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ISBN 978-951-42-8821-0 (Paperback)ISBN 978-951-42-8822-7 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)
U N I V E R S I TAT I S O U L U E N S I S
MEDICA
ACTAD
OULU 2008
D 973
Outi Vierimaa
MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 (MEN1) AND PITUITARY ADENOMA PREDISPOSITION (PAP)IN NORTHERN FINLAND
FACULTY OF MEDICINE, INSTITUTE OF CLINICAL MEDICINE,DEPARTMENT OF CLINICAL GENETICS, DEPARTMENT OF INTERNAL MEDICINE,UNIVERISTY OF OULU;DEPARTMENT OF MEDICAL GENETICS, HAARTMAN INSTITUTE,UNIVERSITY OF HELSINKI
D 973
ACTA
Outi Vierim
aa
D973etukansi.fm Page 1 Wednesday, June 4, 2008 4:08 PM
A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 9 7 3
OUTI VIERIMAA
MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 (MEN1) AND PITUITARY ADENOMA PREDISPOSITION (PAP) IN NORTHERN FINLAND
Academic dissertation to be presented, with the assent ofthe Faculty of Medicine of the University of Oulu, forpublic defence in Auditorium 5 of Oulu UniversityHospital, on June 27th, 2008, at 12 noon
OULUN YLIOPISTO, OULU 2008
Copyright © 2008Acta Univ. Oul. D 973, 2008
Supervised byProfessor Lauri AaltonenProfessor Jaakko LeistiDocent Pasi Salmela
Reviewed byProfessor Marja HietalaProfessor Jorma Viikari
ISBN 978-951-42-8821-0 (Paperback)ISBN 978-951-42-8822-7 (PDF)http://herkules.oulu.fi/isbn9789514288227/ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)http://herkules.oulu.fi/issn03553221/
Cover designRaimo Ahonen
OULU UNIVERSITY PRESSOULU 2008
Vierimaa, Outi, Multiple Endocrine Neoplasia Type 1 (MEN1) and PituitaryAdenoma Predisposition (PAP) in Northern FinlandFaculty of Medicine, Institute of Clinical Medicine, Department of Clinical Genetics,Department of Internal Medicine, University of Oulu, P.O.Box 5000, FI-90014 University ofOulu, Finland; Department of Medical Genetics, Haartman Institute, University of Helsinki,P.O.Box 21, FI-00014 University of Helsinki, Finland Acta Univ. Oul. D 973, 2008Oulu, Finland
AbstractMultiple endocrine neoplasia type 1 (MEN1) is an inherited syndrome characterized byparathyroid, gastroenteropancreatic and pituitary neuroendocrine tumours. In Northern Finland,two founder mutations of the MEN1 gene (1466del12, 1657insC) accounting for the majority ofthe MEN1 cases, have common ancestors born in the 18th and 19th centuries, respectively. Threesmall clusters of familial pituitary adenoma have also been detected, two of which could be linkedby genealogy to a common ancestral couple born in the 18th century.
Clinical evaluation of 82 MEN1 mutation carriers showed that age was a risk factor for mostof the MEN1-related manifestations. In the whole group, nonfunctional pancreatic tumour (NFPT)was more common in the frameshift/nonsense mutation carriers (odds ratio 3.26; 95% confidenceinterval 1.27–8.33, P = 0.014), whereas gastrinoma was more common in the in-frame/missensemutation carriers (OR 6.77, CI 1.31–35.0, P = 0.022). In the founder mutation carriers, the1657insC mutation predicted the risk for NFPT (OR 3.56, CI 1.29–9.83, P = 0.015), while the1466del12 mutation was associated with the risk for gastrinoma (OR 15.1, CI 1.73–131.9,P = 0.014).
The mean ages at death of the 32 obligatory MEN1 founder mutation carriers born between1728 and 1929 were compared to those of the 29 spouses and sex-matched life expectancyestimates derived from Finnish national statistics. The ages at death of the mutation carrier males(61.1 ± 12.0 years) and females (67.2 ± 10.7 years) did not differ from the control groups.
PAP (pituitary adenoma predisposition) locus was mapped in the chromosome region11q12–11q13 by whole-genome single-nucleotide polymorphism genotyping. Combining thelinkage and the gene expression array data, AIP (aryl hydrocarbon receptor interacting protein)was chosen for sequencing. The nonsense mutation Q14X was identified in the affected(acromegaly, gigantism, prolactinoma) family members and in four other patients. Loss ofheterozygosity was detected in pituitary adenomas of AIP mutation carriers.
Mutation analysis of MEN1, HRPT2 (hyperparathyroidism 2), CASR (calcium-sensingreceptor), CDKN1B (cyclin-dependent kinase inhibitor 1B) and AIP genes was performed inprimary hyperparathyroidism patients with features of inherited predisposition. One out of 29patients was found to have the 1466del12 mutation, while no mutations were detected in othergenes.
Keywords: genetic predisposition, multiple endocrine neoplasia type 1, pituitaryadenoma, primary hyperparathyroidism
AcknowledgementsThis study was carried out during the years 1999–2008 at the Departments ofClinical Genetics and Internal Medicine, Oulu University Hospital and Universityof Oulu, and at the Department of Medical Genetics, Haartman Institute,University of Helsinki. I wish to express my sincere gratitude to all those whoparticipated in this project:
Emeritus Professor Jaakko Leisti, my supervisor, for opening my eyes to thefascinating world of clinical genetics. Without his input in the field of hereditarytumour syndromes in the Department of Clinical Genetics I would never havestarted to write this book. Through all these years his guidance has been mostvaluable.
Professor Jaakko Ignatius for giving me the opportunity to work in theDepartment of Clinical Genetics. His support and encouragement to complete thisproject is highly appreciated.
Docent Pasi Salmela, my second supervisor, for his valuable contribution tothis thesis. His profound knowledge on endocrinology and genetics has beenessential to this project to succeed.
Academy Professor Lauri Aaltonen, my third supervisor, for his enthusiasticattitude to my ideas on familial tumour syndromes. His excellence in gene huntinghas been one of the cornerstones in this work.
Professor Jorma Viikari and Acting Professor Marja Hietala for their valuablecomments on the manuscript of this thesis. I also want to thank Anna Vuolteenahofor the careful revision of the language in this thesis, and Helena Saari for theprofessional layout of this book.
All the co-authors and collaborators who made this work possible, RistoBloigu, Ph.D., Docent Jorma Salmi, Dr. Eeva Korpi-Hyövälti, Professor LeoNiskanen, Dr. Anja Orvola, Dr. Anita Gynther, Docent Timo Sane, Professor MattiVälimäki, Marianthi Georgitsi, M.Sc., Rainer Lehtonen, Ph.D, PiaVahteristo,Ph.D., Antti Kokko, Ph.D., Anniina Raitila, M.Sc., Karoliina Tuppurainen, M.Sc.,Professor Ralf Paschke, Dr. Sadi Gündogdu, Dr. Ernesto De Menis, DocentMarkus J. Mäkinen, Docent Virpi Launonen, Docent Auli Karhu, AndreaVillablanca, Ph.D., Andrei Alimov, Ph.D., Professor Catharina Larsson andProfessor Aimo Ruokonen.
All the past and present group members, especially Docent Tapani Ebeling,Docent Soili Kytölä, Liisa Ukkola, M.Sc., and Dr. Eija Eloranta.
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The staff at the Department of Clinical Genetics, especially genetic nursesOuti Kajula and Riitta Mattlar. I am also very grateful to research nurse SiniMarttinen for her prompt assistance with various matters during this work.
All the patients and their families who took part in the study. Without theeffort of the participants this study would never have taken place. Special thanksgo to the family who provided me with carefully researched genealogy data.
My family and friends, especially my parents Mirja and Juha and their spousesfor their support and practical help during all these years. I am also grateful to mysiblings Auli, Maria and Antti for their support and interest in my scientific work. Iam very fortunate to have three wonderful children, Arttu, Lotta and Helka, and acaring husband, Jukka. Their love has given me strength to work with this projectand to get it done.
The financial support of the University of Oulu, Oulu University Hospital andthe cancer Society of Northern Finland is highly appreciated.
Haukipudas, May 2008
Outi Vierimaa
6
AbbreviationsACTH adrenocorticotropic hormoneAHR aryl hydrocarbon receptorAIP aryl hydrocarbon receptor interacting protein geneCASR calcium-sensing receptor geneCDKN1B cyclin-dependent kinase inhibitor 1B geneCGA chromogranin A (CgA)CGB chromogranin B (CgB)CHES1 chekpoint suppressor 1CI confidence intervalCNC Carney complexCS Cushing’s syndromeCT computed tomographyDNA deoxyribonucleic acidECLoma enterochromaffin-like cell tumourEUS endoscopic ultrasoundFHH familial hypocalciuric hypercalcaemiaFIHP familial isolated hyperparathyroidismFIPA familial isolated pituitary adenomaFMTC familial medullary thyroid cancerFSH follicle-stimulating hormoneGEP gastroenteropancreaticGH growth hormoneGHRH growth hormone-releasing hormoneGHRHoma growth hormone-releasing hormone-secreting tumourHPT-JT hyperparathyroidism-jaw tumour syndromeHRPT2 hyperparathyroidism 2 geneIFS isolated familial somatotropinomaIGF-1 insulin-like growth factor 1kD kilodaltonLCCST large-cell calcifying Sertoli cell tumorLH luteinizing hormoneLOH loss of heterozygosityLOD logarithm of oddsMEN1 multiple endocrine neoplasia type 1MEN1 multiple endocrine neoplasia type 1 gene
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MEN2 multiple endocrine neoplasia type 2MEN4 multiple endocrine neoplasia type 4MRI magnetic resonance imagingmRNA messenger ribonucleic acidNFPT nonfunctional pancreatic (neuroendocrine) tumourNLS nuclear localization signalsNM23H1 nonmetastatic protein 23, homolog 1OMIM Online Mendelian Inheritance in ManOR odds ratioPAP pituitary adenoma predispositionPHPT primary hyperparathyroidismPP pancreatic polypeptidePPNAD primary pigmented nodular adrenocortical diseasePRKAR1A regulatory subunit type 1A (RIalpha) of protein kinase A genePRL prolactinPTH parathyroid hormoneTSH thyroid stimulating hormoneUS ultrasoundVHL von Hippel-Lindau diseaseVHL von Hippel-Lindau disease geneVIP vasoactive intestinal peptideVIPoma vasoactive intestinal peptide –secreting tumourvs. versusZES Zollinger-Ellison syndrome
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List of original publicationsThis thesis is based on the following articles, which are referred to in the text bytheir Roman numerals:
I I Vierimaa O, Ebeling TM, Kytölä S, Bloigu R, Eloranta E, Salmi J, Korpi-Hyövälti E, Niskanen L, Orvola A, Elovaara E, Gynther A, Sane T, VälimäkiM, Ignatius J, Leisti J & Salmela PI (2007) Multiple endocrine neoplasia type1 in Northern Finland; clinical features and genotype phenotype correlation.European Journal of Endocrinology 157:285–94.
II Ebeling T, Vierimaa O, Kytölä S, Leisti J & Salmela PI (2004) Effect ofmultiple endocrine neoplasia type 1 (MEN1) gene mutations on prematuremortality in familial MEN1 syndrome with founder mutations. Journal ofClinical Endocrinology and Metabolism 89:3392–6.
III Vierimaa O, Georgitsi M, Lehtonen R, Vahteristo P, Kokko A, Raitila A,Tuppurainen K, Ebeling TM, Salmela PI, Paschke R, Gündogdu S, De MenisE, Mäkinen MJ, Launonen V, Karhu A & Aaltonen LA (2006) Pituitaryadenoma predisposition caused by germline mutations in the AIP gene.Science 312:1228–30.
IV Vierimaa O, Villablanca A, Alimov A, Georgitsi M, Raitila A, Vahteristo P,Larsson C, Ruokonen A, Eloranta E, Ebeling TML, Ignatius J, Aaltonen LA.,Leisti J & Salmela PI. Mutation analysis of MEN1, HRPT2, CASR, CDKN1Band AIP genes in primary hyperparathyroidism patients with features ofgenetic predisposition. Submitted.
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10
ContentsAbstractAcknowledgements
Abbreviations
List of original publications
Contents5 Introduction 136 Review of the literature 17
6.1 Multiple endocrine neoplasia type 1 (MEN1)......................................... 176.1.1 History and definition ................................................................... 176.1.2 Clinical features ............................................................................ 186.1.3 Clinical management of MEN1 mutation carriers ........................ 336.1.4 Mortality ...................................................................................... 346.1.5 Genetics of MEN1 ........................................................................ 35
6.2 Other disorders with inherited predisposition to primary hyperparathyroidism (PHPT) and/or anterior pituitary adenomas.......... 38
6.3 Multiple endocrine neoplasia type 2A (MEN2A)................................... 426.3.1 Hyperparathyroidism-jaw tumour syndrome (HPT-JT) ............... 436.3.2 Familial hypocalciuric hypercalcaemia (FHH)............................. 446.3.3 Familial isolated hyperparathyroidism (FIHP) ............................. 456.3.4 Carney complex (CNC) ................................................................ 456.3.5 Familial isolated pituitary adenoma (FIPA) ................................. 476.3.6 Isolated familial somatotropinoma (IFS) ...................................... 486.3.7 Multiple endocrine neoplasia type 4 (MEN4)............................... 49
7 Purpose of the present study 518 Subjects and methods 53
8.1 Subjects and families............................................................................... 538.1.1 Clinical features and genotype-phenotype correlation of
MEN1 in Northern Finland (I) ...................................................... 538.1.2 Study on Premature Mortality in MEN1 founder mutation
carriers (II) .................................................................................... 538.1.3 Pituitary adenoma predisposition (III) .......................................... 548.1.4 Primary hyperparathyroidism patients with features of
genetic predisposition (IV) ........................................................... 55
11
8.2 Biochemical and imaging studies of MEN1 mutation carriers and PHPT patients with features of genetic predisposition (I, IV) ................ 55
8.3 Obtaining clinical data and causes of death (I) ....................................... 568.4 Comparing mean values of age at death (II) ........................................... 578.5 Histopathology and immunohistochemistry (III).................................... 578.6 SNP array and linkage analyses (III) ...................................................... 578.7 Expression profiling (III) ........................................................................ 598.8 Mutation screening and search for loss of heterozygosity
(LOH) (III, IV) ........................................................................................ 598.9 Ethical issues........................................................................................... 60
9 Results 639.1 MEN1 in Northern Finland; clinical features and genotype-
phenotype correlation (I)......................................................................... 639.2 Effect of MEN1 founder mutations on premature mortality (II)............. 679.3 Characterization of clinical and genetic aspects of pituitary
adenoma predisposition (PAP) (III) ........................................................ 709.4 Mutation analysis of MEN1, HRPT2, CASR, CDKN1B and AIP
genes in PHPT patients with features of genetic predisposition (IV) ..... 7210 Discussion 75
10.1Clinical features of MEN1 and genotype-phenotype correlation (I) ...... 7510.2Mortality in MEN1 (I, II)........................................................................ 7810.3Pituitary adenoma predisposition and AIP (III)...................................... 8210.4PHPT patients with features of genetic predisposition (IV) ................... 85
11 Conclusion and future prospects 89
References
Original publications
12
1 IntroductionMultiple endocrine neoplasia type 1 (MEN1) (Online inheritance in man, OMIM131100) characterized by tumours of the parathyroid, neuroendocrine cells of thepancreas, duodenum and anterior pituitary is one of numerous hereditary tumourpredisposition syndromes (Marx 2001). In addition to MEN1, there are also othertumour predisposition conditions with target organs including endocrine glands.The variety of target organs and clinical manifestations are distinctive to a certainsyndrome, although overlapping exists as well. The feature almost all of thesesyndromes have in common is the mode of inheritance, which is autosomaldominant. Therefore, the offspring of the mutation carrier parent are at 50-percentrisk of inheriting the disease associating gene, and thus at risk of being affected bythe disorder.
The first patient described with presumable MEN1 was reported more than100 years ago (Erdheim 1903), and the gene behind this disorder, MEN1, wasidentified in 1997 (Chandrasekharappa et al. 1997, Lemmens et al. 1997). Despitethe well-characterized clinical features of MEN1, it is noteworthy that thefrequency of the manifestations tends to vary considerably between differentstudies (Table 1). Whether any of this variation of clinical features is dependent onthe genotype of affected heterozygotes is under debate, although the majority ofstudies doubts the existence of genotype-phenotype correlation in MEN1(Agarwal et al. 1997, Bassett et al. 1998, Giraud et al. 1998, Wautot et al. 2002).
The majority of the MEN1-associated manifestations are due to benigntumours of neuroendocrine cells, although those locating in foregut-derived tissues(especially duodenum, thymus, ventricle) and pancreas may be malignant(Falchetti et al. 2008). Several studies suggest that MEN1 patients are at increasedrisk of premature death compared to their non-affected relatives or normalpopulation (Vasen et al. 1989, Wilkinson et al. 1993, Carty et al. 1998, Dean et al.2000, Geerdink et al. 2003). The major causes of MEN1-related deaths havechanged during the past decades. Previously, peptic ulcer disease and renalcomplications of hypercalcaemia due to primary hyperparathyroidism were amongthe most important causes of death in MEN1 patients (Ballard et al. 1964,Majewski & Wilson 1979, Vasen et al. 1989). The most recent studies suggest thatmalignant MEN1-related tumours are the major cause of death in MEN1(Wilkinson et al. 1993, Carty et al. 1998, Doherty et al. 1998, Dean et al. 2000,Geerdink et al. 2003, Ferolla et al. 2005). It is estimated that approximately onethird of the patients die from a MEN1-related cancer (Falchetti et al. 2008).
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Tabl
e 1.
Rep
orte
d pe
rcen
tage
s (%
) of
the
mos
t co
mm
on e
ndoc
rine
tum
ours
in p
atie
nts
with
mul
tiple
end
ocri
ne n
eopl
asia
typ
e 1
(ME
N1)
.
Type
of t
umou
r
Stud
yC
ases
(n)
Par
a-th
yroi
dE
nter
opan
crea
ticA
ntre
rior P
ituita
ryA
dren
alG
astri
cTh
ymic
Bro
nchi
al
(all)
(G)
(NF)
(I)(a
ll)(P
RL)
(NF)
(GH
)
Ben
son
et a
l. 19
8755
8958
2516
942
330
7N
A2
05
Vase
n et
al.
1989
5287
6254
010
278
60a
NA
NA
NA
NA
Trum
p et
al.
1996
220
9541
260.
411
3018
27
6N
Ab
NA
bN
Ab
Bur
gess
et a
l. 19
9816
095
NA
6050
1019
145
036
42
3
Car
ty e
t al.
1998
3482
7447
99
6553
36
120
30
Mar
x et
al.
1998
130
9966
474
1247
2611
316
71
8
Gre
en e
t al.
1999
9594
104
NA
NA
3432
NA
NA
3N
A3
5
Dea
n et
al.
2000
233
9747
287
1047
NA
NA
NA
3N
Ac
NA
cN
Ac
Cle
rici e
t al.
2001
3070
4730
133
30
30
30
33
Kop
p et
al.
2001
2588
6840
2020
6044
120
200
08
Vèr
ges
et a
l. 20
0232
495
5426
NA
742
26N
A4
17N
AN
Ad
NA
d
Lévy
-Boh
bot e
t al.
2004
580
9253
3110
1241
NA
NA
NA
NA
20N
AN
A
Mac
hens
et a
l. 20
07e
258
90N
A38
1821
NA
258
527
37
8
G, g
astri
nom
a; N
F, n
onfu
nctio
nal;
I, in
sulin
oma;
PR
L, p
rola
ctin
-sec
retin
g tu
mou
r; G
H, g
row
th h
orm
one-
secr
etin
g tu
mou
r, N
A; n
ot a
vaila
ble.
a O
ne o
f the
pitu
itary
ade
nom
as w
as a
mix
ed g
row
th h
orm
one-
and
pro
lact
in-p
rodu
cing
tum
our.
b C
arci
noid
(neu
roen
docr
ine)
tum
ours
wer
e fo
und
in 8
(4%
) pat
ient
s.
c C
arci
noid
tum
ours
wer
e fo
und
in 1
0 (4
%) p
atie
nts.
d Th
ymic
or b
ronc
hial
car
cino
ids
wer
e pr
esen
t in
25 (8
%) p
atie
nts.
e Th
e st
udy
popu
latio
n w
as d
ivid
ed in
to 5
birt
h co
horts
and
fig
ures
(pe
netra
nce
in p
erce
ntag
es)
wer
e gi
ven
for
each
coh
ort.
The
figur
es s
how
n he
re a
re t
hem
axim
al p
enet
ranc
e pe
rcen
tage
s of
eac
h tu
mou
r cat
egor
y.
14
It is estimated that approximately 5% of the patients with primaryhyperparathyroidism (PHPT) or pituitary adenoma have a hereditary form of thedisease (Miedlich et al. 2003, Daly et al. 2006). MEN1 is the most commonaetiologic factor for both of these tumours in familial setting. Other syndromesseen in association with dominantly inherited PHPT are multiple endocrineneoplasia type 2 (MEN2), hyperparathyroidism-jaw tumour syndrome (HPT-JT),familial hypocalciuric hypercalcaemia (FHH), and a newly discovered MEN1-likeentity, multiple endocrine neoplasia type 4 (MEN4). The underlying genes in thecorresponding order are RET, HRPT2, CASR and CDKN1B (Mulligan et al. 1993,Pollak et al. 1993, Carpten et al. 2002, Pellegata et al. 2006). Familial isolatedhyperparathyroidism (FIHP) can result from the incomplete expression of asyndromic form of familial hyperparathyroidism, but it can also represent someother entity yet to be determined (Simonds et al. 2002). In addition to MEN1,pituitary tumours are seen in familial setting in Carney complex (CNC), familialisolated pituitary adenoma (FIPA), isolated familial somatotropinoma (IFS), andMEN4 (Daly et al. 2006). Germline mutations in PRKAR1A are found in themajority of CNC patients (Kirschner et al. 2000). The locus for IFS has beenmapped to chromosome 11q13 by several studies (Gadelha et al. 2000, Luccio-Camelo et al. 2004, Soares et al. 2005), but the genetic background for other typesof FIPA families has remained unknown.
In the present work, one of the principal aims was to study the clinical featuresand possible genotype-phenotype correlation in Northern Finnish MEN1 patients.Another purpose of this study was to evaluate the mortality in MEN1 mutationcarriers comparing the age at death to that of non-carrier spouses and to statisticallife expectancy values. In addition, one of the main purposes of this work was todefine the clinical features and genetic background of previously uncharacterizedpituitary adenoma predisposition observed in the area. One further goal of thisstudy was to investigate PHPT patients with features suggestive of geneticpredisposition in order to evaluate the role of different genes in the aetiology ofthis endocrine disorder.
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2 Review of the literature
2.1 Multiple endocrine neoplasia type 1 (MEN1)
2.1.1 History and definition
The main features of multiple endocrine neoplasia type 1 (MEN1) are parathyroidadenoma, enteropancreatic and foregut neuroendocrine tumours and anteriorpituitary adenoma (Marx SJ 2001). Nonendocrine manifestations include lipoma,facial angiofibroma, skin collagenoma, leiomyomas of different sites, meningiomaand ependymoma (Giraud et al. 1997, Pack et al. 1998, McKeeby et al. 2001,Asgharian et al. 2004). Neuroendocrine tumours may cause various symptomsdepending on their hormonal activity. In addition, hormonally silent or activetumours may cause symptoms due to local compression or metastatic growth todistant organs.
The prevalence of MEN1 is estimated to be 10–17.5/100,000 (Betts et al.1980, Eberle & Grun 1981, Watson et al. 1989, Öberg et al. 1989), and accordingto autopsy series as high as 2.5/1,000 (Lips et al. 1962, Lips et al. 1984).
The first description of a MEN1 patient was published in 1903. Erdheimreported the autopsy of a patient with acromegaly, pituitary adenoma, and fourenlarged parathyroid glands (Erdheim 1903). Thirty-six years later, two sisterswere reported with nephrolithiasis in association with tumours of the parathyroidglands and the pancreatic islets (Rossier & Dressler 1939). There was also a familyhistory of peptic ulcer disease on the father’s side of the family; however, it wasnot until 1961 that it could be linked to the sisters’ endocrine tumours (Schmid etal. 1961). The hereditary nature of MEN1 was first suggested in the 1950s byMoldawer and Wermer (Moldawer 1953, Moldawer et al. 1954, Wermer 1954).Prolactinoma was first proposed as a component of MEN1 in 1974 by Cryer et al.The MEN1 syndrome has also been called Wermer’s syndrome and multiple endo-crine adenomatosis type 1 (MEA1).
