growth hormone releasing hormone receptor mutations

Growth Hormone Releasing Hormone Receptor Mutations

Gerhard P. Baumann

Northwestern University

International Symposium on IGF-1, GH and Ghrelin/GHS, Orlando, March 2011

NH2

Extracellular

Intracellular

COOH

Human GHRH ReceptorGPCR Class B

GHRHR Expression

- Pituitary somatotrophs

- Kidney

- Placenta

- Gonads

- Gut

- Hypothalamus

- Widely expressed at low levels in numerous tissues

Several splice variants exist in tumors and some non-tumorous

tissues. Their biological role is unclear.

Regulation of GHRHR Expression

- Pit-1- NF-1

- AP-1

- CREB

- glucocorticoids

- T3

- estrogen (negative)

GHRHR Signaling Pathways

- Gsa - adenylyl cyclase - cAMP - PKA

- Ca2+ - calmodulin

- PLC - DAG - PKC

- Arachidonic acid - eicosanoid

Human Growth Hormone Releasing Hormone (GHRH)

amide -

amide -

GHRH 1-44

GHRH 1-40

GHRH 1-29- amide

Effects of GHRH in somatotropes

- ProliferationResponsible for the major expansion of the somatotrope

population late in pituitary development

- GH biosynthesis

- GH release

The human GHRHR gene (15,511 bp)

Exon 1 2 3 4 5 6 7 8 9 10 11 12 13

3’5’

3’5’

Exon bp 105 103 108 98 98 133 154 61 70 92 130 42 413

Intron bp 4708 126 696 1154 746 1889 290 481 736 560 722 1796

AAAA

5’ 3’

TM # I II III IV V VI VII

1 2 3 4 5 6 7 8 9 10 11 12

7

p

q

14

ATG TAG

13

Signal

NH2

COOH

Asp Glymouse GHRHR

Mutations in human GHRHR (n = 21 mutant alleles, Feb 2011)

1 2 3 4 5 6 7 8 9 10 11 12 13

3’5’

3’5’

5’ 3’

TM # I II III IV V VI VII

1 2 3 4 5 6 7 8 9 10 11 12 13

Signal

–166(ATG) t > c

–164(ATG) t > c

–124(ATG) a > c

IVS1+1 g > a

IVS1+2 t > c IVS3+1 g > a IVS7+1 g > c

c.1146 G > A

( E322E ) IVS12+2 t > a

V10G

Q43X E72X

H137L L144H

A176V

A222E F242C K329E R357C

D4 D5

I I Y

H G V T

S I S

L A V I

F AV

L T II

V A L

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PP

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SES F H S F F D P C

PL

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F A

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A E A

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W L

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WW

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TN

PM

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H M H NH2

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E L G

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PW A

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DP

ELLRTRAKWTTPSRSAAKL VTSMC

Gln StopCanada

Little

GlyAsp

Sindh

Glu StopCanada

HisLeu

Spain

USA

Brazil

GluAla

Pakistan CysPhe

USA

Morocco

GluLys

Portugal

Arg Cys

Israel

D4

Japan

D5

Canada

Glu Glu

Japan

Pakistan

Japan, Portugal, Brazil, Morocco, ItalyPromoter, , Val Gly

Splice donor site mutation

Splice silencer mutation

LeuHis

Canada

GHRHR mutations: Mechanism of inactivation

Nonsense mutations (n=2) Truncation, nonsense-mediated mRNA decay

Splice site mutations (n=5) Intronic readthrough, premature stop

Exonic splicing silencer mutation (n=1) Exon skipping

Promoter mutations (n=3) Decreased Pit-1 binding to P1 or P2 elements

Missense mutations (n=8) Loss of GHRH binding activity (n=6)

Retention in ER (V10G)

Not determined (R357E)

Alba & Salvatori, J Endo 2005

Activating mutations in human GHRHR ?

To date, no gain of function mutations in the GHRHR have

been described.

