Septal contours were assessed by two independent reviewers (JB and SRO) and

genotypic data was kept in a database blinded to all clinical and echocardiographic

data.

75

Results

The demographics of the myofilament genotype negative cohort are shown in Table 1.

As indicated in the study design exclusion criteria, no mutations in the eight genes

underlying myofilament-HCM (beta myosin heavy chain, myosin binding protein C,

etc.) were present in this cohort. This cohort consisted of 239 patients (131 male) with

an average age at diagnosis of 45.1 years old and a mean MLVWT of 20.7 mm. Fifty-

six percent of patients presented with cardiac symptoms, 24% had a family history of

HCM in a first degree relative and 16% had a family history of sudden cardiac death.

Forty percent of patients underwent surgical myectomy because of refractory

symptoms. In comparison, this subset of patients with myofilament genotype negative-

HCM are older, have less hypertrophy, and are less likely to have a reverse curvature

shaped septum compared to the 143 patients with myofilament-HCM (Table 1) [5, 6, 7,

8, 26].

Table 1: Demographics of Myofilament Genotype Negative HCM Cohort

Myofilament Negative(N = 239)

Myofilament Positive(N = 143)

p-value

Male/female 131/108 79/64 NS

Age at diagnosis (years) 45.1 ± 19 35.7 ± 17 < 0.001

MLVWT (mm) 20.7 ± 6 22.8 ± 7 0.002

Mean peak LVOT gradient (mmHG) 48.3 ± 42 45.6 ± 42 NS

Sigmoidal-shaped septal contour 166 (69%) 15 (10%) <0.0001

Presenting w/ cardiac symptoms (%) 55.7% 55.8% NS

Positive family history of HCM* 24% 47% <0.001

Positive family history of SCD* 16% 25% NS

Surgical myectomy 95 (40%) 64 (45%) NS

Pacemaker 40 (17%) 28 (19%) NS

ICD 23 (10%) 37 (25%) <0.001 HCM, hypertrophic cardiomyopathy; LVOT, left ventricular outflow tract; MLVWT, maximum left ventricular wall thickness; SCD, sudden cardiac death; ICD, implantable cardioverter-defibrillator * In first degree relative

76

After analysis of all translated exons of LDB3, CSRP3, TCAP, ACTN2 and VCL, 14

mutations in 13 patients (5%) were discovered. Most mutations were missense

mutations conserved over species and absent in 600 ethnically matched reference

alleles. S196L-LDB3 was identified in two patients. Mutations and their location in the

topology of their respective protein are shown in Figure 1. One patient with the Y468S-

LDB3 missense mutation also harbored a second Z-disc mutation, a frame-shift

mutation (K42 fs/165) in the CSRP3-encoded muscle LIM protein.

Figure 1: Schematic topologies of analyzed genes and the mutations found. The legend behind the gene name directs to the binding domain shown in its partner-protein. For vinculin, the cardiac specific insert that yields metavinculin (exon 19) is shown.

77

78

The clinical phenotype of the 13 patients is shown in Table 2. Overall, the average age

at diagnosis was 42.9 ± 18 years with seven of the twelve patients being male. The

MLVWT is 20.9 ± 9 mm and the LVOT gradient averaged 56.8 ± 49 mmHg. Eight

patients (case 1-3, 5, 6, 11-13) underwent surgical septal myectomy because of

refractory symptoms. Pathological reports of the surgical specimens show at least two

of the three characteristics (cardiomyocyte hypertrophy, endocardial fibrosis and

myofibrillar disarray) of HCM in all cases; half of the specimens showed myofibrillar

disarray.

Ta

ble

2: C

linic

al p

heno

type

of p

atie

nts

with

Z-d

isc

HC

M

Cas

e G

ene

Mut

atio

n Se

xA

geat

Dx

(yrs

) M

LVW

T(m

m)

LVO

T(m

mH

g)

Sept

alsh

ape

Fam

Hx

of H

CM

Fa

m H

x of

SC

D

Trea

tmen

t Pa

thol

ogy

repo

rt

1A

CT

N2

G11

1V

M

31.4

20

10

0 S

igm

oid

No

No

Mye

ctom

y M

arke

d m

yocy

te h

yper

troph

y,

foca

l m

yocy

te d

isar

ray,

en

doca

rdia

l fib

rosi

s

2A

CT

N2

T495

M

M

32.5

16

0

Sig

moi

d N

o N

o M

yect

omy

Mar

ked

endo

card

ial f

ibro

sis,

m

yocy

te h

yper

troph

y, i

nter

stiti

al

fibro

sis

3A

CT

N2

R75

9T

M

17.9

16

12

0 S

igm

oid

No

No

Mye

ctom

y N

o re

port

4C

SR

P3

Q91

L M

44

.5

22

18

Sig

moi

d N

o N

o P

acem

aker

N

A

5T

CA

PR

70W

F

44.2

46

19

S

igm

oid

Yes

N

o M

yect

omy,

P

acem

aker

S

ever

e m

yocy

te h

yper

troph

y,

mod

erat

e in

ters

titia

l fib

rosi

s

6LD

B3

S19

6L

F 73

.0

19

64

Sig

moi

d N

o N

o M

yect

omy

Mar

ked

myo

cyte

hyp

ertro

phy,

m

oder

ate

endo

card

ial f

ibro

sis,

fo

cal m

yocy

te d

issa

ray

7LD

B3

S19

6L

F 63

.8

13

0 A

pica

l N

o N

o R

x N

A

8LD

B3

D36

6N

M

68.5

18

16

S

igm

oid

No

No

Rx

NA

9LD

B3

CS

RP

3Y

468S

, K

42 fs

/165

M

46

.8

18

112

Sig

moi

d N

o N

o R

x N

A

10LD

B3

Q51

9P

F 21

.2

15

55

Sig

moi

d Y

es

No

Rx

NA

11LD

B3

P61

5L

M

28.3

27

12

0 S

igm

oid

No

No

Mye

ctom

y M

oder

ate

myo

cyte

, mild

to

mod

erat

e fo

cal e

ndoc

ardi

al fi

bros

is

12V

CL

L277

M

F 76

20

0

Sig

moi

d N

o N

o M

yect

omy

Myo

cyte

hyp

ertro

phy,

ca

rdio

myo

cyte

dis

arra

y, in

ters

titia

l fib

rosi

s

13V

CL

R97

5W

F 42

.8

22

0 A

pica

l N

o N

o M

yect

omy

Mar

ked

myo

cyte

hyp

ertro

phy,

mild

in

ters

titia

l fib

rosi

s, fo

cal m

yofib

rilla

r di

ssar

ray

In contrast to the patients with myofilament-HCM from our previous study, none of the

patients with Z-disc HCM exhibited reverse septal curvature echocardiographically

(104/143 vs. 0/13, p-value < 0.0001, Figure 2). Instead, 11 of the 13 patients (85%)

had a sigmoidal shaped septum and the other 2 patients had apical-HCM (case 7 and 13). For the entire original cohort of 382 unrelated patients, a putative pathogenic

explanation for sigmoidal-HCM has increased from 8% (myofilament genotype

positive) to now 14% with inclusion of Z-disc mediated disease (Figure 2). The

majority of sigmoidal-HCM remains genotypically unexplained.

Figure 2: Overview of the genotype-phenotype relationships between the two most common septal morphologies (bottom) and the presence of mutations in the cardiac Z-disc (top-middle) or the myofilament (top-sides). Arrows pointing towards the morphologies, represent the frequency of that morphology for a particular genotype. Arrows pointing towards the myofilament or Z-disc represent the number of mutations present when showing a particular septal morphology.

80

Discussion

Due to the hundreds of mutations scattered throughout the genes which encode

proteins of the myofilament, HCM has long been considered a disease of the

sarcomere, more specifically, a disease of the myofilament. With the recent discovery

of HCM associated mutations in genes encoding for proteins of the Z-disc[9, 10, 11,

12, 14] and the distinction whereby HCM associated mutations in PRKAG2 and

LAMP2 have categorized certain cases of glycogen storage disease[27, 28], the

spectrum of genetically-mediated disease pathways continues to expand.

Although specific mutations in particular genes may be rare, the question arises

as to whether there may be significant genotype-phenotype correlations associated

with distinct HCM-yielding pathways such as myofilament-, Z-disc, or metabolic-HCM.

To this end, we further explored our recent discovery that linked reverse curvature

HCM with mutations in genes encoding proteins of the myofilament (i.e. myofilament-

HCM) [5]. Here, we demonstrated that reverse septal curvature was the strongest,

independent predictor of the presence of a myofilament mutation (OR 21, p<0.001)

over age and MLVWT[5].

The cardiac Z-disc as a novel target in the pathogenesis of HCM

Focusing on the myofilament negative subgroup, we extended our investigation to

encompass five cardiomyopathy-susceptibility genes that encode important and

interacting proteins that are key constituents of the cardiac Z-disc architecture. The Z-

disc is an intricate assembly of proteins at the Z-line of the cardiomyocyte sarcomere.

Extensively reviewed, proteins of the Z-disc are important in the structural and

mechanical stability of the sarcomere as they appear to serve as a docking station for

transcription factors, Ca2+-signaling proteins, kinases and phosphatases[23, 24]. In

addition, this assembly of proteins seems to serve as a way station for proteins that

regulate transcription by aiding in their controlled translocation between the nucleus

and the Z-disc[23, 24].

81

With all of these roles, a main implication for the Z-disc is its involvement in the

cardiomyocyte stretch sensing and response systems [25]. While this is a critical task

which is an integral component of Z-disc function in the long term, there is the potential

that the Z-disc may transduce multiple signaling pathways during stress, translating

into hypertrophic responses, cell growth and remodeling [29]. Based on this potentially

important structure-function relationship and its role in the cardiomyocyte stretch

response system, we hypothesized that perturbations in the cardiac Z-disc may confer

susceptibility for the development of sigmoidal-HCM.

Z-disc-HCM is preferentially sigmoidal

Indeed, after extensive analysis of the genes encoding these 5 key Z-disc proteins, we

observed a very strong predilection for sigmoidal disease in the presence of a rare

mutation that disrupts a Z-disc protein. In fact, in contrast to the 79% likelihood for

myofilament-HCM in the setting of reverse curvature-HCM, none of the patients within

this subgroup of Z-disc-HCM displayed reverse septal curvature. Although the vast

majority of sigmoidal HCM in our cohort still is genetically unexplained, the yield of

genetic testing for sigmoidal curvature has nearly doubled by extending the genetic

testing from the 8 myofilament-HCM genes that are tested for commercially to include

these 5 genes associated with Z-disc-HCM. We speculate that Z-disc HCM leads to a

hypertrophic response that is expressed in the areas of highest stress (i.e. LVOT) and

therefore predisposes to a sigmoidal septal contour.

These observations generate several intriguing questions regarding HCM in

association with a sigmoidal septal contour. Whereas in previous morphologic studies,

Lever and colleagues associated sigmoidal-HCM with older age [3], the underlying

genotype rather than age appears to be the predominant determinant of septal

morphology [5]. Given that the vast majority of our patients with sigmoidal HCM still

lack a putative disease-causing mutation, it remains to be determined whether such

patients possess, in fact, congenital HCM (i.e. a primary HCM-predisposing genetic

mutation). It can be speculated that, especially in the sigmoidal septal subgroup, the

sum of all contributors – the presence or absence of a mutation or LVH promoting

polymorphisms [30], an unidentified genetic substrate, environmental factors and

hypertension – culminates in what is clinically labeled as HCM.

82

This multi-factorial model for sigmoidal HCM is supported by the significantly

older age at diagnosis of patients with sigmoidal HCM (49 years) compared to those

with reverse curvature-HCM (32 years)[5]. Furthermore, nearly 20% of patients

classified with sigmoidal-HCM were noted to have mild hypertension [5]. Although

diagnosed with HCM and presently showing co-existent hypertension, a subset of this

group may have a basal septum more sensitive to the pro-hypertrophy trigger of

increased afterload, precipitating basal septal hypertrophy (sigmoidal disease), but

nonetheless culminating in a clinical diagnosis of HCM. In this scenario, a Mendelian

genetic mechanism will not be found.

On the other hand, this novel genotype-phenotype association characterized by

predilection for sigmoidal, basal septal hypertrophy in the setting of perturbations in the

cardiac Z-disc raises the possibility that other constituents of the Z-disc (> 20 proteins)

may host additional HCM-susceptibility mutations in general and sigmoidal-HCM

susceptibility mutations in particular. For example, as one of the central proteins of the

Z-disc, ACTN2 binds to a large number of proteins, including ALP-encoded actinin-

associated LIM-protein [31], CapZ-encoded actin capping protein [32] or S100, of

which the S100B-isoform seems to function as an inhibitor of the hypertrophic

response [33]. ALP, CAPZ and S100 may represent the next tier of HCM candidate

genes to further test our hypothesis that sigmoidal septal shaped HCM is associated

with perturbations in the cardiac Z-disc.

83

Conclusions

Thus far, examination of the five established cardiomyopathy susceptibility genes,

encoding key components of the Z-disc, demonstrate that perturbations in the Z-disc is

a much less common cause for HCM compared to the two most common HCM-

associated genotypes of myosin binding protein C- and beta myosin heavy chain-

HCM. Nevertheless, Z-disc HCM is as common as thin filament-HCM (i.e., troponin T-,

troponin I-, tropomyosin-, or actin-HCM). However, unlike myofilament HCM, Z-disc

HCM is preferentially sigmoidal. Whether a significant proportion of sigmoidal disease

will be explained by perturbations in other components of the cardiac Z-disc awaits

further investigation.

84

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1. Maron BJ. Hypertrophic cardiomyopathy: A systematic review. JAMA 2002; 287(10): 1308-1320. 2. Geisterfer-Lowrance AA, Kass S, Tanigawa G, Vosberg H, et al. A molecular basis for familial hypertrophic cardiomyopathy: A beta cardiac myosin heavy chain gene missense mutation. Cell 1990; 62: 999-1006. 3. Lever HM, Karam RF, Currie PJ, Healy BP. Hypertrophic cardiomyopathy in the elderly. Distinctions from the young based on cardiac shape. Circulation 1989; 79(3) :580-589. 4. Solomon SD, Wolff S, Watkins H, Ridker PM, et al. Left ventricular hypertrophy and morphology in familial hypertrophic cardiomyopathy associated with mutations of the beta-myosin heavy chain gene. J Am Coll Cardiol 1993; 22(2): 498-505. 5. Binder J, Ommen SR, Gersh BJ, Van Driest SL, et al. Echocardiography-guided genetic testing in hypertrophic cardiomyopathy: septal morphological features predict the presence of myofilament mutations. Mayo Clin Proc 2006; 81(4):459-467. 6. Van Driest SL, Jaeger MA, Ommen SR, Will ML, et al. Comprehensive analysis of the beta-myosin heavy chain gene in 389 unrelated patients with hypertrophic cardiomyopathy. JAm Coll Cardiol 2004; 44(3):602-610. 7. Van Driest SL, Vasile VC, Ommen SR, Will ML, et al. Myosin binding protein C mutations and compound herterozygosity in hypertrophic cardiomyopathy. J Am Coll Cardiol 2004; 44(9): 1903-1910. 8. Van Driest SL, Ellsworth EG, Ommen SR, Tajik AJ, et al. Prevalence and spectrum of thin filament mutations in an outpatient referral population with hypertrophic cardiomyopathy. Circulation 2003; 108: 445-451. 9. Bos JM, Poley RN, Ny M, Tester DJ, et al. Genotype-phenotype relationships involving hypertrophic cardiomyopathy-associated mutations in titin, muscle LIM protein, and telethonin. Mol Genet Metab 2006; 88(1): 78-85. 10. Vasile VC, Will ML, Ommen SR, Edwards WD, et al. Identification of a metavinculin missense mutation, R975W, associated with both hypertrophic and dilated cardiomyopathy. Mol Genet Metab 2006; 87(2): 169-174. 11. Hayashi T, Arimura T, Itoh-Satoh M, Ueda K, et al. Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy. J Am Coll Cardiol 2004; 44(11):2192-2201. 12. Geier C, Perrot A, Ozcelik C, Binner P, et al. Mutations in the human muscle LIM protein gene in families with hypertrophic cardiomyopathy. Circulation 2003; 107(10): 1390-1395. 13. Vasile VC, Ommen SR, Edwards WD, Ackerman MJ. A missense mutation in a ubiquitously expressed protein, vinculin, confers susceptibility to hypertrophic cardiomyopathy.Biochem Biophys Res Commun 2006; 345(3): 998-1003. 14. Mohapatra B, Jimenez S, Lin JH, Bowles KR, et al. Mutations in the muscle LIM protein and alpha-actinin-2 genes in dilated cardiomyopathy and endocardial fibroelastosis. Mol Genet Metab 2003; 80(1-2): 207-215. 15. Olson TM, Illenberger S, Kishimoto NY, Huttelmaier S, et al. Metavinculin mutations alter actin interaction in dilated cardiomyopathy. Circulation 2002; 105(4): 431-437.

85

16. Kamisago M, Sharma SD, DePalma SR, Solomon S, et al. Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N Engl J Med 2000; 343(23): 1688-1696. 17. Olson TM, Doan TP, Kishimoto NY, Whitby FG, et al. Inherited and de novo mutations in the cardiac actin gene cause hypertrophic cardiomyopathy. J Mol Cell Cardiol 2000; 32(9): 1687-1694. 18. Olson TM, Kishimoto NY, Whitby FG, Michels VV. Mutations that alter the surface charge of alpha-tropomyosin are associated with dilated cardiomyopathy. J Mol Cell Cardiol 2001; 33(4): 723-732. 19. Gerull B, Gramlich M, Atherton J, McNabb M, et al. Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy. Nat Genet 2002; 30(2): 201-204. 20. Daehmlow S, Erdmann J, Knueppel T, Gille C, et al. Novel mutations in sarcomeric protein genes in dilated cardiomyopathy. Biochem Biophys Res Commun 2002; 298(1): 116-120. 21. Vatta M, Mohapatra B, Jimenez S, Sanchez X, et al. Mutations in Cypher/ZASP in patients with dilated cardiomyopathy and left ventricular non-compaction. J Am Coll Cardiol 2003; 42(11): 2014-2027. 22. Zhou Q, Chu PH, Huang C, Cheng CF, et al. Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy. J Cell Biol 2001;155(4):605-612. 23. Frank D, Kuhn C, Katus HA, Frey N. The sarcomeric Z-disc: a nodal point in signaling and disease. J Mol Med 2006; 84(6): 446-68. 24. Pyle WG, Solaro RJ. At the crossroads of myocardial signaling: the role of Z-discs in intracellular signaling and cardiac function. Circ Res 2004; 94(3): 296-305. 25. Knoll R, Hoshijima M, Hoffman HM, Person V, et al. The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell 2002; 111(7): 943-955. 26. Van Driest SL, Ommen SR, Tajik AJ, Gersh BJ, et al. Yield of Genetic Testing in Hypertrophic Cardiomyopathy. Mayo Clin Proc 2005; 80(6): 739-744. 27. Blair E, Redwood C, Ashrafian H, Oliveira M, et al. Mutations in the gamma(2) subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis. Hum Mol Genet 2001; 10(11): 1215-1220. 28. Arad M, Maron BJ, Gorham JM, Johnson WH, Jr., et al. Glycogen storage diseases presenting as hypertrophic cardiomyopathy. N Engl J Med 2005; 352(4):362-372. 29. Frey N, Katus HA, Olson EN, Hill JA. Hypertrophy of the heart: a new therapeutic target? Circulation 2004; 109(13): 1580-1589. 30. Perkins MJ, Van Driest SL, Ellsworth EG, Will ML, et al. Gene-specific modifying effects of pro-LVH polymorphisms involving the renin-angiotensin-aldosterone system among 389 unrelated patients with hypertrophic cardiomyopathy. Eur Heart J 2005; 26(22): 2457-2462. 31. Xia H, Winokur ST, Kuo WL, Altherr MR, et al. Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs. J Cell Biol 1997; 139(2) :507-515. 32. Papa I, Astier C, Kwiatek O, Raynaud F, et al. Alpha actinin-CapZ, an anchoring complex for thin filaments in Z-line. J Muscle Res Cell Motil 1999; 20(2): 187-197.