The genetic basis of MEN1 began to be resolved in 1988, when Larsson et al.mapped the MEN1 locus to chromosome 11q13 by family studies. They also foundout that the tumours (insulinomas) showed loss of the normal allele of chromo-some 11, providing evidence for MEN1 acting as a tumour suppressor gene (Lars-son et al. 1988). In 1997, the MEN1 gene was characterized almost simultaneouslyby two groups (Chandrasekharappa et al. 1997, Lemmens et al. 1997).
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2.1.2 Clinical features
Primary hyperparathyroidism (PHPT)
Primary hyperparathyroidism (PHPT) is in general a relatively commonendocrinopathy. PHPT is diagnosed by persistent hypercalcaemia in the presenceof inappropriately normal or elevated levels of parathyroid hormone (PTH)(Bilezikian et al. 2002). The prevalence of PHPT in postmenopausal women isestimated to be 21/1,000, or 3/1,000 in the general population with an annualincidence of approximately 20/100,000, depending on the screening methods used(Melton 1991, Adami et al. 2002). It has been estimated that approximately 2–3%of PHPT is associated with MEN1 (Marx 2001). PHPT is the most common andusually the earliest clinical manifestation of MEN1 showing almost total (Table 1)age-dependent penetrance among MEN1 mutation carriers (Marx 2001). A typicalsporadic case of PHPT is a postmenopausal woman, whereas PHPT in MEN1occurs equally in both sexes and is in most cases detectable by the age of 40 years(Burgess et al. 1998, Brandi et al. 2001, Schussheim et al. 2001). In the sporadiccases, PHPT is usually caused by a single adenoma. In MEN1, multipleparathyroid glands are usually affected, although the enlargement is highlyasymmetric (Marx et al. 1991). According to Marx (2001), parathyroid lesions inMEN1 should be regarded as independent clonal adenomas, although an earlyhyperplastic phase has been suggested, but not proven in their development(Brandi et al. 2001). Nevertheless, parathyroid carcinoma is a rare cause of PHPTwith a prevalence of <1% in sporadic cases (Koea & Shaw 1999). In addition,there are only few reported cases of parathyroid carcinoma in association withMEN1 (Shepherd 1985, Sato et al. 2000, Dionisi et al. 2002, Agha et al. 2007).
The classical phrase “painful bones, renal stones, abdominal groans, and psy-chic moans” was used to describe the signs and symptoms of PHPT decades ago,but nowadays the patients are often diagnosed without any symptoms through rou-tine biochemical screening. Modern-day patients may present with subtle neu-rocognitive symptoms including fatigue, lethargy, muscle weakness, depressionand cognitive impairment (Suliburk & Perrier 2007).
PHPT associated with urolithiasis is still a common feature in MEN1 patients.In a recent study on 26 MEN1 patients, 17 (65%) had been diagnosed with urolith-iasis (Christopoulos et al. 2005). In a review of Japanese MEN1 patients from1966 to 1995, 90% of the patients were found to have PHPT, and it was compli-cated by urolithiasis in 35% of cases (Yoshimoto 2000).
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Osteoporosis is an important complication of PHPT, since reduced bone min-eral density (BMD) is a known risk factor for skeletal fracture (Ross 1998). Bur-gess et al. (1999) examined 29 women with MEN1 belonging to a single family inorder to find out the relationship of bone mass to PHPT, fracture risk, and parathy-roidectomy in the setting of MEN1. Osteopoenia and osteoporosis were diagnosedin 41% and 44% of the patients, respectively, and reduced BMD was associatedwith an increased likelihood of skeletal fracture. In addition, parathyroidectomyimproved femoral neck and lumbar spine BMDs by a mean ±SEM of 5.2±2.5%and 3.2±2.9, respectively (Burgess et al. 1999).
Peptic ulcer disease is classically associated with sporadic PHPT (Suliburk &Perrier 2007). According to Bilezikian et al. (2002), asymptomatic PHPT is notassociated with peptic ulcer disease unless the patient is affected by MEN1 at thesame time. It has been shown that PHPT can exacerbate hypergastrinaemia inpatients with Zollinger-Ellison syndrome and MEN1, and the degree of hypergas-trinaemia has been reduced by parathyroidectomy (Norton et al 1987, Metz at al.1994).
Patients with PHPT are also associated with an increased risk of cardiac mor-bidity, hypertension, and metabolic disturbances such as impaired glucose toler-ance, type 2 diabetes mellitus and dyslipidaemia (Mihai et al. 2008). There is alsosome evidence that PHPT in MEN1 patients may be associated with impaired glu-cose intolerance and type 2 diabetes (Furuto-Kato et al. 1994, McCallum et al.2006).
According to some European studies, there is an increased risk of death amongpatients with PHPT persisting years after parathyroidectomy (Hedbäck et al. 1991,Nilsson et al. 2002, Øgard et al. 2004). However, overall mortality has not beendifferent from normal population in patients undergoing parathyroidectomy morerecently (after 1985) (Øgard et al. 2004) or in North American patients (Søreide etal. 1997).
For both sporadic cases and MEN1 patients, the overt symptoms related toPHPT are indications for parathyroid surgery. There are guidelines for parathyroidsurgery in asymptomatic PHPT (Bilezikian et al. 2002, Mihai et al. 2008), butthere is no consensus as to timing or which operation to perform in MEN1 patientswith PHPT (Brandi et al. 2001, Hubbard et al 2006). Due to multiglandularinvolvement in MEN1, either subtotal parathyroidectomy (identifying four glandsand removing at least three but leaving a parathyroid remnant the size of a normalparathyroid in the neck), or total parathyroidectomy (the resection of at least fourglands combined with the transplantation of 10–20 1-mm3 pieces of parathyroid
19
tissue into individual pockets created in the brachioradialis muscle of the forearm)are the methods usually chosen. Common features of MEN1 related parathy-roidectomy are postoperative persistence of PHPT or late recurrence, but on theother hand, hypoparathyroidism as well. (Hellman et al.1992, Hubbard et al.2006.)
Neuroendocrine gastroenteropancreatic (GEP) tumours
Neuroendocrine gastroenteropancreatic tumours are the second most commonmanifestation in MEN1 occurring in 27–74% of the patients (Table 1). Theydevelop from the pancreatic islets and the endocrine cells of the duodenal andgastric mucosa (Tonelli et al. 2005). GEP tumours associated with MEN1 occur atyounger ages and are multicentric compared to sporadic cases (Marx 2001).Typically, microadenomas are diffusely spread in the pancreas and duodenum(Åkerström et al. 2005). GEP tumours may be hormonally active and producesymptoms depending on the hormone in question, or they may be hormonallysilent. Even though non-functional tumours may produce hormonal peptide(s),they do not secrete enough to cause a recognizable syndrome. GEP tumours havegenerally a benign course, although there is a subgroup showing aggressivebehaviour, being a major cause of death among MEN1 patients (Tonelli et al.2005).
Nonfunctional pancreatic tumour (NFPT). Today, it is estimated that nonfunc-tional pancreatic neuroendocrine tumour (NFPT) is the most common neuroendo-crine GEP tumour in MEN1, as well as among sporadic cases (Tonelli et al. 2005,Kazanjian et al. 2006, Triponez et al. 2006a). The frequency of NFPT is steadilyincreasing, probably due to earlier diagnosis after genetic testing and more sensi-tive imaging and laboratory testing (Triponez et al. 2006a, b). However, in thestudies shown in Table 1, gastrinoma was more frequent than NFPT in all (n=14),and insulinoma in eight, respectively. In a prospective endoscopic ultrasonography(EUS) evaluation of 51 MEN1 patients with a median age of 39 years, the fre-quency of NFPTs was 54.9%, and the lesions were multiple with a median of threelesions per patient. It was also shown that the size and number of the NFPTs canincrease over time (Thomas-Marques et al. 2006). In addition, it has been shownthat NFPTs are commonly detectable even at young age; for example, in the Tas-manian MEN1 study population 40% of the subjects in the age range of 10–20years were found to have NFPTs (Burgess et al. 1998), and in the other study (Tho-
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mas-Marques et al. 2006) the youngest patient detected with a NFPT was 16 yearsold.
There is evidence that the tumour size of a NFPT correlates with the risk ofmetastases and death. According to Triponez et al. (2006b), these risks are low forpatients with tumours ≤20 mm. However, the correlation between the size andmetastasis potential has not been observed in some other studies (Lowney et al.1998). The timing and procedure of surgical treatment of NFPTs has not reachedconsensus. Åkerström et al. (2005) consider surgery (enucleation of tumours in thepancreatic head, and concomitant distal 80% subtotal pancreatic resection) when atumour is detectable by EUS, e.g. with size exceeding arbitrarily 5–10 mm, to pre-vent further growth and malignant development. In contrast, Triponez et al.(2006b) concluded that surgery may not be beneficial for MEN1 patients withNFPTs ≤20 mm.
Gastrinoma. Gastrinomas are the most frequent hormonally active type ofGEP tumours, being present in approximately 40% of MEN1 patients (Brandi etal. 2001), although prevalence as high as 60% has been reported (Burgess et al.1998, Table 1). Zollinger-Ellison syndrome (ZES) is a clinical condition caused byhypersecretion of gastrin, which in turn causes overproduction of acid in the pari-etal cells of the ventricle (Marx 2001). The first description of ZES included ful-minating peptic ulcer disease, gastric acid hypersecretion, and non-beta islet celltumours of the pancreas (Zollinger & Ellison 1955). Approximately 20–25% ofpatients with ZES have MEN1 (Cadiot et al. 1999). In a study by Roy et al. (2000)including 261 patients with ZES (58, or 22%, with MEN1 as well), the most com-mon symptoms were abdominal pain and diarrhoea, present in 75% and 73% ofthe patients, respectively, whereas heartburn and weight loss were present in 44%and 17% of the patients, respectively. Gastrointestinal bleeding was the initial pre-sentation in one in every four patients, and an important presenting sign suggestingZES was prominent gastric body folds noted in endoscopy in 94% of the patients.Mean age at onset of ZES was lower in MEN1 patients than in sporadic cases;33.7±1.3 versus 43.2±0.8 years, p<0.0001(Roy et al. 2000). In a study by Trump etal. (1996) including 220 MEN1 patients, it was found that there was a higheroccurrence of gastrinomas above the age of 40 years.
The MEN1-associated gastrinoma is predominantly duodenal in origin andfrequently multicentric, whereas sporadic gastrinomas are usually pancreatic andsingle (Pipeleers-Marichal et al. 1993, Tonelli et al. 2005). Although duodenalgastrinomas of MEN1 patients are usually small in size (<5 mm in diameter), theyare frequently associated with regional lymph node metastases (Tonelli et al. 2005,
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Åkerström et al. 2005). In a prospective study of the natural history of gastrinomain patients with MEN1, 23% (13/57) of the patients were found to have livermetastases, and in 14% (8/57) gastrinomas showed aggressive growth (Gibril et al.2001). The 5-year survival of patients with aggressive disease in which disease-related deaths were assessed was 88% (95% confidence interval, CI, 53–98%)whereas it was 100% (CI, 92–100%) for patients with non-aggressive disease withor without liver metastases (Gibril et al. 2001). In a study by Norton et al. (2001)the 15-year survival was 52% even if the patients had metastatic disease. Gibril etal. (2001) found several factors predictive of aggressive growth of gastrinomasincluding age at MEN1 diagnosis (<35 years), age at ZES onset (27 years) anddiagnosis (33 years), duration of ZES before diagnosis (<2.1 years), fasting gastrinlevels 10,000 pg/ml, pancreatic tumour size >30 mm, presence of liver and bonemetastases, and gastric carcinoids.
Surgery for ZES in MEN1 is controversial. There is conflicting evidence as towhether cure of hypergastrinaemia can be achieved by any kind of surgery(Thompson 1998, Norton & Jensen 2004, Åkerström et al. 2005). On the otherhand, modern antisecretory therapy with proton pump inhibitors, or occasionallyH2 receptor blockers, is efficient in controlling hypergastrinaemia (Brandi et al.2001, Marx 2001). Some surgeons recommend routine surgical exploration todecrease the probability of malignant spread (Skogseid et al. 1996, Lowney etal.1998, Thompson 1998). In this case, the operation includes distal pancreatec-tomy, intraoperative ultrasound and enucleation of tumours in the pancreatic head,duodenotomy, and removal of lymph nodes along the coeliac trunk and hepatic lig-ament (Norton & Jensen 2004). Others perform surgical exploration only when atumour of 20 to 30 mm is imaged (Jensen 1998, Cadiot et al. 1999, Norton et al.1999). Some authors suggest that a more aggressive surgical procedure, pancre-atoduodenectomy, should be adopted more frequently (Tonelli et al. 2005). Inaddition, other treatment options in malignant gastrinoma may include biotherapy(somatostatin analogues, interferon), systemic chemotherapy, selective emboliza-tion of liver metastasis alone or in combination with intra-arterial chemotherapy(chemoembolization) and radiofrequency ablation (Plöckinger et al. 2004).
Insulinoma. Insulinoma is the second most prevalent hormonally active GEPtumour in MEN1, being present in approximately 10–20% of the patients (Marx2001, Table 1). The median age at diagnosis of 56 insulinoma patients with MEN1was 34 years (range, 5–69 years) (Lévy-Bohbot et al. 2004). Typically, patientsaffected with insulinoma suffer from hypoglycaemic attacks and dizziness. A com-bination of hypoglycaemia and hyperinsulinaemia during a fasting test of 48–72
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hours is required for the diagnosis of insulinoma (Marx 2001, Alexakis & Neop-tolemos 2008). In most cases with MEN1 insulinoma, there are multiple islet celltumours, although most of them are nonfunctional and a single tumour of at least 5mm in size is expected to cause the hyperinsulinism. Malignancy rate is higherwith MEN1-associated than with sporadic insulinoma, although 85% of MEN1-related insulinomas are benign (Veldhuis et al. 1997, Åkerström et al. 2005). Sur-gery is the main treatment for all patients with hypoglycaemia due to an insuli-noma (Alexakis & Neoptolemos 2008). During the exploration, any identified isletcell tumours are enucleated, and some surgeons perform concomitant distal (80%)pancreatic resection to minimize the risk of recurrent tumours. Regardless of theoperative method chosen, most patients are cured (Veldhuis et al. 1997, Åkerströmet al. 2005).
Rare functional GEP tumours. There are also some rare functional neuroendo-crine GEP tumours such as glucagonoma, VIPoma, somatostatinoma andGHRHoma that are seen in association with MEN1. Glucagonomas are pancreaticislet tumours that oversecrete glucagon, which is a counter-regulatory hormone inthe insulin control of glucose metabolism. A specific syndrome of diabetes melli-tus, a skin rash (migratory neurolytic erythema), stomatitis, glossitis, cheilosis,hypoaminoacidaemia, cachexia, and a tendency for deep venous thrombosis isassociated with glucagonoma (Doherty 2005). In a study by Lévy-Bohbot et al.(2004) 5/307 (1.6%) of the MEN1 patients with duodenopancreatic lesions or 5/580 (0.86%) of the whole MEN1 cohort had glucagonoma at a median age of 31years (range, 19–51 years). It is estimated that 3% of glucagonoma patients havealso MEN1 (Doherty 2005). Most glucagonomas are usually large with high riskof malignancy, and are suggested to be treated with radical surgery if possible(Åkerström et al. 2005).
VIPomas are mainly pancreatic tumours, with occasional duodenal location.They oversecrete vasoactive intestinal polypeptide (VIP), which is a neuropeptidethat is distributed throughout the nervous system. Peripherally, VIP is located inpeptidergic neurons adjacent to blood vessels throughout the splanchnic nervoussystem, including the small intestine, the colon and the exocrine ducts of the pan-creas. The syndrome associated with the oversecretion of VIP is called Verner-Morrison syndrome, WDHA syndrome (Watery Diarrhoea, Hypokalaemia, Achlo-rhydria) or pancreatic cholera. These patients have very severe, watery, secretorydiarrhoea. Approximately 40% of the tumours are malignant, and 3% are associ-ated with MEN1. (Doherty 2005.) In the study by Lévy-Bohbot et al. (2004) 3/307(0.98%) of the MEN1 patients with duodenopancreatic lesions, or 3/580 (0.5%) of
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the whole MEN1 cohort had VIPomas. It is recommended that VIPomas shouldalso be treated surgically (Åkerström et al. 2005). It is also of note that 80–90% ofVIPoma and glucagonoma patients have a symptomatic response to somatostatinanalogues (Plöckinger et al. 2004).
Somatostatinoma may locate in the pancreas or duodenum. It oversecretessomatostatin, which is a cyclic polypeptide 14 amino acids in length. Somatosta-tin-secreting cells have been described in the hypothalamus, gastrointestinal tractand pancreas. Somatostatin inhibits the release and function of many other hor-mones. The somatostatinoma syndrome consists of diabetes mellitus, cholelithia-sis, and steatorrhoea. Most of the somatostatinomas are malignant, and approxi-mately 1% is associated with MEN1, and of the duodenal somatostatinomas, up to50% are associated with neurofibromatosis type 1 (NF1). (Doherty 2005.) Lévy-Bohbot et al. (2004) found out that 2/307 (0.65%) of the MEN1 patients withduodenopancreatic lesions, or 2/580 (0.34%) of the whole MEN1 cohort hadsomatostatinoma. It is also recommended that somatostatinomas be treated surgi-cally (Åkerström et al. 2005).
GHRHoma is a tumour that oversecretes growth hormone-releasing hormone(GHRH). It stimulates the pituitary to release excessive growth hormone, resultingin acromegaly if untreated. GHRHomas occur in the lung, pancreas, adrenal andintestine. Approximately one third of GHRHomas are associated with MEN1, andthe same proportion are malignant (Marx 2001, Doherty 2005). The treatment con-sists of surgery and medical therapy by somatostatin analogue (Doherty 2005).
Enterochromaffin-like (ECL) cell tumour (ECLoma). Gastric neuroendocrinetumours are found in up to 7% of MEN1 patients (Table 1). Gastric endocrinetumours are commonly referred to as gastric carcinoids, although a more appropri-ate term according to WHO’s classification is neuroendocrine tumours (Kloppel etal. 2004). They are categorized into well- or poorly-differentiated tumours. Well-differentiated tumours are more frequent, and among them, tumours composed ofenterochromaffin-like (ECL) cells are the most common ones. They are also calledECL-cell carcinoids or ECLomas. ECL cells are the main endocrine cells in theoxyntic mucosa in the corpus and fundus of the stomach secreting histamine with akey role in the mechanism of acid secretion (Bordi et al. 2001, Plöckinger et al.2004). Three subtypes of well-differentiated ECL cell tumours are recognized.Type 1 tumours are often multiple and benign, and secondary to hypergastrinaemiaand associated with chronic atrophic gastritis. Type 2 tumours are also multipleand mainly benign lesions associated with hypergastrinaemia in the context ofZES and MEN1. Type 3 is a sporadic tumour, usually solitary and malignant. Less
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than 5% of these tumours can cause the so-called ‘atypical carcinoid syndrome’due to histamine production. (Marx 2001, Plöckinger et al. 2004, Tonelli et al.2005.).
Patients with sporadic ZES rarely develop gastric ECLomas despite hypergas-trinaemia for a long period of time. In contrast, ECLomas occur in 13–30% ofpatients with ZES related to MEN1 (Debelenko et al. 1997). It is thought thathypergastrinaemia is associated with ECL cell stimulation and hyperplasia, andloss of heterozygosity (LOH) at 11q13 detected in type 2 ECLomas may be a pro-moting factor towards tumour transformation (Debelenko et al. 1997).
In addition to fundus and corpus of the stomach, antral mucosa was shown tobe another tissue type to harbour gastric endocrine tumours in MEN1 (Bordi et al.1997). These tumours were not shown to express the phenotype of normal endo-crine cells of the gastric antrum, and in at least two cases they could be identifiedas ectopic ECL cell carcinoids by VMAT-2 (vesicular monoamine transporter)immunostaining (Bordi et al. 1997). In addition, there are at least two descriptionsof MEN1 patients with neuroendocrine tumours in the stomach in the absence ofhypergastrinaemia (Bordi et al. 1997, Hosoya et al. 1999).
Optimal treatment of MEN1 ECLomas is not yet well established (Tonelli etal. 2005). It is recommended by the European neuroendocrine tumour society(ENETS) that small (<10 mm) polyps be surveyed and that >10 mm in size shouldbe endoscopically resected after EUS. If there are > 6 polyps and >10 mm in size,extension to muscular wall or repeated recurrences, either surgical resection orantrectomy may be undertaken (Plöckinger et al. 2004). Partial or total gastrec-tomy with lymph node dissection may be necessary in malignant development, orin recurrences despite local surgical resection (Plöckinger et al.2004).
Anterior pituitary tumours
Pituitary tumours account for approximately 15% of intracranial tumours, and theoverwhelming majority of them are histologically benign, nonmetastaticadenomas arising from the hormone-secreting cells of the anterior lobe (Thapar etal. 1995, Heaney & Melmed 2004). Normal anterior pituitary gland comprises fivedifferent cell types capable of producing prolactin (PRL), growth hormone (GH),adrenocorticotrophic hormone (ACTH), thyroid-stimulating hormone (TSH) andgonadotrophins (luteinizing hormone (LH) and follicle-stimulating hormone(FSH)). In a series of 3,000 surgically removed pituitary adenomas, the mostcommon hormonally active tumours were prolactin cell adenoma (prolactinoma)
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and growth hormone cell adenoma (somatotropinoma), the proportion being 27%and 14%, respectively (Thapar et al. 1995). In addition, different types of pituitaryadenomas producing both prolactin and growth hormone (mixed GH-PRL celladenoma, mammosomatotroph adenoma, acidophil stem cell adenoma) constituted8% of the cases. Approximately one third of the adenomas were hormonally silentor nonfunctional, even though a subset of them might show positiveimmunohistochemical staining for one or more of the anterior pituitary hormones,e.g. silent corticotroph cell adenoma, accounting for 6% of all adenomas. (Thaparet al. 1995.)
Prolactinoma. Among patients with a pituitary tumour, the prevalence ofMEN1 has been 2.7–5%, but among prolactinoma patients as high as 14% (Schei-thauer et al. 1987, Schaaf et al. 1990, Corbetta et al. 1997). Anterior pituitary ade-nomas are the third major group of tumours in MEN1 being present in up to 65%of the patients (Table 1). In a study by Vergès et al. (2002) the age at diagnosis ofpituitary adenoma did not differ between 136 MEN1 patients and 110 control, non-MEN1 patients (38.0±15.3 vs. 36.2±14.6 years). Furthermore, the distribution ofdifferent types of pituitary tumours did not differ between the groups, but clinicalsigns related to tumour size (headache, visual defects) were more common inMEN1 patients (29% vs. 14%, P<0.01) (Vergès et al. 2002).
Prolactinoma is the most common pituitary adenoma among MEN1 patients,with an average prevalence of 22±4% in different studies reviewed by Hao et al.(2004), where the overall prevalence of pituitary adenomas averaged 35±6%. Inthe studies reviewed in Table 1, the prevalence of prolactinoma varies between 0and 53%. The symptoms relating to prolactinoma in women of reproductive ageare galactorrhoea and hypogonadism with menstrual dysfunction and infertility. Inmen, the symptoms are decreased libido, impotence and infertility. At least partlydue to the less recognized symptoms, tumours in men are diagnosed as larger insize after tumour growth causes symptoms of mass effect (Thapar et al. 1995). Ingeneral, women are four times more likely to present with prolactinoma than men,particularly at reproductive age (Thapar et al. 1995). This difference betweensexes seen clinically is not evident in unselected autopsy series where prolactino-mas are identified with equal frequency (McComb et al. 1983).
In a large multicentre study of 324 MEN1 patients including 136 pituitary ade-nomas (85 prolactinomas; 62.5%), an increased female-to-male ration (50% vs.31%, P>0.001) was seen (Vergès et al. 2002). In the same study, a high frequencyof (84%) macroadenomas was found in MEN1-related prolactinomas comparedwith those observed in non-MEN1 patients (24%). MEN1 prolactinomas showed
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worse response to therapy, since normalization of plasma PRL level was signifi-cantly less frequent in MEN1 patients than in non-MEN1 subjects (44% vs. 90%,P<0.001) (Vergès et al. 2002). In addition, Burgess et al. (1996c) concluded thatprolactinomas occurring in MEN1 may behave more aggressively than sporadicprolactinomas; the majority of their untreated prolactinoma patients developedmicro- or macroadenomas during follow-up compared to the literature data bySchlechte et al. (1989) with a rate of 29% of untreated patients with sporadic pro-lactinoma developing radiologically detectable pituitary tumours. In addition, ahigh rate of referral to surgery (for failed treatment with bromocriptine) for mac-roadenomas was noticed (Burgess et al. 1996c). More evidence on behalf of anaggressive natural history of pituitary adenoma in MEN1 patients was provided bythe fact that in their retrospective group of Tasmanian MEN1 patients, 4/34 haddied of pituitary disease (Burgess et al. 1996c).
