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Genetics of Hypogonadotropic Hypogonadism. Lawrence C. Layman, M.D. Professor Chief, Section of Reproductive Endocrinology, Infertility, & Genetics Department of Obstetrics & Gynecology Neurobiology Program The Institute of Molecular Medicine & Genetics The Medical College of Georgia

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Genetics of Hypogonadotropic

Hypogonadism

Lawrence C. Layman, M.D.

Professor

Chief, Section of Reproductive Endocrinology,

Infertility, & Genetics

Department of Obstetrics & Gynecology

Neurobiology Program

The Institute of Molecular Medicine & Genetics

The Medical College of Georgia

Augusta, GA


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Genetics of IHH

  • Normal pubertal milestones

  • Idiopathic hypogonadotropic

  • hypogonadism (IHH)

  • 3. Mutations/phenotype

  • Hypothalamic:

  • KAL1, NROBI, FGFR1, LEP, LEPR

B. Pituitary:

GNRHR, PROP1, HESX1, FSHB, LHB


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Normal H-P-G Axis

HYPOTHALAMUS

GnRH

PITUITARY

FSH

LH

GONAD

Steroids Gametes


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Females:

Males:

Breasts: age 9-11

Pubic hair: 8-9

Growth spurt: 12

Menses: age 12

Testes: age 10-11

Pubic hair: 10-11

Penile growth: 13

Growth spurt: 14

Normal Pubertal Milestones


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Delayed Puberty

1) Females:

No breast development: age 13

No menses: age 15

2) Males:

No testes development: age 14


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Clinical Evaluation

Hypogonadism:

Low sex steroids

No pubertal development

Obtain serum gonadotropins

(LH and FSH)


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H-P-G Axis Dysfunction

HYPO

GnRH

PIT

Hypergonadotropic

Hypogonadism

FSH

LH

  • High FSH & LH

  • Low sex steroids

GONAD

Steroids Gametes


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Idiopathic Hypogonadotropic

Hypogonadism (IHH)

  • Irreversible, delayed puberty

  • Females: age 17 Amenorrhea

  • Males: age 18 Low T (< 100ng/dL)

  • Low FSH, LH

  • No CNS lesion

  • Normal prolactin, thyroid, adrenal function


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H-P-G Axis Dysfunction

HYPO

Hypogonadotropic

Hypogonadism

GnRH

PIT

  • Low FSH & LH

  • Low sex steroids

FSH

LH

GONAD

Steroids Gametes


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Gonadotropins in IHH

  • Gonadotropin responses to exogenous

  • GnRH variable

  • LH Pulsatility Patterns—serial samples

  • (every 10-20 minutes)

  • Apulsatile

  • Decreased frequency

  • Decreased amplitude

  • Nocturnal prepubertal pattern


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Prospects for Fertility

Hypogonadotropic Hypogonadism:

  • Induce secondary sex characteristics with

  • steroids (estrogen or testosterone)

  • Hypothalamic or pituitary

  • If pituitary failure, replace pituitary

  • hormones

  • Supply missing gonadotropins or GnRH

  • Good prognosis depending upon age

  • (20%/cycle)


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OMIM Entries with IHH (>40)

215470 Chorioretinal dystrophy, spinocerebellar

ataxia & HH

253320 Multicore myopathy with mental retardation,

short stature, & HH

212840 Cerebellar ataxia & HH

176270 Prader-Willi syndrome

176270 Fertile eunich syndrome

235200 Hemochromatosis (HFE)

602390 Hemochromatosis type (HFE2)

157900 Moebius syndrome

209900 Bardet-Biedl syndrome (BBS1-6)


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FGFR1

KAL1

HYPOTHALAMUS

GnRH

LEP/LEPR

NROB1

PITUITARY

PROP1

HESX1

FSH

LH

GONAD

Steroids

Gametes

GNRHR

FSHB

LHB


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GNRH1 Gene

  • Pivotal gene in reproduction

  • Expressed in:

1. Hypothalamus

2. Pituitary

3. Placenta

4. Ovary

5. Breast

  • Deficiency: hypogonadotropic hypogonadism


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Weiss et al. J Clin Endocrinol Metab 1989;69:299.