86

33. Tsoporis JN, Marks A, Kahn HJ, Butany JW, et al. Inhibition of norepinephrine-induced cardiac hypertrophy in s100beta transgenic mice. J Clin Invest 1998; 102(8): 1609-1616.

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88

Chapter 5

Relationship Between Sex, Shape, and Substrate in Hypertrophic Cardiomyopathy

J. Martijn Bos, Jeanne L. Theis, A. Jamil Tajik, Bernard J. Gersh, Steve R. Ommen, Michael J. Ackerman

Am Heart J 2008; 155(6): 1128 – 34

Abstract

Background: Hypertrophic cardiomyopathy (HCM) is a disease characterized by

substantial genetic, morphologic and prognostic heterogeneity. Recently, sex-related

differences in HCM were reported with females being older at diagnosis and exhibiting

greater left ventricular outflow tract obstruction than men. We sought to evaluate the

influence of sex on the HCM phenotype in a large cohort of unrelated patients with

genetically and morphologically classified HCM.

Methods: Comprehensive genotyping of 13 HCM-susceptibility genes encoding

myofilament and Z-disc proteins of the cardiac sarcomere was performed previously

on 382 unrelated patients with HCM. Blinded to the genotype, the septal morphology

was graded as reverse curvature-, sigmoidal-, apical-, or neutral contour-HCM by

echocardiography.

Results: Overall, females were a) significantly older at diagnosis (45.1 ± 20 vs. 35.8 ±

17 years; p<0.001), b) had greater left ventricular outflow tract obstruction (53.5 ± 45

vs. 41.7 ± 42 mmHg; p = 0.009), c) were more likely to have concomitant hypertension

(19% vs. 11%, p = 0.02), and d) had a higher rate of surgical myectomy (49% vs. 36%,

p = 0.01) than men. Interestingly, these sex-based differences were apparent only

among patients with sigmoidal-HCM (p < 0.001).

Conclusions: In this largest cohort of comprehensively genotyped and

morphologically classified patients with clinically diagnosed HCM, we observed that the

striking sex-related differences in the clinical phenotype are confined largely to the

subset of mutation negative, sigmoidal-HCM. Whereas mutations within the sarcomere

appear to dominate the disease process, in their absence, sex has a significant

modifying effect, specifically noted in cases of sigmoidal-HCM.

Keywords Sex, hypertrophy, hypertrophic cardiomyopathy, septum, echocardiography

90

Abbreviations FH Family history

HCM Hypertrophic cardiomyopathy

LVEDD Left ventricular end-diastolic dimension

LVH Left ventricular hypertrophy

LVOT(O) Left ventricular outflow tract (obstruction)

MLVWT Maximum left ventricular wall thickness

SCA Sudden cardiac arrest

91

Background

Affecting 1 in 500 persons, hypertrophic cardiomyopathy (HCM) is a disease or

diseases characterized by marked genetic and prognostic heterogeneity[1].

Characterized by unexplained myocardial hypertrophy in the absence of precipitating

factors, HCM is the most common cause of sudden death in young athletes[1, 2].

Since the sentinel discovery of the first locus linked to familial HCM[3] and the first

HCM-associated mutations identified in the MYH7-encoded -myosin heavy chain[4],

hundreds of mutations scattered among 16 HCM-associated genes encoding

sarcomeric proteins have been identified.

In a pre-genomics study by Lever et al, a striking correlation between the

echocardiographically classified reverse- and sigmoidal septal contour, and age of

onset was described[5]. This observation was followed by an early shape-genetic

substrate analysis by Seidman et al. showing a correlation between reverse septal

curvature and the presence of an HCM-associated MYH7 mutation[6]. Recently, a

strong relationship between the genetic substrate comprised by all 8 myofilament

genes underlying HCM and the morphological subtype was elucidated in a large cohort

of genotyped patients with HCM[7]. The morphology of the left ventricle and septum

were much more closely related to the presence or absence of an underlying

myofilament mutation than to the age of the patient. In fact, multivariate analysis

revealed reverse septal contour to be the strongest independent predictor of a

myofilament mutation, with an odds ratio of 21[7].

Over the past several years, several studies have described sex differences in

HCM[8, 9, 10, 11]. Most recently, significant sex related differences were reported in a

large cohort of American and Italian patients with HCM. This study, in which women

were underrepresented, showed that women were older and more symptomatic at the

time of initial diagnosis[11]. Furthermore, the aforementioned study noted that women,

usually with left ventricular outflow tract obstruction (LVOTO), were more likely to

progress to advanced heart failure and stroke[11]. The relative contributions between

sex, genetic substrate, and anatomical shape could not be ascertained because this

analysis was performed on a cohort of genetically undefined and morphologically

unclassified patients with HCM. Due to the heterogeneous nature both at the level of

the genotype as well as the specific anatomical morphology, we sought to further

evaluate the influence of sex on the HCM phenotype in a large cohort of unrelated

patients with genetically and morphologically classified HCM.

92

Methods

Between April 1997 and December 2001, a total of 382 unrelated patients (210 male,

mean maximum left ventricular wall thickness (MLVWT, 21.5 ± 6mm) underwent

clinical evaluation including echocardiography in Mayo Clinic’s HCM Clinic, a tertiary

referral center for HCM and surgical septal myectomies. Furthermore, comprehensive

genetic testing for 8 myofilament- and 5 Z-disc-associated, HCM-susceptibility genes

was completed for all patients in Mayo Clinic’s Windland Smith Rice Sudden Death

Genomics Laboratory[12, 13, 14, 15, 16, 17, 18]. Informed consent for this IRB-

approved study was obtained from all patients or parents, if underage.

Evaluation of septal curvature and cavity contour was previously performed and

blinded to genotype, patients were classified morphologically into sigmoidal-, reverse

curve-, apical-, and neutral contour-HCM[7]. The diagnosis of HCM was based on the

echocardiographic demonstration of increased left ventricular wall thickness in the

absence of clear etiology. Data on symptomatic status at initial visit (angina, dyspnea)

was collected and scored in severity using the New York Heart Association (NYHA) -

class, while the overall NYHA-class was assessed as well.

As hypertension is a common disease in the US population, some patients in

this cohort also had mild concomitant hypertension. In these cases, the diagnosis of

HCM was felt to be the appropriate diagnosis, by experienced clinicians dedicated to

the care of patients with HCM, as the severity of hypertrophy was out of proportion to

the concomitant hypertension. As a reference, 317 patients were referred to the Mayo

HCM clinic during this time period and were felt to have either significant hypertension

or aortic valve stenosis rather than HCM, and were therefore not included in this

cohort.

93

Statistical analysis

Student’s t tests and Fisher’s exact tests were applied to calculate overall differences

between the males and females, as well as sex differences for the four difference

morphological subgroups using JMP Statistical Software (JMP 6.0, SAS Institute Inc.

2005). For characteristics with multiple levels, multivariate analyses ( 2) were

performed to assess the distribution of the given character between sexes, and

therefore a single p-value was reported. Multiple logistic and linear regression

analyses which included the sex-by-shape interaction effect were used to assess

whether the difference between sexes were, in fact, dependent on morphology. A p-

value <0.05 was considered statistically significant.

94

Results

The demographics of the entire cohort as well as the independent analysis of males

and females are shown in Table 1. Overall, there were 382 patients (210 male)

diagnosed at an average age of 41.5 ± 19 years with females being significantly older

at diagnosis than males (45.1 ± 20 vs. 35.8 ± 17 years; p < 0.001). As inquired during

the interview, one third of patients had a family history of HCM and 20% of patients

had a family history of sudden cardiac arrest (SCA). Fifty-two patients (14%) were

found to have concomitant hypertension at their evaluation at Mayo Clinic (mean

systolic blood pressure, SBP, 123 ± 17 mmHg), which was more common in women.

Nineteen percent of women (31/172) had concomitant hypertension compared to 11%

of males (21; p = 0.02). Clinically, women were more symptomatic at diagnosis with

respect to dyspnea (p = 0.002) and overall NYHA-class (p = 0.0006). During mean

follow-up of 24 months (range 0.1 – 88 months), 25 patients died of HCM-associated

causes, but no sex-differences were observed in these small numbers.

95

Table 1: Sex differences among patients with clinically diagnosed HCM

Total Male Female p-valueN 382 210 172 Age at Dx (years) 41.5 ± 19 35.8 ± 17 45.1 ± 20 < 0.001 Age > 50 (%) 125 (33) 55 (26) 70 (41) 0.003 Angina* n(%) 151 (40) 80 (38) 71 (45) 0.2 Dyspnea* n(%) 250 (65) 126 (60) 134 (78) 0.002 NYHA-class n(%) Class I 116 (30) 82 (39) 34 (20) Class II 74 (19) 48 (23) 26 (45) Class III 164 (43) 76 (36) 88 (51) Class IV 8 (2) 4 (2) 4 (2)

0.0006

FH HCM (%)§ 117 (31) 62 (30) 55 (32) 0.7 FH SCA (%)§ 53 (14) 34 (17) 19 (11) 0.2 Hypertension (%) 52 (14) 21 (11) 31 (19) 0.02 SBP (mmHg) 123 ± 17 122 ± 16 124 ± 19 0.4 DBP (mmHg) 72 ± 11 73 ± 11 71 ± 12 0.1 Septal myectomy (%) 159 (42) 75 (36) 84 (49) 0.01 Septal ablation (%) 15 (4) 5 (2) 10 (6) 0.1 Echocardiography MLVWT (mm) 21.5 ± 6 21.7 ± 6 21.4 ± 7 0.7 Patients w/ obstruction (%) 294/382 (77) 153 (73) 141 (82) 0.04 Resting gradient (mm Hg) 47.3 ± 42 41.7 ± 40 53.5 ± 45 0.009 EF (%) 72.7 ± 8 72.5 ± 8 73.1 ± 8 0.4 Morphology Sigmoid 181 (47) 102 (49) 79 (46) Reverse 131 (35) 69 (33) 62 (36) Apical 37 (10) 22 (10) 15 (9) Neutral 33 (8) 17 (8) 16 (9)

0.83

Genotype positive 157 (41) 86 (41) 71(41) 1.0 Mutation location Thick filament (%) 57 (15) 23 (11) 34 (20) Intermediate filament (%) 57 (15) 37 (18) 20 (11) Thin filament (%) 12 (3) 9 (5) 3 (2) Z-disc (%) 12 (3) 7 (3) 5 (3) Multiple (%)# 19 (5) 10 (4) 9 (5)

0.06

Dx, diagnosis; DBP, diastolic blood pressure; DT, deceleration time; EF, ejection fraction; HCM, hypertrophic cardiomyopathy; LA, LVEDD, left ventricular end-diastolic dimension; MLVWT, maximum left ventricular wall thickness; SBP, systolic blood pressure SCA, sudden cardiac arrest defined as unexpected death, nocturnal or within one hour of witnessed collapse * Symptomatic status as classified by NYHA-class, data shown are class II, III and IV combined; §In a first-degree relative; #Patients harboring more than one HCM mutation, (double/compound heterozygotes)

96

Although there was no difference in mean MLVWT between men and women

(21.7 ± 6 vs. 21.4 ± 7 mm; p = 0.7), a slightly greater portion of women than men

(141/172 (82%) vs. 153/210 (73%); p = 0.04) had obstructive HCM with a significantly

higher LVOT gradient (53.5 ± 45 vs. 41.7 ± 40 mm Hg; p = 0.009). Overall, sigmoidal-

HCM (181 patients, 47%) and reverse curve-HCM (131 patients, 35%) represented the

two major morphological subtypes (Figure 1); only 37 patients (10%) had apical-HCM

and 33 patients (8%) had neutral contour-HCM. As shown previously, only 14% of

patients with sigmoidal-HCM had a probable disease causing mutation following

comprehensive open reading frame/splice site genetic testing of the 13 HCM-

susceptibility genes compared to 79% of the patients with reverse curve-HCM[7, 17].

Overall, there was no statistical difference in the distribution of each morphological

subtype of HCM or distribution of mutations between men and women.

Figure 1: Two most common morphologic subtypes of HCM. Echocardiographic picture and graphic depiction of the 2 most common morphologic subtypes of HCM: sigmoidal-HCM (47%) and reverse curve-HCM (35%). Gene + = presence of HCM-associated mutation.

97

To investigate the influence of septal contour, we subdivided the cohort into the

four septal contour subgroups and further analyzed the sex-related based differences

of the two major sub-groups of sigmoidal- and reverse curve-HCM. Strikingly, the

effect of sex on clinical phenotype that was first observed for the cohort at large was

present only among patients with sigmoidal-HCM (Table 2). Akin to the initial

observations gleaned from the entire cohort, women with sigmoidal-HCM were older at

diagnosis (56.0 ± 15 vs. 42.6 ± 16 years old; p < 0.001), were more likely to show

obstructive HCM (75/79, 95% vs. 83/102, 84%; p = 0.007), had higher LVOT gradient

(63.9 ± 40 vs. 49.7 ± 42 mm Hg; p = 0.02), and were more likely to have concomitant

hypertension (p = 0.05) compared to men with sigmoidal-HCM. Although not

statistically significant, more women (52%) than men (40%) underwent surgical septal

myectomy (p = 0.1).

In contrast to the overall observation, no statistical differences were seen in

symptomatic status (angina, dyspnea and overall NYHA-class) between sexes and the

two morphological subgroups. Specifically, the clinical presentation in women was

similar between obstructive sigmoidal HCM and obstructive reverse curve-HCM

suggesting that symptoms stem from the degree of obstruction regardless of the

morphological substrate for that obstruction. No statistical differences were observed

between men and women in MLVWT (p = 0.6), EF (0.08) or the presence or location of

a HCM-associated mutation (p = 0.1). Sex had no demonstrable effect for patients with

reverse curve-HCM.

To assess whether the observed differences between sexes were dependent

specifically on the morphology, multiple linear and logistic regression analyses were

performed. For women, age at diagnosis (p = 0.01), systolic blood pressure (p =

0.008), and presence of LVOTO (p = 0.04) were in fact directly dependent on the

sigmoidal morphology whereas prevalence of myectomy no longer achieved statistical

significance.

98

Table 2: Sex differences in the two most common morphological subgroups

Sigmoidal-HCM (n = 181)

Reverse Curve-HCM (n = 131)

Male Female p Male Female p

Multiregr.

model(p)

N 102 79 69 62 Age at Dx (years) 42.6 ±16 56.0 ± 15 <0.001 29.2 ± 16 33.3 ± 18 0.2 0.01 Age > 50yrs (%) 36 (35) 47 (59) 0.002 5 (7) 10 (16) 0.2 0.9 Angina* n(%) 49 (48) 35 (44) 0.8 20 (29) 27 (44) 0.4 0.2 Dyspnea* n(%) 73 (72) 67 (85) 0.1 35 (51) 43 (69) 0.03 0.4 NYHA-class n (%) Class I 29 (28) 10 (13) 33 (48) 17 (27) Class II 23 (23) 21 (26) 18 (26) 19 (31) Class III 49 (48) 47 (59 16 (23) 25 (40) Class IV 1 (1) 2 (3)

0.07

2 (3) 1 (2)

0.07 0.7

FH HCM (%)* 25 (25) 14 (18) 0.4 29 (42) 30 (48) 0.5 0.2 FH SCA (%)* 13 (13) 5 (6) 0.2 15 (22) 10 (16) 0.5 0.5 Hypertension (%) 13 (13) 20 (26) 0.05 2 (3) 4 (7) 0.4 0.1 SBP (mmHg) 124 ± 16 131 ± 30 0.01 118 ± 17 115 ± 13 0.2 0.008 DBP (mmHg) 73 ± 10 73 ± 11 0.6 71 ± 11 69 ± 11 0.3 0.7 Septal myectomy 41 (40) 41 (52) 0.1 27 (39) 26 (42) 0.9 0.4 Echocardiography MLVWT (mm) 19.8 ± 5 19.4 ± 5 0.6 24.5 ± 7 24.6 ± 7 0.9 0.9

Patients w/ obstruction (%) 83 (84) 75 (95) 0.007 51 (74) 47 (76) 0.8 0.04

Resting gradient (mmHg) 49.7 ± 42 63.9 ± 40 0.02 38.1 ± 37 50.3 ± 49 0.1 0.9

EF (%) 73.1 ± 6 74.6 ± 5 0.08 71.7 ± 9 72.6 ± 9 0.6 0.8 Genotype positive 15 (15) 10 (13) 0.8 58 (84) 47 (76) 0.3 0.6 Mutation location Thick filament (%) 0 (0) 4 (5) 0.04 17 (25) 23 (37) 0.1

Intermediate filament (%) 6 (6) 2 (3) 0.5 26 (38) 14 (23) 0.09

Thin filament (%) 1 (1) 1 (1) 1.0 7 (10) 2 (3) 0.2 Z-disc (%) 7 (7) 3 (4) 0.5 0 (0) 0 (0) - Multiple (%)# 1 (1) 0 (0) 1.0 8 (12) 8 (13) 1.0

0.3

Dx, diagnosis; DBP, diastolic blood pressure; DT, deceleration time; EF, ejection fraction; HCM, hypertrophic cardiomyopathy; LA, left atrial LVEDD, left ventricular end-diastolic dimension; MLVWT, maximum left ventricular wall thickness; SBP, systolic blood pressure SCA, sudden cardiac arrest, defined as unexpected death, nocturnal or within one hour of witnessed collapse. * Symptomatic status as classified by NYHA-class, data shown are class II, III and IV combined §In a first-degree relative; #Patients harboring more than one HCM mutation, (double/compound heterozygotes)

99

Our prior demonstration that reverse curve-HCM is predominantly genotype

positive whereas sigmoidal-HCM is mostly genotype negative, prompted us to further

homogenize the two most common subsets of morphological/genetic HCM by

comparing patients with mutation positive/reverse curve-HCM (N = 105) to patients

with mutation negative/sigmoidal-HCM (N = 156). Herein, sex-based differences in age

at diagnosis, LVOT gradient and presence of concomitant hypertension were

significantly higher among women than men for the largest subtype of HCM, i.e.

mutation negative/sigmoidal-HCM (Figure 2).