The management of MEN1-related prolactinoma does not differ from that oftheir sporadic counterparts. In most cases dopamine agonists are the first-linetreatment, and in some cases surgery is necessary. Radiotherapy is needed onlyrarely to control the tumour expansion (Beckers et al. 2003).
Nonfunctional pituitary adenoma. Nonsecreting or nonfunctional pituitaryadenomas may present with mass effect (hypopituitarism, pituitary apoplexy,visual compromise) (Marx 2001). Earlier, occasional cases of nonfunctional pitu-itary adenomas had been noted in MEN1, but Burgess et al. (1996c) found that 5out of 41 (12%) patients studied had nonfunctional pituitary adenoma. In the otherstudies (Table 1) the prevalence of nonfunctional pituitary adenoma has variedbetween 0–11%. If treatment is needed, surgery by a transsphenoidal approach isthe first choice in most cases (Beckers et al. 2003).
GH-secreting pituitary adenoma. Hypersecretion of GH results in gigantismand excessive linear growth prior to closure of the epiphyseal growth plates, andafter that, in acromegaly (Eugster & Pescovitz 1999). Hypersecretion of GH isusually caused by pituitary adenoma, except for rare cases where the primarycause is oversecretion of GHRH from a hypothalamic or ectopic neuroendocrine(primarily pancreas or bronchus) tumour (Ayuk & Sheppard 2006). Gigantism isextremely rare. Acromegaly has an estimated annual incidence of three to fourcases per million and a current estimated prevalence of 40–60 cases per million(Colao et al. 2004, Kauppinen-Mäkelin et al. 2005, Ayuk & Sheppard 2006).
Clinical features of acromegaly include direct effects of the tumour (headache,visual impairment, hyperprolactinaemia, hypopituitarism) and systemic effects ofGH/IGF-1 (insulin-like growth factor type 1) excess. The latter include soft tissue
27
and skin changes (acral enlargement, increased skin thickness and soft tissuehyperplasia, increased sweating, skin tags, acanthosis nigricans), cardiovasculardisorders (cardiomyopathy, hypertension, arrhythmias), metabolic features(impaired glucose and lipid metabolism, increased nitrogen retention), respiratorydistress (upper airway obstruction, sleep apnoea, macroglossia), bone and jointfractures (increased articular cartilage thickness, arthropathy, carpal tunnel syn-drome, osteopoenia), and other endocrine consequences (multinodular thyroid goi-tre, thyrotoxicosis, hypercalciuria, hyperparathyroidism) (Colao et al. 2004).Acromegaly has been associated with a 2- to 3-fold increased mortality, but thereis evidence that the normalization of post-treatment basal GH concentration (lessthan 2.5–1 µg/l) is associated with a normal life-span (see Kauppinen-Mäkelin etal. 2005).
In the early reports on MEN1 the majority of the pituitary lesions were associ-ated with acromegaly (Erdheim 1903, Underdahl et al. 1953, Ballard et al. 1964).In the more recent study, GH-secreting tumours were present in 12/324 (3.7%) ofthe MEN1 patients in the France-Belgium multicentre study, or 12/136 (8.8%) ofthose with pituitary adenomas (Vergès et al. 2002). The mean age at diagnosis was43.6±16.1 years. In the other series, the prevalence of GH-producing adenoma hasranged from 0 to 7% (Table 1). It is of note that the records of 124 MEN1 patientsfrom the large Tasman 1 kindred were reviewed, and the authors did not find anycases of acromegaly or gigantism (Burgess et al. 1996b). In addition, a subset of44 patients was screened for elevated levels of serum IGF-1, with no abnormallevels found in any of them (Burgess et al. 1996b,c).
The first-line treatment of GH-producing adenoma is transsphenoidal surgery,which is sometimes combined with somatostatin analogues. In cases of mixed GH-PRL-secreting adenomas or in the 10–20% of cases resistant to somatostatin ana-logues, dopamine agonists may be used. (Beckers et al. 2003.) The GH receptorantagonist pegvisomant is a new treatment option in acromegaly (Stewart 2003).
ACTH-secreting pituitary adenoma. ACTH-secreting pituitary adenomas(functioning corticotroph adenomas) constituted 8% of the tumours in a series of3,000 surgically treated pituitary adenoma patients (Thapar et al. 1995). In MEN1patients the prevalence in different studies has ranged from 1.7 to 7% (Trump et al.1996, Marx et al. 1998, Vergès et al. 2002). Excessive secretion of ACTH leads tohypercortisolism and Cushing’s syndrome. Approximately 80% of the Cushing’ssyndrome (CS) cases are ACTH-dependent (secondary to a tumour hypersecretingACTH), whereas 20% are ACTH-independent (in most cases adrenal tumours).An ACTH-secreting pituitary tumour is present in approximately 80% of ACTH-
28
dependent CS referred to as Cushing’s disease in this case. ACTH may also besecreted from a non-pituitary tumour (small cell lung carcinoma, bronchial neu-roendocrine tumours), constituting CS referred to as ectopic CS or occult ectopicCS. Hypercortisolism has multiple, diverse effects on tissues and metabolic func-tions. The most prevalent signs are obesity, plethora, moon face, hypertension,bruising, red-purple striae, muscle weakness and ankle oedema. The most com-mon symptoms include weight gain, menstrual disturbances, hirsutism, psychiatricdisturbances, backache, muscle wasting, fractures and scalp hair loss. (Makras etal. 2006.) In most cases, the treatment of pituitary adenomas secreting ACTH con-sists of surgery, and in addition, in some cases radiotherapy (Beckers et al. 2003).
Rare functional pituitary adenomas. Pituitary tumours secreting LH/FSH orTSH are among the least common hormonally active pituitary tumour types ingeneral, and in association with MEN1 as well (Thapar et al. 1995, Marx 2001).Gonadotroph adenoma may cause ovarian hyperstimulation, but in most cases thesecretion of adenoma is insufficient to cause a recognizable clinical syndrome(Välimäki et al. 1999). Thus, most gonadotroph adenomas appear clinically asnonfunctional, and are only found through immunohistochemical staining(Daneshdoost et al. 1993). In the series of Thapar et al. 6.4% of the tumours weregonadotrophinomas. Virtually all gonadotropinomas are macroadenomas at pre-sentation (Thapar et al. 1995). Only very recently it has been shown that clinicallyapparent gonadotroph adenoma may exist in association with MEN1 (Benito et al.2005). TSH secreting adenoma is the least common pituitary tumour type account-ing for only 1% of all pituitary adenomas. It is a rare cause of hyperthyroidism inclinical practice. As their diagnosis is often delayed, these tumours are mostlydiagnosed as macroadenomas (Foppiani et al. 2007). Primary therapy for gonadot-roph adenomas is neurosurgery, but there may be a limited role for medical therapyin patients with residual disease (Shomali & Katznelson 1999). TSHomas are besttreated by transsphenoidal surgery. Radiotherapy is indicated for inoperable orincompletely resected tumours. Octreotide administration is a useful adjunct forreducing tumor size preoperatively and for the medical management of surgicaltreatment failures (McDermott & Ridgway 1998).
Adrenal lesions
Adrenal involvement in MEN1 has recently been getting more attention due to thewide range of prevalence (3–55%) in different series of patients (Table 1 and 2).The first reports referred to autopsy studies or to incidental findings at laparotomy,
29
and the impact of adrenal involvement was underestimated (Waldmann et al.2007). In general, clinically silent adrenal masses discovered by imaging studiesperformed for unrelated reasons are called adrenal incidentalomas. They havebecome a rather common finding in clinical practice, with a prevalence of 0.5–2%at abdominal CT scan, and in most cases, these masses are non-hypersecreting andbenign (reviewed in Barzon et al. 2003). An even higher prevalence rate wasfound in a prospective evaluation on subjects undergoing high-resolution CT scanof the chest in a lung cancer screening programme; 23 out of 520 (4.4%) subjectswith adrenal masses were identified; 21 adrenal adenomas, 1 myelolipoma, and 1metastasis of lung cancer (Bovio et al. 2006). The prevalence of clinicallyunapparent adrenal masses detected at autopsy is less than1% for ages <30 years,and increases to 7% in those 70 years of age or older (Anonymous 2002). The riskof malignancy over time for masses defined as benign at diagnosis is estimated tobe 1/1,000, even though 5–25% of the masses increase in size during follow-up.Hormonal hyperfunction develops in about 1.7% (0–11%) of cases, the risk beinghigher in patients with lesions larger than 30 mm and/or with unilateral radiotraceruptake at scintigraphy (Barzon et al. 2003).
In most cases, MEN1-associated adrenal lesions consist of diffuse or nodularhyperplasia, or adenomas. In studies specially analysing adrenal involvement(Table 2) in MEN1, Cushing syndrome was present in 0 to 5% of the entire patientpopulation, or in 0 to 17% of those with adrenal involvement. Pheochromocytomawas found less frequently with a prevalence of 0 to 3% in the whole study popula-tion, or 0 to 6% in those with adrenal involvement. Adrenal cortical carcinoma(ACC) was most common in the series by Skogseid et al. (1995) with a prevalenceof 7% in the whole study group. Among the patients with adrenal lesions, ACCwas most common in the study by Langer et al. (2002) with a prevalence of 22%.In addition, primary hyperaldosteronism due to adrenal adenoma has beenreported in association with MEN1 (Beckers et al. 1992).
There is no consensus on MEN1-related adrenal lesions, but based on theirown experience Waldmann et al. (2007) recommend surgery in MEN1 patientswith adrenal tumours exceeding 30 mm, and a closer follow-up by EUS in patientswith newly diagnosed adrenal lesions.
30
Table 2. Studies with special interest on adrenal lesions in multiple endocrineneoplasia type 1 (MEN1) 1 patients.
Thymic neuroendocrine carcinoma
Thymic neuroendocrine carcinoma (carcinoid) is a rare type of tumour with about170 cases reported after its first description in 1972 (Rosai & Higa 1972, Teh et al.1998c). MEN1-related thymic neuroendocrine carcinomas constituteapproximately 25% of all cases (Teh et al. 1998c), and they occur in 1–8% ofMEN1 patients (Marx et al. 1998, Gibril et al. 2003, Table 1). It is the mostaggressive tumour in MEN1, diagnosed on average in the fifth decade, and has aclear male predominance in both sporadic and MEN1-related cases (Gibril et al.2003). Sporadic thymic carcinoids are associated with Cushing’s syndrome in onethird of the cases, but ACTH or other hormone-secreting thymic carcinoids areexceptions in association with MEN1 (Yano et al. 2006). The majority of patientsare asymptomatic until late stage of the disease, when they may present with chestpain or discomfort and dyspnoea (Teh et al. 1998c). MEN1-associated thymicneuroendocrine carcinoids are often seen in familial clusters and the patients areoften heavy smokers or have been exposed to other carcinogens (Teh et al. 1998a,1998c, Ferolla et al. 2005).
It is suggested that cervical thymectomy at the time of parathyroid surgerymight prevent thymic neuroendocrine carcinoma development (Brandi et al.2001). However, it has been recognized that this procedure does not totally elimi-nate, but rather reduces the risk of subsequent formation of thymic carcinoid (Bur-gess et al. 2001, Lim et al. 2006). In case of thymic carcinoid, cure is uncommoneven with early surgery. Therefore, Gibril et al. (2003) suggest that perioperativeradiation should be considered.
Study Adrenal lesions Bilateral CS,hyper-
cortisolism
Pheo-chromo-cytoma
Adreno-cortical cancer
n (%) n (%) n (%) n (%) n (%)
Skogseid et al. 1995 17 (40) 7 (41) 0 (0) 0 (0) 3 (18)
Burgess et al. 1996d 12 (36) 5 (42) 0 (0) 0 (0) 0 (0)a
Barzon et al. 2001 7 (35) 6 (86) 0 (0) 0 (0) 0 (0)
Langer et al. 2002 18 (27) 8 (44) 3 (17) 1 (6) 4 (22)
Waldmann et al. 2007 21 (55) 9 (43) 2 (10) 1(5) 1 (5)
CS, Cushing’s syndrome. a In the same report, a review of medical records for Tasmanian 1 pedigree members dying before 1984 indicated that adrenocortical cancer had occurred in 3 patients with MEN1.
31
Bronchial neuroendocrine tumour
Bronchial neuroendocrine tumours (bronchopulmonary carcinoids) are present inapproximately 5% of MEN1 patients (Table 1), but prevalence as high as 31% hasbeen suggested (Sachithanandan et al. 2004). In contrast to thymic carcinoids,bronchial tumours usually show a much more indolent natural history and have afemale predominance. They also are multicentric and develop both synchronouslyand metasynchronously in the same patient with a mean age of diagnosis of 37years (range 20–55) in a study by Sachithanandan et al. (2004). They are usuallynonfunctional, but as thymic carcinoids may occasionally cause atypical carcinoidsyndrome with facial flushing, lacrimation, headache, and bronchoconstriction, thebiochemical cause remains unknown (Marx 2001). It is suggested bySachithanandan et al. (2004) that radiological surveillance may be sufficient for aproportion of patients who have pulmonary nodules identified during MEN1screening; clinical symptoms and size of the tumour (for example 30 mm or rapidgrowth) could serve as possible criteria for surgery.
Nonendocrine manifestations
Multiple facial angiofibromas were exclusively associated with tuberous sclerosisuntil Darling et al. (1997) observed these lesions in 88% (28/32) of theirconsecutive MEN1 patients, whereas collagenomas were found in 72% (23/28),and lipomas in 34% (11/32). The authors also suggested that confetti-likehypopigmented macules (2/28; 6%), and multiple gingival papules (2/28; 6%) arecutaneous manifestations of MEN1 (Darling et al. 1997). Facial angiofibromaswere found in 43% 12/28) of the Japanese patients with MEN1 (Sakurai et al.2000). LOH studies have shown allelic deletion of the MEN1 gene inangiofibromas, collagenomas, and lipomas of MEN1 patients, thus providingfurther evidence for these tumours being manifestations of MEN1 (Pack et al.1998, Vortmeyer et al. 1998). Also, leiomyomata of different sites has beenoccasionally documented in MEN1 patients, and 11q13 LOH has been detected inoesophageal and uterine smooth muscle tumours in association with MEN1(Vortmeyer et al. 1999, McKeeby et al. 2001). Spinal ependymoma is estimated tobe an infrequent (<1%) manifestation of MEN1 (Marx 2001). One of the firstdescriptions of a patient with spinal ependymoma in association with MEN1 wasreported in 1996 by Kato et al. Giraud et al. (1997) reported another case alongwith the results of genetic studies showing allelic loss of the wild-type MEN1 in
32
the ependymoma. In addition, 6/74 (8%) of the MEN1 patients in a prospectivestudy by Asgharian et al. 2004 were found to have meningiomas. They alsoobserved LOH at 11q13 in a resected meningioma, thus concluding that MEN1plays a role in its pathogenesis (Asgharian et al. 2004).
2.1.3 Clinical management of MEN1 mutation carriers
MEN1 Mutation testing can be offered to index case with MEN1 or with atypicalMEN1 and to their relatives (Brandi et al. 2001). In approximately 10–20% ofindex cases for familial MEN1 cases, no mutation in the MEN1 gene can be found(Brandi et al. 2001). In such cases, haplotype analysis around the MEN1 locus atchromosome 11q13 can allow screening for MEN1 carrier status (Brandi et al.2001). MEN1 genetic testing of an unaffected child is rarely justified, since it willnot have a major effect on their immediate care (Marx 2001, American Society ofHuman Genetics Board of Directors and the American College of MedicalGenetics Board of Directors 1995). Karges et al. (2002) suggest that genetictesting of family members (with the possible result of starting clinical follow-up ofmutation-positive individuals) is generally not advisable before the age of 10–12years.
Table 3 shows the screening programme suggested by the international con-sensus statement reported by Brandi et al. (2001). Biochemical screening (Table 3)allows the detection of MEN1-associated tumours one to two decades prior to clin-ically overt disease. Therefore, medical or surgical treatment will start earlier,which may result in reduced rate of morbidity and mortality, although this has notbeen proven (Clerici et al. 2001, Brandi et al. 2001). The screening programme forMEN1 mutation carriers includes annual clinical examination together with patienthistory and biochemical testing, and imaging testing every 3–5 years (Brandi et al.2001, Karges et al. 2002, Kopp et al. 2001). Based on the patient’s detailed history,physical examination, or basic laboratory and imaging findings, additional diag-nostic procedures may be required (Karges et al. 2002). Some authors would con-sider starting the screening protocol at a later age than suggested by Brandi et al.(2001), e.g. Johnston et al. (2000) recommended that the age to start could be 10–15 years, and Öberg and Skogseid (1998) suggest starting screening from the onsetof adolescence. Some groups have also measured pancreatic polypeptide (PP) as anon-specific marker of islet cell tumours, and performed a standardized meal stim-ulation test for gastrin and PP for detecting GEP tumours (Öberg & Skogseid1998, Kopp et al. 2001). In the OUH the genetic counselling for MEN1 mutation
33
testing is usually offered when the person at risk of MEN1 reaches adulthood. Inonly rare cases, the mutation testing and/or screening have been started earlier. Allthe MEN1 mutation-positive individuals are offered follow-ups in order to screenMEN1 manifestations by endocrinologist. The frequency of biochemical testingand imaging studies depends on the clinical findings of the MEN1 heterozygote.(Unpublished data, original publication I.).
Table 3. Screening programme for follow-up of MEN1 (multiple endocrine neoplasiatype 1) mutation carriers according to the consensus statement by an internationalgroup consisting mostly of clinical endocrinologists (modified after Brandi et al. 2001).
2.1.4 Mortality
Several studies suggest that MEN1 patients are at increased risk of prematuredeath compared to their non-affected relatives or to normal population (Vasen etal. 1989, Wilkinson et al. 1993, Carty et al. 1998, Dean et al. 2000, Geerdink et al.2003, Table 4). In a study by Dohorty et al. (1998) the MEN1-specific deaths(n=27) occurred at a younger age (median 47 years) than either MEN1 patients’nonendocrine deaths (n=32) (median 60 years, p<0.02) or all non-MEN1-relateddeaths in kindred members (n=44) (median 55 years, p<0.05). However, there wasno difference in survival between MEN1 carriers and unaffected kindred members.The authors concluded, however, that there is a substantial subset of MEN1carriers who die early, often from malignant endocrine neoplasms. Therefore theysuggest aggressive screening programmes to identify and initiate treatment ofmalignancies in the setting of MEN1 (Doherty et al. 1998). In 1993 Wilkinson etal. published a retrospective analysis of the natural history of untreated MEN1 in
Tumours screened for Age to begin (year)testing
Biochemical tests annually
Imaging testsevery 3 years
Parathyroid adenomaGastrinoma
820
Calcium, PTHGastrin, gastric acid outputa;secretin stimulated gastrin
NoneNone
InsulinomaOther enteropancreatic
520
Fasting glucose; insulinChromogranin-A; glucagon;proinsulin
111In-DTPA octreotide scan; CT or MRI
Anterior pituitaryForegut carcinoidsb
520
PRL; IGF-1None
MRICT
a Gastric acid output measured if gastrin is high; secretin-stimulated gastrin measured if gastrin is high or if gastric acid output is high. b Stomach best evaluated for carcinoids (ECLomas) incidental to gastric endos-copy. Thymus removed partially at parathyroidectomy in MEN1. PTH, parathyroid hormone; PRL, prolac-tin; IGF-1, insulin-like growth factor type 1; 111In-DTPA octreotide, Indium-111-diethylenetriaminepenta-acetic acid-octreotide; CT, computerized tomography, MRI, magnetic resonance imaging.
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one of the Tasmanian MEN1 kindreds with data available over a period of 130years. Most cases were unrecognized as MEN1 at the time of the patient’s death.They concluded that MEN1 leads to premature death and that neoplasia rather thanpeptic ulcer disease is the main cause of death. A retrospective review of allMEN1 patients treated at the Mayo Clinic during the period 1951–1997 showedthat the overall 20-year survival of MEN1 patients was 64% (95% confidenceinterval was 56–72%), and that of an age and gender matched upper Midwestpopulation 81% (P<0.001).
In the earlier reports the major cause of death among MEN1 patients was pep-tic ulcer disease (Ballard et al. 1964, Majewski & Wilson 1979, Vasen et al. 1989).Other repeatedly seen causes of MEN1-related death were renal complications ofhypercalcaemia and pituitary tumour. Today, malignant neuroendocrine tumours,mainly islet cell tumours and thymic carcinoids, have become the most importantcause of death among MEN1 patients (Wilkinson et al. 1993, Carty et al. 1998,Doherty et al. 1998, Dean et al. 2000, Geerdink et al. 2003, Ferolla et al. 2005,Table 4).
Table 4. Published studies indicating causes of death related to multiple endocrineneoplasia type 1 (MEN1), modified from Geerdink et al. (2003).
2.1.5 Genetics of MEN1
MEN1 gene and its product, menin
The MEN1 gene was identified in 1997; it is located at chromosome 11q13, andconsists of 10 exons that span approximately > 9 kb of genomic DNA, and
Reference
Cases
Causes of deathPepticUlcer
Disease
Malignant tumours RenalCompli-cationsa
PituitaryTumour
OtherGEP Carcinoids
n % % % % % %Ballard et al. 1964 8 88 13 0 0 0 0Majewski & Wilson 1979 91 9 0 0 0 0 0Vasen et al. 1989 15 73 13 0 13 0 0Wilkinson et al. 1993 20 10 15 20 30 15 10Doherty et al. 1998 27 22 44 22 11 0 0Carty et al. 1998 6 0 83 0 0 0 17Dean et al. 2000 17 6 59 18 0 0 12Geerdink et al. 2003 17 24 35 35 6 0 0 GEP (gastroenteropancreatic). a Due to hypercalcaemia caused by primary hyperparathyroidism.
35
encodes a 610-amino acid protein referred to as menin (Chandrasekharappa et al.1997, Lemmens et al. 1997). The main transcript of the MEN1 gene is a 2.8-kbmRNA, although at least seven alternative transcripts have been reported (Lemos& Thakker 2008). Menin is ubiquitously expressed and is located predominantlyin the nucleus in nondividing cells (Guru et al. 1998), but in dividing cells it isfound mainly in the cytoplasm (Huang et al. 1999). It has no homology to anyknown proteins or sequence motifs. Menin has at least three nuclear localizationsignals (NLSs) and five putative guanosine triphosphatase (GTPase) sites (La et al.2006, Lemos & Thakker 2008). Menin has been shown to interact with severalproteins that are involved in transcriptional regulation, genome stability, celldivision and proliferation (see Lemos & Thakker 2008). Menin has also beenshown to bind to a broad range of gene promoters suggesting that it functions as ageneral transcriptional regulator (Scacheri et al. 2006).
MEN1 gene acts as a tumour suppressor gene. The presumed mechanism fortumour formation in MEN1 involves loss of menin functions in a tumour precursorcell. The first hit according to Knudson’s “two-hit” hypothesis is the inheritedMEN1 gene defect and therefore it is present in every cell of the body. When thefirst hit is combined with a somatic loss of the normal allele of the MEN1 gene (thesecond-hit; loss of heterozygosity, LOH) in one cell, neoplastic clonal expansionfrom that cell is initiated (Brandi et al. 2001, Pannett & Thakker 2001).
MEN1 mutations
The mutations are scattered throughout the entire coding region and splice-sites ofthe MEN1 gene. According to a recent review article by Lemos and Thakker(2008) a total of 1,133 germline mutations and 203 somatic mutations of theMEN1 gene have been published. They consist of 459 different germline and 167different somatic mutations. A total of 61 of the germline mutations were alsofound to occur as somatic mutations, thus the total number of different MEN1mutations was 565.The 1,133 germline mutations consisted of 41% frameshiftdeletions or insertions, 23% nonsense mutations, 20% missense mutations, 9%splice site mutations, 6% in-frame deletions or insertions, and 1% whole or partialgene deletions (Lemos & Thakker 2008). Mutations at four sites accounted for12.3% of all mutations (c.249_252delGTCT, deletion at codons 83–84;c.1546_1547insC, insertion at codon 516; c.1378C>T (Arg460Ter); andc.628_631delACAG, deletion at codons 210–211), thereby indicating potentialmutational hotspots (Lemos & Thakker 2008). In addition to the mutational
36
hotspots, some of the recurrent mutations are due to a founder effect, which hasbeen reported e.g. from Tasmania, Newfoundland and Northern Finland (Olufemiet al. 1998, Burgess et al. 2000, Kytölä et al. 2001). In Northern Finland, twodifferent founder mutations (c.1356_1367del12 and c.1546insC, originallydesignated as 1466del12 and 1657insC, respectively) have been found (Kytölä etal. 2001).