Layman et al. Fertil Steril 1992;57:42.

Nakayama et al. J Clin Endocrinol Metab 1990;70:1233.

IHH

1. Hypogonadal mouse:

Gnrh1 gene deletion

Mason et al. Science 1986;234:1372.

2. Human IHH: no GNRH1 gene

mutations


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  • IHH

  • Anosmia

  • Neurologic abnormalities:

synkinesia

visual abnormalities

  • Renal anomalies

  • Midfacial defects

Kallmann syndrome

X-linked recessive: males


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1. Anosmia

2. GnRH deficiency

Kallmann syndrome

  • GnRH & olfactory neurons migrate

    • from olfactory placode to hypothalamus

  • KAL1 gene: protein directs migration, so

  • if mutations

Franco et al. Nat 1991;353:529.

Legouis et al. Cell 1991;67:423.


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Kallmann Syndrome

  • KAL1 gene mutations in ~50% X-linked

  • families

  • Half of males with KAL1 mutations have

    • unilateral renal agenesis

(Hardelin et al. Hum Mol Genet 1993;2:373)

  • About 5% or less of unselected K.S.

    • males have KAL1 gene mutations

Bick et al. N Eng J Med 1992;326:1752.

Georgopoulos et al. J CEM 1997;82:213.

Layman et al. J Soc Gynecol Invest 1998


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Kallmann syndrome

ExpressionPhenotype

Olfactory bulb Anosmia

Cerebellum Nystagmus

Ataxia

Spinal cord (cort/spinal) Synkinesia

Oculomotor nucleus Eye movement

abnormalities

Retina Visual defects

Meso- & meta-

nephros Renal agenesis

Facial mesenchyme Cleft palate

Cartilage & Limb bud Club foot


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Prevalence of KAL1 Mutations

KAL1 mutations

  • Familial KS: 3/21 (14%)

  • Sporadic KS: 4/38 (11%)

  • Normosmic IHH: 0/42

Total KS: 7/59 (12%)

Total IHH patients: 7/101 (7%)

Oliveira et al. JCEM 2001;86:1532-8.


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Franco et al. Nat 1991;353:529.

Legouis et al. Cell 1991;67:423.

Kallmann syndrome

  • KAL1 on pseudoautosomal Xp

  • Inactive pseudogene on Yq

  • Encodes anosmin-1, a protein with

  • neural cell adhesion properties

  • Orthologs in chicks, zebrafish, C.

  • elegans, Drosophila

  • Not cloned in murine species yet, but

  • human Abs detect its presence

MacColl et al. Neuron 2002:34:675-8.


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Anosmin-1

  • C elegans ortholog (CeKal1) cloned

  • Required for ventral enclosure & male

  • ray (tail) formation during embryogenesis

  • Modulates branching of neurites

  • Human KAL1 cDNA can compensate for loss

  • of worm CeKal1 indicating function conserved

(Ruglari et al. Devel 2002;129:1283-94.)

  • Secreted molecule that binds via heparan sulfate

  • proteoglycan to its receptor to induce axon

  • branching and misrouting

Bulow et al PNAS 2002;99:6346-51.


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Kallmann syndrome

1) Absent LOT branches causes anosmia

2) Lack of GnRH neurons to forebrain causes

IHH

3) May be anosmia also because of lack of

primary contacts between olfactory axons

& OB anlage

Hypothesis: anosmin-1 in OB area exerts

attractive effect of olfactory receptor neurons

to create contact

Soussi-Yanicostas et al. Cell 2002;109:217-28.


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Zanaria et al. Nat 1994;372:635.

Muscatelli et al. Nat 1994;372:672.