Figure 2: Male-female comparisons among patients with genotype positive, reverse curve-HCM and patients with genotype negative, sigmoidal-HCM. Bar diagrams showing the sex differences between males and females with HCM in the specific subgoups of reverse-curve, genotype positive (gene +), reverse curve-HCM and genotype negative (gene-), sigmoidal-HCM on age at diagnosis (top left panel), resting left ventricular outflow tract gradient (top right panel), percentage with surgical myectomy (bottom right panel), and percentage with mild hypertension (bottom left panel).

100

To investigate the potential confounding influence of concomitant hypertension,

the analysis of sex-differences per septal subgroup was repeated excluding the

patients diagnosed with concomitant hypertension. As shown in Table 3, all previously

observed, statistically significant differences that were confined to the sigmoidal-HCM

subgroup – age at diagnosis, number of patients with obstruction, degree of LVOT

obstruction and rate of surgical myectomies – persisted, and no new statistically

significant differences were seen (data not shown). Again, logistic regression models

showed a clear female sex-sigmoidal shape dependence with respect to age at

diagnosis (p = 0.006) and presence of LVOTO (p = 0.02). Overall, patients with

sigmoidal-HCM and concomitant hypertension were less likely to undergo surgical

myectomy than patient without hypertension (21% vs. 52%; p < 0.001), explaining the

increase of significance in surgical myectomies when hypertension was excluded from

the analysis.

Table 3: Sex differences in the two most common morphological subgroups after exclusion of patients with mild hypertension

Sigmoidal-HCM(n = 148)

Reverse Curve-HCM (n = 125)

Male Female p Male Female p

Multiple regr.

models (p)

N 89 59 - 67 58 - -

Age at Dx 40.9 ± 16 53.8 ± 15 0.001 29.4 ± 16 31.6 ± 16 0.5 0.006

Patients w/ obstruction n(%) 72 (81) 57 (97) 0.005 50 (75) 44 (75) 1.0 0.02

Resting gradient (mm Hg) 50.0 ± 42 66.1 ± 39 0.02 38.4 ± 37 49.0 ± 49 0.2 0.06

Septal myectomy n(%) 37 (42) 38 (64) 0.008 26 (39) 24 (41) 0.9 0.1

101

Discussion

Long considered a disease of the sarcomere or, more specifically, a disease of the

myofilament, the discovery of mutations in multiple proteins outside the myofilament

has caused an expansion of the spectrum of genetically-mediated pathways

culminating in the disease phenotype that clinicians diagnose as HCM. Currently, over

18 HCM-susceptibility genes have now been published, with 8 of these genes

encoding the essential cardiac myofilaments for which HCM genetic testing is now

commercially available. With this large number of putative pathogenetic genes, a

variety of genes yielding rare mutations, it is intriguing that 30 to 50% of adults with

clinically diagnosed HCM remain genetically unexplained[19]. Binder et al. recently

described an important genotype-phenotype relationship linking the genotypic

substrate to the morphological shape. The analysis of a large cohort of genotyped –

and echocardiographically characterized patients reveal that nearly 80% of patients

with reverse curve-HCM have a positive genetic test for myofilament-HCM whereas

the same genetic test is positive in fewer than 10% of patients clinically diagnosed with

HCM but having a sigmoidal contour (i.e. sigmoidal-HCM) [7]. More recently, the yield

for sigmoidal-HCM increased from 8% to 14% with the inclusion of 5 Z-disc associated

genes[17]. Conversely, myofilament-HCM preferentially yields reverse curve-HCM

whereas Z-disc-HCM predisposes to the development of sigmoidal-HCM[20].

Now, this present study examines the influence of sex in the interplay between

genetic substrate and anatomical shape. Overall, this cohort mirrors previously

published studies that examined the effect of sex on a presumably heterogeneous

cohort of HCM lacking both morphological and genetic sub-classification[9, 10, 11].

Moreover, the data presented herein suggest that the differences are largely confined

to sigmoidal-HCM which constitutes the anatomical phenotype of nearly half of the

patients with clinically diagnosed HCM in our institution. Furthermore, it shows that

specifically for age at diagnosis, systolic blood pressure and presence of LVOTO,

there is a direct sex-by-morphology interaction for women with sigmoidal-HCM.

102

These observations among distinct subtypes of HCM generate several

questions regarding the influence of sex in the phenotypic expression of disease. More

specifically, it suggests that gender does not seem to be a significant modifier in

reverse curve-HCM. This is supported by our original morphological data that the

underlying genotype rather than sex appears to be the predominant determinant of

septal morphology[7]. Thus, in reverse curve-HCM, the presence of a structural

myofilament mutation is the driver of the phenotype, whereas in sigmoidal-HCM, a

multi-factorial process culminates in a clinical expression of HCM, with significant male

– female differences.

The presence of mild, concomitant hypertension may be a contributing factor in

the pathogenesis of sigmoidal-HCM in women as 1 of 4 females within this

morphological classification of HCM were mildly hypertensive. Several studies have

shown that in response to pressure-overload, sex differences in the hypertrophic

response patterns can be seen. Krumholz et al. showed that in isolated hypertension,

significant sex differences can be observed in cardiac adaptation. In contrast to our

findings, they show females predominantly develop a concentric hypertrophy, whereas

a more eccentric pattern was observed in men[21]. Similar patterns of sex-dependent

hypertrophy were observed in aortic stenosis[22, 23] and as a response to

hemodynamic overload after myocardial infarction[24]. Furthermore, the presence of

concomitant hypertension could mean there has always been a presence of low-grade

hypertension and therefore a higher afterload in these patients. These factors

combined with a (undefined) genetic susceptibility for HCM by means of a

pathogenetic mutation or a LVH-promoting polymorphism [25], or endocrine factors

[26] could all converge in the phenotype of clinically diagnosed, sigmoidal-HCM.

Although recent studies have shown that the prevalence of LVOTO is far more

prevalent than previously believed [27, 28], our study may be biased with its higher

prevalence of patients with obstructive HCM at rest due to our role as tertiary referral

center for the surgical treatment of HCM. This is reflected in higher prevalence of

patients with resting LVOTO (75% vs. ~35-40% in other published HCM cohorts)[27,

28] as well as a higher rate of surgical myectomies (42% vs. ~5-10% in other

published HCM cohorts)[29]. Our observations might therefore be less applicable to a

broader spectrum of patients with HCM, particularly non-obstructive HCM. On the

other hand, the conclusions regarding these important sex-substrate differences

appear robust for the subset of patients with obstructive disease.

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Conclusions

In this large cohort of comprehensively genotyped and morphologically classified,

unrelated patients with clinically diagnosed HCM, we observed that the striking and

previously noted sex-related differences in HCM are confined largely to the subset of

patients with mutation negative, sigmoidal-HCM. Sex does not appear to be a

significant genetic modifier in myofilament-HCM.

Acknowledgements

We are indebted to the statisticians from Mayo Clinic’s NIH funded Center for

Translational Science Activities (CTSA) for their help with the proper study design and

statistical analyses.

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17. Theis JL, Bos JM, Bartleson VB, Will ML, et al. Echocardiographic-determined septal morphology in Z-disc hypertrophic cardiomyopathy. Biochem Biophys Res Commun 2006; 351(4): 896-902. 18. Bos JM, Poley RN, Ny M, Tester DJ, et al. Genotype-phenotype relationships involving hypertrophic cardiomyopathy-associated mutations in titin, muscle LIM protein, and telethonin. Mol Genet Metab 2006; 88(1): 78-85. 19. Van Driest SL, Ommen SR, Tajik AJ, Gersh BJ, et al. Sarcomeric genotyping in hypertrophic cardiomyopathy. Mayo Clin Proc 2005; 80(4): 463-469. 20. Bos JM, Ommen SR, Ackerman MJ. Genetics of hypertrophic cardiomyopathy: one, two, or more diseases? Curr Opin Cardiol 2007; 22(3): 193-199. 21. Krumholz HM, Larson M, Levy D. Sex differences in cardiac adaptation to isolated systolic hypertension. Am J Cardiol 1993; 72(3): 310-313. 22. Carroll JD, Carroll EP, Feldman T, Ward DM, et al. Sex-associated differences in left ventricular function in aortic stenosis of the elderly. Circulation 1992; 86(4): 1099-1107. 23. Kostkiewicz M, Tracz W, Olszowska M, Podolec P, et al. Left ventricular geometry and function in patients with aortic stenosis: gender differences. Int J Cardiol 1999; 71(1): 57-61. 24. Jain M, Liao R, Podesser BK, Ngoy S, et al. Influence of gender on the response to hemodynamic overload after myocardial infarction. Am J Physiol Heart Circ Physiol 2002; 283(6): H2544-2550. 25. Perkins MJ, Van Driest SL, Ellsworth EG, Will ML, et al. Gene-specific modifying effects of pro-LVH polymorphisms involving the renin-angiotensin-aldosterone system among 389 unrelated patients with hypertrophic cardiomyopathy. Eur Heart J 2005; 26(22): 2457-2462. 26. Malhotra A, Buttrick P, Scheuer J. Effects of sex hormones on development of physiological and pathological cardiac hypertrophy in male and female rats. Am J Physiol 1990; 259(3 Pt 2): H866-871. 27. Maron MS, Olivotto I, Zenovich AG, Link MS, et al. Hypertrophic cardiomyopathy is predominantly a disease of left ventricular outflow tract obstruction. Circulation 2006; 114(21): 2232-2239. 28. Shah JS, Tome Esteban MT, Thaman R, Sharma R, et al. Prevalence of exercise induced left ventricular outflow tract obstruction in symptomatic patients with non-obstructive hypertrophic cardiomyopathy. Heart 2008; 94(10): 1288-94. 29. Maron BJ. Controversies in cardiovascular medicine. Surgical myectomy remains the primary treatment option for severely symptomatic patients with obstructive hypertrophic cardiomyopathy. Circulation 2007; 116(2): 196-206; discussion 206.

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Chapter 6

TGFß-Inducible Early Gene-1 (TIEG1):A Novel Hypertrophic Cardiomyopathy-

Susceptibility Gene

J. Martijn Bos, Malayannan Subramaniam, John R. Hawse, I. Christiaans, Steve R. Ommen, Arthur A.M. Wilde, Thomas C. Spelsberg, Michael J. Ackerman

Manuscript in preparation

Abstract

Background: Hypertrophic cardiomyopathy (HCM) is the most common heritable

cardiovascular disease and the most common cause of sudden cardiac death in the

young. Over 24 genes have been implicated in the pathogenesis of HCM. However, for

about half of patients with HCM, the genotypic substrate remains elusive. A recent

study showed that male TGF Inducible Early Gene-1 (TIEG1) knock-out (TIEG1-/-)

mice develop HCM after 16 months. Microarray analysis on the mice hearts showed a

13-fold up-regulation of PTTG1-encoded pituitary tumor-transforming gene 1. We

therefore speculated that TIEG1 could be a novel candidate gene in the pathogenesis

of genotype negative HCM, possibly through a loss of its repression on PTTG1

expression.

Methods: For this study, we analyzed a cohort of 923 unrelated patients from two

independent cohorts of patients with HCM (664 male, age at diagnosis 47.6 ± 18,

mean left ventricular wall thickness (MLVWT) 20.0 ± 7mm). All patients were genotype

negative with respect to the 9 genes responsible for myofilament/sarcomeric-HCM.

Open reading frame/splice site mutational analysis of TIEG’s 4 translated exons was

performed using DHPLC and direct DNA-sequencing. Site directed mutagenesis was

performed to clone novel variants. The effect of wild type and mutant TIEG1 on the

PTTG1 and SMAD7 promoters was studied using transient transfection and luciferase-

assays. Cardiac HCM tissue was studied by immunohistochemistry to determine levels

of PTTG1 protein expression.

Results: Six novel missense mutations (A12T, M27T, T216A, E137K, A204T and

S225N) in TIEG1 were discovered in 6/923 patients (2 males/4 females, mean age at

diagnosis 56.2 ± 23 years, MLVWT 20.8 ± 4 mm). Each missense mutation was

absent in 800 ethnically-matched reference alleles and involved residues that were

conserved across species. Compared to the 50% repression of PTTG1 promoter

function by wild type TIEG1, 5 TIEG1 mutants had this repression significantly

attenuated resulting in marked accentuation of PTTG1 promoter function similar to the

TIEG1-/- KO-mice. One TIEG1 mutant significantly altered TIEG1-function on SMAD7-

expression. By immunohistochemistry, PTTG1-protein expression was increased in

myectomy specimens from all patients with HCM, irrespective of TIEG1 mutation

status, compared to normal hearts.

108

Conclusions: This is the first paper to associate mutations in TIEG1 to human

disease with the discovery of 6 novel, HCM associated variants. Functional assays

suggest a role for PTTG1 and SMAD7 in the pathogenesis of TIEG1-mediated HCM.

Up-regulation of PTTG1 could be a final common pathway response in HCM. Future

studies are needed to elucidate the precise role of PTTG1 in the pathogenesis of

TIEG1-HCM as well as HCM in general.

Keywords

Hypertrophic cardiomyopathy, TIEG1, KLF10, genes, PTTG1

109

Background

In the last two decades, over 24 disease-susceptibility genes have been elucidated for

hypertrophic cardiomyopathy (HCM), a disease characterized by unexplained cardiac

hypertrophy that affects approximately 1 in 500 individuals [1, 2]. Currently over 80% of

reverse-curve HCM and 10% of sigmoidal-HCM is explained by mutations in genes

encoding the myofilaments of the cardiac sarcomere[3], making a large portion of

sigmoidal HCM genetically elusive. More recently, rare mutations in genes encoding Z-

disc proteins and calcium handling proteins have been linked to the pathogenesis of

HCM[1], but the search for novel HCM-causing genes continues.

TIEG1-encoded TGF -inducible early gene-1 (TIEG1)(also referred to a

KLF10-encoded Kr ppel-Like Factor 10) was discovered originally as an early

response gene following TGF treatment of human osteoblast and is expressed in

many tissues, including cardiac myocardium[4, 5]. It is a member of the Krüppel-like

family of transcription factors, which are known to be involved in anti-proliferative and

apoptotic inducing functions following TGF -induction[6]. Subsequent studies in

engineered TIEG1-knock out (TIEG-/- ) mice[7] showed that male mice develop HCM

with significant, but relatively late-onset cardiac hypertrophy at 16 months of age[8].

Microscopic examination of TIEG-/--male mice hearts showed characteristic hallmarks

of HCM: cardiomyocyte hypertrophy, fibroblast hyperplasia and myocyte disarray[8].

Furthermore, microarray analysis revealed a significant up-regulation of PTTG1-

encoded pituitary tumor transforming gene-1, demonstrating that TIEG1 plays an

important role in the repression of proliferative and hypertrophic pathways, possibly

through the actions of PTTG1. Based on these findings, we hypothesized that TIEG1

could be a novel HCM-susceptibility gene.

110

Methods

Study cohort

Our study cohort consisted of 923 unrelated patients with HCM from two large cardiac

referral centers – Mayo Clinic (Rochester, MN USA) and the Academic Medical Center

(AMC, Amsterdam, The Netherlands). All patients were genotype negative for

mutations in the 9 HCM-associated genes, currently included in commercially available

genetic tests (MYBPC3, MYH7, TNNT2, TNNI3, TNNC1, TPM1, MYL2, MYL3, and

ACTC). Clinical data were collected on all patients, including pertinent personal and

family history (especially with regard to HCM or sudden cardiac arrest (SCA), and an

echocardiogram to determine maximum left ventricular wall thickness (MLVWT) and

resting left ventricular outflow tract gradient (LVOT). Clinical diagnosis of HCM was

made when subjects had a MLVWT over 13mm in the absence of hypertrophy

inducing conditions such as aortic stenosis or hypertension.

Genetic analysis

DNA of all patients was extracted from peripheral blood lymphocytes (Gentra Inc,

Minneapolis, MN). After design of intronic primers, for each patient all 4 translated

exons of TIEG1 (Nm_005646) were amplified by polymerase chain reaction (PCR) and

subsequently analyzed for genetic variations by denaturing high performance liquid

chromatography (DHPLC)(WAVE®, Transgenomic, Omaha, NE). Abnormal DHPLC-

elution profiles were subjected to direct DNA sequencing (ABI Prism 377, Applied

Biosystems, Foster City, CA) to determine the nature of nucleotide substitution. All

translated exons were analyzed for 800 Caucasian reference alleles from ostensibly

healthy, ethnically-matched controls to distinguish novel HCM-associated mutations

from rare or common polymorphisms.

Site-directed mutagenesis and luciferase assays

After design of sequence specific primers, identified mutations and control variants

were created using site-directed mutagenesis, cloned into the pcDNA4.0 expression

vector (Invitrogen, Carlsbad, CA) and transformed into XL-10 ultracompetent cells

(Quickchange® II, Stratagene, La Jolla, CA). DNA was purified from bacteria

(Miniprep®, Qiagen, Valencia, CA) and all constructs were confirmed by direct DNA

sequencing.

111

Effects of the various TIEG1 mutations on PTTG1-promoter activity (PTTG1-

promoter including the 5’-flanking region (-1,321 to -3) cloned in front of a luciferase

reporter as previously described[8]) were studied using luciferase assays. Blinded to

the observer, the PTTG1-promoter construct (1 g) was transfected into AKR2B mouse

embryo fibroblasts along with 1 g of empty expression vector, wild-type (WT) TIEG1-

expression vector or the various mutant TIEG1 expression vectors. Following 24h of

transfection, cell lysates were prepared and analyzed for luciferase activity. Luciferase

assays were repeated at least 3 times and values were normalized to total protein

concentrations and expressed as fold-change relative to empty-vector promoter

activity.