Nonsense and frameshift mutations would presumably cause truncated MEN1proteins that would lack at least one of the NLSs and other functional domains, orthey could also result in loss of the translated protein because of nonsense-medi-ated mRNA decay (NMD) (see Lemos & Thakker 2008). Missense mutations mayaffect functionally critical amino acid residues involved in protein interactions,thus leading to inactivation of menin. In addition, some missense mutations havebeen found to alter the capacity of menin to regulate the target promoters (La et al.2006), or to result in the reduction of protein stability and enhanced proteolyticdegradation (Yaguchi et al. 2004). The splice-site mutations are predicted to leadto accumulation of unspliced or incompletely spliced precursor mRNA, or theappearance of aberrantly processed mRNA from the use of alternative normallyoccurring splice sites, or novel, or cryptic splice sites (see Lemos & Thakker2008). There are examples of splice site mutations leading to frameshift and result-ing in a premature termination codon (Turner et al. 2002, Lemos et al. 2007).
Genotype-phenotype correlation
Most reports doubt the existence of any kind of genotype-phenotype correlation inthe MEN1 syndrome, implying that the underlying mutation would not predict theclinical picture of affected heterozygote. Neither the location of the mutation alongthe gene nor the mutation type seems to have any effect upon the phenotype(Agarwal et al. 1997, Bassett et al. 1998, Giraud et al. 1998, Wautout et al. 2002).There are some exceptions or variants that argue against the statement of lackinggenotype-phenotype correlation. First of all, MEN1 mutations have been reportedin 42 families with isolated hyperparathyroidism (FIHP), and 38% of these aremissense mutations that are less likely to result in a truncated, inactivated protein.This proportion of missense mutations among FIHP families contrastssignificantly (P<0.01) with the situation in MEN1 patients in whom 20% aremissense mutations (Lemos & Thakker 2008). Second, there seems to be anoverrepresentation of mutations leading to truncated menin among patients withthymic carcinoids (Lim et al. 2006). In a review of 22 separate MEN1 families
37
with thymic carcinoids, all but two (91%) had mutations coding for a truncatedprotein, even though, when compared with the prevalence of truncating mutationsin all reported MEN1 mutations, it was not statistically significant (P=0.39) (Limet al. 2006). Third, there is also a prolactinoma variant of MEN1 called Burin(Newfoundland) variant of MEN1, which is characterized by a high prevalence ofprolactinoma and a low prevalence of gastrinoma (Olufemi et al. 1998). Thereported families with prolactinoma variant of MEN1 have been associated withtruncating mutations, if a mutation has been found (Olufemi et al. 1998, Hao et al.2001, Kong et al. 2001). Nevertheless, in a large Tasmanian MEN1 pedigree witha splice-site mutation c.446–3C>G, prolactinoma was found commonly (50%) intwo branches but uncommonly in others, indicating that there are other geneticdeterminants inherited separately from the MEN1 gene that modify the clinicalfeatures (Burgess et al. 1998a, Burgess et al. 2000). Fourth, there is a report byBartsch et al. (2000) including 21 MEN1 patients that claims that patients withtruncating mutations in the N-or C-terminal region (exons 2, 9 or 10) of the MEN1gene had a significantly higher rate of malignant pancreatic endocrine tumours(PET) (55% vs 10%; P<0.05) and tended to have shorter disease-free intervals (26vs 92 months, P=0.11) than patients with other mutations. Fifth, Kouvaraki et al.(2002) found that all (100%) of the 21 patients with known PET and MEN1mutation status who had frameshift mutations had PETs, whereas 22 (79%) of 28patients with all other types of MEN1 mutations had PETs (P=0.03). In addition,14 of the 29 patients with pituitary tumours had frameshift mutations in exon 2(representing 2 of 9 kindreds); frameshift mutations in exon 2 were not found inany of the 20 patients without pituitary tumours (P<0.001). In addition, all 4glucagonomas were associated with frameshift mutation in exon 2 (P=0.004),while bronchial and thymic carcinoids were more frequently associated withmutations in exon 10 (Kouvaraki et al. 2002).
2.2 Other disorders with inherited predisposition to primary hyperparathyroidism (PHPT) and/or anterior pituitary adenomas
Table 5 shows disorders of genetic tumour predisposition featuring endocrinegland involvement. Apart from MEN1 that has already been discussed, entitiesinvolving predisposition to primary hyperparathyroidism and/or anterior pituitaryadenomas are reviewed in more detail in this chapter. It is estimated that familialforms constitute approximately 5% of PHPT showing autosomal dominant modeof inheritance. An inherited condition should be suspected in younger patients
38
with PHPT (<40 years old), in patients with multiple parathyroid adenomas orhyperplasia at surgery (indicative of MEN1 or familial hypocalciurichypercalcaemia), atypical parathyroid adenomas or parathyroid carcinoma(indicative of hyperparathyroidism-jaw tumour syndrome), and with a family orpast medical history pointing to any of the syndromes which can cause PHPT(MEN1 and 2A, hyperparathyroidism-jaw tumour syndrome, familialhypocalciuric hypercalcaemia) (Miedlich et al. 2003, Table 5). Similarly toparathyroid tumours, most pituitary adenomas occur sporadically. It is suggestedthat hereditary tumour syndromes may be associated with approximately 5% ofpituitary tumours (Daly et al. 2006). A hereditary form of pituitary adenoma maybe suspected in patients with features of any of the known syndromes associatedwith pituitary adenoma (MEN1, Carney complex, isolated familialsomatotropinoma, familial pituitary adenoma; Table 5) or a family historyindicative of these. As with familial forms of PHPT, young age has been associatedwith GH-secreting tumours in the setting of isolated familial somatotropinoma(Soares & Frohman 2004).
39
Table 5. Disorders of genetic tumour predisposition featuring endocrine glandinvolvement (excluding entities with predisposition to mainly gonadal tumours). Themajor references are found through OMIM numbers at the website http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim.
Disorder (OMIM) Genetic background a
(chromosome loca-tion)
Major manifestations andendocrine gland involvement
Beckwith-Wiedemann sdr, BWS(130650)
CDKN1C (11p15.5)NSD1 (5q35)KCNQ10T1b (11p15.5)H19 b (11p15.5)11p15 locus alter-ations c
Overgrowth of various organsExomphalosWilms tumourAdrenocortical cytomegaly,carcinomaNeuroblastomaHepatoblastoma
Carney complex 1, CNC1(160980)
PRKAR1A (17q23-q24)
Spotty skin pigmentationPPNADMyxoma(tosis), various sitesAcromegaly; pituitary adenomaLCCSCTThyroid nodules or carcinomaPsammomatous melanotic schwannomaBlue (epitheloid) nevusBreast ductal adenomaOsteochondromyxoma
Carney complex 2, CNC2(605244)
gene? (2p16) Same as CNC1
Carney-Stratakis syndrome,CSS (606864)
SDHB (1p36.1-p35)SDHC (1q21)SDHD (11q23)
Paraganglioma, multicentricGIST
Carney triad, CT(604287)
not known ParagangliomasGISTPulmonary chondromasAdrenocortical adenomas
Cowden syndrome, CS d
(158350)PTEN (10q23.31) Lhermitte-Duclos disease
Mucocutaneous lesionsBreast cancer(Follicular) thyroid cancerMacrocephalyEndometrial carcinoma
Familial isolated hyperparathy-roidism,FIHP (145100)
gene? (2p13.3-14)MEN1 (11q13)HRPT2 (1q31.2)CASR (3q13.3-q21)
Parathyroid tumours
Familial isolated pituitary ade-noma, FIPA (102200)
not identified Various types of pituitary adenomas, functional and nonfunctional
Familial medullary thyroid can-cer, FMTC (155240)
RET (10q11) Medullary thyroid cancer
Familial papillary thyroid cancer/papillary renal cancer, FPTC/PRN1 e (605642)
gene? (1q21) Papillary thyroid cancerPapillary renal neoplasiaMultinodular goitre
Hyperparathyroidism-jaw tumour syndrome, HPT-JT (145001)
HRPT2 (1q31.2) Parathyroid adenoma/carcinomaOssifying fibromasKidney cysts and tumours
40
Table 5. ContinuedDisorder (OMIM) Genetic background a
(chromosome loca-tion)
Major manifestations andendocrine gland involvement
Isolated familial somatotropino-mas, IFS (102200)
gene ? (11q13.1-q13.3)
Growth hormone producing pituitary adenoma (acromegaly, gigantism)
Li-Fraumeni syndrome, LFS(151623, 609265, 609266)
TP53 (17p13.1)CHK2 (22q12.1)gene? (1q23)
Bone and soft-tissue sarcomasBreast carcinomaBrain tumoursAdrenocortical carcinomaLeukeamias
McCune-Albright, MAS(174800)
GNAS1 f (20q13.2) Polyostotic fibrous dysplasiaCafé-au-lait spotsPrecocious pubertyThyrotoxicosisPituitary gigantism with or without an adenomaHyperprolactemiaCushing’s syndrome
Multinodular goitre, MNG1 e (138800)
gene? (14q) Multinodular goitrePapillary thyroid cancer
Multiple endocrine neoplasia type 1, MEN1 (131100)
MEN1 (11q13) Parathyroid adenoma Neuroendocrine gastroentropancreatic tumoursPituitary adenomaAdrenal lesions
Multiple endocrine neoplasia type 2AMEN2A (171400)
RET (10q11) Medullary thyroid cancerPheochromocytomaParathyroid adenoma
Multiple endocrine neoplasia type 2BMEN2B (162300)
RET (10q11) Medullary thyroid cancerPheochromocytomaCharacteristic facesThickening of the corneal nervesHyperplasia of the intrinsicautonomic ganglia in the wall of the intestine
Multiple endocrine neoplasia type 4, MEN4 (610755)
CDKN1B (12p13) Pituitary adenomaParathyroid tumours
Neurofibromatosis type 1, NF1(162200)
NF1 (17q11.2) Café-au-lait spotsNeurofibromasAxillary and inguinal frecklingPseudoarthrosisLisch nodulesScoliosisNeoplasia, mainly CNS (rarely duodenal carci-noid, somatostatinoma, pheochromocytoma)
Nonmedullary thyroid cancer 1,NMTC1 e (606240)
gene? (2q21) Papillary thryroid cancer, especially follicular variant
Primary pigmented nodularadrenocortical disease 2, PPNAD2(610475)
PDE11A (2q31) Adrenocortical hyperplasia, adenomaCushing’s syndrome
Paragangliomas 1, PGL1(168000)
SDHD (11q23) Head and neck paragangliomasPheochromocytoma
Paragangliomas 2, PGL 2(601650)
gene? (11q13.1) Head and neck paragangliomas
41
2.3 Multiple endocrine neoplasia type 2A (MEN2A)
Multiple endocrine neoplasia type 2 (MEN2) is a rare autosomal dominantdisorder of tumour formation and developmental abnormalities affectingapproximately 1 out of 30,000 individuals (Ponder 2001). MEN2 can be dividedfurther into three distinct phenotypes; MEN2A, MEN2B (formerly MEN3) andFMTC with genetic defects in the RET gene in all of them (Table 5, Ponder 2001).The most common type is MEN2A, accounting for 65% of the families (Eng et al.1996), whereas MEN2B is probably the least common form of MEN2 (Ponder2001). The latter is also the most distinctive variant of MEN2 (Table 5, Ponder2001). All variants of MEN2 show a high and early penetrance for medullary
Table 5. ContinuedDisorder (OMIM) Genetic background a
(chromosome loca-tion)
Major manifestations andendocrine gland involvement
Paragangliomas 3, PGL3(605373)
SDHC (1q21) Head and neck paragangliomas
Paragangliomas 4, PGL4(115310)
SDHB (1p36.1-p35) PheochromocytomaHead and neck paragangliomasIncreased frequency of extra-adrenal and malig-nant pheochromocytoma
Peutz-Jeghers syndrome, PJS(175200)
STK11/LTB1 (19p13.3)
Hamartomatous polyps of gastrointestinal tractMucocutaneous hyperpigmentationSCTATAdenocarcinoma of cervixLCCSCTIncreased risk for various other tumours
Thyroid tumours with cell oxyph-ilia, TCO1 e (603386)
gene? (19p13.2) Thyroid papillary cancer, especially with cell oxyphilia Multinodular goitre
Von Hippel-Lindau syndrome, VHL g
(193300)
VHL (3p26-25) Hemangioblastomas of the retina and CNSClear cell renal cell cancerPheochromocytomaPancreatic islet cell tumoursEndolymphatic sac tumoursRenal, pancreatic, epididymiccysts
a A mutation in a gene unless otherwise mentioned. b Methylation abnormalities (epigenetic changes).c Cytogenetic duplication, translocation or inversion, or uniparental disomy. PPNAD, primary pigmented nodular adrenocortical disease; LCCSCT, large cell calcifying sertoli cell tumour; GIST, gastrointestinal stromal tumour. d Bannayan-Riley-Ruvalcaba syndrome (BRRS), Proteus syndrome (PS), and Proteus-like syndrome (PSL) are allelic to CS, although CS is the only PTEN hamartoma tumour syndrome (PHTS) with a clearly documented predisposition to malignancies (Gustafson et al. 2007). Lhermitte-Duclos dis-ease is defined as presence of a cerebellar dysplastic gangliocytoma. In CS, Mucocutaneous lesions include multiple facial trichilemmomas, acral keratoses and papillomatous papules (Lachlan et al. 2007).e These entities are part of familial nonmedullary thyroid cancer FNMTC (188550). f Mutations occur as somatic mosaicism and are not inherited. SCTAT, sex cord tumours with annular tubules. CNS, central nervous system. g Clinical phenotype 1 of VHL includes no pheochromocytoma, but it is present in types 2A-2C; renal cancer is absent or rare in types 2A and 2C; type 2B includes both pheochromocytoma and renal cancer (Maher 2006).
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thyroid carcinoma, but PHPT is present only in MEN2A with a prevalence ofmultigland parathyroid tumours in 20–30% of the patients (Brandi et al. 2001).Thyroidectomy during childhood is recommended to prevent medullary thyroidcancer in all MEN2 variants. In addition, pheochromocytoma is found in 50% ofthe MEN2 patients (Brandi et al. 2001). Therefore, clinically MEN2A is readilydifferentiated from familial isolated hyperparathyroidism or other forms offamilial PHPT.
Unlike most other genes underlying tumour predisposition conditions, RET isnot a tumour-suppressor gene. Instead, it acts as an oncogene through gain of func-tion in the context of MEN2. Interestingly, loss of activity mutations in RET resultin Hirschsprung disease of the colon and rectum (Ponder 2001). In addition, RETmutations show clear correlations with the variant of the MEN2 phenotype (Marx2005). The majority (90%) of the RET mutations in MEN2A occur in 6 cysteinesin a small 25-amino-acid domain in the extracellular region, whereas almost allRET mutations in MEN2B are confined to one cytoplasmic amino acid Met918Thr(Marx 2005). Instead, in FMTC most RET mutations overlap with those inMEN2A, although there are other mutations seemingly specific to FMTC in aminoacid 533 of the extracellular domain and in amino acids 791–891 of the cytoplas-mic domain (Marx 2005).
2.3.1 Hyperparathyroidism-jaw tumour syndrome (HPT-JT)
The hyperparathyroidism-jaw tumour (HPT-JT) syndrome is a rare autosomaldominant disorder characterized by the occurrence of parathyroid tumours andossifying fibromas usually affecting the mandible or the maxilla (Bradley et al.2006, Table 5). Some HPT-JT patients may also develop uterine tumours and renallesions including Wilms tumours, hamartomas and polycystic disease (Teh et al.1996, Bradley et al. 2005, Bradley et al. 2006), In HPT-JT, PHPT is the mostcommon and usually the first manifestation with a prevalence of approximately90%. PHPT in HPT-JT is usually due to a solitary adenoma, although multiglanddisease may also occur and approximately 15% of the parathyroid tumours aremalignant (Simonds et al. 2002, Bradley et al. 2005, Bradley et al. 2006). Theprevalence of jaw tumours is approximately 35%, and these may appear as early as13 years of age (Cavaco et al. 2001). In addition, other tumours, including Hurtlecell thyroid adenomas, pancreatic adenocarcinomas and testicular mixed germ celltumours have also been observed in one or two patients with HPT-JT (Haven et al.2000). Mutations in HRPT2 are detected in the majority of patients with HPT-JT
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(Carpten et al. 2002). It is of note that germline mutations in HRPT2 can be foundin patients with apparently sporadic parathyroid carcinoma (Shattuck et al. 2003).
2.3.2 Familial hypocalciuric hypercalcaemia (FHH)
Unlike other disorders predisposing to PHPT, familial hypocalciuric hyper-calcaemia (FHH) is not a hereditary tumour syndrome. Instead, it is an autosomaldominant condition with an underlying loss-of-function mutation in the CASR(calcium-sensing receptor) gene in the majority of the cases (Pollak et al. 1993,Thakker 2004). However, there is evidence that FHH is a heterogenetic conditionwith two other loci identified with yet undiscovered genes at chromosome 19p(OMIM 145981) and 19q (OMIM 600740) (Heath et al. 1993, Lloyd et al. 1999).Secretion of PTH is regulated by extracellular calcium through a G protein-cou-pled calcium-sensing receptor (Miedlich et al. 2003). An allelic disorder to FHH isneonatal severe primary hyperparathyroidism (NSHPT) that is a life-threateningdisorder characterized by severe neonatal hypercalcaemia, failure to thrive, bonyundermineralization, multiple fractures and ribcage deformity (Thakker 2004).NSHPT results from homozygous loss-of-function mutations in CASR, althoughpatients with sporadic NSHPT have been reported to be associated with de novoheterozygous CASR mutations (Pearce et al. 1995).
Normally, PTH secretion is inhibited by small increments in extracellular cal-cium levels above normal. Subsequently, calcium uptake from the intestine, renalabsorption and release of calcium from bone are reduced to re-establish the normalvalue (Miedlich et al. 2003). In case of heterozygous CASR mutation, inactivationof the calcium-sensing receptor results in half-maximal inhibition of PTH secre-tion only at increased concentrations of serum calcium (calcium set-point)(Miedlich et al. 2003, Thakker 2004).
The clinical features of FHH include mild to moderate hypercalcaemia, nor-mal or mildly elevated serum PTH concentrations, mild hypermagnesaemia andinappropriately low urinary calcium excretion (calcium clearance to creatinineclearance ratio <0.01) (Thakker 2004). Hypercalcaemia in FHH is highly penetrantat all ages, even in the perinatal period (Marx et al. 1981, Simonds et al. 2002). Itis also typical that hypercalcaemia is not cured by parathyroid surgery, which infact is almost always contraindicated in FHH (Miedlich et al. 2003). At surgery,hyperplasia of the parathyroid glands can be found (Thorgeirsson et al. 1981),although somewhat conflicting findings do exist (Law et al. 1984). There is onekindred reported with a highly atypical phenotype including family members with
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hypercalciuria, hyperphosphaturia, inappropriately high serum PTH levels andrenal stones resembling mild PHPT (Carling et al. 2000). In addition, normocal-caemia after parathyroidectomy and the presence of nodular hyperplasia or ade-noma in some of the enlarged parathyroid glands were features not typically seenin FHH (Carling et al. 2000). Even though patients with FHH are usually asymp-tomatic, chondocalcinosis, pancreatitis and gallstones may be rare associations(Davies et al. 1981, Heath 1989, Thakker 2004).
2.3.3 Familial isolated hyperparathyroidism (FIHP)
Familial isolated hyperparathyroidism (FIHP) is a subgroup of familialhyperparathyroidism that can result from the incomplete expression of asyndromic form of familial hyperparathyroidism, or from full expression of other,genetically yet undetermined entities (Simonds et al. 2002; table 5). The diagnosisof FIHP is made in kindreds with PHPT in the absence of clinical, biochemical,and radiological evidence of MEN1, HPT-JT or FHH (Cetani et al. 2006). It issuggested that the distinction between FIHP and the syndromic categoriesarbitrarily depends on the sensitivity of diagnostic tests used to detect thesyndrome (Simonds et al. 2002). Cetani et al. (2006) reviewed the overall resultsof genetic studies in FIHP families, which showed that in total 18% (range, 0–57%) of the kindreds showed mutations in MEN1, 12% (range, 0–18%) in CASR,and 5% (range, 0–29%) in HRPT2. In the majority of the FIHP kindreds thegenetic cause remains unknown (Teh et al. 1998b, Kassem et al. 2000, Simonds etal. 2004, Cetani et al. 2006). Warner et al. (2006) has provided evidence foranother locus for familial isolated hyperparathyroidism finding linkage to a 1.7Mb region on chromosome 2p13.3-14. The gene has not yet been identified.
2.3.4 Carney complex (CNC)
Carney complex (CNC) is a rare autosomal dominant condition that has beendescribed in about 500 people to date. In more than 60% of the patients it is causedby inactivating mutations in the gene encoding for the protein kinase A (PKA)type 1A regulatory (R1) subunit (Kirschner et al. 2000, Boikos & Stratakis 2007,Table 5). Another locus on chromosome 2 (2p16) has been reported, but the genepossibly underlying CNC2 has not yet been identified (Stratakis et al. 1996). Thediagnosis of CNC is made if two of the main manifestations (Table 5) of thesyndrome are present, confirmed by histology, biochemical testing or imaging.
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Supplemental criteria include affected first-degree relative and inactivatingmutation of the PRKAR1A gene (Boikos & Stratakis 2007). The median age atdiagnosis of CNC is 20 years (Stratakis et al. 2001). It is suggested that patientswith CNC and/or germline PRKAR1A mutations should be followed up for themanifestations of the disease annually to improve their prognosis (Boikos &Stratakis 2007)
The spotty pigmentation or lentiges in CNC appear as small, brown to blackmacules that have a typical distribution including the lips, conjunctiva and inner orouter canthi, vaginal and penile mucosa. The characteristic pigmentation typicallydevelops shortly before, or during puberty, although it may also be the first sign atbirth (Stratakis et al. 2001). Other pigmented lesions such as blue or other nevi,café-au-lait spots and depigmented lesions may appear at different ages (Carney etal. 1998).
Primary pigmented nodular adrenocortical disease (PPNAD) and cardiacmyxomas seem to have a bimodal distribution among CNC patients; the majorityof patients present with PPNAD or cardiac myxomas in the second and thirddecade of life, but a second group of patients may be diagnosed with either type oftumour (or rarely, both) in the first 2–3 years of life (Boikos & Stratakis 2007).Overall, Cushing’s syndrome due to PPNAD is observed in 25–30% of patientswith CNC. Since CNC associated Cushing’s disease is often cyclical and atypical,developing progressively over the years, it is difficult to diagnose (Stratakis et al.1998, Stratakis et al. 2001). Cardiac myxomas are the most common among thenoncutaneous lesions found in CNC patients (Stratakis et al. 2001). They areresponsible for more than 50% of the disease-specific mortality among CNCpatients (Stratakis et al. 2000). Skin myxomas are found especially in the eyelid,external ear canal, breast and nipples. Other possible sites to find myxomasinclude the oropharynx, the female genital tract and the pelvis. Breast myxomato-sis may be found through fat-suppressed magnetic resonance imaging (Boikos &Stratakis 2007).
Large-cell calcifying Sertoli cell tumours (LCCSCT) and thyroid nodulesoften appear during the first decade of life and progress gradually to replace thenormal testicular and thyroid follicular tissue, respectively. In addition, malignantgrowth in later years is possible (Boikos & Stratakis, 2007). LCCSCTs are almostalways found by ultrasonography in postpubertal male patients in CNC, thereforedetection of testicular microcalcifications by this method can be used as a diagnos-tic tool in CNC (Boikos & Stratakis, 2007). LCCSCTs are usually benign in CNC,and they rarely have any endocrine consequences due to increased P-450 aro-
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matase expression leading to gynaecomastia and precocious puberty (Boikos &Stratakis 2007). Thyroid gland nodules are common among patients with CNC; byultrasonography up to 75% of patients have cystic or multinodular disease(Stratakis et al. 2000). Follicular or thyroid cancer may develop in up to 10% ofCNC patients with pre-existing thyroid pathology (Stratakis et al. 1997, Boikos &Stratakis 2007).