Extensive screening of over 130 somatotrope tumors for

somatic activating mutations in GHRHR, as a potential

basis for constitutive proliferative drive, has not discovered

any such mutations.

However, two somatic inactivating mutations (W250X and

G294R) have been described in GH-producing pituitary

tumors.

A number of polymorphisms without clinical significance are

known to exist.

Clinical manifestations

Dr. Aguiar-Oliveira and the Itabaianinha cohort

Clinical manifestations (cont.)

Mode of inheritance: autosomal recessive (homozygous or compound heterozygous).

Very high penetrance

Two large kindreds with high degrees of consanguinity exist in

Pakistan (Dwarfs of Sindh) and in Itabaianinha, Brazil.

Founder mutations are implicated in both.

Heterozygous carriers are physically normal and biochemically

intermediate to near-normal.

Phenotype: Isolated GH deficiency type IB

– postnatal growth retardation → dwarfism

– some basal GH production

– other pituitary functions normal

– delayed puberty, menopause at normal age

– other features typical for GH deficiency (some exceptions)

Pituitary hypoplasia (anterior lobe)

Relative microcephaly (Head circumference -4.1 ± 0.2 SDS)

No. of cases reported: ~ 180 (~100 in Itabaianinha; ~45 in Pakistan/India)


Short stature: Full impact of mutation seen only in adults

Adult height (Sindh): Male 130 ± 10.6 cm (118-148) -8.9 ± 1.2 SDS

Female 113.5 cm (113-114) -8.4 SDS

(Brazil): Male 127 ± 7.6 cm (110-140) -7.6 ± 0.9 SDS

Female 119 ± 8.5 cm (107-129) -7.8 ± 0.9 SDS

Upper-lower segment and height-arm span ratio: Normal

Waist-hip ratio (Sindh): 0.92 ± 0.05 (elevated)

(Brazil): 0.97 ± 0.02 (controls 0.90 ± 0.02)

Bone age delayed

Anthropometric and clinical data

- No or minimal dysmorphism

- No microphallus

- No hypoglycemia

Biochemical Data

Sindh Itabaianinha

Fasting GH: 0.070 ± 0.054 ng/ml (low-normal) 0.102 ± 0.063 ng/ml

IGF-I: 5.2 ± 0.4 ng/ml (very low) 6.7 ± 2.8 ng/ml

IGF-II 86 ± 22 ng/ml (low) 142 ± 14 ng/ml

IGF-BP3: 0.42 ± 0.13 mg/ml (very low) 0.43 ± 0.17 mg/ml

IGF-BP2: 440 ± 123 ng/ml (high) 661 ± 30 ng/ml

ALS: --- (very low) 916 ± 73 ng/ml


Anterior Pituitary Hypoplasia

Lin & Rosenfeld, Nature 1993

Little mouse

Ant. pituitary volume

330 ± 96 mm3

103 ± 21 mm3

normal

Is there a genotype-phenotype relationship?

Insufficient data

- Extensive information only available for 2 severely inactivating

(null) mutations

- Other, potentially milder mutations only phenotyped in 1-3 patients

- Patients phenotyped at varying ages are difficult to compare

- Functional differences shown in vitro (e.g., cAMP generation)

not always borne out in vivo

Probably

Novel insights gained into GH biology

Regulation of GH secretion

- Generation of secretory pulses

- Role of GHS

Natural history of life-long, severe, untreated, isolated GH deficiency

- Cardiovascular disease

- Bone and skeletal disease

- Quality of life

- Life span

Clock Time

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SLEEP

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Patient 1

GH Secretion Rate

GH Secretion Profiles in GHRH-Receptor Deficiency

GH Secretion in GHRH-Receptor Deficiency

Patient 24 h mean Number of Half-life GH productionnumber plasma GH peaks/24 h rate

( ng/ml) (min) (ng/ml/24 h)