To assess the effect of mutations on the cardiac expressed SMAD7-promoter,

1 g of empty expression vector, and either WT-TIEG1 or TIEG1 mutants were

transfected into H9c2 rat cardiocytes along with a SMAD7-promoter construct (1ug).

Eight hours after transfection, culture media (DMEM + 10% horse serum (HS)) was

replaced with DMEM with 1% HS to induce cardiocyte differentiation. Thirty-six to forty-

eight hours after transfection, cells were lysed, and luciferase assays were performed

and analyzed as described above.

Immunohistochemistry

To determine expression of PTTG1-protein, cardiac tissue obtained following surgical

myectomy in patients with HCM and from non-failing left ventricular hearts at autopsy

(controls) was stained with monoclonal PTTG1-antibody (Epitomics, Burlingame, CA).

Formalin fixed, paraffin embedded tissue was retrieved from 2 TIEG1-genotype

positive patients, 2 genotype negative HCM patients, and 2 autopsy-negative, non-

cardiac death subjects. Paraffin-blocks were sectioned at 5 m for

immunohistochemical staining. Deparaffinization with xylene and subsequent

rehydration with graded ethanol preceded heat induced epitope retrieval with EDTA

buffer (pH 8) in a Lab Vision PT Module (Fremont, CA). The staining procedure was

carried out by an automated immunohistochemistry-staining machine (DAKO

Techmate 500, DAKO, Denmark) using the Envision program. Titration for correct

dilution of antibody was performed and after review, a dilution of 1:75 was selected for

all assays. We compared expression of PTTG1-protein in tissue of TIEG1-genotype

positive patients to genotype negative HCM patients as well as cardiac tissue of two

autopsy negative, non-cardiac death subjects.

112

Statistical analyses

Statistical analyses were performed using JMP 7.0 statistical software (JMP, Cary, NC)

using analysis of variance (ANOVA) and Student’s t-test. A p-value <0.05 was

considered statistically significant.

113

Results

Demographics of the study cohort are summarized in Table 1. Overall 923 patients

with HCM (664 male) were enrolled in this study – 739 from Mayo Clinic (USA) and

184 from Academic Medical Center (AMC) (NL). Patients had an average age at

diagnosis of 47.6 ± 18 years and mean MLVWT of 20.0 ± 7 mm. Twenty-six percent of

patients reported a family history of HCM and 15% of patients had a family history of

SCA. Thirty percent of patients had undergone surgical myectomy and 14% of patients

had received an ICD. The specific demographics of each cohort (Mayo and AMC) are

detailed in Table 1. Overall, patients from the AMC cohort had a lower MLVWT (17.5

± 5.4 vs. 17.5 ± 5.4 (p =0.03) and were more likely a family history of HCM (35% vs.

25%, p = 0.005) or sudden cardiac arrest (SCA) (47% vs. 15%, p < 0.001). Patients at

Mayo were likely to have undergone surgical septal myectomy (38% vs, 6%, p<0.001),

reflecting the referral bias for Mayo Clinic as a surgical center for treatment of

obstructive HCM.

Table 1: Demographics of study cohort

Total Mayo AMC

N 923 739 184

Sex (male/female) 664/359 441/298 123/61

Age at Dx, years 47.6 ± 18 47.4 ± 18 48.3 ± 19

Septal thickness, mm 20.0 ± 7 20.6 ± 8 17.5 ± 5.4*

LVOT gradient, mm Hg 45.1 ± 40 45.2 ± 44 44.7 ± 29

Family history of HCM, n (%) 249 (26) 184 (25) 65 (35)*

Family history of SCA, n (%) 199 (22) 113 (15) 86 (47)*

Myectomy, n (%) 289 (31) 281 (38) 8 (6)*

ICD, n (%) 126 (14) 103 (14) 23 (14)

Dx, diagnosis; HCM, hypertrophic cardiomyopathy; ICD, implantable cardioverter defibrillator; LVOT, left ventricular outflow tract; SCA, sudden cardiac arrest. *, p<0.05

114

Genetic results

Genetic analysis of TIEG1’s open reading frame (exome) revealed 6 novel missense

mutations (A12T, M27T, E137K, A204T, T216A and S225N) in 6 patients with HCM

that were absent in 800 reference alleles (Figure 1). One novel variant (Q10H) was

discovered in HCM patients as well as healthy controls at similar frequencies (allelic

frequency 0.4%). One previously reported rare polymorphism (S249F, rs4734653) was

seen in 3 HCM-patients, but not in our cohort of 400 ethnically matched controls. This

variant, however, was previously described in 0.8% of healthy Europeans.

Figure 1: Topology of TIEG1-protein. Schematic representation of 480 amino acid containing TIEG1 protein with HCM-associated (black circles) and control variants/polymorphisms (white circles) identified in two cohorts of HCM patients.

115

All variants and surrounding residues found in patients with HCM (Figure 2A) as well as control variants (Figure 2B) were conserved across species and the mutant

residues were not seen in other species. In certain species, general sequence

homology was poor or even absent for the first 90 residues of TIEG1.

Figure 2: Sequence conservation. Shown is the conservation across species of (a) novel, HCM-associated TIEG1-mutations (top panel) and (b) novel and previously described control variants (bottom panel).

116

117

Patient characteristics

Patient characteristics for TIEG1-positive patients are summarized in Table 2. Each

mutation was found once in 2 male and 4 female patients. All patients were of

Caucasian ethnicity. Overall, there did not seem to be a specific phenotype associated

with TIEG1-mediated HCM, although in most cases – except for case 2 - HCM was

late onset (mean age at diagnosis 56.2 ± 23 years). The mean MLVWT of TIEG1-

positive patients was 20.8 ± 4 mm and family history of HCM was found in 2/6 (cases 3 and 6) and SCA in 1 case (case 6). Three patients had undergone surgical septal

myectomy for relief of symptoms (cases 2-4), a number relatively high compared to

the annual average rate of myectomy in HCM (~5-10%). The most severely affected

patient (case 2) was a man with M27T-TIEG1. He was diagnosed at 15 years of age,

with extreme hypertrophy (MLVWT, 26mm) and obstruction (117 mmHg gradient) for

which a surgical myectomy was performed. To date, both parents and most siblings do

not meet the diagnostic criteria of HCM following frequent echocardiographic screening

suggesting variable penetrance of the disease or a de novo mutation in this patient.

His family, as well as the others’, have been contacted but have declined or have not

yet enrolled for co-segregation studies.

Tabl

e 2:

Pat

ient

cha

ract

eris

tics

CO

PD

, ch

roni

c ob

stru

ctiv

e pu

lmon

ary

dise

ase;

Dx,

dia

gnos

is;

HC

M,

hype

rtrop

hic

card

iom

yopa

thy;

HF,

hea

rt fa

ilure

; IC

D,

impl

anta

ble

card

iove

rter

defib

rilla

tor;

LV

OT,

left

vent

ricul

ar o

utflo

w tr

act;

SC

A, s

udde

n ca

rdia

c ar

rest

.

Cas

eC

ohor

tN

ucle

otid

ech

ange

Mut

atio

nSe

xA

ge a

t D

x(y

rs)

MLV

WT

(mm

)LV

OT

grad

ient

(mm

Hg)

FH HC

MFH SC

ATr

eatm

ent

Oth

er

1A

MC

c.

GC

G>A

CG

p.

A12

T M

65

17

13

N

o N

o -

-

2M

ayo

c.A

TG>A

CG

p.

M27

T M

15

26

11

7 N

o N

o M

yect

omy

Par

ents

+ m

ost

sibl

ings

ech

o ne

gativ

e

3M

ayo

c.G

AA

>AA

A

p.E

137K

F

48

20

55

Yes

N

o M

yect

omy,

IC

D

Two

sons

echo

neg

4A

MC

c.

AC

A>G

CA

p.

T216

A

F 58

16

20

N

o N

o M

yect

omy

Fath

er d

ied

sudd

enly

at a

ge

63 u

nkno

wn

caus

e 5

May

o c.

GC

T>A

CT

p.A

204T

F

80

20

49

No

N

o -

6M

ayo

c.A

GT>

AA

T p.

S22

5N

F 71

26

16

Y

es

Yes

-

Pat

ient

dec

ease

d at

age

76

of H

F an

d C

OP

D

SMAD7 promoter activity in H9c2-cardiocytes

To study the effect of TIEG1 on SMAD7-promoter expression in a more native

environment, we performed a luciferase assay in H9c2 rat cardiocytes. Thirty-six to

forty-eight hours after transfection, cardiocytes were lysed and analyzed for luciferase

activity as described above. As expected, WT TIEG1 repressed SMAD7-promoter

activity by ~70%. Four of the 6 HCM-associated variants as well as the two control

variants exhibited normal TIEG1-like function relative to SMAD7 (Figure 3). Two of the

putative TIEG1-HCM mutations – A12T and S225N – altered SMAD7-promoter

expression with S225N-TIEG1 showing significantly increased expression of SMAD7

promoter activity as compared to wild-type (Figure 3).

Figure 3: SMAD7-promoter activity in H9c2-cardiocytes. Bar diagram showing SMAD7-promoter activity in H9c2-cardiocytes. Wild-type TIEG1 (WT) repressed SMAD7-expression. Four of six mutations as well as control variants act like WT, whereas S225N-TIEG1 significantly alters TIEG1-function on SMAD7-expression.

119

PTTG1 promoter activity in AKR2B-fibroblasts

Twenty-four hours after transfection with the PTTG1 promoter and either WT-TIEG1,

mutant TIEG1, or control variant expression constructs, AKR2B-cells were lysed and

analyzed for luciferase activity. As expected, WT TIEG1 repressed PTTG1-promoter

activity by ~55% (Figure 4). Akin to data from TIEG1-/- -mice, 5 of the 6 putative

TIEG1-HCM mutations resulted in luciferase activity that was significantly higher than

that of WT (p < 0.05 compared to WT), and in the case of T216A-TIEG1, up to 2 fold

higher than vector control (Figure 4). In contrast, PTTG1-promoter activity seen in

Q10H-TIEG1 control variant was identical to WT TIEG1 effect, while on the other hand,

S249F-TIEG1 showed increased PTTG1 activity.

Figure 4: PTTG1-promoter activity in AKR2B-fibroblasts. Bar diagram showing PTTG1-promoter activity in AKR2B-fibroblasts. Wild-type TIEG1 (WT) repressed PTTG1-expression, where 5 of 6 mutations altered PTTG1-expression significantly.

120

PTTG1 protein expression in cardiac tissue

In order to determine the expression of PTTG1 protein in HCM patients, we performed

immunohistochemical analysis on paraffin embedded tissue sections derived from

patients with TIEG1-HCM (cases 2 and 3), genotype negative-HCM and autopsy

normal hearts (Figure 5). Characteristic hallmarks of HCM, such as myofibrillar

disarray as well as fibrosis could be seen in all myocardial specimens from all 4

patients with HCM (Figure 5C-F). Only mild to no PTTG1-protein expression was seen

in normal heart tissue (Figure 5A+B) as previously described[9]. In contrast, PTTG1

protein expression was dramatically increased in tissue of both HCM genotype

negative patients (Figure 5C + D) as well as the two patients (Cases 2 and 3) with

TIEG1-HCM (Figure 5E + F). PTTG1 localized mostly to cytoplasm and myofibers of

cardiomyocytes and strongest expression was seen in TIEG1-mediated disease. This

data suggests PTTG1 could be a biomarker for HCM in general, although more data is

needed to determine whether there is a different expression of PTTG1 between

genotype negative and TIEG1-mediated HCM.

121

Figure 5: Immunohistochemistry staining of PTTG1 in cardiac tissue. Immunohistochemical staining for PTTG1 protein expression demonstrates little PTTG1-protein expression in autopsy normal hearts (A and B), but severely increased levels of PTTG1 protein in myocardial specimens derived from patients with genotype negative-HCM (C and D) and TIEG1-HCM. Characteristic hallmarks of HCM – myofibrillar disarray and fibrosis – can also be seen in HCM tissues (C-F).

122

Discussion

Over the past two decades, multiple genes encoding proteins involved in various

processes in the cell have been implicated in the pathogenesis of HCM. Since the

discovery of the first gene, MYH7-encoded -myosin heavy chain for HCM in 1989,

over 24 HCM-susceptibility genes have been reported and commercial genetic testing

is now available for a large subset of these genes[1, 10]. However, for many patients,

the underlying genetic cause remains elusive and research continues to discover novel

HCM-associated genes. Recently, male TIEG1-/- mice exhibited features of late-onset

HCM, including asymmetric cardiac hypertrophy, increased ventricular size at 16

months of age, increased heart weight to body weight ratio, increased fibrosis and

increased wall thickness compared to WT mice[8]. Furthermore, Masson’s Trichrome-

staining demonstrated evidence of myocyte disarray and fibrosis which led us to

hypothesize that TIEG1 could be a candidate gene in the pathogenesis of HCM.

Herein, we analyzed 923 unrelated patients with HCM from the USA and the

Netherlands. After comprehensive analysis of all translated regions of TIEG1, we

discovered 6 novel, HCM-associated missense mutations in patients which were

absent in 800 ethnically-matched Caucasian healthy control subjects. Furthermore, we

discovered a novel control variant. The Q10H-variant was seen in our patients as well

as in our 800 reference alleles (allelic frequency 0.4%). Notably, S249F-TIEG1

(rs4734653) was seen in our patients (Allelic frequency 0.5%), was absent in our

reference alleles; but was seen in 0.8% of European controls.

Overall, no TIEG1-specific phenotype seemed to be associated with human

TIEG1-HCM, although similar to the TIEG -/- mice, the patient’s cardiac hypertrophy

was of late-onset and obstructive in most cases. While HCM-phenotype was seen

exclusively in male- TIEG -/- mice, no gender predilection could be demonstrated

among the small cohort of patients with putative TIEG1-HCM. Female TIEG1-/- mice

are known to have skeletal defects which have been characterized as osteopenia[11].

Careful examination of TIEG1-mutation positive patients’ charts – especially women -

revealed no specific skeletal problems in our patients, although it must be

acknowledged that most patients were specifically referred to a cardiologist for HCM

evaluation.

123

Transforming growth factor- (TGF ) is a key mediator of cardiac adaptations

to hemodynamic overload and plays a critical role in induction of cardiac hypertrophy,

heart failure and fibrosis[12]. This is caused by TGF -induced expression of collagen

mRNA and subsequent collagen deposition in fibroblasts, induced expression of

factors of the fetal gene program (MYH7 and ACTC) in cardiomyocytes, and through

activation of the MAPK signaling pathway and p38-induced transcription factors[13,

14]. TIEG1 plays a critical role in the regulation of TGF in multiple cell types. First, it

was demonstrated that expression of TIEG1 is increased within 30 minutes following

TGF treatment in osteoblast cells[4]. Normal TIEG1 function then subsequently

attenuates TGF -signaling through either activation of SMAD2 or repression of the

inhibitory co-factor SMAD7[15, 16, 17].

SMAD7 is a member of the SMAD-family of proteins involved in TGF -

signaling, and with SMAD6, comprises the subgroup of inhibitory SMADS that

antagonize TGF -family members [18, 19]. While most mice devoid of the

indispensible MH2-domain of SMAD7 die in utero, surviving mice have impaired

cardiac functions (such as ejection – and shortening fraction) and cardiac

arrhythmias[20]. Because of its known role in TIEG1-TGF -signaling, as well as the

mutant mouse phenotype, we sought to examine the effects of our novel HCM-

associated TIEG1-variants on the activity of the SMAD7-promoter. We found that two

of the 6 mutants and none of the control variants alter TIEG1-function on SMAD7, with

one variant showing significantly altered function compared to WT TIEG1.

Further studies of TIEG1-/- male mice demonstrated the mice develop

characteristic features of HCM during aging with a marked upregulation of PTTG1[8].

PTTG1 is typically overexpressed in a variety of endocrine-related tumors, especially

pituitary, thyroid, breast, ovarian, and uterine tumors as well as non-endocrine tumors.

PTTG1 functions in cell replication, proliferation, DNA damage/repair, organ

development, and metabolism (reviewed in [21]). In vitro luciferase assays studies

demonstrated that TIEG1 acts directly on the promoter of PTTG1 causing a 60-70%

drop in PTTG1’s promoter activity suggesting that the observed myocyte hypertrophy

and fibrosis in male TIEG-/- mice may be mediated by loss of TIEG1’s normal inhibition

over PTTG1 and consequential accentuation in PTTG1 gene expression [8]. Akin to

these observations, our current study showed that 5 of our 6 TIEG1 mutations resulted

in a significant increase in PTTG1 promoter activity relative to WT TIEG1.

124

In addition, protein levels of PTTG1 in surgically resected, hypertrophic

myocardium of patients with TIEG1-HCM, was markedly increased. Further,

accentuation in PTTG1 might be a final common pathway hypertrophic response as

two patients with genotype negative HCM also displayed this finding. These data

suggest that these putative HCM-associated TIEG1 mutations dysregulate TIEG1’s

normal repressive control over either SMAD7 or PTTG1 and that PTTG1 protein

expression might be a ‘final common pathway’ biomarker in HCM[22, 23]. Further

studies are therefore needed to dissect the biological role of PTTG1. Conceivably,

gain-of-function PTTG1 mutations in humans or transgenic overexpression of PTTG1

in mice could precipitate HCM.

125

Conclusions

This is the first report to associate mutations in the TIEG1 gene with human disease.

We have identified 6 novel, HCM associated TIEG1 missense mutations and have

demonstrated that a number of these variants have abnormal function with regard to

mutant TIEG1’s ability to regulate either the PTTG1 or SMAD7 promoters, two genes

known to be associated with hypertrophic pathways. Furthermore, tissue expression of

PTTG1 seemed to be associated with HCM in general with highest expression seen in

TIEG1-mediated HCM suggesting PTTG1 might be a biomarker for HCM. While these

studies have implicated TIEG1 in human HCM, additional in vitro and in vivo functional

studies are needed to further elucidate the exact pathway(s) leading to HCM in TIEG1-

genotype positive patients. Furthermore, studies are needed to examine the potential

role of PTTG1 as a biomarker in the pathogenesis of HCM.