Biochemical acromegaly with elevation of growth hormone and IGF-I levelsand subtle hyperprolactinaemia may be present in up to 75% of patients, althoughclinically evident acromegaly is a relatively infrequent manifestation of CNC(Boikos & Stratakis 2006). Most CNC patients do not have any tumorous findingsin pituitary imaging studies (Boikos & Stratakis 2006). Similar to patients withMc-Cune Albright disease, pituitary hyperplasia appears to precede tumour devel-opment in CNC. Also, most patients in CNC have mild hypersomatomammotropi-naemia starting in adolescence (Boikos & Stratakis 2006). Acromegaly is charac-terized by a slow, progressive course in CNC, with pituitary tumours beingdetected from the third decade of life. Also, in many cases acromegaly has notbecome apparent until after operation for Cushing’s syndrome (Boikos & Stratakis2007). There is also evidence that pituitary tumours may be multicentric in CNC,supporting the hypothesis of hyperplasia developing into an adenoma (Pack et al.2000).
Psammomatous melanotic schwannoma (PMS) is very rare as a sporadic, iso-lated tumour. In association with CNC it may become malignant. PMS may befound anywhere in the central and peripheral nervous system, but its most frequentsite has been the gastrointestinal tract and the paraspinal sympathetic chain inpatients with CNC (Boikos & Stratakis 2007). An unusual mammary tumour,breast adenoma, and osteochondromyxoma of bone are additional tumours associ-ated with CNC (Boikos & Stratakis 2007).
2.3.5 Familial isolated pituitary adenoma (FIPA)
Pituitary adenomas of all types can occur in a familial setting in the absence ofMEN1 and CNC, the phenotype being termed as familial isolated pituitaryadenoma (FIPA, Table 5). Before an international and multicentric study reportedby Daly et al. (2006), there were only a few reports on other familial isolatedpituitary adenomas apart from isolated familial somatotropinoma (IFS) (Gardneret al. 1989, Links et al. 1993, Berezin & Karasik 1995).
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In the report by Daly et al. (2006) there were 64 FIPA families including 138affected individuals with 55 prolactinomas, 47 somatotropinomas, 28 nonsecretingadenomas and 8 ACTH-secreting tumours. A first-degree relative of 76% of theaffected individuals was affected. A single tumour phenotype occurred in 30 fami-lies (homogenous families), whereas different tumour types occurred in the rest(heterogeneous families). FIPA cases were younger at diagnosis than sporadiccases (38.4± 16.3 vs. 41.9±15.1 years, respectively; P=0.015). Prolactinomas fromheterogeneous families were larger and had more frequent suprasellar extensionthan their sporadic or homogenous counterparts. Instead, prolactinomas in homog-enous families were similar to sporadic prolactinomas in respect to the finding that71% of the cases were females with microprolactinomas. One of the male patientsfrom a heterogeneous FIPA family developed a malignant prolactinoma describedearlier by Petrossians et al. 2000 (Daly et al. 2006). Familial cases with non-func-tioning tumours were also younger at diagnosis (P=0.03) and had more frequentlyinvasive tumours (P=0.024) than sporadic ones. The demographic and clinicalcharacteristics of the familial and sporadic Cushing’s disease groups did not differsignificantly from one another (Daly et al. 2006).
2.3.6 Isolated familial somatotropinoma (IFS)
Isolated familial somatotropinoma (IFS) is defined as the occurrence of at leasttwo cases of acromegaly/gigantism in a single family in the absence of CNC orMEN1 (Daly et al. 2006, Table 5). Approximately 100 cases of IFS in more than40 different families have been reported (Luccio-Camelo et al. 2004, Soares &Frohman 2004).
According to a recent review (Soares & Frohman 2004) the clinical manifesta-tions of IFS were similar to those in patients with sporadic somatotropinomas. Theincidence of macroadenomas in patients with sporadic acromegaly varies from 60–90%, while it was 88% in IFS. Gigantism was present in 21 of the 96 cases (12%)on which information was available. Immunohistochemical studies were per-formed on 35 tumours, of which 20 (57%) exhibited prolactin immunostaining.Many of these patients were mildly hyperprolactinaemic (Soares & Frohman2004). The histological types of the adenoma vary from somatotroph (most cases)to mammosomatotroph even within the same family (Verloes et al. 1999). It wasalso noted that there was a preponderance of invasive macroadenomas at the timeof diagnosis among the familial cases of somatotropinoma (Verloes et al. 1999, DeMenis & Prezant 2002). In addition, IFS patients (n= 26, 12 families) representing
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19% of the FIPA cases and families analysed by Daly et al. (2006), had moreaggressive tumours, with extrasellar (P=0.023) and suprasellar extension(P=0.043) occurring more frequently than in heterogeneous somatotropinomafamilies. The median age at diagnosis was 26 years in IFS cases, whereas in spo-radic cases of acromegaly the average age at onset is 40–50 years (Soares & Fro-hman 2004). IFS patients analysed by Daly et al. (2006) were diagnosed at a meanage of 33.8±18.9 years, which was more than 10 years younger than somatotropi-noma patients from either heterozygous familial isolated pituitary adenoma fami-lies (49.3±16.3, P= 0.002) or sporadic cases (44.1±13.5, P=0.0023). In addition, allfive patients with gigantism belonged to IFS families (Daly et al. 2006). The sub-optimal results of surgical treatment in reported IFS patients compared to those ofsporadic GH-secreting tumours were pointed out by De Menis & Prezant (2002).
Most IFS pedigrees are small, including only two affected relatives (Verloes etal. 1999), and the disease was expressed in only a single generation in 60% of thefamilies and across multiple generations in the others reviewed by Soares & Fro-hman (2004). It has been suggested that IFS is inherited as an autosomal dominantdisease with reduced or incomplete penetrance (Verloes et al. 1999, Soares & Fro-hman 2004, Soares et al. 2005). Loss of heterozygosity at chromosome 11q13, thelocus of the MEN1 gene, has been reported in both sporadic and familial soma-totropinomas, although the MEN1 sequence and expression has appeared normalin most cases (Gadelha et al. 2000). In 2000, Gadelha et al. provided evidence fora linkage of the IFS locus to chromosome 11q13.1-q13.3 and for a potential sec-ond locus at 2p16-12. The latter was excluded in subsequent studies in several dif-ferent families (Soares & Frohman 2004), but the hypothesis of the chromosomeregion 11q13 as harbouring a potential IFS locus was supported by further studies(Luccio-Camelo et al. 2004, Soares et al. 2005).
2.3.7 Multiple endocrine neoplasia type 4 (MEN4)
Multiple endocrine neoplasia type 4 was only recently brought to the OMIMdatabase on the basis of a single report on humans (Table 5). Pellegata et al. (2006)reported a 48-year-old female patient who developed acromegaly and had a 3-cmpituitary tumour removed when she was 30. Histologically the tumour was aninvasive pituitary adenoma with growth hormone hyperproduction, high mitoticactivity, and cell atypia. Sixteen years later she was diagnosed with PHPT. At thetime of the study she had not yet undergone surgery, although the authorssuggested that PHPT in this patient was likely to be caused by parathyroid
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hyperplasia or adenoma. The patient was found to carry a germline heterozygousnonsense mutation (c.G692A) in CDKN1B (cyclin-dependent kinase inhibitor 1B)encoding the cyclin-dependent kinase inhibitor p27Kip1. The proband’s older sister,also a mutant carrier, was diagnosed at age 55 with renal angiomyolipoma. Her sondeveloped testicular cancer at age 28, but his mutational status, as well as that ofthe proband’s father with acromegaly, is unknown (Pellegata et al. 2006).
Ozawa et al. (2007) analysed the CDKN1B gene in 16 cases with sporadictumours both in the parathyroids and the pituitary, and another 18 cases withrelated features of familial tumours (an index case with parathyroid and/or pitu-itary tumours and one first-degree relative with the other in that tumour pair). Theyfound no pathogenic alterations in the CDKN1B gene in any of the patients, whichled to the conclusion that the MEN1 variant with sporadic parathyroid tumours,sporadic pituitary tumours, and no identified MEN1 mutation is usually not causedby CDKN1B germline mutations (Ozawa et al. 2007).
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3 Purpose of the present study1. To study the clinical features of Northern Finnish MEN1 mutation carriers and
to evaluate whether there is genotype-phenotype correlation in relation to aparticular mutation and the resulting phenotype.
2. To study whether MEN1 mutation carriers are at increased risk of prematuremortality.
3. To study the familial cluster of pituitary adenoma presumably due to foundermutation effect of an unknown gene and to describe the clinical features andgenetic background of this new clinical entity.
4. To study the possible role of different genetic factors in the aetiology ofprimary hyperparathyroidism in patients with features of hereditarypredisposition.
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4 Subjects and methods
4.1 Subjects and families
4.1.1 Clinical features and genotype-phenotype correlation of MEN1 in Northern Finland (I)
MEN1 mutation carriers, identified at the Department of Clinical Genetics in OuluUniversity Hospital (OUH) during 1982–2001 and belonging to the MEN1families originating from Northern Finland were included in the study. Initially, 25index patients received genetic counselling for MEN1, which was also offered totheir relatives at risk. After genetic counselling, a total of 159 subjects opted forfurther studies. Prior to 1997, the ascertainment of heterozygotes was based onboth biochemical and radiological information, combined later with MEN1 linkageanalysis. All these cases were confirmed by direct mutation analysis when itbecame possible in 1997. From then on all the new cases have been directlyanalysed by MEN1 mutation testing. Among the 159 individuals studied, theMEN1 gene analysis revealed 87 heterozygotes including 25 index patients and 62newly diagnosed family members. Of the newly diagnosed heterozygotes, fivewere excluded from the analysis for various reasons (two were tested as newborns,two did not enter the screening programme, and one was living outside Finland).Thus, the study population consisted of 82 MEN1 mutation carriers.
4.1.2 Study on Premature Mortality in MEN1 founder mutation carriers (II)
All the families with one or more MEN1 probands with the two founder mutations(1466del12 and 1657insC) were included. There were altogether 15 probands in13 families with the MEN1 syndrome. In the families, 40 cases have beendocumented to have the 1466del12 mutation (9 families) and 30 the 1657insCmutation (4 families). The pedigrees of probands made during the initial visits atthe department of Clinical Genetics at OUH were further extended by genealogicalstudies. The names, dates, and places of birth of the ancestors and the dates ofdeath were traced using Finnish church records. Practically all the roots goingback to 1700 were evaluated to find common ancestors and many even further.The lines of inheritance back to the MEN1 patients were studied to evaluate the
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obligatory carriers and their spouses with respect to lifespan, including allindividuals born before 1929.
4.1.3 Pituitary adenoma predisposition (III)
Three small clusters of familial pituitary adenoma in Northern Finland wereincluded in the study. The first cluster displayed two cases of acromegaly and onecase of gigantism. The second cluster consisted of two patients, one diagnosedwith acromegaly, and the other with prolactinoma. The third cluster consisted oftwo distantly related patients with gigantism. Genealogy data reaching back to thethe 18th century had been generated by family members based on publicly availableofficial population registries. The first two clusters could be linked by genealogyto a large pedigree (Figure 1, family 1), and the third appeared as separate (Figure2, family 2). Additional pituitary adenoma patients belonging to the pedigree wereidentified by using data on a previously characterized population-based cohort of54 patients diagnosed with GH-secreting pituitary adenoma between 1980 and1999 at OUH (Kauppinen-Mäkelin et al. 2005), patient interviews, and acomputerized search for all cases with archived samples of pituitary adenomas atOUH from 1978 to 2000.
Samples of 13 affected individuals and 7 obligatory carriers as well as 4 keyunaffected relatives (parents) were obtained from families 1 and 2 (Figure 1 and2). The sample material was either blood or paraffin-embedded tissue. For the pop-ulation-based study 45 blood or paraffin-embedded samples were obtained fromOUH. These patients with sporadic GH-secreting pituitary adenoma were diag-nosed between 1980 and 1999. Genomic normal DNA from one German individ-ual and one Turkish individual, as well as from one Italian sibling pair with soma-totropinoma, was included in this study. In addition, DNA from 10 unselectedFinnish sporadic acromegaly patients was available. For the controls of the FinnishAIP mutations, 219 anonymous Finnish blood donors from the Oulu region wereused. For the Italian AIP mutation 52 anonymous Italian blood donors, 94 UKCaucasians (Human Random Control DNA panel, Porton Down, Salisbury, Wilt-shire, UK), and 109 unrelated Caucasian CEPH (Centre d'Etude du Polymor-phisme Humain) individuals were used as controls.
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4.1.4 Primary hyperparathyroidism patients with features of genetic predisposition (IV)
Medical records of 286 patients treated for PHPT during 1974–2001 in OuluUniversity Hospital were reviewed. The patient was included in the study when atleast one of the following inclusion criteria was met: multiglandular, recurrent orpersistent PHPT, another MEN1-related manifestation (enteropancreatic, pituitary,adrenal or foregut neuroendocrine tumour), age at onset of PHPT 40 years orunder, or PHPT/MEN1-related manifestation(s) in the family. The firstdocumented finding of hypercalcaemia or a urinary tract stone were used todetermine the age at onset of PHPT. Twenty of the patients were excluded sincethey had already been diagnosed as having MEN1 based on genetic or clinicalevidence. Altogether, 56 patients meeting the inclusion criteria were invited to thestudy by a written invitation, and 29 of them were willing to take part in the study.
4.2 Biochemical and imaging studies of MEN1 mutation carriers and PHPT patients with features of genetic predisposition (I, IV)
All MEN1 mutation carriers had regular follow-ups at the closest endocrinologyunit for screening of the MEN1-related manifestations. The basic follow-up visitevery 1 to 3 years consisted of biochemical screening of serum sample (ionizedcalcium, PTH, pancreatic polypeptide (PP), gastrin, chromogranin A (CGA) andprolactin). Radiological imaging (ultrasound (US), computed tomography (CT),magnetic resonance imaging (MRI)) of the abdomen was performed every 1–3years and of the pituitary (CT, MRI) every 5–10 years. In cases of active disease,the patients were seen more frequently and a wide range of different diagnosticmethods was used, including somatostatin-receptor scintigraphy to detectneuroendocrine tumours of various sites. SPSS 12.0.1 (SPSS Inc., Chigaco, IL,USA) was used for statistical analyses.
PHPT patients with features of genetic predisposition were screened for ion-ized calcium, PTH, PP and prolactin in order to identify MEN1 suspected patientssince approximately 10–20% of cases are missed in MEN1 mutation analysis(Brandi et al. 2001). A clinical evaluation by an endocrinologist and more detailedbiochemical screening was offered to patients with marked elevation of serum PPand/or prolactin, or evidence of recurrent PHPT. The prolactin concentration wasanalysed by automated chemiluminescence system (Advia Centaur, Bayer Corpo-ration, NY, USA) and PTH (Nichols Institute Diagnostics, San Clemente, CA,
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USA) and PP (Euro-Diagnostica, Arnhem, the Netherlands) by radioimmunoassayfollowing the instructions of the manufacturers. Testing of serum cortisol, gastrin,ionized calcium, FSH, LH, testosterone, free thyroxine (FT4), TSH, and plasmaCGA, was done by standard automated techniques on clinical service basis forthose with marked elevation of serum PP and/or prolactin, or evidence of recurrentPHPT.
4.3 Obtaining clinical data and causes of death (I)
The medical records of the MEN1 mutation carriers were reviewed for thepresence and timing of the MEN1-related manifestations, any other tumours anddeaths. In addition, the death certificates and the autopsy reports were studied, ifavailable, to evaluate the causes of death.
The onset and diagnosis of PHPT was defined as the first incidence of hyper-calcaemia with an inappropriately elevated serum PTH level. The classification ofGEP tumours was based on clinical evidence of excess hormone secretion or thelack thereof, and in operated patients it was confirmed by histology using theWHO criteria (Kloppel et al. 2004). For nonfunctional pancreatic tumours (NFPT),the diagnosis was based on either constantly elevated tumour markers (S-PP and/or P-CGA) or visualization of the tumours by imaging examinations in the absenceof other pancreatic hormone oversecretion and clinical syndrome. The diagnosis ofgastrinoma was based on elevated serum gastrin levels, in most cases confirmedby an appropriate rise during secretin test/or increased gastric acid output. Gastros-copy and measurement of parietal cell autoantibodies were used to rule out thepossibility of atrophic gastritis. Pituitary adenomas were also classified accordingto the presence of hormonal activity or the lack thereof on clinical and biochemicalbasis. They were considered microadenomas when less than 10 mm and macroad-enomas when equal to or greater than 10 mm in diameter (Hardy 1973). Adrenalglands were registered as affected when evidence of hyperplasia or tumours wasseen in imaging studies, and as hormonally active if indicated by biochemical test-ing (1.5 mg overnight dexamethasone test, measurement of 24-h urine normeta-nephrines and metanephrines, and in hypertensive patients measurement of plasmarenin and serum aldosterone). The follow-up time was counted from the first visitto a health care institution for MEN1 suspicion in the patient or family. The lastpoint of observation period was counted as the last information concerning MEN1-associated documentation available by the end of August 2003 or the patient’sdeath by that time.
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Mean ages were reported as mean ± 1 S.D. Binary logistic regression analysiswas used for analysing the covariates affecting the likelihood of having differentMEN1 manifestations. The study cohort was analysed in two groups containingeither all the ascertained heterozygotes or just the founder mutation carriers. Cova-riates included in all of the analyses were age at the end of the follow-up, genderand mutational background (mutation class when analysing all the cases, and theexact mutation when analysing the founder mutation carriers). Confidence inter-vals (CI) of 95% are reported in parenthesis after odds ratio (OR) values. P<0.05was considered significant. Penetrance of the major manifestations by age was cal-culated by Kaplan-Meier analysis.
4.4 Comparing mean values of age at death (II)
SPSS for Windows (Release 10.0.7) was used for statistical analyses. Forcomparing mean values of ages in the groups, Student’s t test was used, andLevene’s homogeneity-of-variance test was used to ascertain appropriateness ofthe analysis. Using revised (1881–1990) and abridged (1751–1880) life tables forFinland, an estimate of expected lifetime was applied for each obligatory MEN1carrier and their spouses (Kannisto & Nieminen 1996, Turpeinen & Kannisto1997). These tables give sex- and birth-year category-specific life expectancyvalues in years at a certain age. In this survey, life expectancy at the age of 25years was used. Birth-year categories in earlier years were in 10-year and later in5-year stratification periods. The significance of the differences in causes of deathwas tested by Person 2 test and Fisher’s exact test.
4.5 Histopathology and immunohistochemistry (III)
Immunohistochemistry of familial and sporadic adenomas for hypophysealhormones (ACTH, GH, FSH, LH, TSH, and PRL) was performed according tostandard laboratory procedures using LabVision Autostainer 480 (LabVisionCorp., Fremont CA).
4.6 SNP array and linkage analyses (III)
To identify the predisposing locus for pituitary adenoma, genomic DNA wasisolated from blood by the PureGene DNA isolation kit (Puregene, gentraSystems, Minneapolis, MN). SNP genotyping was performed for 16 individuals
57
from family 1 using Human Mapping 50K Xba 240 SNP array (Affymetrix, SantaClara, CA). Signal intensity data was analysed using the GeneChip DNA analysissoftware v. 3.0.2.8 (GDAS) (Affymetrix). The genotyping data were convertedinto appropriate linkage format by the ALOHOMORA software (Ruschendorf &Nurnberg 2005). Because current linkage programmes are not suitable foranalysing large numbers of markers in large pedigrees (Lingaas 2003), family 1had to be divided into 3 separate branches for the genome-wide parametric andnon-parametric multipoint calculations with the Allegro software (Gudbjartsson etal. 2000, Gudbjartsson et al. 2005). In these analyses LOD scores for threebranches were calculated separately and added together by loci.
In order to obtain maximum information from the linkage data, the entire fam-ily 1 data was reanalysed as one pedigree in regions displaying LOD scores over 3with the SimWalk2 2.91 software (Sobel & Lange 1996, Sobel et al. 2001) usingsets of 10–20 markers per analysis. Suitable analysis files for SimWalk2 were cre-ated with the Mega2 data handling programme (Mukhopadhyay et al. 2005). Inorder to evaluate the effect of linkage disequilibrium (LD) in regions with LODscores over 3, the data were further analysed with reduced marker density (<0.1cM) using SimWalk2 software.
The most likely haplotypes were constructed with the Allegro and SimWalk2software and the results were visualized with HaploPainter V.024beta (Thiele &Nurnberg 2005). Fine mapping studies were performed on genomic DNA isolatedfrom blood or paraffin-embedded normal tissue. Thirty published and novel micro-satellite markers from chromosomal region 11q12.2-11q13.3 (physical location61.4–69.0 Mb), as well as the six most informative SNP-markers from HumanMapping 50K Xba 240 SNP array mapping to the region of interest were utilized.Analyses of microsatellites and SNP-markers were carried out using ABI3730sequencer (Applied Biosystems, Foster City, CA). Microsatellite data were analy-sed with Genemapper software v. 3.7 (Applied Biosystems). The most informativemarkers in the shared region were used for the overall parametric linkage calcula-tion in families 1 and 2 by the SimWalk2 software with default general parameters.The disease model parameters were as follows: phenocopies for acromegaly orgigantism 0.0002, and for any pituitary adenoma 0.01, penetrancies for homozy-gous and heterozygous disease genotypes 0.1 and the disease allele population fre-quency 0.001. All the other individuals except the affected ones were consideredunknown phenotype.
58
4.7 Expression profiling (III)
Expression profiles were obtained for 16 individuals, 9 affected/obligatory carriersand 7 controls. As controls, 5 unrelated spouses from families 1 and 2 were used.Total RNA was extracted from whole blood with the PAXgene Blood RNA Kit(PreAnalytiX, Hombrechtikon, Switzerland) and concentrated with RNeasyMinElute Cleanup columns (Qiagen, Valencia, CA). The quality of RNA wasanalysed using a spectrophotometer and an Agilent 2100 Bioanalyzer (AgilentTechnologies, Palo Alto, CA). Biotin-labelled and fragmented cRNA was preparedfrom 5 µg of total RNA with procedures recommended for the Human GenomeU133 Plus 2.0 array expression analysis (Affymetrix). The HG-U133 Plus 2.0Chips were hybridized according to manufacturer’s instructions, and scanned withGeneChip® scanner 3000 (Affymetrix). GeneChip Operating Software (GCOS)(Affymetrix) was used to obtain quantitative expression information for the probesets. The quantitative expression data were normalized by scaling all chips to theaverage gene expression data of all 16 chips, and the expression of each probe setwas divided by the mean expression of that probe across all the samples so that theresulting mean expression for every probe was 1. The normalized expression datawere filtered to remove expression values below detectable levels using theAffymetrix Detection Algorithm assigned flag cells. Subsequent analyses wererestricted to probe sets with detectable expression in a sufficient number ofsamples to allow a Student’s t-test between the affected and control groups. Dataprocessing and analyses were performed with GeneSpring 7.0 software (siliconGenetics, Redwood City, CA).
4.8 Mutation screening and search for loss of heterozygosity (LOH) (III, IV)
Mutation screening of the candidate gene for pituitary adenoma predisposition,AIP, was done from genomic DNA isolated either from blood of paraffin-embedded normal tissues. Primers (Original article III, Supported online material,Table S4) were designated to amplify the coding region and exon-intronboundaries of AIP. PCR protocols are available upon request. Sequencing wasperformed using the Big Dye 3.1 termination chemistry and ABI3730 DNAsequencer (Applied Biosystems). LOH analyses were done from paraffin-embedded tumour samples by sequencing the corresponding mutation position.
59
In the study including PHPT patients with features of genetic predisposition,peripheral blood samples were used as a source of constitutional DNA in thegenetic analyses. Mutation screening of the MEN1, HRPT2, CASR, CDKN1B andAIP genes was performed by sequencing the entire coding region and exon-intronboundaries. For MEN1, HRPT2, CASR and CDKN1B the primers used for amplifi-cation and sequencing, as well as the annealing temperatures for the amplifica-tions, have been published previously (Villablanca et al. 2002, Villablanca et al.2004, Georgitsi et al. 2007) or are available upon request, and for AIP as men-tioned above. For MEN1, HRPT2 and CASR the sequencing was done with the BigDye Terminator cycle sequencing kit (Perkin Elmer) and run in an automatedsequencer ABI377 (Perkin Elmer / Applied Biosystems, Foster City, CA). ForCDKN1B the sequencing was done with the Big Dye 3.1 termination chemistryand an ABI3730 DNA sequencer (Applied Biosystems, Foster City, CA).
4.9 Ethical issues
Appropriate research permissions have been obtained from the Ethical Board ofthe Northern Ostrobothnia Health Care District and the Ethical Board of HelsinkiUniversity Hospital. All patients gave their informed consent for blood specimens.Tumour samples were studied under the authorization of the National Authorityfor Medicolegal Affairs if the patient’s authorization was not available. Relativesof patients were only contacted through patients. Genetic counselling was offeredto the patients and relatives if a mutation was detected in any of the genes studied.
60
Fig.