1 0.052 18 14.6 3.41

2 0.020 --- ---- ----

3 0.021 16 16.1 1.42

4 0.046 12 15.7 2.95

Mean 0.035 15.3 15.5 2.59

Normal mean 1.394 19.0 16.5 80.2

Response to hexarelin in GHRH-R Deficiency

Minutes

-15 0 15 30 45 60 90 120

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#1

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Hexarelin

Cardiovascular risk factors in Itabaianinha cohort

Abdominal fat increased

Blood pressure increased

LDL-cholesterol increased

C-reactive protein increased

No carotid intima-media thickening

No increased cardiovascular deaths

GH treatment increases carotid intima-media thickening

Bone mineral density

Areal BMD at lumbar spine, femoral neck, forearm and total body

is decreased by DEXA (mean Z scores -1.7 to – 3.7) (Sindh)

BMD at calcaneus is decreased by quantitative ultrasound (mean Z score -1.15)

(Itabaianinha).

No evidence for an increased fracture rate.

GH therapy for 6 months mildly improved BMD (Z score -0.78)

Potential artifact due to small bone size is not accounted for.

Lumbar BMD in Genetic GHRH-Receptor Deficiency

Patient Areal BMD Z score Volumetric BMD* Z scorenumber (g/cm2) (g/cm3)

1 0.564 –4.8 0.100 –1.90

2 0.820 –2.5 0.139 1.62

3 0.703 –3.5 0.117 –0.61

4 0.845 –2.2 0.142 1.88

Mean 0.733 –3.25 0.124 0.25

SD 0.128 1.20 0.020 1.80

Normal mean 0.159

* Volumetric BMD (BMAD) = BMC/(area)3/2

Quality of Life in Itabaianinha cohort

QoL measured by Life Satisfaction Hypopituitarism Module (QLS-H)

- No difference between GH deficient and control subjects

- No improvement in QoL after 6 months of GH treatment

(exception: satisfaction with improved physical endurance)

- No difference after 12 months washout time

Life span in GH deficiency

- GH-deficient and GH-resistant rodents have a prolonged life span.

(little mouse, GHR-KO mouse, Snell, Ames mouse, spontaneous dwarf rat, etc.)

- The Krk dwarfs (harboring a PROP-1 mutation) are said to live to unusually old age.

- Acquired adult GH deficiency is associated with a shortened life span.

- GH is frequently portrayed as an anti-aging agent → beneficial?

- A Swiss patient cohort with a GH-1 gene deletion had a shortened life span.

- How to reconcile these different observations?

Longevity in untreated GHRHR Deficiency

Aguiar-Oliveira et al, JCEM 2010

All ages Age >20

Summary: Genetic GHRH-Receptor Deficiency

Twenty-one mutant alleles reported, all are inactivating

Recessive inheritance, 100% penetrance

May account for ~10% of familial isolated GH deficiency

Heterozygotes show normal height and mild biochemical and body composition abnormalities

Phenotype:

- Isolated GH deficiency type IB

- Ultradian GH rhythm persists, with severely decreased pulse amplitudes, but normal pulse frequency

- Good response to GH therapy

- Proportionate short stature

- Relative eumorphism

- Relative microcephaly, apparent normal intelligence


Phenotype, continued:

- Anterior pituitary hypoplasia (somatotrope deficiency)

- Absence of childhood hypoglycemia

- Characteristic high-pitched, raspy voice in males

- Absence of microphallus

- Delayed puberty (2-3 years)

- Fertility and lactation appear normal

Parity is low, possibly because of late pairing and obstetricalconsiderations

- Probably normal or near-normal bone mineral density

- Systolic hypertension (Itabaianinha people)


Phenotype, continued:

- Increased % fat (abdominal), low lean body mass

- Increased LDL cholesterol, C-reactive protein

- Normal carotid intima/media thickness

- No phenotypic evidence of GHRH resistance in extrapituitary tissues, despite widespread expression of GHRH and the GHRH-receptor (exception: relative microcephaly)

growth hormone releasing hormone receptor mutations

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