126

References 1. Bos JM, Towbin JA, Ackerman MJ. Diagnostic, prognostic, and therapeutic implications of genetic testing for hypertrophic cardiomyopathy. J Am Coll Cardiol 2009; 54(3): 201-211. 2. Maron BJ. Hypertrophic cardiomyopathy: A systematic review. JAMA 2002; 287(10): 1308-1320. 3. Binder J, Ommen SR, Gersh BJ, Van Driest SL, et al. Echocardiography-guided genetic testing in hypertrophic cardiomyopathy: Septal morphological features predict the presence of myofilament mutations. Mayo Clin Proc 2006; 81(4): 459-467. 4. Subramaniam M, Harris SA, Oursler MJ, Rasmussen K, et al. Identification of a novel tgf-beta-regulated gene encoding a putative zinc finger protein in human osteoblasts. Nucleic Acids Res 1995; 23(23): 4907-4912. 5. Subramaniam M, Hefferan TE, Tau K, Peus D, et al. Tissue, cell type, and breast cancer stage-specific expression of a TGF-beta inducible early transcription factor gene. J Cell Biochem 1998; 68(2): 226-236. 6. Dang DT, Pevsner J, Yang VW. The biology of the mammalian kruppel-like family of transcription factors. Int J Biochem Cell Biol 2000; 32(11-12): 1103-1121. 7. Subramaniam M, Gorny G, Johnsen SA, Monroe DG, et al. TIEG1 null mouse-derived osteoblasts are defective in mineralization and in support of osteoclast differentiation in vitro. Mol Cell Biol 2005; 25(3): 1191-1199. 8. Rajamannan NM, Subramaniam M, Abraham TP, Vasile VC, et al. TGFbeta inducible early gene-1 (tieg1) and cardiac hypertrophy: Discovery and characterization of a novel signaling pathway. J Cell Biochem 2007; 100(2): 315-325. 9. Chiriva-Internati M, Ferraro R, Prabhakar M, Yu Y, et al. The pituitary tumor transforming gene 1 (PTTG-1): An immunological target for multiple myeloma. J Transl Med 2008; 6: 15. 10. Geisterfer-Lowrance AA, Kass S, Tanigawa G, Vosberg H, et al. A molecular basis for familial hypertrophic cardiomyopathy: A beta cardiac myosin heavy chain gene missense mutation. Cell 1990; 62: 999-1006. 11. Hawse JR, Iwaniec UT, Bensamoun SF, Monroe DG, et al. TIEG-null mice display an osteopenic gender-specific phenotype. Bone 2008; 42(6): 1025-1031. 12. Brand T, Schneider MD. The TGF beta superfamily in myocardium: Ligands, receptors, transduction, and function. J Mol Cell Cardiol 1995; 27(1): 5-18. 13. Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signaling pathways. Nat Rev Mol Cell Biol 2006; 7(8): 589-600. 14. Parker TG, Packer SE, Schneider MD. Peptide growth factors can provoke "Fetal" Contractile protein gene expression in rat cardiac myocytes. J Clin Invest 1990; 85(2): 507-514. 15. Johnsen SA, Subramaniam M, Janknecht R, Spelsberg TC. TGFbeta inducible early gene enhances tgfbeta/smad-dependent transcriptional responses. Oncogene 2002; 21(37): 5783-5790. 16. Johnsen SA, Subramaniam M, Katagiri T, Janknecht R, et al. Transcriptional regulation of SMAD2 is required for enhancement of TGFbeta/SMAD signaling by TGFbeta inducible early gene. J Cell Biochem 2002; 87(2): 233-241.

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17. Johnsen SA, Subramaniam M, Monroe DG, Janknecht R, et al. Modulation of transforming growth factor beta (TGFbeta)/SMAD transcriptional responses through targeted degradation of TGFbeta-inducible early gene-1 by human seven in absentia homologue. J Biol Chem 2002; 277(34): 30754-30759. 18. Heldin CH, Miyazono K, ten Dijke P. TGFbeta signalling from cell membrane to nucleus through smad proteins. Nature 1997; 390(6659): 465-471. 19. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003; 113(6): 685-700. 20. Chen Q, Chen H, Zheng D, Kuang C, et al. SMAD7 is required for the development and function of the heart. J Biol Chem 2009; 284(1): 292-300. 21. Vlotides G, Eigler T, Melmed S. Pituitary tumor-transforming gene: Physiology and implications for tumorigenesis. Endocr Rev 2007; 28(2): 165-186. 22. Bowles NE, Bowles KR, Towbin JA. The "Final common pathway" Hypothesis and inherited cardiovascular disease. The role of cytoskeletal proteins in dilated cardiomyopathy. Herz 2000; 25(3): 168-175. 23. Olivotto I, Cecchi F, Poggesi C, Yacoub MH. Developmental origins of hypertrophic cardiomyopathy phenotypes: A unifying hypothesis. Nat Rev Cardiol 2009; 6(4): 317-321.

128

Chapter 7

Diagnostic, Prognostic and Therapeutic Implications of Genetic Testing for Hypertrophic Cardiomyopathy

J. Martijn Bos, Jeffrey A. Towbin, Michael J. Ackerman

J Am Coll Cardiol 2009; 54(3): 201 – 211 [Review]

Purpose of Review

Over the last two decades the pathogenic basis for the most common heritable

cardiovascular disease, hypertrophic cardiomyopathy (HCM), has been investigated

extensively. Affecting approximately 1 in 500 individuals, HCM is the most common

cause of sudden death in young athletes. In recent years, genomic medicine has been

moving from the bench to the bedside throughout all medical disciplines including

cardiology. Now, genomic medicine has entered clinical practice as it pertains to the

evaluation and management of patients with HCM. The continuous research and

discoveries of new HCM-susceptibility genes, the growing amount of data from

genotype-phenotype correlation studies, and the introduction of commercially available

genetic tests for HCM make it essential that the modern-day cardiologist understand

the diagnostic, prognostic, and therapeutic implications of HCM genetic testing.

130

Affecting 1 in 500 people, hypertrophic cardiomyopathy (HCM) is a disease marked by

phenotypic and genotypic heterogeneity and is the most prevalent, heritable

cardiovascular disease. HCM is the most common cause of sudden cardiac death in

young athletes[1]. HCM can manifest negligible to extreme hypertrophy, minimal to

extensive fibrosis and myocyte disarray on microscopy, absent to severe left

ventricular outflow tract (LVOT) obstruction, and distinct septal morphologies such as

reverse curve-, sigmoidal-, and apical-HCM. The clinical course varies extremely as

well, ranging from an asymptomatic lifelong course to dyspnea/angina refractory to

pharmacotherapy to sudden death as the sentinel event. Fully described for the first

time by Teare in 1958, HCM was regarded as ‘asymmetrical hypertrophy of the heart

in young adults’[2]. It has since been referred to by an array of names – idiopathic

hypertrophic subaortic stenosis[3], muscular subaortic stenosis[4] and hypertrophic

obstructive cardiomyopathy[5] - reflecting its clinical heterogeneity and its relatively

uncommon occurrence in daily cardiologic practice.

131

Diagnostic Implications of HCM Genetic Testing

Identification of HCM-Susceptibility Genes

Nearly 20 years ago, the first chromosome locus for familial HCM and subsequently

mutations involving the MYH7-encoded -myosin heavy chain were elucidated as the

pathogenic basis for HCM[6, 7]. Since then several hundreds of mutations scattered

among at least 27 putative HCM-susceptibility genes encoding various sarcomeric,

calcium-handling and mitochondrial proteins have been identified (Table 1, 2). The

most common genetically-mediated form of HCM is myofilament (sarcomeric)-HCM

with hundreds of disease-associated mutations in 9 genes encoding proteins

(myofilaments) critical to the cardiac sarcomere. This includes -myosin heavy chain

(MYH7)[7], regulatory - (MYL2) and essential myosin light chains (MYL3)[8], myosin

binding protein C (MYBPC3)[9], cardiac troponin T (TNNT2), -tropomyosin (TPM1)

[10], cardiac troponin I (TNNI3)[11], cardiac troponin C (TNNC1)[12] and actin

(ACTC)[13, 14]. Complete screening through a large cohort of patients has not been

performed, yet targeted screening of giant sarcomeric TTN-encoded titin, which

extends throughout half of the sarcomere, has thus far revealed only 1 mutation[15].

132

Table 1: Summary of HCM-Susceptibility Genes

Gene Locus Protein Frequency (%)

Myofilament-HCM

TTN 2q24.3 Titin < 1

MYH7 14q11.2-q12 -myosin heavy chain 15 – 25

MYH6 14q11.2-q12 -myosin heavy chain < 1

MYL2 12q23-q24.3 Ventricular regulatory myosin light < 2

MYL3 3p21.2-p21.3 Ventricular essential myosin light < 1

MYBPC3 11p11.2 Cardiac myosin-binding protein C 15 – 25

TNNT2 1q32 Cardiac troponin T < 5

TNNI3 19p13.4 Cardiac troponin I < 5

TPM1 15q22.1 -Tropomyosin < 5

ACTC 15q14 -Cardiac actin < 1

TNNC1 3p21.3-p14.3 Cardiac troponin C < 1

Z-disc HCM

LBD310q22.2-

q23.3

LIM binding domain 3

(Alias: ZASP) 1 – 5

CSRP3 11p15.1 Muscle LIM protein < 1

TCAP 17q12-q21.1 Telethonin < 1

VCL 10q22.1-q23 Vinculin/metavinculin < 1

ACTN2 1q42-q43 Alpha-actinin 2 < 1

MYOZ2 4q26-q27 Myozenin 2 < 1

Calcium-handling HCM

JPH2 20q12 Junctophilin-2 < 1

PLN 6q22.1 Phospholamban < 1

Bolded genes are available as commercial genetic test

133

Expanding the scope of proteins involved in the pathogenesis of HCM, the

spectrum of HCM-associated genes has moved outside the myofilaments of the

sarcomere to encompass additional subgroups that could be classified as ‘Z-disc-

HCM’ and ‘calcium-handling HCM’ (Table 1). Due to its close proximity to the

contractile apparatus of the myofilament, its specific structure-function relationship with

regards to cyto-architecture, as well as its role in the stretch-sensor mechanism of the

sarcomere, attention subsequently focused on the cardiac Z-disc. This focus has been

fueled by the fact that HCM and DCM are partially allelic disorders, in which mutations

in the same genes – especially the Z-disc – can be responsible for both

cardiomyopathic phenotypes[16, 17, 18, 19, 20, 21, 22, 23, 24]. The first Z-disc

mutations associated with HCM were described in muscle LIM protein encoded by

CSRP3[21] and telethonin encoded by TCAP[23]. Recently, LDB3-encoded LIM

domain binding 3, ACTN2-encoded alpha actinin 2, VCL-encoded

vinculin/metavinculin[24, 25, 26] and MYOZ2-encoded myozenin-2[27] have been

added to that list. In another demonstration of mutations in one gene causing multiple

diseases, MYPN-encoded myopalladin (MYPN) mutations were implicated in the

pathogenesis of DCM, HCM and restrictive cardiomyopathy (RCM) - via disturbed

myofibrillogenesis, abnormal gene expression, and/or abnormality in assembly of Z-

disc and intercalated disc (Purevjav et al. unpublished data).

In yet another signal transduction pathway, proteins involved in calcium

induced calcium release and the hypothesis that errors in this process may lead to

compensatory hypertrophy have always been of high interest in the pathogenesis of

HCM. Mutations have been described in the promoter and coding region of PLN-

encoded phospholamban, an important inhibitor of cardiac muscle sarcoplasmic

reticulum Ca(2+)-ATPase (SERCA)[28, 29]. Recently, mutations in JPH2-encoded

junctophilin 2, which helps approximate the sarcoplasmic reticulum calcium release

channels and plasmalemmal L-type calcium channels, may cause HCM [30].

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Table 2: HCM phenocopies

Gene Locus Protein Syndrome

TAZ Xq28 Tafazzin (G4.5) Barth syndrome/LVNC

DTNA 18q12 -dystrobrevin Barth syndrome/LVNC

PRKAG2 7q35- q36.36 AMP-activated protein kinase WPW/HCM

LAMP2 Xq24 Lysosome-associated membrane

protein 2 Danon’s syndrome/WPW

GAA 17q25.2-q25.3 -1,4-glucosidase deficiency Pompe’s disease

GLA Xq22 -galactosidase A Fabry’s disease

AGL 1p21 Amylo-1,6-glucosidase Forbes disease

FXN 9q13 Frataxin Friedrich’s ataxia

PTPN11 12q24.1 Protein tyrosine phosphatase,

non-receptor type 11, SHP-2

Noonan’s syndrome,

LEOPARD syndrome

RAF1 3p25 V-RAF-1 murine leukemia viral

oncogene homolog 1

Noonan’s syndrome,

LEOPARD syndrome

KRAS 12p12.1 v-Ki-ras2 Kirsten rat sarcoma viral

oncogene homolog Noonan’s syndrome

SOS1 2p22-p21 Son of sevenless homolog 1 Noonan’s syndrome

LVNC, left ventricular non-compaction; WPW, Wolff-Parkinson-White syndrome. Bolded genes are available as commercial genetic test

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New insights and approaches to genomics of cardiac hypertrophy and

HCM

Not only in molecular genetics but also in other fields of “-omics”, novel pathways

underlying the pathophysiology of cardiac hypertrophy and HCM have been identified

using several new techniques to study large-scale transcriptional changes[31, 32, 33].

A transcriptomic approach can be performed by using microarray, a technique that

enables to give a snapshot view of gene expression that, combined with complex

analytic tools, can identify genes that are differentially regulated or seem to be co-

regulated and thereby form a transcriptional network of genes and pathways.

Microarray-chips can hold over tens of thousands of genes and can be utilized to

compare expression levels in certain disease states with healthy controls.

In 2002, Hwang et al. studied RNA from heart failure patients with either HCM

or DCM, and found almost 200 genes to be up-regulated and 51 genes down-

regulated in both conditions, as well as several genes differentially expressed between

the two diseases providing information on different pathways and genes involved in the

pathogenesis[34]. Rajan et al. performed microarray analysis on ventricular tissue of

two previously developed transgenic, 2.5 months old HCM-mice ( -TM175 and -

TM180) carrying mutations in alpha-tropomyosin (TPM1). Studying 22,600 genes, 754

differentially expressed genes between transgenic and non-transgenic mice were

detected, of which 266 were differentially regulated between the 2 different mutant

hearts showing most significant changes in genes belonging to the

‘secreted/extracellular matrix’ (up-regulation) and ‘metabolic enzymes’ (down-

regulation) [35].

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Another emerging field is that of microRNA’s (miR’s) and their role in cardiac

development and (hypertrophic) heart disease. These fundamental cellular regulators

were first described by Lee et al in 1993[36] and consist of approximately 22 non-

coding RNA molecules that silence genes through posttranscriptional regulation.

MicroRNA’s play an important role in cardiac development as well as in orchestrating

organogenesis and early embryonic patterning processes[37, 38]. Furthermore, these

non-coding RNA molecules seem to play an important role in cardiac remodeling and

the development of hypertrophy as initially reported by Van Rooij et al in 2006 (42).

Utilizing 2 mouse models of pathological hypertrophy – transverse aortic constriction

(TAC) and calcineurin transgenic mice, 6 miR’s were up-regulated, which in vitro,

were sufficient to induce hypertrophic growth of cardiomyocytes[39]. Furthermore, a

transgenic mouse model over-expressing one of these miR’s (miR-195) showed that a

single miRNA could induce pathological hypertrophy and heart failure[39]. Over the

last year, multiple studies have been published with miRNA expression profiles in

different settings, in vivo and in vitro, of cardiac hypertrophy[37, 39, 40, 41, 42, 43].

Lastly, PMAGE (polony multiplex analysis of gene expression) is a technique

that detects messenger RNAs (mRNAs) as rare as one transcript per three cells [44].

Using this new technique, early transcriptional changes preceding pathological

manifestations were identified in mice with HCM-causing mutations, including low-

abundance mRNA encoding signaling molecules and transcription factors that

participate in the disease pathogenesis[44].

The development and implementation of these new techniques as well as their

applications in research and clinical models of cardiac hypertrophy and HCM will over

the years teach us more about the pathophysiology of normal and pathologic

hypertrophy as well as HCM. This in turn might lead to discovery of novel disease

causing genes, involved pathways and possible novel therapeutic targets.

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HCM Genetic Testing in Clinical Practice

Recently, HCM genetic testing has matured from its two-decade long residence in

research laboratories into the realm of clinically available, diagnostic testing for

physicians evaluating and treating patients with this disease [(Harvard Partners,

Correlagen, PGxHealth, and GeneDx. These companies now offer testing for the 8

most common myofilament associated genes; additional genes offered by some are

the genes involved in the glycogen storage diseases or the recently discovered HCM-

associated gene troponin C encoded by TNNC1. The HCM-susceptibility genes

available for commercial genetic testing are highlighted in bold in Table 1 and 2.

Although some of the new HCM-susceptibility genes may surpass the

prevalence of mutations found in some of the myofilament proteins, MYBPC3 and

MYH7 remain by far the most common HCM-associated genes, with an estimated

prevalence of 15 to 25% for both genes. Among the 9 HCM-associated, myofilament

encoding genes, the prevalence of myofilament-HCM has ranged from 35 to 65% in

several different, international cohorts of unrelated patients who met the clinically

accepted definition of HCM[45, 46].

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Echo-guided genetic testing

While several phenotype-genotype relationships have emerged to enrich the yield of

genetic testing, most of these patient profiles have not been particularly clinically

informative. Recently, the possibility of echo-guided genetic testing has been

explored[47]. Noting a predominance of sigmoidal-HCM among the elderly, Lever et al

suggested over 2 decades ago that there was a strong age-dependence with the

various septal morphologies of HCM, where septal contour was classified as reverse

curve-, sigmoidal-, apical-, and neutral contour-HCM (Figure 1)[48]. In the early 1990s,

Solomon et al observed that patients with mutations in the beta myosin heavy chain

(MYH7-HCM) generally had reversed curvature septal contours (reverse curve-HCM)

[49].

Figure 1: Septal morphologies in HCM. Shown are the most common septal morphologies in HCM. The distribution of septal morphologies among a large cohort of patients with HCM is shown along the top while the yield of genetic testing for each morphological subgroup is shown along the bottom of the figure.

139

Subsequently, a large genotype-phenotype analysis correlating the septal

morphology with the underlying genotype was conducted. After extensive analysis of

the echocardiograms of nearly 400 unrelated patients, sigmoidal HCM (47% of cohort)

and reverse curve-HCM (35% of cohort) represented the two most prevalent

anatomical subtypes of HCM (Figure 1). In this study, the yield of genetic testing for

myofilament-HCM (8 genes) was 80% in reverse curve-HCM but only 10% in patients

with sigmoidal-HCM and septal contour was the strongest predictor of a positive HCM

genetic test, regardless of age (odds ratio 21, p <0.0001) [47]. These observations

may facilitate echo-guided genetic testing by enabling informed genetic counseling

about the pre-test probability of a positive genetic test based upon the patient’s

expressed anatomical phenotype (Figure 2).