1. P
edig
ree
of t
he f
amily
1 w
ith p
ituita
ry a
deno
ma.
Indi
vidu
als
avai
labl
e fo
r th
e st
udy
are
indi
cate
d by
A2,
A5,
etc
. Num
bers
with
in d
iam
onds
indi
cate
num
bers
of c
hild
ren.
Cir
cles
, fem
ales
; squ
ares
, mal
es; s
lash
es th
roug
h sy
mbo
ls, d
ecea
sed.
Bla
cken
edsy
mbo
ls,
som
atot
ropi
nom
a; h
oriz
onta
l lin
es,
prol
actin
oma;
bla
cken
ed s
ymbo
ls a
nd h
oriz
onta
l lin
es,
mix
ed p
ituita
ry a
deno
ma.
Gen
erat
ions
are
indi
cate
d by
Rom
an n
umer
als
on th
e le
ft. G
ener
atio
n I i
s fr
om th
e 18
th c
entu
ry (O
rigi
nal p
ublic
atio
n III
).
61
Fig. 2. Pedigree of the family 2 with pituitary adenoma. Individuals available for thestudy are indicated by A8 and A34. Numbers within diamonds indicate numbers ofchildren. Circles, females; squares, males; slashes through symbols, deceased.Blackened symbols, somatotropinoma. Generations are indicated by Roman numeralson the left (Original publication III).
62
5 Results
5.1 MEN1 in Northern Finland; clinical features and genotype-phenotype correlation (I)
Out of the 82 individuals (35 men, 47 women) found to be MEN1 heterozygouscarriers 68 (83%) were found to carry either the mutation 1466del12 or 1657insC,which are the founder mutations in Northern Finland as described earlier (Kytöläet al. 2001). The mutation 1466del112 was found in 39 individuals belonging to 9families, and 1657insC was found in 29 individuals from 4 families. Threeadditional mutations D418N, G156R and R527X were found in 9, 3 and 2individuals, respectively, from three different families. For the 82 mutation-positive individuals, the mean age at onset of the follow-up was 40.8 ± 14.6 yr(range, 11–70 yr) and the mean age at the end of the observation period was 48.1 ±15.4 yr (range, 18–78 yr). The mean duration of the follow-up time was 7.3 ± 4.5yr (range, 0–18 yr). Age-dependent penetrance of the major manifestationsaccording to Kaplan-Meier analysis is shown in Figure 1 in the originalpublication I. The prevalence, mean age at diagnosis and risk factors of the majorMEN1 manifestations among the 82 MEN1 heterozygotes are shown in Table 6.
PHPT was detected in 31 males and 45 females (n=76; 93%) (Table 6). Onlysix patients (7%) remained normocalcaemic during the follow-up. For these sixpatients without PHPT the mean age at the end of the observation period was 33.7± 13.6 yr (range, 18–54 yr). A total of 43 patients (52%) were operated on theparathyroid glands at least once. All of the removed glands showed benignhistology except for one patient who was found to have metastasized parathyroidcarcinoma affecting two glands at the age of 38 years. Genotype was not found topredict the risk of PHPT.
In this patient cohort, four types of GEP tumours could be found:nonfunctional pancreatic tumours, gastrinomas, other hormonally activepancreatic tumours and neuroendocrine tumours of the gastrointestinal tract. GEPtumours were found in 26 males and 34 females (n= 60 or 73%) (Table 6). Meanage at the end of the follow-up for the patients not having any signs of GEPtumours was 34.0 ± 12.4 yr (range, 18–65 yr). Of the patients with GEP tumours,18 (30%) had undergone an operation to remove or reduce the size of the tumour.For the whole group of GEPs, no genotype-phenotype correlation was found.
63
Tabl
e 6.
Pre
vale
nce,
mea
n ag
e at
det
ectio
n an
d ri
sk fa
ctor
s of
the
maj
or m
ultip
le e
ndoc
rine
neo
plas
ia ty
pe 1
(ME
N1)
man
ifest
atio
nsam
ong
the
82 h
eter
ozyg
otes
in re
latio
n to
diff
eren
t mut
atio
ns fo
und.
Bin
ary
logi
stic
regr
essi
on a
naly
sis
was
use
d fo
r ana
lysi
ng th
eco
vari
ates
aff
ectin
g th
e lik
elih
ood
of h
avin
g di
ffer
ent
ME
N1
man
ifest
atio
ns.
The
stud
y co
hort
was
ana
lyse
d in
tw
o gr
oups
cont
aini
ng e
ither
all
the
asce
rtai
ned
hete
rozy
gote
s (n
=82)
or
just
the
foun
der
mut
atio
n ca
rrie
rs (n
=68)
. Cov
aria
tes
incl
uded
in a
ll of
the
anal
yses
wer
e ag
e at
the
end
of th
e fo
llow
-up,
gen
der a
nd m
utat
iona
l bac
kgro
und
(mut
atio
n cl
ass
whe
n an
alys
ing
all t
he c
ases
,an
d th
e ex
act m
utat
ion
whe
n an
alys
ing
the
foun
der m
utat
ion
carr
iers
). C
onfid
ence
inte
rval
s (C
I) of
95%
are
repo
rted
in p
aren
thes
isaf
ter
odds
rat
io (
OR
) va
lues
. P
<0.0
5 w
as c
onsi
dere
d si
gnifi
cant
. M
utat
ion
clas
s 1
(MC
1)
(n=
51)
incl
udes
in-
fram
e/m
isse
nse
mut
atio
ns (1
466d
el12
, D41
8N, G
156R
) and
mut
atio
n cl
ass
2 (M
C 2
) (n=
31)
incl
udes
fram
eshi
ft an
d no
nsen
se m
utat
ions
(165
7ins
C,
R52
7X).
Mut
atio
n 1
(M 1
) is
1466
del1
2, m
utat
ion
2 (M
2) i
s 16
57in
sC.
Tum
our
Mea
n ag
e of
dete
ctio
n ±
1 S
D, r
ange
Mut
atio
ns14
66de
l12
(n=
39)
%
1657
insC
(n=
29)
%
D41
8N(n
= 9)
%
G15
6R(n
= 3)
%
R52
7X (n
= 2)
%
Tota
l(n
= 82
)%
Tota
l gro
up(n
= 82
) R
isk
fact
or, O
R (C
I), P
Foun
der m
utat
ions
car
riers
(n=
68)
Ris
k fa
ctor
, OR
(CI),
PP
HP
T 39
.8 ±
13.
5 (1
6–69
)95
90
8910
010
093
Age
, 1.0
8 (1
.01–
1.17
), 0.
031
No
risk
fact
ors
GE
P all
met
asta
sis
NF
G Oth
er
GI
46.8
± 1
2.8
(21–
69)
55.1
± 1
2.4
(28–
70)
45.3
± 1
2.5
(23–
69)
51.6
± 1
1.4
(34–
70)
NA
55.4
± 9
.9 (4
4–68
)
74 23 39 39 0 5
72 14 69 3 3 7
67 0 44 22 0 11
67 0 33 0 33 0
100 0 50 50 0 0
73 16 50 23 2 6
Age
, 1.1
2 (1
.06–
1.19
), 0.
000
Age
, 1.0
7 (1
.02–
1.12
), 0.
008
Mal
e, 4
.27
(1.0
3–17
.6),
0.04
5M
C 2
, 3.2
6 (1
.27–
8.33
), 0.
014
Age
, 1.0
9 (1
.04–
1.15
), 0.
001
MC
1, 6
.77
(1.3
1–35
.0),
0.02
2N
AN
A
Age
, 1.1
1 (1
.05–
1.17
), 0.
000
Age
, 1.0
8 (1
.03–
1.15
), 0.
005
M 2
, 3.5
6 (1
.29–
9.83
), 0.
015
Age
, 1.0
8 (1
.03–
1.14
), 0.
004
M 1
, 15.
1 (1
.73–
131.
9), 0
.014
NA
NA
Pitu
itary
all
PR
LN
FM
ixed
40.2
± 1
4.4
(12–
62)
40.9
± 1
4.5
(22–
62)
41.1
± 1
4.8
(12–
54)
NA
26 18 8 0
31 21 7 3
22 22 0 0
67 67 0 0
50 0 50 0
29 21 7 1
No
risk
fact
ors
No
risk
fact
ors
NA
NA
No
risk
fact
ors
No
risk
fact
ors
NA
NA
Thym
ic51
.3 ±
5.0
(45–
55)
07
00
504
NA
NA
Adr
enal
53.7
± 1
0.6
(32–
70)
3924
4433
100
35A
ge, 1
.09
(1.0
5–1.
14),
0.00
0A
ge, 1
.09
(1.0
4–1.
14),
0.00
0P
HP
T, p
rimar
y hy
perp
arat
ryro
idis
m i
s co
nsid
ered
as
a si
gn o
f pa
rath
yroi
d tu
mou
r (h
yper
plas
ia o
r ad
enom
a);
GE
P, g
astro
ente
ropa
ncre
atic
tum
our;
NF
nonf
unct
iona
l tu
mou
r, G
, ga
strin
oma;
Oth
er G
EP,
ins
ulin
oma
(165
7ins
C),
grow
th h
orm
one-
rele
asin
g-ho
rmon
e se
cret
ing
tum
our
GH
RH
oma
(G15
6R);
GI,
gast
roin
test
inal
tra
ct t
umou
rs (
othe
r th
an d
uode
nal g
astri
nom
a);
PR
L, p
rola
ctin
sec
retin
g pi
tuita
ry a
deno
nom
a, T
he m
ixed
ade
nom
a (in
165
7ins
C m
utat
ion
carr
ier)
sec
rete
d bo
th p
rola
ctin
and
TS
H. N
A, n
ot a
pplic
able
due
to s
mal
l num
ber o
f sub
ject
s.
64
The most prevalent type of GEP tumours was a nonfunctional pancreatic tumour(NFPT) present in 17 males and 24 females (n= 41 or 50%). Of these, 20 (49%)had the 1657insC, 15 (37%) the 1466del12, 4 (10%) the D418N, 1 (2%) theR527X and G156R mutation. In the whole patient cohort, the mutation classincluding the frameshift and nonsense mutations was found to increase the risk ofhaving a NFPT with an OR of 3.26 (CI, 1.27–8.33), P= 0.014. In the foundermutations group, the 1657insC mutation gave an OR of 3.56 (CI, 1.29–9.83), P=0.015.
Gastrinomas were suspected clinically and biochemically in 8 males and 11females (n=19 or 23%). Of these patients, 15 (79%) had the 1466del12 foundermutation. Two other patients (11%) had the D418N mutation, one patient (5%) hadthe 1657insC founder mutation, and another one (5%) the R527X mutation.Gastrinoma patients were found in 7/9 (78%) of the families carrying the1466del12 mutation. In the whole study group, the mutation class including the in-frame deletion and missense mutations predicted the risk for gastrinoma with an6.77 (CI, 1.31–35.0), P=0.022, and in the founder mutations group the 1466del12mutation gave an OR of 15.1 (CI, 1.73–131.9), P=0.014.
An insulinoma was found in one female patient (age 31 years), and apancreatic growth-hormone-releasing-hormone secreting tumour resulting inacromegaly was seen in one male patient (age 21 years). Neuroendocrine tumoursof the gastrointestinal tract (other than duodenal gastrinomas) were found in 5female patients, associating with hypergastrinemia in 3. All of these five patientswere also affected by pancreatic endocrine lesions. All the patients withmetastasized GEP tumours had either the 1466del12 (9 cases) or the 1657insC (4cases) mutation. Lymph node metastases were found in 3 males and 3 females, andliver metastases in 5 males and 2 females.
Pituitary adenomas were found in 24 (29%) patients (7 males, 17 females)(Table 6). Mean age for those without a tumour was 47.5 ± 16.0 yr (range 18–78yr) at the end of the follow-up. Among the patients with pituitary adenomas, 17(71%) had prolactinoma, 6 (25%) had a nonfunctional tumour, and one patient hada mixed adenoma secreting both prolactin and TSH. At imaging studies, 12 (50%)were classified as macroadenomas, and 11 (46%) were microadenomas. Oneprolactinoma could not be visualized. Of the patients with pituitary adenomas, 12(50%) had received some therapy. Dopamine agonist treatment had been given to10 (42%), and 5 had also been operated on. Another patient with prolactinomareceived only operative treatment, and one further patient with a nonfunctionaladenoma had received radiation therapy. In addition to the patients with adenomas,
65
there was one further patient with sellar tumour diagnosed at 9 years of age.According to the histology report, the removed tumour was angiosarcoma. Thetumour sample was not available for review. The patient received radiation therapyand was followed up by an endocrinologist for panhypopituitarism. At 37 years ofage, he was found to be a MEN1 heterozygote through family screening. Twoyears after entering the follow-up he died of metastasized parathyroid cancer.Genotype had no effect on the risk of pituitary adenoma.
Neuroendocrine tumours beyond the gastrointestinal tract were all thymic inorigin (thymic carcinoids or neuroendocrine carcinoma of thymus) and they werefound in three male patients (Table 6). The patients were two brothers with the1657insC mutation at ages 45 and 52 years, and another male patient with theR527X mutation at 55 years. By the end of the follow-up the brothers haddeveloped local recurrences at 47 and 55 years of age, and the former was alsofound to have skeletal metastases in the spine before his death at 51 years of age.The third patient did not show any signs of thymic neuroendocrine carcinoma sixmonths after the operation.
Adrenal lesions were present in 29 (35%) of the patients (12 males, 17females) (Table 6). All the adrenal lesions were considered benign and hormonallyinactive, except for two patients, who had subclinical hypercortisolism. Of thepatients with adrenal lesions, 28 (97%) were also affected by one or more GEP-tumours including 15 nonfunctional pancreatic tumours (52%), 12 gastrinomas(41%) and 1 with both (3%). The likelihood of adrenal lesions was not associatedwith a certain genotype.
Other tumours found in the study population included 5 cases of thyroidcarcinoma (4 papillary, 1 medullary type), 4 cases of renal tumours (2angiomyolipomas, 2 carcinomas; multifocal papillary carcinoma and clear cellcarcinoma), 2 breast carcinomas (both invasive ductal carcinoma), 3 uterinetumours (leiomyoma and leiomyolipoma in one patient, mesodermal mixedtumour in another). One patient was diagnosed with prostate adenocarcinoma andtransitional cell carcinoma of bladder. In addition to uterus, leiomyomas werefound in the ileum, ventricle and epididymis. Systematic search for cutaneoustumours or uterine leiomyomas was not performed. Meningioma was found in twopatients. Four of the thyroid cancers appeared in two families carrying either1657insC or D418N mutation (2 cases in each). The fifth was found in a1466del12 mutation carrier who had also had clear cell renal cancer and breastcancer.
66
During the observation period 9 men and 6 women died at a mean age of 59.0± 12.7 yr (range 39–72 yr) and 62.9 ± 15.0 yr (range, 36–79 yr), respectively.MEN1 was considered to be a direct cause of death in 7 cases. Of these, 6 patients’death was associated with MEN1-related malignancy. A metastasized GEP tumourwas the direct cause of death in 5 patients, including two pancreatic gastrinomaswith liver metastases, two NFPTs with liver and the other one with local lymphnode metastases, and one neuroendocrine carcinoma of the stomach and theduodenum with multiple metastases. One of the patients died of parathyroid cancerwith multiple widely spread metastases. In addition, for one patient diagnosed ashaving gastrinomas 15 years earlier, the cause of death was haemorrhage from aduodenal ulcer. He had refused to attend the follow-up visits 8 years earlier. MEN1was a contributing factor to death in 3 cases presenting as a metastasizedpancreatic neuroendocrine tumour in 2 patients and as a thymic neuroendocrinecarcinoma in one patient. Five patients died of non-MEN1-related causes.
5.2 Effect of MEN1 founder mutations on premature mortality (II)
With one exception, common ancestors for all the MEN1 patients were found. Thepedigrees of the founder mutations are illustrated in Figures 3 and 4. The initialgroup consisted of 34 obligatory gene carriers, 16 males and 18 females, (MEN1positive in later text) and of 33 spouses, 18 males and 15 females, born between1728 and 1929. Two male spouses were excluded from the analyses because ofinsufficient information (IX:11, IX:17 in Figure 3). Thus, the final study groupconsisted of 34 heterozygotes and 31 spouses. Four female subjects were stillalive, two gene positive (IX:12 born1927 and IX:16 born 1924 in Figure 3) andtwo spouses (IX:3 born 1922 in Figure 3 and III:8 born 1928 in Figure 4). In the1466del12 mutation pedigree, there were 28 heterozygotes (15 males and 13females) and 25 spouses (11 males and 14 females), and in the 1657insC mutationpedigree, six heterozygotes (one male and five females) and six spouses (fivemales and one female). The living subjects were excluded from true lifespananalysis, but a subanalysis using their age as lifespan was made.
MEN1 obligate carrier males (n=16) died at a mean age of 61.1 ± 12.0 years(range, 39–89) and MEN1 obligate carrier females (n= 16) at a mean age of 67.2 ±10.7 years (range, 42–79). Table 7 summarizes the results of comparative lifespananalyses, which showed no significant differences between any of the groupsanalysed.
67
Fig.
3. P
edig
ree
of 1
466d
el12
mut
atio
n-po
sitiv
e fa
mili
es w
ith M
EN
1 ca
se(s
) or
igin
atin
g fr
om c
omm
on a
nces
tors
. Onl
y ob
ligat
ory
gene
car
rier
s (a
ffec
ted,
clo
sed
sym
bols
) and
thei
r sp
ouse
s ar
e ill
ustr
ated
in d
esce
ndin
g ge
nera
tions
. Sub
ject
s bo
rn 1
930
or la
ter
are
excl
uded
. Bir
th y
ears
of
ance
stor
s ar
e sh
own.
Sub
ject
s ar
e nu
mbe
red
by g
ener
atio
n (R
oman
num
bers
) an
d by
ord
er in
the
gene
ratio
n (A
rabi
c nu
mbe
rs).
Sub
ject
s IX
:3,
IX:1
2, a
nd I
X:1
6 w
ere
aliv
e as
of
Dec
embe
r 14
, 20
01.
Sub
ject
IX
:9 w
as n
ot m
arri
ed(O
rigi
nal p
ublic
atio
n II,
© T
he E
ndoc
rine
Soc
iety
, 200
4).
VIII:
5
VII:3
VII:4
VI:1
VI:2
VIII:
6
VII:1
VII:2
VIII:
1VI
II:3
VIII:
2
IX:1
VIII:
4
VII:5
VI:3
VI:4
VIII:
7
VII:6
VII:7
VIII:
8
V:1
V:2
III:1
III:2
II:1
II:2
I:1I:2
IV:1
IV:2
III:3
III:4
VIII:
9
IX:2
IX:3
IX:4
VIII:
10
IX:5
IX:6
VIII:
11
IX:7
IX:8
VIII:
12
IX:9
VII:9
VIII:
13VI
II:14
IX:1
0IX
:11
IX:1
2IX
:13
VIII:
15
IX:1
4IX
:15
VIII:
16
IX:1
6IX
:17
1705
1709
1466
del1
2-m
utat
ion
68
Fig. 4. Pedigree of 1657insC mutation-positive families with MEN1 case(s) originatingfrom common ancestors. Only obligatory gene carriers (affected, closed symbols) andtheir spouses are illustrated in descending generations. Subjects born 1930 or later areexcluded. Birth years of ancestors are shown. Subjects are numbered by generation(Roman numbers) and by order in the generation (Arabic numbers). Subject III:8 wasalive as of December 14, 2001 (Original publication II, © The Endocrine Society, 2004).
Table 7. Mean ages of death (years) of 32 obligatory MEN1 founder mutation carriers(1466del12 and 1657insC) compared to the mean ages of death of 29 unaffectedspouses in sex groups. The comparison was also done between life expectancy andmean age of death in each group (affected and unaffected subjects in sex groups).Lifespan analysis included deceased subjects from the founder mutation pedigreesborn between 1728 and 1929. Subjects from the 1466del12 mutation pedigree included26 affected/heterozygous (15 males and 11 females) and 24 unaffected spouses (11males and 13 females). Subjects from the 1657insC mutation pedigree included 6affected/heterozygous (1 male and 5 females) and 5 unaffected male spouses.
Subjects Mean age (range) Comparison of mean age at death, P-value
Affected (m) (n=16)Life expectancy
61.1 ± 12.0 (39–89)61.7 ± 3.1
affected (m) vs. life expectancy, 0.841(NS)
Unaffected (m) (n=16)Life expectancy
65.8 ± 15.3 (34–84)62.2 ± 1.7
affected (m) vs. unaffected (m), 0.342 (NS)unaffected (m) vs. life expectancy, 0.377 (NS)
Affected (f) (n=16)Life expectancy
67.2 ± 10.7 (42–79)66.5 ± 3.3 affected (f) vs. life expectancy, 0.806 (NS)
Unaffected (f) (n=13)Life expectancy
67.7 ± 14.7 (46–87)63.7 ± 3.5
affected (f) vs. unaffected (f), 0.917, (NS)unaffected (f) vs. life expectancy, 0.272 (NS)
M, male; f, female; vs., versus; NS, nonsignificant, P-value <0.05 was considered significant.
I:1 I:2
II:2II:1 II:4II:3
III:2 III:4 III:6 III:7III:1 III:3 III:5 III:8
1844 1846
1657insC-mutation
69
In addition, in linear regression analysis with the age of death as a dependentvariable and pedigree group, MEN1 trait, and sex as explaining variables andexpected lifespan and birth year as covariates, no significant associationsexplaining death age were encountered.
The causes of death of 55 out of 61 subjects were found, and causes of deathof six subjects were not attained (one proband and five spouses). Reliability of thecauses was classified by the investigator as certain (36%), quite certain (13%), andas only based on church records (51%). The main causes of death werecardiovascular disease (41.8%), infection (16.4%, mainly tuberculosis), ageing(12.7%), trauma (5.5%), withering (5.5%), malignancy not classically associatedwith MEN1 (5.5%), MEN1-associated causes of death (9.1%, often classified ascancer in the abdomen) and MEN1 causes (3.6%, such as gastroenteropancreatictumor). Withering (synonyms wilting, fading, and wasting away) as a cause ofdeath is a descriptive term and could imply a chronic disease, often malignancy,causing death. When the last four (withering, malignancy, MEN1-associated causeof death, and MEN1 cause) were classified as possible MEN1 causes and others asnot MEN1 causes, a statistically significant difference was found betweenprobands and spouses in females (Fisher’s exact test, P = 0.003) but not in males(Fisher’s exact test, P= 0.355). In female subjects, out of 15 probands, eight (53%)died of a possible MEN1 cause, whereas of the 12 female spouses, none died of apossible MEN1 cause. Excluding 1657insC mutation from the analyses did notabolish significance, but in the subgroup of 1657insC mutation families, statisticalsignificance could not be estimated because of small numbers.
5.3 Characterization of clinical and genetic aspects of pituitary adenoma predisposition (PAP) (III)
In the genome-wide search, the highest parametric LOD score of 3.9 inchromosome 11 at 68.4–73.6 cM (Xba240deCode genetic map, Haldane sex-average cM) was detected for the high-stringency phenotype (acromegaly orgigantism, 6 individuals). The haplotype construction of family 1 individualsplaced the PAP locus between SNPs rs174449 and rs 1938685 (chromosome 11,61.7–69.0 Mb, Ensembl, version 36, December 2005), harbouring 295 genes.Families 1 and 2 shared the linked haplotype, which segregated perfectly withacromegaly. The added maximum LOD score for these two families was 7.1 withhigh-stringency (acromegaly or gigantism) criteria (6.3 for family 1 and 0.8 forfamily 2) and 8.3 (7.5 for family 1 and 0.8 for family 2) with the low-stringency
70
(any pituitary adenoma) criteria. Two individuals with prolactinoma appeared torepresent phenocopies (A9 and A10).
Expression profiling revealed 172 probe sets that mapped in the linked region.The two lowest P values were obtained for the two separate probe setsrepresenting AIP (also known as XAP2 and ARA9, Genbank no U78521.1) (P=0.00026 and P= 0.00114). Thus AIP was chosen as the prime candidate formutation analysis. One other gene, galectin-12 (LGALS12) was also chosen on thebasis of decreased expression and an association of galectin-3 to pituitarytumorigenesis (Riss et al. 2003). No difference was detected in MEN1 expression.The coding region of AIP was sequenced, and a nonsense mutation Q14X (whereQ is Gln), perfectly segregating with the GH-secreting adenoma phenotype infamilies 1 and 2, was identified (Figure 5). The mutation was absent in 209 localblood donors. LGALS12 and MEN1 analyses were negative. In the population-based material, including 4 cases of families 1 and 2, six displayed Q14X, and onedisplayed IVS3-1G>A, affecting the splice acceptor site of exon 4. The latterchange was screened among 219 local blood donors with negative results. Theclinical characteristics of affected AIP mutation carriers are shown in Table 8. Theage at diagnosis, sex, and size of adenoma were compared between PAP (n=7) andAIP mutation-negative (n=38) patients. Differences in tumour size or sexdistribution were not observed. PAP patients were significantly younger thanmutation negative patients (24.7 ± 10.7 versus 43.6 ± 11.9 years, P= 0.0003). Inaddition, 10 unselected Finnish sporadic acromegaly patients from whom DNAand appropriate authorization was available were screened for AIP mutations. Twopatients were found to carry the Q14X mutation (Table 8). Three families with twoaffected individuals from other countries were also studied. While no mutationwas detected in the German and Turkish sample, the Italian siblings displayed anonsense mutation R304X (where R is Arg) in exon 6 (Table 8). The change wasabsent in 203 Caucasian controls from the United Kingdom and the CEPH, as wellas in 52 local (Trevisio) blood donors. The phenotype in the siblings resembledthat seen in Finns; young age at onset and no visible evidence of autosomaldominant transmission.
LOH-analysis was possible in eight tumours from mutation-positiveindividuals, including five somatotropinomas, one mixed-type tumour, and twoprolactinomas; loss of the wild-type allele was detected in all cases, showing thatthese tumours were null with respect to AIP.
71
Fig. 5. Schematic figure of AIP. FK506-binding protein (FKBP)-homology region andtetratricopeptide repeats (TPRs) are shown as colored boxes. The regions necessaryfor interaction with AHR and HSP90 proteins are shown with black lines. AIP germlinemutations identified in this study are shown in corresponding regions (Originalpublication III).
5.4 Mutation analysis of MEN1, HRPT2, CASR, CDKN1B and AIP genes in PHPT patients with features of genetic predisposition (IV)
The mean age of onset of PHPT in the 29 patients (20 females, 9 males) was 38.4 ±10.6 yr (range, 19–56 yr) and mean age at diagnosis of PHPT 42.0 ± 10.2 (range,20–61 yr). More than one of the inclusion criteria was met by 7 (24%) patients.Eighteen patients (13 females, 5 males) met the inclusion criterion of age being 40years or less at the time of onset of PHPT and their mean age was 31.3 ± 5.2 yr(range, 19–39 yr) at onset, and 36.5 ± 8.1 yr (range, 20–55 yr) at diagnosis. Elevenpatients (38%) had multiglandular disease; 9 with two affected glands and 2 withthree hyperplastic glands. Histology of removed parathyroid gland(s) showedhyperplasia in 14 patients (48%), adenoma in 12 patients (41%), both hyperplasiaand adenoma in two (7%) and hyperplasia or adenoma in one (3%). Four patients(14%) met the inclusion criterion of recurrent or persistent PHPT. One patient wasregarded as having another MEN1 lesion in addition to PHPT; patient 1617 hadhad bilateral adrenal enlargements diagnosed as incidentalomas and pancreaticcysts. Family history of PHPT was present in 3 patients (1143; a sister, diagnosedat 72 years, 1237; mother at 73 years and a sister at 38 years, 2037; aunt at 51years). Three patients had MEN1 suspected lesions in the family (1211; mother,liver metastasis of a carcinoid tumour at 79 years, 1241; several relatives atmother’s side affected by abdominal cancer, 1605; mother, pulmonary carcinoid
1 2 3 4 5 6
Q14X R304X
FKBP12 TPR TPR TPRTPR4 5 62 3
AHRHSP90
nonsensesplice site
1 2 3 4 5 6
Q14X R304X
FKBP12 TPR TPR TPRTPR4 5 62 3
AHRHSP90
nonsensesplice site
IVS3-1G>A
72
Tabl
e 8.
Clin
cal c
hara
cter
istic
s of
the
PAP
pat
ient
s w
ith a
ger
mlin
e A
IP m
utat
ion
(Ori
gina
l pub
licat
ion
III).
Pat
ient
AIP
Mut
atio
nS
exC
linic
al
Dia
gnos
isA
ge a
t Dia
gnos
is
(yea
rs)
His
topa
thol
ogic
al D
iagn
osis
Ele
vate
d P
ituita
ry
Hor
mon
e(s)
IHC
of a
deno
ma
Ade
nom
a D
iam
eter
(m
m)
Sur
gery
on
Ade
nom
a
A2
Q14
XM
Acr
omeg
aly
13N
AN
AN
AN
Aye
s
A6a
Q14
XM
Gig
antis
m11
Som
atot
roph
ade
nom
aG
HG
H+,
c30
yes
A8
Q14
XM
Gig
antis
m20
NA
NA
NA
NA
no
A13
aQ
14X
MA
crom
egal
y21
Mam
mos
omat
otro
ph a
deno
ma
GH
, PR
LG
H++
, PR
L+, c
28ye
s
A14
aQ
14X
MA
crom
egal
y34
Mam
mos
omat
otro
ph a
deno
ma
GH
GH
++, P
RL+
, c22
yes
A20
Q14
XM
Pro
lact
inom
a19
Pro
lact
inom
aP
RL
GH
-, P
RL+
+, c
30ye
s
A21
Q14
XF
Pro
lact
inom
a18
NA
PR
LN
A15
no
A31
Q14
XF
Acr
omeg
aly
22N
AG
HN
AN
Aye
s
A33
Q14
XM
Gig
antis
m17
NA
GH
NA
11ye
s
A34
aQ
14X
MG
igan
tism
14M
amm
osom
atot
roph
ade
nom
aG
HG
H+,
PR
L+, c
33ye
s
A38
aQ
14X
FA
crom
egal
y24
Mam
mos
omat
otro
ph a
deno
ma
GH
, PR
LG
H++
, PR
L++,
c12
yes
A61
aQ
14X
MA
crom
egal
y42
Som
atot
roph
ade
nom
aG
HG
H++
, c10
yes
A82
Q14
Xb
FA
crom
egal
y22
NA
NA
NA
NA
yes
A85
Q14
XM
Pro
lact
inom
a62
Pro
lact
inom
aP
RL
GH
-, P
RL+
+, c
30ye
s
A97
Q14
Xb
MA
crom
egal
y20
NA
NA
NA
NA
no
A70
IVS
3-1G
>AF
Acr
omeg
aly
26M
amm
osom
atot
roph
ade
nom
aG
H, P
RL
GH
++, P
RL+
, c7
yes
A78
R30
4XF
Acr
omeg
aly
18S
omat
otro
ph a
deno
ma
GH
GH
++10
yes
A79
R30
4XM
Gig
antis
m18
Som
atot
roph
ade
nom
aG
HG
H++
8ye
sa B
elon
g to
the
popu
latio
n-ba
sed
coho
rt, b be
long
to u
nsel
ecte
d ac
rom
egal
y pa
tient
s. c A
dren
ocor
ticot
roph
ic h
orm
one
(AC
TH),
thyr
oid
stim
ulat
ing
horm
one
(TS
H),
folli
cle
stim
ulat
ing
horm
one
(FS
H) a
nd lu
tein
izin
g ho
rmon
e (L
H) n
egat
ive,
NA
not
ava
ilabl
e; G
H, g
row
th h
orm
one;
PR
L, p
rola
ctin
; IH
C, i
mm
unoh
isto
chem
istry
.
73
tumour). In addition, two patients (1370, 1276) who were invited to the study fortheir young age at onset were found to be cousins. Furthermore, the mother andsister of patient 1156, and the daughter of patient 1198 have been found to havePHPT after starting this study.
Patient 1237 was found to have the 1466del12 (c. 1356_1367del12) foundermutation in the MEN1 gene (Kytölä et al. 2001). No other disease causingmutations were detected in the MEN1, HRPT2 or CASR genes in the rest of thepatients (n= 28). CDKN1B and AIP genes were analysed in 27/28 patients (patient1609 withdrew her consent at this point) with no pathogenic alterations found.Common polymorphic sequence variations were detected in all genes studied.According to biochemical screening, 5 patients had hypercalcaemia (1142, 1153,1155, 1198, 1237), 4 of which (all but 1142) were found to have recurrent PHPTon a subsequent evaluation by endocrinologist. They were also rescreenedbiochemically for MEN1 manifestations with no positive findings, except for thepatient with the MEN1 mutation. The level of PP was markedly elevated in fivepatients (1211, 1216, 1237, 1377, 1617). Three of them were evaluated andexamined by endocrinologist with no further evidence of pancreatic tumours. Thelevel of serum PP in the patient with the MEN1 mutation has varied from normalrange to approximately 2-fold to normal in control screenings, whereas serumgastrin level has not been elevated. Imaging studies have not been performed yet.One of the patients (1617) was not willing to undergo further examinations. Noneof the patients (n=29) had elevated levels of prolactin.
74
6 Discussion
6.1 Clinical features of MEN1 and genotype-phenotype correlation (I)
Two founder mutations enriched in Northern Finland and resulting in a cluster ofMEN1 patients (Kytölä et al. 2001), as well as a well-organized health care systemoffer a good opportunity to study this disease within an unbiased material onpopulation basis. In the present study, we found 15 (38%) patients with gastrinomaamong 39 individuals carrying the 1466del12 mutation, and only one (3%) patientwith gastrinoma among 29 individuals carrying the 1657insC mutation. Thisseems to indicate clearly that the 1466del12 carrier patients are more prone todevelop gastrinomas. On the other hand, the prevalence of nonfunctionalpancreatic tumours among the 1466del12 mutation carriers was significantlylower, 15 (38%) vs. 20 (69%), when compared to 1657insC mutation carriers.Resembling our findings, enteropancreatic lesions showed intrafamilialhomogeneity in a 10-year prospective screening study published from Sweden(Skogseid et al. 1991). In their study, insulin-proinsulin excess was significantlyoverrepresented in a family with a pronounced malignant profile of theirpancreatic tumours, whereas in another family all pancreatic lesions were benign,and only the Zollinger-Ellison syndrome was displayed. Contrary to the study bySkogseid et al. (1991), we could not find any difference between familiesaccording to malignant behaviour of GEP tumours. Instead, male gender seemedto be a risk factor for developing metastasized GEP tumours.
Hao et al. (2004) described two kindreds (A and B) with a low prevalence ofgastrinoma (10%) and high prevalence of prolactinoma (40%). The authorspointed out that the difference from typical MEN1 was more striking forgastrinoma than for prolactinoma. Patients from kindred B harboured a nonsensemutation Y312X, while no MEN1 mutation was identified in kindred A (Hao et al.2004). Patients with prolactinoma variant from Newfoundland had an R460Xnonsense mutation (Olufemi et al. 1998). A common finding between cases withprolactinoma variant and 1657insC mutation carriers is the low prevalence ofgastrinoma. In addition, all three mutations led to truncated proteins, like most ofthe MEN1 mutations (Chandrasekharappa & Teh 2003). In the present study themutation class leading to truncated proteins (frameshift insertion/nonsensemutation) was found to be a risk factor for nonfunctional pancreatic tumour, whilemutation class leading to presumably lesser changes in the menin protein (in-frame
75
deletion/missense mutations) predicted the risk for gastrinomas. In a paper ofgenotype/phenotype correlation of MEN1 mutations in sporadic gastrinomas(Goebel et al. 2000) the authors combined their data with results taken from othercases of sporadic gastrinomas (Wang et al. 1998). They stated that 40% (11/28) ofthe mutations described (8 missense and 3 in-frame deletions) altered the aminoacid sequence of menin, while 60% (17/28) resulted in a truncated protein (Goebelet al. 2000). In familial MEN1 the majority (73%) of the mutations led to atruncated protein (Agarwal et al. 1997, Bassett et al. 1998, Giraud et al. 1998).
The 1466del12 mutation, located in exon 10, is an in-frame deletion that ispredicted to shorten the menin protein by 4 amino acids at codons 453–456. Thedeleted region locates in the area that is required for interaction with PEM(polymorphic epithelial mucin), NM23H1 (nonmetastatic protein 23, homolog 1)and CHES1 (chekpoint suppressor 1) proteins (Chandrasekharappa & Teh 2003,Lemos & Thakker 2008). The frameshift mutation 1657insC at codon 516 ispredicted to cause a premature stop codon 14 codons upstream (Turner et al.2002). This would affect the binding site with SMAD3 (SMA- and MAD-relatedprotein 3), ASK (activator of s-phase kinase) and CHES1, and two of the nuclearlocalization signals (NLSa, NLS2) would be cut off. It is also possible that theframeshift mutation results in loss of the translated protein because ofnonsense-mediated mRNA decay (NMD). (Chandrasekharappa & Teh 2003,Lemos & Thakker 2008). It is not known whether these changes in interaction dueto MEN1 mutations have any role in the tumorigenesis of gastrinomas ornonfunctional pancreatic tumours.
While the issue of genotype-phenotype correlation in MEN1 is under debate,it is a widely recognized feature in von Hippel-Lindau disease (VHL) (Table 5).The association of pheochromocytoma with missense mutations in the VHL genewas discovered more than ten years ago (Crossey et al. 1994, Chen et al. 1995).Moreover, Ong et al. (2007) studied 537 individuals with VHL disease, and theydiscovered that missense mutations resulting in substitution of a surface aminoacid conferred a higher pheochromocytoma risk than those that resulted in thedisruption of structural integrity. Currently, it is not known what the key factors arefor pheochromocytoma development in VHL disease. However, increased JunBexpression secondary to altered atypical protein kinase C signalling has beenlinked to failure of developmental apoptosis in adrenal medullary progenitor cellsin VHL disease as well as other causes of familial pheochromocytomas. (Lee et al.2005, Ong et al. 2007). Ong et al. (2007) also pointed out that even though VHLdisease has been considered to demonstrate a classical “two-hit” model of
76
tumorigenesis, phenotypically normal renal cells from VHL patients demonstratealtered gene expression patterns compared to normal control kidney cells(Stoyanova et al. 2004). These findings suggest that the presence and nature of thegermline mutation (“first-hit”) may influence cell function and susceptibility totumorigenesis.
In the present study, the likelihood of having a GEP tumour was increasedwith advancing age with an OR of 1.12 in the entire group, and 1.11 in thesubgroup of the founder mutation carriers. According to Kaplan-Meier analysisthe penetrance of GEP tumours is 100% by 70 years of age (Original publication I,Figure 1). Similarly to the findings in a Tasmanian kindred study (Burgess et al.1998), however, when nonfunctional pancreatic tumours were analysed alone, theydid not associate with advancing age. Our findings of age-related penetrance ofother various MEN1 lesions are in agreement with previous studies (Skogseid etal. 1991, Burgess et al. 1998, Geerdink et al. 2003, Hao et al. 2004). In the presentstudy, the peak of the prevalence of pituitary tumours was reached around 60 yearsof age (Original publication I, Figure 1), and advancing age was not found to be arisk factor for pituitary tumours. A similar finding with prolactinoma was alsoseen in the Tasmanian kindred (Burgess et al. 1998). In addition, in our study,mutational background or gender did not predict the risk for pituitary adenoma orprolactinoma. In a large multicentre study, Vergès et al. (2002) found that 85% ofthe tumours were macroadenomas, whereas in our study the proportion ofmacroadenomas was 50%. This difference may reflect the higher proportion ofnewly diagnosed heterozygotes with early diagnoses in our study. Furthermore, inthe study of Vergès et al. (2002), there were tumours secreting GH or ACTH in 4%and 2%, and cosecreting and nonsecreting tumours in 4% and 6% of the cases. Thealmost total lack of other functioning pituitary adenomas than prolactinomas in ourstudy may be due to a lower overall number of pituitary tumours as compared tothe number of pituitary adenomas (n= 136) in the big multicentre study (Vergès etal. 2002). Nevertheless, the lack of GH-secreting adenomas has also been shownin the Tasmanian kindred where 124 patients were reviewed (Burgess et al.1996b).
The only identifiable risk factor for developing PHPT was advancing age withan OR of 1.08 per year. According to Kaplan-Meier analysis the penetrance ofPHPT would be 100% by 70 years of age (Original publication I, Figure1). Onepatient was found to have multiglandular parathyroid cancer, which is extremelyrare either in sporadic form or associated with MEN1. It is not known whether thepast radiation therapy on presumed sellar angiosarcoma, or peculiar family history
77
of different tumours (mother; thyroid papillary carcinoma, renal clear cellcarcinoma, breast cancer, leiomyoma and leiomyolipoma of uterus, mother’ssister; mesodermal mixed tumour of uterus) had any aetiological impact on thedevelopment of parathyroid cancer.
The prevalence of adrenal tumours (35%) in this study is one of the highestreported in the literature (Table 1). This could be due to the high frequency ofimaging studies we used. There are also reports including adrenal carcinoma andpheochromocytoma in varying numbers of patients (Table 2). We were only able tofind benign lesions, and only two were associated with subclinicalhypercortisolism. Similarly, an indolent course of adrenal lesions has also beenreported elsewhere (Burgess et al. 1996d, Barzon et al. 2001).
The occurrence of neuroendocrine gastrointestinal tumours, earlier known ascarcinoids, only in women in the present study may be a coincidence.Nevertheless, we noted thymic neuroendocrine tumours in this study only in menin accordance with previous reports (Teh et al. 1998a,c, Ferolla et al. 2005).Although we did not find any bronchial neuroendocrine tumours in our study, theyare usually seen more frequently in women (Sachithanandan et al. 2005).Regarding to these data, it is apparent that there exists some clinical variation inMEN1 according to the gender of the affected heterozygote.
There were five cases of thyroid cancer in this study. The aetiology of theseand other tumours outside the classical MEN1 tumour spectrum noted in our studyis not known. However, others have shown by LOH studies that leiomyomas of theoesophagus and uterus, meningioma and lipoma may be part of the MEN1syndrome (Pack et al. 1998, McKeeby et al. 2001, Asgharian et al. 2004). It is alsoof note that medullary thyroid carcinoma is typically associated with MEN2 andits occurrence in our patient with MEN1 is probably a coincidence.
6.2 Mortality in MEN1 (I, II)
Of the deaths caused directly by MEN1 among the 82 heterozygotes, six out ofseven (86%) were caused by metastasized tumours associated with MEN1 (5patients with a GEP tumour, one with parathyroid cancer), and only one wascaused by complications of peptic ulcer disease. In three other cases MEN1 was acontributing factor with a malignant tumour (two patients with GEP, one with aneuroendocrine tumour of the thymus). This is in accordance with some otherrecent studies showing that malignant islet cell tumours and malignant carcinoidsare the major causes of MEN1-associated death, whereas in older studies peptic
78
ulcer disease was the most common MEN1-associated cause of death (Table 3).Due to the small overall number of deaths in this patient cohort no further analysison mortality was undertaken. Instead, the main result of our genetically establishedgenealogical survey was that the lifespan of the subjects considered to be MEN1heterozygotes did not show significant differences in comparison with the group oftheir spouses in sex groups or general life expectancy estimates.
We have had an opportunity to observe two family trees with a foundermutation. To ascertain that the true common ancestors are found, practically all thepedigree roots to the 18th century were evaluated, and many were evaluated evenfurther. Nonpaternity is a possibility always worth bearing in mind, and this isprobably the case in the 1466del12 mutation-positive family, which could not beconnected to ancestors despite the same mutation. Haplotype analysis around themutant MEN1 allele in our families has shown that the area is quite conserved, andthus recent common ancestors within 10 generations could be expected to be found(Kytölä, S, unpublished data). One of the advantages in our retrospective survey ofMEN1 heterozygotes and their spouses is that gene carriers can also be included,thus completing the picture. In addition, the evaluation period is almost 300 years,a feature unsurpassed by modern prospective studies for centuries. One of thedrawbacks in the present genealogical survey is the assumption that the MEN1gene does not cause mortality before the age of 25 years. According to theliterature, this is quite rare (Brandi et al. 2001), also according to our ownunpublished data. Thus, the use of life expectancy tables for 25-year-olds can beregarded as acceptable. Another selection bias in our study is that these personswere practically all ancestors in the study group, and fertility is obligatory to be anancestor. MEN1 positivity can cause infertility, for example throughhyperprolactinaemia, and thus omit subjects from this survey (Schuppe et al.1999). Pituitary adenomas, most of them prolactinomas, are present inapproximately 22±4% of patients with MEN1 (Hao et al. 2004). The family treesof the two MEN1 pedigrees imply that heterozygosity has not, at least markedly,reduced fertility and the number of children. Heterozygote advantage cannot beexcluded when family trees of expanding MEN1 families are looked at. Thegenetic fitness is close to 100%. Including siblings was not generally possiblebecause MEN1 status could not be judged in them.
The birth and death dates are reliable, grounded on a sound tradition of churchrecords kept by clergy and initially legislated by the king of Sweden. The causes ofdeath have also often been recorded by the clergy, but up until the 20th century they
79
were quite descriptive because of unprofessional classification and lack ofautopsies.
When evaluating whether excess mortality exists, the death rate of the wholepopulation to be compared with is a crucially important factor. In the years 1700–1880, death rates of the population of Finland were remarkably high, resemblingthose of present-day underdeveloped countries, until a sustained decline inmortality began in the decade 1870–1880 (Turpeinen & Kannisto 1997). Lifeexpectancy was much higher at age 15 than at birth, demonstrating a highmortality in childhood. This is why the life expectancy value changes in adultswere minor or moderate compared with major changes in children’s lifeexpectancy when approaching the year 1900 (Turpeinen & Kannisto 1997). Birthyear and life expectancy showed a strong positive mutual correlation in the wholegroup and subgroups of men and women as can be expected, because theyrepresent population value estimates. Between the years 1750 and 1885, the lifeexpectancy of 25-year-olds was very variable and did not show any consistenttrends, but was rather a plateau. During that period, the life expectancy mean of25-year-old males was 59.5 yr (range, 55.2–62.7 yr) and that of females 61.0 yr(range, 57.2–64.5 yr). After 1885, there was a clear, strong positive linearcorrelation between life expectancy and birth year. During that phase, lifeexpectancy reached modern lifespan levels, and the rise from 1885 was 13 yr inmales and 18 yr in females.
When comparing the lifespan of the obligatory gene carriers to the lifespan ofimaginary reference subjects with the same sex and birth year in the same 5- to10-yr period, the results confirmed the data obtained from the comparison of genecarrier group to spouse group. Assumptions that genetic screening will reducemorbidity as well as mortality in MEN1 in the future have been postulated inscientific literature (Clerici et al. 2001). The findings here, which are in contrastwith previous studies, could be related to differences in genotypes orgene-environment interactions. Still, the most probable reason that ourgenealogical survey did not show excess premature mortality in MEN1 patientscan be mainly explained by high mortality in the whole population because ofperiods of famine, infectious diseases, and wars. Presymptomatic diagnosis hasnot been shown to yield clear clinical benefit in patients with MEN1 (Arnold2008). Our retrospective view did not show a harmful effect of MEN1 gene traitpositivity to life expectancy, but it can be speculated whether the situation is thesame nowadays, when life expectancy is from 13 yr (in men) to 18 yr (in women)higher. The historical study population did not show any significant associations
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with the age of death when pedigree group, MEN1 trait, and sex were used asexplaining variables, and expected lifespan and birth year as covariates. The trendof increasing lifespan did not come up when subjects born in 1860 or later wereanalysed in subgroups of sex and probands and spouses separately, perhaps due tothe small number of subjects. All four subjects who were excluded from the mainanalysis because they were still living had already exceeded the expected lifespan[two MEN1 subjects (+1.1 and 6.1 yr) and two spouses (+8.2 and 0.8 yr)], andincluding them in the analyses with age as lifespan did not change results.
A large multigenerational MEN1 family with clinical expression suggestive ofanticipation was reported by Giraud et al. (1997). Anticipation is a phenomenon inwhich severity increases and age of onset decreases in successive generations. It ismost commonly found in neuropsychiatric disorders and associated withtrinucleotide expansions. In the family described by Giraud et al. (1997) twoobligate gene carriers died at the ages of 85 and 76 without the history of MEN1,whereas two other living gene carriers above the age of 65 had had no clinicalevidence of MEN1. In the fourth generations four out of eight affected membershad severe MEN1-related and atypical malignancies. In the fifth generation, allfive patients were below the age of 22 when the disease was detected. Repeatedexpansion detection studies were carried out but failed to detect any expansion.The authors suggested that anticipation is uncommon in MEN1, and thatcosegregation of other modifying genes might be involved, resulting in moresevere phenotypes in subsequent generations (Giraud et al. 1997). The hypothesisof anticipation could theoretically explain our finding that the lifespan of theMEN1 obligate heterozygotes did not show significant differences in comparisonwith the controls, but further studies are needed to explore the issue.