Role of HCM genetic testing for both index cases and relatives

Although there may be some prognostic relevance presently and therapeutic relevance

futuristically to the HCM genetic test in the index case who already clinically manifests

the disease, the principal role for index case genetic testing is diagnostic. It can

however, as we will show later on, be of significant importance to the approach and

screening of relatives.

Figure 2 provides a flow chart for clinicians, in which the 2 pathways are

described when an index case (HCM proband) is identified. It must be recognized that,

before a diagnosis of HCM in the proband is made, a full history, examination,

including extensive family history should be performed. This way clues can be picked

up to expose other causes of unexplained LVH –aortic stenosis, hypertension or the

presence of a phenocopy – as being responsible for the patient’s symptoms. For

example, signs of ventricular pre-excitation might point to a PRKAG2-mediated

glycogen storage disease or an inheritance pattern that strictly affects males might

suggest LAMP2-mediated disease. If the phenotype is HCM, echocardiography may

inform genetic counseling by providing an a priori probability for a positive genetic test

and advice on how to proceed with further evaluation and family screening (left arm of

algorithm). If genetic testing of the major genes remains negative, the presence of a

phenocopy with pure cardiac involvement should be considered.

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Figure 2: Genetic- and echocardiographic-based screening in HCM. Flow-chart showing a possible decision tree to follow in genetic- and echocardiography-based screening in HCM. Noted is the a priori probability for a positive genetic test result based on the echocardiographic scored septal contour, as well as the steps to follow if a patient chooses not to pursue genetic testing.

141

As it stands now, genetic testing of the index case for the index case has the

potential of providing the diagnostic gold standard for his/her offspring, siblings, and

parents and more distant relatives. A positive genetic test would then enable

systematic scrutiny of the index case’s relatives to separate the “haves” from the “have

nots” (positive versus negative test). In other words, the genetic testing of the index

case risk stratifies the family enabling 2 very different courses to be charted: 1) close

surveillance of the genotype-positive, pre-clinical individual and 2) casual observation

or dismissal of the genotype-negative/phenotype-negative relative and his/her future

progeny.

In general and irrespective of genetic testing, once a diagnosis of HCM has

been rendered, all first degree relatives and probably “athletic” second degree relatives

to the index case should be screened by an ECG and echocardiogram. Annual

screenings are recommended for young adults (age 12 to 25 yrs) and athletes and

thereafter every 3 to 5 years. As intimated previously, if an HCM-causing mutation is

established for the index case, first degree relatives should then have confirmatory

genetic testing for that particular HCM-causing mutation. Depending on the established

familial versus sporadic pattern, confirmatory genetic testing should proceed in

concentric circles of relatedness.

For example, if the index case’s mutation is present in his/her father, then the

paternal grandparents and paternal aunts/uncles should be tested. The index case’s

first degree cousins may or may not need genetic testing depending on the results of

the testing among the aunts and uncles and so forth. If a phenotype negative family-

member tests negative for the index case’s mutation, then future cardiologic

evaluations for that relative and his/her progeny may not be necessary. However, a

decision to cease surveillance for HCM in a relative hinges critically on the certainty of

the identified gene/mutation and its causative link as well as the complete absence of

any traditional evidence used to clinically diagnose HCM (i.e. asymptomatic and

normal echocardiogram).

142

Aside from the role of genetic testing described above, there is still concern

among patients and practitioners on social and economic aspects of knowing ones

genetic make-up. For example, genetic testing may or may not be covered by an

individual’s health insurance plan. Some payors view HCM genetic testing as a bona

fide clinical test while others view it as an “investigational” one. Secondly, if genetic

testing is performed, a patient might feel anxiety about the potential clinical dangers of

hosting an HCM-predisposing mutation. Also, there might be fears that the presence of

this information in one’s medical records might influence health insurance premiums

and employment opportunities, although this last issue has been addressed recently

by former President Bush’s signing of the Genetic Information Nondiscrimination Act

(GINA) into law. This is an important step to provide protection against genetic

discrimination. Besides discussing the differential impact in terms of follow-up for a

genotype positive relative compared to a genotype negative relative as described

earlier, these important issues should also comprise the genetic counseling

Novel imaging techniques for early detection of HCM disease gene

expression

With growing knowledge on genetics and other pathogenetic pathways in HCM, it

would be very helpful if parameters could be found that suggest pre-clinical, pre-

hypertrophic expression of the genetic substrate. Tissue Doppler (TD)

echocardiography studies in transgenic rabbit models of HCM[50, 51, 52] and in

humans have shown reduced myocardial Doppler velocities in genotype positive

subjects without LVH[50, 53, 54, 55]. Nagueh et al. provided the first evidence in 2001

demonstrating that myocardial contraction and relaxation velocities as detected by TD

are reduced in familial, mutation positive HCM. In 2002, Ho et al. showed that

abnormalities of diastolic function as assessed by Doppler tissue imaging precede

development of LVH in patients with MYH7-mutations where a combination of Ea

velocity and ejection fraction (EF) was highly predictive of affected phenotype in

patients without hypertrophy[55]. Cardiac MRI (CMR) is also showing potential to

become an important tool in the diagnosis of HCM as it has capacity to acquire images

with tissue contrast and border definition that is often superior to echocardiography[56,

57, 58, 59]. In 2006, Germans et al. performed CMR on 16 mutation positive HCM

patients and detected pre-hypertrophic crypts in the inferoseptal LV wall, possibly

representing early pathological alterations stemming from the pathogenic

substrate[60].

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Prognostic Implications of HCM Genetic Testing

From the early-beginnings of the genomic-era and since the description of the first

HCM-causing mutation, investigators have attempted to correlate genotypes to

particular clinical phenotypic expressions. Stemming from earlier pedigree studies,

specific missense mutations were associated with a markedly unfavorable prognosis

whereas others had an uneventful natural history. These observations resulted in

specific mutations being designated as either “malignant” mutations or “benign”

mutations [61, 62, 63, 64, 65, 66, 67, 68]. The first study of its kind was published by

Watkins et al. in 1992 in which they described mutations in MYH7 found in 12 out of 25

families with HCM[61]. They concluded that the MYH7-R403C mutation was

associated with a significantly shorter life expectancy, and could therefore be

considered a ‘malignant’ mutation. In contrast, a non-charge change mutation (V606M)

was associated with nearly normal survival and therefore was considered ‘benign’[61].

In 2003, Woo et al. analyzed mutations in functional domains in 15 (out of 70) MYH7-

positive probands and concluded that their may be prognostically informative

domains[69].

Initial reports on the clinical expression from the most common subtype of HCM

– MYBPC-mediated HCM – seems to show a slower, but progressive clinical disease

course, with later onset, milder disease characteristics[68, 70, 71]. Investigators in the

Netherlands and South-Africa have discovered founder mutations in MYBPC3 with

mild phenotypic expression that are present in at least 30% of their cases[72].

Similarly, certain genotype-phenotype correlations were attributed to TNNT2 (troponin

T)-HCM. Far less common than MYBPC-HCM or MYH7-HCM, TNNT2-HCM (affecting

< 5% of patients) was associated with less severe left ventricular wall thickness, but a

higher incidence of premature sudden cardiac death[64, 73, 74]. Overall, these

TNNT2-HCM patients who suddenly died had less hypertrophy and less fibrosis, but

more myocyte disarray, which may have provided the substrate for malignant

arrhythmias[74].

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Overall, these observations have been gleaned from small cohorts involving

larger families with penetrant disease expression whereas genotype-phenotype

studies involving large cohorts of unrelated patients have indicated that great caution

must be exercised with assigning particular prognostic significance to any particular

mutation[75, 76, 77]. In one such cohort, only 2% hosted one of those formally

annotated ‘benign’ mutations and moreover, these particular hosts displayed a severe

clinical phenotype with all 5 patients requiring surgical myectomy, 3 of the 5 having a

family history of sudden cardiac death, and 1 adolescent requiring an orthotopic heart

transplant[77]. In contrast, 3 patients hosting a so-called ‘malignant’ mutation displayed

a heretofore mild phenotype[75]. Furthermore, these studies have demonstrated that

the two most common forms of genetically mediated HCM – MYH7-HCM and

MYBPC3-HCM – are phenotypically indistinguishable[78].

More recently, in one of the first studies of its kind for HCM, a longitudinal study

in a large cohort of unrelated Italian patients with HCM have shown an increased risk

of cardiovascular death, non-fatal stroke or progression to New York Heart Association

functional class III/IV among patients with a positive HCM genetic test involving any of

the myofilament genes compared to those patients with a negative genetic test (25%

vs. 7%, respectively; p = 0.002) (Figure 3A); multivariate analysis showed myofilament

positive HCM (i.e. a positive genetic test) to be the strongest predictor of an adverse

outcome (hazard ratio 4.27 (CI 1.43 – 12.48), p = 0.008)[79]. Furthermore, patients

with myofilament genotype-positive-HCM had greater probability of developing severe

LV systolic dysfunction (p = 0.021; Figure 3B) and restrictive LV filling (p = 0.018;

Figure 3C).

Lastly, it has been observed that patients with multiple mutations (i.e.

compound or double heterozygotes), detected in about 3-5% of genotype positive

patients, have a more severe phenotype and increased incidence of sudden death[78,

80, 81], suggesting a gene-dosage effect might contribute to disease severity.

Interestingly, in the majority of cases of compound heterozygosity, one of the

mutations usually involves MYBPC3[78]. In their longitudinal study, Olivotto et al.

observed a similar trend showing that patients with double mutations (of which 1 was

usually MYBPC3) had greater disease severity than myofilament negative patients or

patients with a single MYBPC3 -, thick filament – or thin filament mutation combined (p

< 0.05; Figure 3D).

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Figure 3: Relation of genetic test status to outcome in patients with hypertrophic cardiomyopathy. Follow-up data shows that patients harboring a myofilament mutation (i.e. a positive genetic test) progress to CV Death, ischemic stroke or NYHA-Class III-IV more rapidly than patients with a negative genetic test (A). Furthermore, patients with a myofilament mutation are more likely to develop systolic dysfunction (B) or a restrictive filling pattern (C), independent of the genotype involved (D).

In summary, although clinical prognostication must be rendered with great

caution for specific gene domains or specific genetic mutations, a positive HCM

genetic test in general portends a greater likelihood for disease progression,

particularly as it pertains to systolic and diastolic dysfunction and propensity to develop

symptoms. As such, clinical genetic testing may thereby aid in the prognostication of a

patient’s disease outcome.

146

Interpretation of rare variants and phenocopies

One group of patients that pose an intriguing challenge for clinicians is that of patients

with seemingly unexplained LVH that mimics the HCM-phenotype. These diseases are

usually referred to as phenocopies and the most important ones are listed in Table 2. Phenocopies or rare variants pose a tough dilemma for the clinician. If the phenotype

does not look like typical HCM, other symptoms like ventricular pre-excitation or

muscle weakness are present, the presence of an underlying multi-system disease

should be considered and additional testing should be performed. On the other hand,

if myofilament genetic testing does not reveal an HCM-associated mutation, testing for

mutations in the metabolic genes can reveal that the LVH is the primary presentation

of a multi-system disease process.

A different cardiomyopathy and phenocopy that can present itself with

seemingly unexplained hypertrophy is that of left ventricular non-compaction (LVNC) –

a primary cardiomyopathy characterized by a severely thickened 2-layered

myocardium, numerous prominent trabeculations, and deep intertrabecular

recesses[82, 83]. Although genetically still largely unexplained, mutations in the TAZ-

encoded tafazzin (G4.5) – also associated with Barth syndrome –, DTNA-encoded -

dystrobrevin and LDB3 have been associated in the pathogenesis of LVNC[84, 85, 86].

Recently, Klaassen et al. systematically analyzed a cohort of 63 unrelated patients with

LVNC for mutations in 6 myofilament encoding genes, identifying nine distinct

heterozygous mutations in 11 patients in MYH7, ACTC and TNNT2[87] suggesting that

there might be a shared etiology for the myofilament forms of the common allelic

cardiomyopathies of HCM, DCM and LVNC.

Some diseases presenting chiefly with cardiac hypertrophy turn out to have

clearly distinct underlying pathophysiologies. In 2001, 2 independent groups

discovered PRKAG2 mutations being involved in families with cardiac hypertrophy and

ventricular pre-excitation, conduction abnormalities and signs of Wolff-Parkinson-White

(WPW) syndrome[88, 89]. In 2005, Arad et al. also described mutations in lysosome-

associated membrane protein-2 encoded by LAMP2 and PRKAG2 and found that

underlying glycogen storage diseases mimicked the clinical phenotype of HCM [88, 89,

90, 91]. Yang et al. showed that LAMP2 mutations may account for a significant

portion of patients diagnosed with pediatric- or juvenile onset HCM, especially when

skeletal myopathy and/or WPW are present[92].

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Role of modifiers in HCM

The role of modifiers of the HCM phenotype, either by the presence of common

polymorphisms or founder-mutations, has become the subject of recent investigations.

The most important subgroup of polymorphisms, studied to date, involve the major

components of the renin- angiotensin-aldosterone system (RAAS). Polymorphisms in

the RAAS-pathway [angiotensionogen-I converting enzyme (ACE), angiotensin

receptor 1 (AGTR1), chymase 1 (CMA), angiotensin I (AGT) and cytochrome P450,

polypeptide 2 (CYP11B2)): DD-ACE, CC-AGTR1, AA-CMA, T174M- and M235T-AGT,

and CC-CYP11B2] appear to influence the HCM phenotype, in particular the severity

of LVH[93, 94]. Among patients with the DD-ACE genotype, there was greater LVH

than among those with an ID or II genotype[95]. Furthermore, a combined ‘pro-LVH’

profile of five RAAS-genes was associated with higher degree of LVH in one particular,

founder MYPBC3-HCM pedigree [93] and in a large cohort of myofilament positive

patients[94].

In 2008, sex hormone polymorphisms were shown to modify the HCM

phenotype(104). Fewer CAG repeats in AR-encoded androgen receptor were

associated with thicker myocardial walls in male subjects (p = 0.008) and male carriers

of the A-allele in the promoter of ESR1-encoded estrogen receptor 1 (SNP rs6915267)

exhibited a 11% decrease in LV wall thickness (p = 0.047) compared to GG-

homozygote male subjects[96]. HCM modifier polymorphisms like these could

contribute to the clinical differences observed between men and women with HCM[97,

98]. The release of the complete human genome sequence and the enormity of

variation in individuals show a growing role for modifier genes and the search for effect

by genome-wide studies. In 2007, Daw et al. performed the first study of this kind for

HCM and they identified multiple loci with suggestive linkage. Effect sizes on left

ventricular mass on this cohort of 100 patients ranged range from ~8g shift from one

locus for the common allele to 90g shift for another locus’ uncommon allele[99].

148

Therapeutic Implications of HCM Genetic Testing

Pharmacogenomics

Currently, there is no available therapy specifically designed to target specific HCM-

causing gene mutations or particular HCM genotypes. Further, no therapies have

been shown to reverse the hypertrophic process in humans. One of the first studies of

its kind was performed in transgenic MYH7-R403Q mice models (designated

MHC403/+). In a randomized trial MHC403/+ -mice treated with diltiazem, a L-type

calcium inhibitor, showed significant improvement as compared to mice treated with

placebo in terms of cardiac systolic function as measured by increased end-diastolic

and end systolic volumes, decreased dP/dTmax values and end-systolic

elastance[100]. Furthermore, diltiazem-treated mice showed significantly less

hypertrophy at 30 and 39 weeks than age-matched MHC403/+-untreated mice as

well as less fibrosis and myocyte disarray on microscopy[100]. Recently, Westermann

et al. showed in a different transgenic mouse model (TNNT2–I79N) – that diltiazem

improved diastolic function and prevented diastolic heart failure and sudden cardiac

death compared to untreated mice[101].

As previously discussed, RAAS polymorphisms modify the phenotype of HCM,

particularly MYBPC3-HCM[93, 94] and there is now growing evidence that ACE-

inhibitors especially combined with low doses of aldosterone receptor blockers may

attenuate the progression of hypertrophy and fibrosis [102, 103, 104, 105, 106]. In

early mouse-models of transgenic cardiac troponin T (cTnT-Q92) that exhibit myocyte

disarray and fibrosis, a randomized, blinded trial comparing losartan (an angiotensin-II

blocker) or placebo demonstrated that losartan significantly reversed fibrosis and

expression of collagen 1 (I) and TGF -1 in the transgenic mice[107]. In a similar

study involving the same transgenic mice, losartan produced a 50% reduction in

myocyte disarray compared to mice treated with placebo as well as complete

normalization of the collagen volume fraction[108].

149

Lastly, another group of drugs, statins, may favorably modify the phenotype of

hypertrophic cardiomyopathy. A study involving 24 transgenic mice harboring the

MYH7 R403Q-mutation showed a regression of hypertrophy and fibrosis, improved

cardiac function and reduced ERK1/2 activity after treatment with simvastatin

compared to 12 non-transgenic mice[109]. Similar results were observed in transgenic

rabbits with this mutation who were treated with atorvastatin[110]. However, a small,

randomized control pilot study failed to show an effect on humans with HCM[111].

Therefore, one can envision that, with increasing knowledge of the patient’s

pathogenic substrate and polymorphism profile, specific therapies may someday

emerge. In other cases for example, the proper and prompt recognition of an HCM

phenocopy such as cardiac Fabry’s disease can facilitate gene-specific

pharmacotherapy such as enzyme-replacement therapy. Albeit rare, such clinical

sleuthing can enable early treatment and prevent the progression of the disease.

Recently, human trials such as the “DELIGHT” (DiltiazEm Long-term In Genotype-

positive Hypertrophic cardiomyopathy as preclinical Treatment) trial have begun and

are examining whether calcium channel inhibitors like diltiazem can prevent the

development of hypertrophy among patients with genotype positive/LVH negative-

HCM.

150

Conclusions

Genomic medicine, as it pertains to HCM, has moved from the bench to the bedside,

but caution is needed to interpret and manage the genetic portfolio of a patient.

Although some prognostic forecasts may be gleaned from the HCM genetic test,

therapeutic decisions regarding use of a defibrillator should not be dictated by the

genetic test result. Instead, knowledge of the genetic background in subjects with HCM

has significant diagnostic implications and echocardiography may help guide genetic

testing by providing anticipatory guidance and a pre-test probability of a positive

genetic test result. Clearly, knowledge of disease-causing mutations in an index case

enables rapid genetic testing and diagnosis of potentially at-risk relatives thereby

providing improved and informed follow-up and treatment decisions for such family

members. The information gained in these subjects can define risk status and, in those

subjects with negative genetic screening, less close follow-up and testing over time

and psychological freedom.