In our genealogically formed study group, there was a significant difference incauses of death between female probands and female spouses (P =0.003), but thesame was not true in males. In female subjects, out of 15 probands, eight (53%)died of a possible MEN1 cause, whereas of the 12 female spouses, none died of apossible MEN1 cause. This means that female obligatory gene carriers showed adifferent pattern of causes of death from female controls. The difference insignificance between females and males could partly be explained by lower lifeexpectancy in males, and thus competing death causes could have intervenedbefore MEN1 causes. A retrospective classification of old causes of death intoMEN1-related and other causes yields only a crude estimate for the evaluation ofthe effect of MEN1 gene positivity, as MEN1-related causes of death includedmainly causes that could be regarded as resulting from a malignancy. Reliability of
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the initial causes of death in the documents was classified by the investigator ascertain in 36% of the cases.
6.3 Pituitary adenoma predisposition and AIP (III)
Combining chip-based technologies with genealogy data we identified germlinemutations in the aryl hydrocarbon receptor interacting protein (AIP) gene inindividuals with pituitary adenoma predisposition (PAP). A nonsense mutationQ14X was found in affected members of families 1 and 2. In addition, we hadDNA samples from 45 of the 54 acromegaly patients belonging to thepopulation-based study cohort, including four cases from families 1 and 2. Out of45 patients from the population-based cohort, 6 displayed Q14X, and onedisplayed IVS3-1G>A. These alterations were absent in more than 200 local blooddonors. It is of note that six out of the fifteen patients diagnosed under 35 years ofage in the population-based series had AIP mutation. Thus, in a population-basedseries from Northern Finland, two AIP mutations account for 16% of all patientsdiagnosed with pituitary adenomas secreting growth hormone and for 40% of thesubset of patients who were diagnosed when they were younger than 35 years ofage. Another series of 10 unselected Finnish sporadic acromegaly patients werescreened and Q14X was found in two of them, which is compatible with findingsin the population-based cohort. The possible role of AIP in pituitary adenomapredisposition in other populations was confirmed by our finding that Italiansiblings with somatotroph adenoma displayed a nonsense mutation R304X.
The clinical diagnosis of the PAP patients identified during this study includesacromegaly (n= 10), gigantism (n= 5), and prolactinoma (n= 3). Histopathologicaldiagnosis of the GH-secreting adenomas was either mammosomatotroph adenomaor somatotroph adenoma. Loss of the wild-type allele was detected in all of thesetumour types, strengthening the notion that PAP is associated with predispositionto both prolactinomas and GH-secreting pituitary adenomas. Mammosomatotrophadenomas are bihormonal tumours secreting both GH and prolactin (Thapar et al.1995). In accordance with this, three out of five patients diagnosed withmammosomatotroph adenoma had had elevated levels of GH and prolactin. In atleast one of the patients (A34) serum prolactin was not measured before surgery onpituitary adenoma. Young age at onset seems to be a characteristic finding inpatients with PAP; AIP mutation carriers (n=7) were significantly younger thanmutation negative patients (24.7 ± 10.7 versus 43.6 ± 11.9 years, P = 0.0003) in thepopulation-based acromegaly cohort. Thus, for identification of PAP patients,
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young age at onset is a useful indicator. Differences in tumour size or sexdistribution were not observed.
The PAP phenotype with very-low-penetrance susceptibility to GH-secretingpituitary adenoma and prolactinoma does not fit well to any of the known familialpituitary adenoma syndromes, including MEN1, Carney complex, familial isolatedpituitary adenoma and isolated familial somatotropinoma (Frohman & Eguchi2004, Heaney & Melmed 2004, Daly et al. 2006). These syndromes are familial,and the low penetrance of PAP appeared unique. However, there are somesimilarities when comparing the PAP phenotype to all of these syndromes. Forinstance, in addition to PAP, prolactinoma is found commonly in MEN1 and FIPApatients (Table 1). Similar bihormonal overproduction of GH and prolactin may beseen especially in patients with Carney complex, IFS and PAP (Daly et al. 2006,Soares & Fohman 2004). Younger age at diagnosis of pituitary adenoma has alsobeen found in FIPA patients compared to sporadic cases (38.4 vs. 41.9 years) in astudy by Daly et al. (2006). Also in the same study, IFS patients were diagnosed ata mean age of 33.8 years, which was more than 10 years younger thansomatotropinoma patients from either heterozygous FIPA families (49.3 years) orsporadic cases (44.1 years). Even younger age at onset, 26 years, was found bySoares and Frohman (2004) in a review of 46 IFS families reported in theliterature. Also, in CNC, biochemical findings of mammosomatotroph hyperplasiacan be found in most patients starting from adolescence (Boikos & Stratakis 2007).A common finding between IFS and PAP is also that both show reduced orincomplete penetrance, although this phenomenon seems to be even more strikingin our PAP patients with only one affected sibling pair found in the Finnishpatients. Other AIP mutation positive cases were more distant relatives or did nothave any family history suggestive of pituitary adenoma. Some of the IFS familiesmay share the same aetiology as PAP patients, since the locus for IFS has beenlinked to 11q13.1-q13.3 (Gadelha et al. 2000, Luccio-Camelo et al. 2004, Soareset al. 2005). Following our initial discovery of pituitary adenoma predispositionwith AIP gene germline mutations (Original paper III), it has been estimated that15% of FIPA patients and 50% of IFS patients carry AIP mutations (Beckers &Daly 2007).
It has not been previously realized that genetic predisposition to pituitaryadenoma, in particular the GH-oversecreting type, can account for a substantialproportion of cases. Our study not only reveals this aspect of the disease, but alsoprovides molecular tools for efficient identification of predisposed individuals.Without preexisting risk awareness, the patients are typically diagnosed after years
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of delay, leading to substantial morbidity. Simple tools for efficient clinicalfollow-up of predisposed individuals are available, underlining the importance ofour findings. Our results suggest that inherited tumor susceptibility may be morecommon than previously thought. The identification of the PAP gene indicates thatit is possible to identify the causative genetic defects in low-penetrance conditionseven in the absence of a strong family history.
AIP (OMIM 605555) was originally identified by its interaction with thehepatitis B virus X protein and thus designated XAP2 (hepatitis B virusX-associated protein) (Kuzhandaivelu et al. 1996) (Figure 5). AIP consists of 330amino acids with regions of amino acid sequence similarity to the 52-kDaFK506-binding protein known to be associated with the glucocorticoid receptor(Figure 5). AIP forms a complex with the aryl hydrocarbon receptor (AHR) andtwo 90-kD heat-shock proteins (HSP90). (Carver & Bradfield 1997.) The R304Xmutation removes the AHR binding region (Bell & Poland 2000, Petrulis &Perdew 2002). ARH is a ligand-activated transcription factor that regulates avariety of xenobiotic metabolizing enzymes (Meyer & Perdew 1999). Dioxin-likechemicals display high affinity to AHR, which mediates most of the toxicresponses of these agents. AHR also participates in cellular signalling pathways(Marlowe & Puga 2005). AIP modulates the subcellular localization of AHR andprevents the AHR from undergoing nucleocytoplasmic shuttling (Pollenz &Dougherty 2005). Nuclear translocation of AHR is also regulated by a complexformed by AIP and PDEA2 (phosphodiesterase type 2A) (de Oliveira et al. 2007).AIP also binds to and attenuates the activity of PDE4A5 (cyclic AMP-specificphosphodiesterase type 4 isoform A5) as well as PPAR (peroxisomeproliferator-activated receptor alpha) (Bolger et al. 2003, Sumanasekera et al.2003). It has also been found recently by Kang and Altieri (2006) that AIPassociates directly with survivin, which is a multifunctional member of the IAP(inhibitor of apoptosis) family, thus preserving protein stability and a cytosolicanti-apoptotic threshold. Survivin is over-expressed in foetal tissues and humancancers, but it is almost undetectable in normal tissues. Overexpression of survivinhas also been observed in benign central nervous system tumours includingpituitary adenomas. (Hassounah et al. 2005, Wasko et al. 2005). It has also beenreported that AIP functions as a cytosolic factor that mediates preprotein importinto mitochondria by binding to both translocases of the outer membrane ofmitochondria (Tom) 20 and preproteins (Yano et al. 2003). Recently, it was alsoshown by Froidevaux et al. (2006) that AIP is necessary for T3-independentactivation of TRH transcription mediated by TR1 (thyroid hormone receptor 1).
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The mechanism by which AIP exerts its tumour-suppressive action in the pituitaryremains to be determined. Further work on the functional role of AIP should proveinformative in revealing key cellular processes involved in the genesis of pituitaryadenomas, including potential drug targets.
6.4 PHPT patients with features of genetic predisposition (IV)
This is the first study where the five major genes (MEN1, HRPT2, CASR,CDKN1B and AIP) known to cause PHPT or related disorders have beensystematically studied in a well-defined cohort of patients. We have studiedextensively 29 selected patients in order to estimate the aetiological role ofgermline mutations in these genes in PHPT predisposition. Only one patient (3%)with a germline mutation (MEN1, 1466del12) was found. However, this finding isin line with our hypothesis that due to the two founder mutations in the district ofOulu University Hospital, there may be more undiscovered MEN1 patients in thearea. We were not able to study all the potential cases of PHPT, since only 52% ofthe invited patients participated in the study. In addition, incomplete family historyin the medical records may have hampered identification of some of the familialcases.
The inclusion criteria were chosen based on known features of hereditaryparathyroid tumour predisposition (Simonds et al. 2002). With the exception of therepeatedly high level of S-PP in patient 1237, not a single patient in this cohort hadendocrine features that would have met the definition of MEN1 syndrome, whichrequires the existence of 2 of the 3 main MEN1-related tumours (parathyroidadenoma, entero-pancreatic neuroendocrine tumour, and pituitary adenoma)(Brandi et al. 2001). The clinical features of patient 1617 (adrenal incidentalomas,pancreatic cysts) were suggestive of MEN1, but she refused further clinicalevaluation. The elevated level of PP of other patients was not regarded as a sign ofpancreatic neuroendocrine tumour in subsequent evaluations by endocrinologists.It is recognized, however, that these patients with elevated levels of PP mayrequire surveillance for pancreatic tumour development. In addition, patient 1235had had pheochromocytoma which could be suggestive of MEN2, but the absenceof medullary thyroid carcinoma at the age of 66 years makes this diagnosisunlikely. Even though it has also been suggested that atypical presentations ofMEN1 may include pheochromocytoma (Dackiw et al. 1999), the absence ofevidence of other major tumours prevents setting the diagnosis of MEN1. Eventhough MEN1 features were dominating in our inclusion criteria, obvious cases of
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other familial forms of PHPT should have been picked up since family history ofPHPT was one of the inclusion criteria. It should also be noticed that the mutationdetecting methods used in this study do not necessarily find all possible pathogenicalterations in the studied genes, such as large deletions or mutations in promoter orother regulatory regions of the genes.
In the entire PHPT patient population reviewed at least 7% (21/286) of thecases were affected by MEN1. The total number of PHPT patients examined in theOulu University Hospital during 1978–2001 was approximately 350, leading to aprevalence of 6% of MEN1 in this patient group. Earlier, it had been estimated thatthe fraction of MEN1 in PHPT would be 2–3% (Marx 2001). By the year 2001there were more than 20 known MEN1 mutation carriers with PHPT in the area.Not all of them were caught in the computerized hospital records search for PHPT,probably because presymptomatically identified MEN1 carriers usually have mildforms of PHPT, and thus clinicians may choose not to use the respective diagnosiscode used as a tag in the search. Since it is likely that many of thepresymptomatically diagnosed carriers of MEN1 are not included in the examinedcohort of PHPT the present work also represents more accurately the naturalsetting of PHPT patients in general.
The frequency of somatic MEN1 mutations has previously been studied insporadic parathyroid tumours with mutations found in 12–21% of the tumours(Heppner et al.1997, Carling et al. 1998, Farnebo et al. 1998). In these studies nogermline mutations have been found. In contrast, Uchino et al. (2000) studiedparathyroid tumours of 112 unselected patients and peripheral blood leukocytesfrom 64 of the patients. MEN1 mutation was found in 25/112 (22%) of the tumourspecimens, and three of the mutations could also be found in the correspondingblood sample DNA (Uchino et al. 2000). In addition, several studies on selected(increased risk of being a MEN1 mutation carrier) groups of PHPT patients havebeen reported, with varying MEN1 mutation frequencies. In five different studiesincluding patients with sporadic PHPT with no other MEN1-related lesions, MEN1mutation was detected in 0 to 40% of cases (mean 14%) (Cupisti et al. 2000,Langer et al. 2003, Cardinal et al. 2005, Ellard et al. 2005, Tham et al. 2007). Infour studies including patients with sporadic PHPT and at least one otherMEN1-related manifestation, the MEN1 mutation detection rate varied between 0and 50% (mean 21%) (Roijers et al. 2000, Cardinal et al. 2005, Ellard et al. 2005,Tham et al. 2007). Family history of hyperparathyroidism alone (FIHP) wasassociated with a 22 to 57% (mean 38%) detection rate of MEN1 mutation, andfamily history of MEN1-related lesions with a 73 to 100% (mean 83%) detection
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rate of MEN1 mutation (Cardinal et al. 2005, Ellard et al. 2005, Tham et al. 2007).If our patients are divided into corresponding groups, the figures for MEN1mutation detection rate are as follows: 0% (0/19) in sporadic PHPT patients, 0%(0/1) in sporadic PHPT patients with at least one MEN- related manifestation, 14%(1/7) in familial PHPT, and 0% (0/3) in PHPT patients with family history ofMEN1-related lesions. Our low MEN1 mutation rate in the patients examined mayat least partly be due to the fact that we deliberately excluded known patients withMEN1.
In two of the studies reviewed above the fraction of multiglandular diseaseamong MEN1 mutation negative and positive could be estimated. In the study byCupisti et al. (2000) two of the five patients with germline mutations hadmultiglandular disease, whereas one of the patients with double adenoma had amutation in the tumour tissue, but DNA from healthy tissue was not available forfurther studies. Two of the mutation negative patients diagnosed at 13 and 18 yearshad single adenomas. In the study by Langer et al. (2003) all the 15 PHPT patientswere diagnosed at 40 years or less, and two (13%) of the patients withmultiglandular disease were found to have MEN1 mutation, whereas the rest (n=13, 87%) with solitary adenoma were MEN1 mutation negative. Of the studiesreviewed above, three concluded that patients with young age (<35 or 40 years)and/or multiglandular disease (Cupisti et al. 2000, Roijers et al. 2000) orhyperplasia (Tham et al. 2007) would be suitable for MEN1 mutation screening,whereas two of the studies (Langer et al. 2003, Cardinal et al. 2005) recommendedpatients with young age (<30 or 40 years) and multiglandular disease for MEN1mutation screening. In our study, there were three patients with multiglandulardisease and young age at onset, including the patient with the 1466del12 MEN1mutation; therefore, according to the above-mentioned criteria she would havebeen recommended for mutation testing even without the knowledge of familyhistory. This is an important issue since approximately 10% of MEN1 mutationsarise de novo, the affected patients having no remarkable family history.
Somatic HRPT2 mutations are found in the majority of parathyroidcarcinomas, but very rarely in sporadic adenomas (Howell et al. 2003, Cetani et al.2004). In addition, germline mutations of HRPT2 can be found in patients withapparently sporadic parathyroid carcinoma (Shattuck et al. 2003). Interestingly,germline HRPT2 mutations are found on average in 5% (range 0–29%) of FIHPkindreds (Cetani et al. 2006). Similarly, the rate of CASR germline mutations inFIHP families has been on average 12%, ranging from 0 to 18% (Cetani et al.2006). Probably due to the low number of proven familial cases in this study and
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the overall low frequency of HRPT2 and CASR mutations in FIHP kindreds, it isnot surprising that we did not find any mutations in these genes.
Pellegata et al. (2006) reported the first case with a germline CDKN1Bmutation. She was a 48-year-old Caucasian woman with acromegaly diagnosed at30 years. At age 46, she was diagnosed with PHPT, but she had not yet beenoperated on (Pellegata et al. 2006). The second reported patient with CDKN1Bmutation and MEN1-like phenotype was diagnosed with PHPT at the age of 47years (Georgitsi et al. 2007). She had been diagnosed with small-cellneuroendocrine cervical carcinoma at the age of 45 years and ACTH-secretingpituitary adenoma at the age of 46 years. The prevalence of CDKN1B mutations inPHPT patients without prominent syndromic features has not been studied earlier.Similar to our findings, the absence of pathogenic alterations in CDKN1B genewas found in a study by Ozawa et al. (2007), where they examined patients with aMEN1 variant with sporadic parathyroid and pituitary tumours and no identifiedMEN1 germline mutation.
AIP gene defects were not detected in PHPT patients in this study. This is inaccordance with our recent study where somatic AIP mutations were not found in79 sporadic endocrine (non-pituitary) tumours including 6 parathyroid adenomasand 2 carcinomas (Raitila et al. 2007). Instead, 2 prolactinomas out of 9 werefound to harbour the Northern Finnish founder mutation Q14X (Raitila et al.2007). The PHPT study population in this study originates from the same area asthe PAP patients with the Q14X mutation. Therefore, the absence of Q14Xmutation carriers in this study leads to the conclusion that AIP gene defects maynot have a role in the pathogenesis of PHPT.
Based on our study and those reviewed above, it appears that sporadic andfamilial PHPT patients with multiglandular hyperplasia and young age at onset(below 40 years) are good candidates for MEN1 mutation screening. It also seemsthat CDKN1B and AIP may not have a significant contribution to PHPTpredisposition.
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7 Conclusion and future prospectsThe major observations and conclusions of this study are as follows:
1. Clinical features in Northern Finnish MEN1 patients were similar to thosereported by other studies (Table 1 and 6). PHPT was present in 93%, GEPtumours in 73%, pituitary adenomas in 29% and adrenal lesions in 35% of theMEN1 mutation carriers. Subgroups of different GEP tumours were unevenlydistributed among carriers of the two MEN1 founder mutations: gastrinomawas present in 38% of the 1466del12 mutation carriers and in 3% of the1657insC mutation carriers. Instead, NFPT was found in 39% of the1466del12 carriers, and in 69% of the 1657insC mutation carriers. In thefounder mutations group (n= 68) the OR for gastrinoma was 15.1 (CI, 1.73–131.9; P= 0.014) in the 1466del12 mutation carriers compared to the 1657insCmutation carriers. In the whole study group (n= 82) the OR for gastrinoma was6.77 (CI, 1.31–35.0; P= 0.022) in the mutation class including in-frame andmissense changes compared to the mutation class including frameshift andnonsense mutations. The 1657insC mutation carriers appeared to be at risk forNFPT with an OR of 3.56 (CI, 1.29–9.83, P= 0.015), whereas the mutationclass including the frameshift and nonsense mutations predicted the risk forNFPT with an OR of 3.26 (CI, 1.27–8.33; P= 0.014). Our study indicates thatthere is some variation in the risks for certain types of GEP tumours betweenpatients with different MEN1 mutations. Whether this phenomenon is causedby mutation per se and subsequent variations of mutated menin products indifferent transcriptional interactions or some other factor, such as a modifiergene cosegregating with MEN1 gene, needs further studying. Furthermore,more research is needed before extending these findings from our NorthernFinnish families to other families carrying the same MEN1 gene mutations. Itis also important to update the data concerning the Northern Finnish MEN1patient cohort and test whether our findings in this study can be repeated.
2. The MEN1 pedigree study population of subjects born between 1728–1929gives information on the survival of gene heterozygotes in a historical context.The lifespan of MEN1 positive subjects was equal to their spouse group andalso similar to the life expectancy estimates derived from Finnish nationalstatistics. Thus, the MEN1 gene did not show any harmful effect on survival inour analyses. More prospective studies are needed to assess if the MEN1 gene
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has a statistically significant negative effect on survival in modern times,when life expectancy has generally increased in the population.
3. The clinical features and genetic background of previously uncharacterizedpituitary adenoma predisposition, PAP, were defined in this study. Linkageanalysis in families 1 and 2 placed the locus for PAP in chromosome 11q12-11q13, a region previously implicated in IFS and including the MEN1 gene.On the basis of gene expression data in the linked area, AIP was chosen forfurther analysis. A nonsense mutation Q14X was found perfectly segregatingwith the acromegaly/gigantism phenotype. Out of 45 patients from thepopulation-based acromegaly cohort 6 had Q14X mutation and one had anucleotide substitution IVS3-1G>A affecting the splice acceptor site of exon4. In the population-based cohort of patients with GH-secreting adenoma, PAPpatients (n=7) were significantly younger than mutation negative patients(n=38) (24.7 ± 10.7 versus 43.6 ± 11.9 years, P= 0.0003), but no differences intumour size or sex distribution were observed. In the population-based series,six of the fifteen patients diagnosed under 35 years of age had AIP mutation.Thus, in a population-based series from Northern Finland, two AIP mutationsaccount for 16% of all patients diagnosed with pituitary adenomas secretinggrowth hormone and for 40% of the subset of patients who were diagnosedwhen they were younger than 35 years of age. Another series of 10 unselectedFinnish sporadic acromegaly patients were screened and Q14X was found intwo of them, which is compatible with findings in the population-basedcohort. In addition, germline AIP mutation (R304X) was also found in Italiansiblings with GH-secreting pituitary adenoma. Loss of the wild-type allele wasdetected in eight tumours (five GH-secreting, one secreting both GH andprolactin, two prolactinomas) from mutation-positive individuals available forLOH-studies. This finding strengthened the notion that PAP is associated withpredisposition to prolactinoma, GH-secreting adenoma and bihormonal GHand prolactin secreting tumours. The results of LOH analyses also indicatedthat AIP is likely to act as a tumour suppressor.
Further work on the functional role of AIP should prove informative inrevealing key cellular processes involved in the genesis of pituitary adenomas,including potential drug targets. Future goals also include studying thephenotypic variation and penetrance of PAP, and the role of AIP mutations inpatients with both sporadic and familial pituitary adenomas outside Northern
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Finland. Predictive testing of AIP mutation carrier status will also need furtherstudying.
4. We examined 29 PHPT patients with features of genetic predisposition bymutation screening of 5 different genes including MEN1, HRPT2, CASR,CDKN1B and AIP and by serum screening of ionized calcium, PTH, prolactinand PP. Of the 29 patients, only one was found to carry a mutation in one ofthese genes. A female patient diagnosed with PHPT due to multiglandulardisease at 38 years of age and having recurrent PHPT and a family history ofPHPT displayed the founder mutation 1466del12 of MEN1. Based on ourstudy and those reported by others it appears that sporadic and familial PHPTpatients with multiglandular hyperplasia and young age at onset (below 40years) are good candidates for MEN1 mutation screening. It also seems thatCDKN1B and AIP may not have significant contribution to PHPTpredisposition. Our future work will focus on expanding our familial PHPTmaterial with subsequent genealogical and molecular analyses forpredisposition gene identification, and this and similar efforts elsewhereshould shed new light on the molecular basis of PHPT susceptibility.
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Original publicationsI I Vierimaa O, Ebeling TM, Kytölä S, Bloigu R, Eloranta E, Salmi J, Korpi-
Hyövälti E, Niskanen L, Orvola A, Elovaara E, Gynther A, Sane T, VälimäkiM, Ignatius J, Leisti J & Salmela PI (2007) Multiple endocrine neoplasia type1 in Northern Finland; clinical features and genotype phenotype correlation.European Journal of Endocrinology 157:285–94. © Society of the EuropeanJournal of Endocrinology (2007)
II Ebeling T, Vierimaa O, Kytölä S, Leisti J & Salmela PI (2004) Effect ofmultiple endocrine neoplasia type 1 (MEN1) gene mutations on prematuremortality in familial MEN1 syndrome with founder mutations. Journal ofClinical Endocrinology and Metabolism 89:3392–6. © The Endocrine Society(2004)
III Vierimaa O, Georgitsi M, Lehtonen R, Vahteristo P, Kokko A, Raitila A,Tuppurainen K, Ebeling TM, Salmela PI, Paschke R, Gündogdu S, De MenisE, Mäkinen MJ, Launonen V, Karhu A & Aaltonen LA (2006) Pituitaryadenoma predisposition caused by germline mutations in the AIP gene.Science 312:1228–30.
IV Vierimaa O, Villablanca A, Alimov A, Georgitsi M, Raitila A, Vahteristo P,Larsson C, Ruokonen A, Eloranta E, Ebeling TML, Ignatius J, Aaltonen LA.,Leisti J & Salmela PI. Mutation analysis of MEN1, HRPT2, CASR, CDKN1Band AIP genes in primary hyperparathyroidism patients with features ofgenetic predisposition. Submitted.
The permission of Society of the European Journal of Endocrinology (I), TheEndocrine Society (II) and The American Association for the Advancement ofScience (AAAS) (III) to reprint the original papers is gratefully acknowledged.
Original publications are not included in the electronic version of the dissertation.
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