Increasingly, clinical care in HCM and other genetic-based disorders includes

the wise use and wiser interpretation of genetic tests. Therefore, understanding the

genetic underpinnings of disease and the risk placed on these subjects will be

imperative for all patients and their families. The 21st century clinician must be

cognizant of the state-of-the-art of translational genetics in order to best care for their

patients and families, as well as to help to define new clinical guidelines over the next

decade.

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79. Olivotto I, Girolami F, Ackerman MJ, Nistri S, et al. Myofilament protein gene mutation screening and outcome of patients with hypertrophic cardiomyopathy. Mayo Clin Proc 2008; 83(6): 630-638. 80. Ingles J, Doolan A, Chiu C, Seidman J, et al. Compound and double mutations in patients with hypertrophic cardiomyopathy: Implications for genetic testing and counseling. JMed Genet 2005; 42(10): e59. 81. Ho CY, Lever HM, DeSanctis R, Farver CF, et al. Homozygous mutation in cardiac troponin T: Implications for hypertrophic cardiomyopathy. Circulation 2000; 102: 1950-1955. 82. Oechslin EN, Attenhofer Jost CH, Rojas JR, Kaufmann PA, et al. Long-term follow-up of 34 adults with isolated left ventricular noncompaction: A distinct cardiomyopathy with poor prognosis. J Am Coll Cardiol 2000; 36(2): 493-500. 83. Jenni R, Rojas J, Oechslin E. Isolated noncompaction of the myocardium. N Engl J Med 1999; 340(12): 966-967. 84. Bleyl SB, Mumford BR, Thompson V, Carey JC, et al. Neonatal, lethal noncompaction of the left ventricular myocardium is allelic with Barth syndrome. Am J Hum Genet 1997; 61(4): 868-872. 85. Ichida F, Tsubata S, Bowles KR, Haneda N, et al. Novel gene mutations in patients with left ventricular noncompaction or Barth syndrome. Circulation 2001; 103(9): 1256-1263. 86. Vatta M, Mohapatra B, Jimenez S, Sanchez X, et al. Mutations in Cypher/ZASP in patients with dilated cardiomyopathy and left ventricular non-compaction. J Am Coll Cardiol 2003; 42(11): 2014-2027. 87. Klaassen S, Probst S, Oechslin E, Gerull B, et al. Mutations in sarcomere protein genes in left ventricular noncompaction. Circulation 2008; 117(22): 2893-2901. 88. Gollob MH, Green MS, Tang AS, Gollob T, et al. Identification of a gene responsible for familial wolff-parkinson-white syndrome. N Engl J Med 2001; 344(24): 1823-1831. 89. Blair E, Redwood C, Ashrafian H, Oliveira M, et al. Mutations in the gamma(2) subunit of amp-activated protein kinase cause familial hypertrophic cardiomyopathy: Evidence for the central role of energy compromise in disease pathogenesis. Hum Mol Genet 2001; 10(11): 1215-1220. 90. Arad M, Benson DW, Perez-Atayde AR, McKenna WJ, et al. Constitutively active amp kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy. JClin Invest 2002; 109(3): 357-362. 91. Arad M, Maron BJ, Gorham JM, Johnson WH, Jr., et al. Glycogen storage diseases presenting as hypertrophic cardiomyopathy. N Engl J Med 2005; 352(4): 362-372. 92. Yang Z, McMahon CJ, Smith LR, Bersola J, et al. Danon disease as an underrecognized cause of hypertrophic cardiomyopathy in children. Circulation 2005; 112(11): 1612-1617. 93. Ortlepp JR, Vosberg HP, Reith S, Ohme F, et al. Genetic polymorphisms in the renin-angiotensin-aldosterone system associated with expression of left ventricular hypertrophy in hypertrophic cardiomyopathy: A study of five polymorphic genes in a family with a disease causing mutation in the myosin binding protein c gene. Heart (British Cardiac Society) 2002; 87(3): 270-275. 94. Perkins MJ, Van Driest SL, Ellsworth EG, Will ML, et al. Gene-specific modifying effects of pro-LVH polymorphisms involving the renin-angiotensin-aldosterone system among 389 unrelated patients with hypertrophic cardiomyopathy. Eur Heart J 2005; 26(22): 2457-2462.

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The Growing Field of Genetic Contributors to the Pathogenesis of Hypertrophic Cardiomyopathy

or

About ‘lumpers’ and ‘splitters’: McKusick revisited

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In 1969, Victor McKusick, a medical geneticist, wrote a paper entitled “On lumpers and splitters, or the nosology of genetic disease”[1]. In this paper, McKusick disclosed the two leading principles in genetic nosology (study of classification of disease): ‘pleiotropism’ (multiple effects of a single etiologic factor) and ‘genetic heterogeneity’ (existence of two or more fundamentally distinct entities with essentially one and the same phenotype). And, already in 1969, McKusick articulated that against man’s naturally tendency to ‘lumping’ (recognize similarities and lump a disease under one umbrella), geneticist are forced to be ‘splitters’ instead[1]. This 40-year old paper seems prophetic for the current state of genetics of hypertrophic cardiomyopathy (HCM). Characterized by not only profound genetic heterogeneity, but also vast phenotypic and pathological diversity, for years the field of HCM tried to fit this disease in one, two or more diagnostic corners and continuing genetic discoveries still raise the question whether we should still be ‘lumpers’ or ‘splitters’.

Clinically, since Teare and Brock first described HCM as ‘asymmetrical hypertrophy of the heart in young adults’ [2, 3], this disease has been known by many names, such as idiopathic hypertrophic subaortic stenosis[4], muscular subaortic stenosis[5] and hypertrophic obstructive cardiomyopathy[6]. It wasn’t until 1995 that it was agreed upon to ‘lump’ all these syndromes under one name[7]. Accordingly, HCM is now described as left and/or right ventricular hypertrophy, usually asymmetric and involving the interventricular septum with predominant autosomal dominant inheritance involving sarcomeric contractile proteins[7]. Pathologically, HCM has been characterized by microscopic features of cardiomyocyte hypertrophy, myofibrillar disarray and interstitial fibrosis, although recent research has shown an emerging role of myocardial ischemia, coronary microvascular dysfunction and myocardial bridging[8, 9]. And genetically, over 24 HCM-susceptibility genes have been elucidated with mutations found in genes encoding proteins of the cardiac myofilament, Z-disc and calcium-handling pathways as well as multiple genes involving syndromes mimicking the HCM phenotype [10]. Also, numerous HCM modifier genes have been identified [11, 12, 13].

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And lastly, based on previous studies and observations described in this thesis, we have seen a strong genotype-morphology correlation between reverse-curve HCM and the presence of a myofilament mutation[14] and subsequently observed that Z-disc HCM is predominantly sigmoidal[15], suggesting a strong link between ventricular septal morphology and HCM-substrate. With all these different observations and possible clinical, pathological, genetic or morphological groupings, we can ask ourselves whether we should start ‘splitting’ HCM into for example morphologic and genetic subgroups of HCM, or keep ‘lumping’ and focus our research back onto understanding a proposed ‘final common pathway’ for HCM[16, 17].

This thesis has uncovered some new insights in this disease with the discovery of novel HCM-associated genes located to the cardiac Z-disc (Chapter 2 and 3) [18, 19], a morphologic predilection for sigmoidal morphology and relationships between sex, shape, and genetic substrate (Chapters 4 and 5) [15, 20] as well as the discovery of a novel HCM-susceptibility gene (TIEG1) and possible biomarker (PTTG1) for HCM (Chapter 6). However, multiple challenges and research questions remain, whose answers will help us further understand the pathogenesis of this disease, the role of modifiers, epigenetic factors and environmental influences, and novel therapeutic strategies.

First of all, with 30-50% of HCM genetically unexplained, the number of HCM-associated genes is expected to rise either by candidate gene selection, linkage analysis of large families with unexplained HCM, or large genome wide association studies. It remains to be seen whether a gene explaining a large portion of HCM (like MYH7-HCM and MYBPC3-HCM) will be found, but it is a certainty that novel disease-associated genes will be discovered. However, with the mutations expected to be found at a low frequency, it will raise the question whether the variants are really pathogenic or just rare, innocuous amino-acid alterations. Before such variants are published as disease causing/contributing, it will call for increasingly stringent clinical, familial, functional and bioinformatics studies.

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With the 1000-genome project in its final stages cataloguing the full genetic code and its interpersonal variations of 1000 ostensibly healthy volunteers, a lot more knowledge on normal genetic variation will enter the public databases to help answer an important part of this question. But, especially in a fairly common disease like HCM, one still has to wonder: what is normal? Expressing sometimes at a later age, it can be envisioned that some people participating in the 1000-genome project were considered healthy upon entry in the study, but still develop hypertrophy and HCM later on in life. More than ever, especially in the current age of clinical genetic testing for HCM, it is important to separate the real from the rare. Research programs should be performing appropriate functional assays into possible pathogenic variants and genetic testing companies should be performing their due diligence determining the possible disease associated role of a finding (pathogenic or innocuous?) before reporting back to the clinician and patient.

Secondly, since most research thus far has been focused on translated portions of the genome, a huge part of our genetic code remains poorly understood from the standpoint of HCM pathogenesis. Promoter regions, splice sites, enhancers and other intronic functional and non-functional domains might end up playing an important role in the pathogenesis of many diseases including HCM. Next-generation sequencing techniques will make it easier to read much larger portions of the genome with the possibility to identify novel, disease associated variants located to potentially functional domains of the intronic genome (“introme’). Although putatively pathogenic, novel assays to prove its pathogenicity will have to be developed to add the satisfactory level of clinical significance to these variants. Also on the level of translation, post-translational processes and epigenetics (DNA methylation, chromatin remodeling, RNA transcription etc), a lot remains to be discovered. Not only just amino-acid altering variations or insertions/deletions might contribute to disease development, but also translational and posttranslational modifications that take place in the human cell.

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Steps in this direction have for example already been taken by the discovery and description of the role of microRNA’s (miR’s) in hypertrophic heart disease and their role as negative regulators of gene expression. MiR’s are small, non-coding RNA-molecules that inhibit translation or promote degradation of target mRNAs. Previous studies on two mouse models of pathological hypertrophy – transverse aortic constriction (TAC) and calcineurin transgenic mice – demonstrated 6 miR’s were up-regulated, which in vitro, were sufficient to induce hypertrophic growth of cardiomyocytes[21]. A transgenic mouse model over-expressing miR-195 showed that even a single miR could induce pathological hypertrophy and heart failure[21]. Multiple studies in vivo and in vitro, have since been published with miR expression profiles in different settings of cardiac hypertrophy demonstrating the potential role of miR’s and possible therapeutic target in (hypertrophic) heart disease and HCM [21, 22, 23, 24, 25, 26]. Comprehensive genetic analyses of the genes encoding these miRs or their respective target sites have yet to be performed in a large collection of patients with HCM. Also, no studies have yet demonstrated expression profiles of miRs in human HCM samples.

Thus far, no therapeutic option exists to treat, prevent or reverse cardiac hypertrophy in patients with genotype positive HCM or HCM in general, although animal studies do suggest that disease prevention/regression is a possibility. In a randomized trial, diltiazem (a L-type calcium channel blocker) treated MYH7-HCM transgenic mice showed significant improvement of cardiac systolic function, significantly less hypertrophy at 30 and 39 weeks as well as less fibrosis and myocyte disarray compared to untreated, transgenic mice [27]. These findings were replicated in a troponin T-HCM transgenic mouse model also [28]. Currently, human clinical trials are near completion of their first stages evaluating treatment with diltiazem and the progression of hypertrophy in genotype positive patients without signs of hypertrophy. Similar animal studies have been published showing that other drugs like ace-inhibitors, aldosterone receptor blockers, angiotensin-II blockers or statins might attenuate progression of hypertrophy, fibrosis and myofibrillar disarray in transgenic mouse models, but small human trials have been inconclusive or have yet to be finalized[29, 30, 31, 32, 33, 34, 35]. Prevention of hypertrophy or at least slowing its progression should change its natural history although it remains to be seen whether these or other drugs could also prevent the incidence of sudden cardiac death.

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Even with novel techniques, identification of novel putative pathogenic variants in unexplored regions or discovery of novel pathways, it’s possible that a subset of patients will remain without a genetic explanation of their disease. Furthermore, as HCM is characterized by profound heterogeneity, even within patients of families with the same genetic background, the genetic, epigenetic, environmental basis for incomplete penetrance and variable expressivity remains poorly understood. Already demonstrated by previous modifier and gender studies, one can envision that certain intrinsic or environmental factors such as gender, (low-grade) hypertension, race or smoking amongst others, trigger one of the pro-hypertrophic pathways leading to a disease diagnosed by clinicians as HCM, but studies have yet to study and identify specific environmental factors that influence the disease.

Lastly, research can be focused on understanding the pathogenic pathways of cardiomyopathies as allelic diseases. Particularly when one looks at the HCM-associated genes encoding Z-disc proteins, it is striking that most are also associated in the pathogenesis of DCM with both cardiomyopathic phenotypes sometimes seen in the same family [36, 37, 38, 39, 40, 41, 42, 43, 44]. This poses the question how mutations in one gene can lead to two divergent compensatory responses and whether these pathways are induced by the underlying genotype or that exogenous factors determine whether one develops HCM or DCM. Looking at the role of Z-disc genes in the heart and functional capacities in the stretch-sensor and mechanoreceptor function of the heart, one can see where pathways of possible stretch-induced hypertrophy and dilatation of these diseases overlap, but studies are still needed to dissect the exact pathways whereby these divergent diseases develop. Not only are similar genotypic background seen between HCM and DCM; recently for example sarcomeric genes have been associated in the pathogenesis of left ventricular non-compaction (LVNC) [45] and restrictive cardiomyopathy (RCM)[45, 46]. Presently, the McKusick ‘pleiotropism’ encompasses 4 distinct cardiomyopathies capable of emanating from sarcomeric perturbations.

Thus, in a time where we have gained more and more knowledge on the extent and breadth of clinical, pathological, genetic and morphological heterogeneity of HCM and have witnessed the expansion of cardiomyopathic pleiotropism, perhaps we should heed McKusick’s 40-year old admonition to resist the urge to lump and be a ‘splitter’ instead.

167

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170

Summary

172

Hypertrophic cardiomyopathy (HCM) is a disease characterized phenotypically by

unexplained left ventricular hypertrophy in the absence of an underlying cause. With a

prevalence of 1 in 500 individuals, HCM is the most common heritable cardiac disease

and the most common cause of sudden cardiac death in young adults, especially

athletes. Pathologically, HCM is characterized by microscopic cardiomyocyte

hypertrophy, myofibrillar disarray and focal, interstitial fibrosis. Genetically, HCM was

considered a disease of the sarcomere, or more specifically the cardiac myofilaments,

with most mutations thus far found in genes encoding cardiac myofilaments. However,

among different international cohorts, the yield of genetic testing for these genes, now

equivalent to commercial genetic tests, ranged between 30 and 70%. More recently, it

was demonstrated that there was a strong correlation between ventricular septal

morphology and the underlying genotype with 80% of reverse-curve HCM being

genotype positive and only 8% of sigmoidal-HCM harboring a pathogenic mutation.

Based on these findings, we set out to study the genetic basis of genotype negative

HCM and study the genotype-phenotype correlations of newly discovered HCM-

susceptibility genes.

Chapter 1 of this thesis is a general introduction on HCM which briefly reviews

the clinical and genetic history of HCM. Furthermore, the state of genetics of HCM in

2007 and recent observation of correlation between septal contour and underlying

genetic substrate are discussed. It also sets the stage for most chapters in this thesis

and the discussion whether HCM is one disease or maybe a disease that can be

categorized in various clinical, genetic or morphological subgroups.

Chapter 2 describes the discovery and subsequent genotype-phenotype

analyses of mutations in three HCM-susceptibility genes that encode key

cytoskeletal/scaffolding proteins of the Z-disc. As a large percentage of patients

remained without genetic explanation for their disease, the research field moved from

the myofilament proteins of the sarcomere to the sarcomere’s Z-disc and its vast array

of proteins as mutations had previously been published in dilated cardiomyopathy

(DCM) or animal models supported a role for certain genes in the pathogenesis in

HCM. We discovered that 4% of unrelated patients with HCM harbored mutations in

CSRP3-encoded muscle LIM protein (MLP) and TCAP-encoded telethonin (TCAP).

Phenotypically these patients mirrored the phenotype seen in myofilament-HCM and

patients were more affected than patients who continued to be genetically elusive.

173

Chapter 3 provides the first description of mutations in ANKRD1-encoded

ankyrin repeat domain 1 (ANKRD1) (also known as cardiac ankyrin repeat protein

(CARP)) associated with HCM. In this collaborative effort with our colleagues in Japan

led by Dr. Akinori Kimura, we describe the discovery of 3 novel mutations in ANKRD1

in two independent cohorts of unrelated patients with HCM. Furthermore, two

mutations were found in the N2A-domain of the large sarcomere-stretching protein titin

encoded by TTN. Functional analyses of these mutations demonstrated increased

binding of titin to ANKRD1 as well as altered localization of ANKRD1-mutants in

cardiomyocytes. Compared to wild type ANKRD1, which localizes to the striated

pattern at the Z-I-bands, mutant ANKRD1 showed increased localization within or at

nuclear membrane in approximately 60% of mature cardiomyocytes.

Chapters 4 and 5 describe genotype-phenotype correlations between the

previously discovered, novel Z-disc associated HCM mutations and their relationship to

sex (Chapter 5) and/or their underlying genotypic substrate. Akin to the strong

correlations between reverse curve-HCM and the presence of a myofilament mutation,

we found that Z-disc HCM is preferentially sigmoidal. Described in Chapter 4, we found

that among the 13 patients with Z-disc HCM (including mutations in the novel HCM-

associated gene alpha-actinin 2 encoded by ACTN2), 85% had a sigmoidal septal

contour. None of the patients demonstrated the myofilament-associated reverse septal

morphology. These findings led us to speculate that the pathogenic mechanism behind

Z-disc HCM might predispose patient to the more sigmoidal septal bulge at the left

ventricular outflow tract.

Chapter 5 focuses on this same subset of patients, but specifically focuses on

the sex-related differences within this cohort. In a previous study involving genetically

uncharacterized patients with HCM from the USA and Italy, women i) were older and

more symptomatic at diagnosis, ii) had more left ventricular outflow tract obstruction,

and iii) were more likely to progress to advanced heart failure and stroke. In our study,

we performed a similar analysis on patients that were genotyped for mutations in the

myofilament genes and scored for septal morphology on echocardiography. Similar to

previous studies, we found striking differences between men and women, but these

differences were confined largely to the subgroup of women with mutation-negative,

sigmoidal HCM.

174

Multiple and linear regression models demonstrated that, for women, age at

diagnosis, systolic blood pressure and presence of left ventricular outflow tract

obstruction were directly dependent on sigmoidal morphology. These observations

demonstrated that whereas mutations within the sarcomere appear to dominate the

disease process, in their absence, sex has a significant modifying effect among

patients with genotype negative, sigmoidal HCM.

In an effort to further explain genotype negative HCM, we subsequently moved

away from the cardiac Z-disc to analyze a novel candidate gene, which in knock-out

mice showed late onset HCM and a distinct gender predilection. Described in Chapter

6 is the discovery of 6 novel HCM-associated mutations in TIEG1-encoded TGF -

inducible early gene-1 in two independent cohorts of unrelated patients with HCM from

the Academic Medical Center (Amsterdam, NL) and the Mayo Clinic (Rochester, MN

USA). Subsequent in vitro studies showed that, akin to data from the TIEG1 knock-out

mouse, 5 out of 6 TIEG1-mutations significantly altered TIEG1 function on the PTTG1-

promoter resulting in a significant upregulation of PTTG1-promoter activity.

Immunohistochemistry analyses showed increased PTTG1-protein staining in HCM

patients in general and even more in patients with TIEG1-HCM suggesting that up-

regulation of PTTG1 might be a final common pathway in HCM and a potential disease

biomarker.

Chapter 7 is a recent review article describing the diagnostic, prognostic and

therapeutic implications of genetic testing for HCM. Besides the past and present state

of HCM genetics, it describes how genetic testing has now moved from the laboratory

bench into the physician’s hands at the patient’s bedside. It provides physicians an

algorithm on when to consider genetic testing and discusses guidelines for screening

family members of patients with HCM. It also discussed the particular situation of

screening children or athletes related to patients with HCM. Lastly, it provides insights

in the current and future options for patients with genotyped HCM and examines where

research is making strides to delineate the underpinnings of this disease.

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In conclusion, the understanding of HCM has matured from its cornerstone as a

disease of the sarcomere to a compendium of diseases with various clinical, genetic

and morphologic substrates. Research has provided us more insights into i) the

pathogenetic development of HCM, ii) the possible pro-hypertrophic pathways that are

triggered, and iii) the modifiers that influence the disease phenotypes. These findings

have opened the door for individualized medicine and the development of therapeutic

trials aimed at disease prevention or slowing of disease progression in patients with

genotype positive, hypertrophy negative HCM. However, far more research is needed

to understand its exact pathogenic pathways, discover novel drug targets or

understand and prevent its devastating feature of sudden cardiac death.

176

Samenvatting

178

Hypertrofische cardiomyopathie (HCM) is een aandoening gekarakteriseerd door

hypertrofie van de linker ventrikel in de afwezigheid van een aanwijsbare,

onderliggende oorzaak. Met een prevalentie van 1 op 500 is het de meest

voorkomende, erfelijke hartaandoening en de belangrijkste oorzaak voor plotselinge

hartdood in jong volwassenen, voornamelijk atleten. Karakteristieke pathologische

veranderingen zijn hypertrofie van de cardiomyocyt, disorganizatie van de myofibrillen

en focale, interstitiele fibrose. Op genetisch vlak werd HCM gezien als een ziekte van

de sarcomeer, waarin de meeste mutaties tot nu toe zijn gevonden in genen die

coderen voor eiwitten in het contractiele apparaat van de hartspiercel. Echter, meta-

analyses lieten zien dat de frequentie van mutaties in verschillende cohorten lag

tussen de 30 en 70%. Recent onderzoek toonde een sterke correlatie tussen de vorm

van het interventriculare septum van het hart (het “fenotype”) en het onderliggende

genotype; 80% van de patiënten met septum hypertrofie, waarbij hypertrofie de linker

hartkamer inbuigt (reverse curve HCM) had een mutatie, in tegenstelling tot slechts 8%

van de patiënten met hypertrofie rond het uitstroom traject van de linker ventrikel

(sigmoïdal-HCM). Gebaseerd op deze bevindingen, besloten we het onderzoek te

richten op de genetische basis van HCM in patienten waarbij geen afwijking in

sarcomeer genen werd gevonden en genotype-fenotype correlaties van nieuw

ontdekte genen.

Hoofdstuk 1 is een algemene introductie in HCM, waarin de klinische en

genetische geschiedenis van HCM worden besproken. Verder wordt de status van de

genetica van HCM in 2007 besproken alswel de recente observatie van een sterke

correlatie tussen de vorm (de morfologie) van het septum en het onderliggende

genetische substraat. Daarbij is het een opzet voor de meeste hoofdstukken van dit

proefschrift en de discussie of HCM één ziekte is dan wel een ziekte die onderverdeeld

zou moeten worden in verschillende klinische, genetische en/of morfologische

subgroepen.

179

In hoofdstuk 2 worden de ontdekkking en genotype-fenotype relaties

besproken van mutaties in 3 Z-disc geassocieerde genen. Aangezien na analyse van

de sarcomeer genen in een groot gedeelte van HCM patiënten geen mutatie werd

gevonden werd de aandacht gericht op de aanliggende elementen van de Z-disc;

reeds eerder waren mutaties gevonden in dilaterende cardiomyopathie (DCM) en

verscheidende diermodellen onderschreven een potentiele rol van de Z-disc genen in

de pathogenese van HCM. In ons onderzoek beschrijven we de ontdekking dat 4%

van patiënten met HCM een mutatie heeft in de Z-lijn eiwitten muscle LIM-protein

(MLP, gecodeerd door CSRP3) en telethonin (gecodeerd door TCAP). Fenotypisch

gezien leken patiënten met een Z-disc mutatie op patiënten met een mutatie in een

van de myofilament genen en waren ze meer aangedaan dan patiënten zonder HCM

mutatie.

Hoofsdtuk 3 beschrijft de ontdekking van mutaties in Ankyrin repeat domain 1

(gecodeerd door ANKRD1) in patiënten met HCM. In samenwerking met collega’s in

Japan geleid door Dr. Akinora Kimura beschrijven we de ontdekking van 3 nieuwe

mutaties in ANKRD1 in twee onafhankelijke cohorten van HCM-patiënten. Daarnaast

werden twee mutaties gevonden in het N2A-domein van het zeer grote, sarcomeer

eiwit titin (gecodeerd foor het gen TTN). Functionele studies van deze mutaties

vertoonden toegenomen binding van titin en ANKRD1 alswel dislocalisatie van

ANKRD1-mutanten in cardiomyocyten. In tegenstelling tot normaal ANKRD1,

normaalgesproken gelocaliseerd in de Z-I band van de sarcomeer, bleek gemuteerd

ANKRD1 gelocaliseerd te zijn in of rond het membraan van de nucleus in ongeveer

60% van volwassen cardiomyocyten.

In hoofdstuk 4 en 5 worden de genotype-fenotype relaties beschreven tussen

eerder ontdekte, myofilament mutaties en de relatie tot sexe (hoofdstuk 5) en/of de

onderliggende morfologische vorm van het interventriculaire septum. Gelijk aan de

correlatie gezien tussen reverse-curve HCM en de aanwezigheid van een myofilament

mutatie, bleek na analyse Z-disc HCM voornamelijk sigmoïd van vorm te zijn. Van de

13 patiënten met een mutatie in een van de Z-disc genen, bleek 85% een sigmoïdale

contour op echo te hebben; geen van de patiënten had tekenen van reverse-curve

HCM. Deze bevindingen leidden ons tot de speculatie dat het pathogenetische

mechanisme van Z-disc patiënten preferentieel sigmoïdale hypertrofie veroorzaakt in

het uitstroom traject van de linker ventrikel.

180

Hoofdstuk 5 richt zich op dezelfde subgroep, met een speciaal focus op sexe-

verschillen tussen de patiënten. Een eerder gepubliceerde studie op patiënten zonder

bekend genotype met HCM uit de VS en Italie liet zien dat vrouwen over het algemeen

ouder waren bij hun diagnose met HCM, meer obstructie hadden van het linker

ventriculaire uitstroom traject en vaker hartfalen ontwikkelden. Voor onze studie

ondernamen we een soortgelijke analyse op patiënten met HCM, gegenotypeerd voor

mutaties in de sarcomeer genen en gescoord voor de morfologsiche vorm van het

septum. Gelijk aan de voorgaande resulaten zagen wij significante verschillen tussen

mannen en vrouwen, maar verschillen werden alleen gezien in de subgoup van

vrouwen met mutatie-negatief, sigmoïd HCM. Multi en lineair statistische regressie

modellen wezen uit dat, voor vrouwen, diagnose leeftijd, systolische bloeddruk en

aanwezigheid van linker ventrikel uitstroom obstructie direct afhankelijk waren van

sigmoïde hypertrofie. Deze bevindingen demonstreerden dat in HCM patiënten met

een myofilament mutatie de mutatie het ziekteproces lijkt te domineren, en dat in

afwezigheid van een mutatie, sexe een grote invloed lijkt te hebben op het fenotype

van HCM.

In een poging om genotype negatief HCM verder te verklaren, ondernamen we

vervolgens een stap weg van de Z-disc om een nieuw kandidaat gen te bestuderen,

dat in knock-out muizen een beeld liet zien van HCM met een specifieke voorkeur

voor mannetjes muizen. In hoofdstuk 6 beschrijven we de ontdekking van 6 nieuwe

HCM-mutaties in TIEG1 (TGF -inducible early gene-1 gecodeerd door het gen TIEG1)

in twee onafhankelijke cohorten van HCM patiënten van het Academisch Medisch

Centrum in Amsterdam (AMC) en de Mayo Clinic in Rochester, MN in de Verenigde

Staten. In vitro analyses van de gevonden mutaties, liet, gelijk aan studies in de knock-

out muizen, een modificatie van TIEG1 zien op de promoter van PTTG1 uitschrijven

svp voor 5 van de 6 gekloonde mutaties resulterend in een significante overexpressie

van de PTTG1-promoter activiteit. Immunohistochemische analyse van hartweefsel liet

een toename zien van PTTG1-eiwit expressie zien in HCM-patiënten en zelfs meer in

patiënten met een TIEG1-mutatie, aanwijzingen dat PTTG1 wellicht een biomarker is

voor HCM.

181

Hoofdstuk 7 is een recent review artikel waarin de diagnostische,

prognostische en therapeutische implicaties voor genetisch testen in HCM worden

besproken. Naast de geschiedenis en huidige stand van zaken in genetica van HCM,

wordt besproken hoe genetisch testen voor HCM zich heeft verplaatst van het

laboratorium naar de handen van de arts aan het bed van de patiënt. Het geeft artsen

een algoritme dat tot hulp is in de keuze voor genetisch testen en bespreekt de huidige

richtlijnen voor screenen van familieleden van patiënten met HCM; screening van

kinderen en athleten worden tevens besproken. Het biedt inzichten in de huidige en

toekomstige therapeutische opties voor patiënten met een HCM-mutatie en beschrijft

de meest recente ontwikkelingen in het onderzoek omtrent HCM.

Concluderend, de genetische basis van HCM heeft zich de laatste jaren

uitgebreid van een ziekte van de sarcomeer naar een ziekte met verscheidene

klinische, genetische en morfologische verschijningsvormen. Onderzoek heeft ons

nieuwe inzichten gegeven in de pathogenetische ontwikkeling van de ziekte, mogelijke

pathways die aangezet worden en identificatie van modificerende factoren die de

verschillen fenotypes zouden kunnen beinvloeden. Deze bevindingen hebben de deur

geopend naar patiënt-gerichte geneeskunde en de ontwikkeling van trials gericht op

preventie of remming van ziekte progressie in genotype positieve patiënten zonder

hypertrofie. Meer onderzoek is echter nodig om te exacte pathogenetische aspecten te

ontcijferen, nieuwe targets te vinden voor medicijnen en naar het voorkomen van

plotselinge hartdood.

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List of publications

188

Peer reviewed publications

1. Bos JM, Poley RN, Ny M, Tester DJ, Xu X, Vatta M, Towbin JA, Gersh BJ, Ommen SR, Ackerman MJ. Genotype-phenotype relationships involving hypertrophic cardiomyopathy-associated mutations in titin, muscle LIM protein, and telethonin. Molecular Genetics and Metabolism 2006; 88(1): 78 – 85.

2. Bos JM, Hagler DJ, Silvilairat S, Cabalka A, O’Leary P, Daniels O, Miller FA, Abraham TP. Right ventricular function in asymptomatic individuals with a systemic right ventricle. Journal of the American Society of Echocardiography 2006; 19(8): 1033 – 1037.

3. Theis JL*, Bos JM*, Bartleson VB, Will ML, Binder J, Vatta M, Towbin JA, Gersh BJ, Ommen SR, Ackerman MJ. Echocardiographic-determined septal morphology in Z-disc hypertrophic cardiomyopathy. Biochemical and Biophysical

Research Communications 2006; 351(4): 896 – 902 (*co-equal first author).

4. Landstrom AP, Weisleder N, Batalden KB, Bos JM, Tester DJ, Ommen SR, Wehrens XH, Claycomb WC, Ko JK, Hwang M, Pan Z, Ma J, Ackerman MJ. Mutations in JPH2-encoded junctophilin-2 associated with hypertrophic cardiomyopathy. Journal

of Molecular and Cellular Cardiololgy 2007; 42(6): 1026 – 1035

5. Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, Martinelli S, Pogna EA, Schackwitz W, Ustaszewska A, Landstrom AP, Bos JM, Ommen SR, Esposito G, Lepri F, Faul C, Mundel P, Siguero JPL, Tenconi R, Selicorni A, Rossi C, Mazzanti L, Torrente I, Marino B, Digilio MC, Zampino G, Ackerman MJ, Dallapiccola B, Tartaglia M, Gelb BD. Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nature Genetics 2007; 39(8): 1007 – 1012.

6. Olivotto I, Girolami F, Ackerman MJ, Nistri S, Bos JM, Zachara E, Ommen SR, Theis JL, Vaubel RA, Re F, Armantano C, Poggessi C, Torricelli F, Cecchi F. Myofilament protein gene mutation screening and outcome in hypertrophic cardiomyopathy. Mayo Clinic Proceedings 2008; 83(6): 630 – 638.

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7. Bos JM, Theis JL, Tajik AJ, Gersh BJ, Ommen SR, Ackerman MJ. Relationship between sex, shape and substrate in hypertrophic cardiomyopathy. American Heart Journal 2008; 155: 1128 – 1134.

8. Landstrom AP, Parvatiyar MS, Pinto JR, Marquardt ML, Bos JM, Ommen SR, Potter JD, Ackerman MJ. Defining TNNC1-Encoded Cardiac Troponin C as Novel Target Gene for Hypertrophic Cardiomyopathy. Journal of Molecular and Cellular

Cardiology. 2008; 45(2):281 – 288.

9. Maron MS, Finley JJ, Bos JM, Hauser TH, Manning WJ, Haas TS, Lesser JR, Udelson JE, Ackerman MJ, Maron BJ. Prevalence, clinical significance, and natural history of left ventricular apical aneurysms in hypertrophic cardiomyopathy. Circulation.

2008; 118(15): 1541 – 1549.

10. Arimura T*, Bos JM*, Sato A, Kubo T, Okamoto H, Nishi H, Harada H, Koga Y, Moulik M, Doi Y, Towbin JA, Ackerman MJ, Kimura A. Cardiac ankyrin repeat protein gene (ANKRD1) mutations in hypertrophic cardiomyopathy. Journal of the American

College of Cardiology 2009; 54(4): 334 – 342 (*co-equal first author).

11. Theis JL, Bos JM, Theis JD, Miller DV, Dearani JA, Schaff HV, Gersh BJ, Ommen SR, Moss RL, Ackerman MJ. Expression Patterns of Cardiac Myofilament Proteins – Genomic and Protein Analysis of Surgical Myectomy Tissue from Patients with Obstructive Hypertrophic Cardiomyopathy. Circulation: Heart Failure 2009; 2: 325 – 333.

12. Rubinshtein R, Glockner JF, Ommen SR, Araoz PA, Ackerman MJ, Sorajja P, Bos JM, Tajik AJ, Valeti US, Nishimura RA, Gersh BJ. Characteristics and Clinical Significance of Late Gadolinium Enhancement by Contrast-Enhanced Magnetic Resonance Imaging in Patients with Hypertrophic Cardiomyopathy. Circulation: Heart

Failure. 2009 Oct 22. [Epub ahead of print]

13. McLeod CJ, Bos JM, Theis JL, Edwards WD, Gersh BJ, Ommen SR, Ackerman MJ. Histologic characterization of hypertrophic cardiomyopathy with and without myofilament mutations. American Heart Journal. 2009; 158(5): 799 – 805.

190

Editorial comments, review articles

1. Ackerman MJ, Van Driest SL, Bos M. Are longitudinal, natural history studies the next step in genotype-phenotype translational genomics in hypertrophic cardiomyopathy? Journal of the American College of Cardiology 2005; 46(9): 1744 – 1746 [Editorial comment].

2. Bos JM, Ommen SR, Ackerman MJ. Genetics of hypertrophic cardiomyopathy: one, two, or more diseases? Current Opinion in Cardiology 2007; 22(3): 193 – 199 [Review].

3. Bos JM, Towbin JA, Ackerman MJ. Diagnostic, Prognostic and Therapeutic Implications of Genetic Testing for Hypertrophic Cardiomyopathy. Journal of the

American College of Cardiology 2009; 54(3): 201 – 211 [Review].

Book Chapters

1. Menon SC, Bos JM, Ommen SR, Ackerman MJ. Arrhythmogenic Malignancies in Hypertrophic Cardiomyopathy. In: Electrical Diseases of the Heart: Genetics, Mechanisms, Treatment, Prevention (Chapter 40). Springer 2007. ISBN: 978-1846288531.

2. Bos JM, Ommen SR, Ackerman MJ. Hypertrophic Cardiomyopathy in the Era of Genomic Medicine. In: Genomic and Personalized Medicine (Chapter 61).Academic Press 2008. ISBN: 978-0123694201.

3. Bos JM, Ommen SR, Ackerman MJ. Hypertrophic Cardiomyopathy in the Era of Genomic Medicine. In: Essential Genomic and Personalized Medicine (Chapter 28).Academic Press 2009. ISBN: 978-0123749345.

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