Adrenal Hypoplasia Congenita (AHC)

Hypogonadotropic Hypogonadism (HH)

  • Adrenal failure in infancy to age 10

  • If survive, have delayed puberty (HH)

  • X-linked recessive

  • NROB1 gene (formerly DAX1) mutations,

  • steroid receptor, cause both AHC/HH

  • Adrenal, hypothalamic, pituitary develop

  • DSS region on Xp


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Del1219nt & Gly329Glu

  • Normal response to GnRH

  • (suggesting hypothalamic defect)

  • Minimal LH response during GnRH

  • priming (suggesting pituitary)

GGAT duplication codon 418

  • Normal FAS, no response to GnRH

  • (suggesting pituitary)

NROB1 (DAX1) Heterogeneity

Habiby et al. JCI 1996;98:1055.


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NROB1 (DAX1) in IHH

  • 106 IHH males (85 sporadic; 21 familial)

  • DNA sequencing of the coding region

No mutations

Conclusion: NROB1 mutations uncommon

in IHH patients without AHC

Achermann et al. JCEM 1999;84:4497-4500.


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NROB1 (DAX1) in Females

  • Mutation in female with HH (no AHC), who

  • had with skewed X-inactivation

  • Variabile expression within the family (both

  • males had HH/AHC)

Merke et al. NEJM 1999;340:1248-1252.

  • Female with HH & missense mutation? in NH2

ASHG 2002 meeting 10/02

  • Conditional KO: not ovarian determinant, but

  • instead important for spermatogenesis

Yu et al. Nat Genet 1998;20:353-357.


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Leptin Deficiency

Leptin deficient ob/ob mouse:

  • Obesity

  • Hyperinsulinemia

  • Infertility (20 to HH)

  • Hypothermia

  • Cold intolerance

  • Hypercortisolemia

Zhang et al. Nat 1994;372:425-432.


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(Montague et al. Nat 1997;387:903-908)

  • Causes obesity & HH

(Strobel et al. Nat Genet 1998;18:214-215.)

Human Leptin Deficiency

  • Normally: + correlation of BMI & leptin

  • Leptin deficiency rare in obesity


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LEP Gene Mutations & HH

Obese Male:

  • BMI = 55.8 kg/m2

  • Low serum leptin (0.9ng/mL)

  • Autosomal recessive

  • 2 sibs with similar phenotype

  • Mutant not secreted from cell

Strobel et al. Nat Genet 1998;18:214-215.


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Leptin Receptor Gene Mutation

  • Obesity and HH

  • Homozygous G to A in splice donor site

  • (exon skipping exon 16)

  • Protein truncated (lack transmembrane

  • intracellular domains)

Clement et al. Nat 1998;392:398-401


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FGFR1 Mutations

  • Autosomal dominant Kallmann syndrome

  • (IHH & anosmia)

  • Loss of function mutations in fibroblast

  • growth factor receptor 1 (FGFR1)

  • Also termed KAL2

  • Gain of function mutations cause cranio-

  • synostosis (Pfeiffer syndrome) & cranio

  • facial-skeletal dysplasia (Jackson-Weiss)

  • syndrome

Dode et al. Nat Genet 2003;33:463-465.


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FGFR1 Mutations

  • Identified 10-11Mb region on 8p11.2-p12 via

  • 2 patients with contiguous gene deletion

  • syndromes, who also had KS

  • Region had three genes—FGFR1 candidate

  • None of 43 patients had deletions (Southern)

  • 12/129 (9.3%) unrelated patients with KS

  • (91 males; 38 females) had mutations

  • Reduced penetrance & variable expressivity

  • Some patients with cleft palate/lip,

  • dentogenesis, synkinesis

Dode et al. Nat Genet 2003;33:463-465.


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FGFR1 & KAL1 Relationship

  • Could anosmin-1 (KAL1 protein) be the ligand

  • for FGFR1?

  • FGF interacts with the FGFR1 and heparan

  • sulfate proteoglycans (HSPGs)—necessary

  • for receptor dimerization &

  • autophosphorylation

  • Anosmin-1 binds to HSPGs

  • KAL1 expressed in olfactory bulbs & Fgfr1 is

  • expressed in rostral forebrain & required

  • for olfactory bulb evagination in mouse

Dode et al. Nat Genet 2003;33:463-465.


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GNRHR Gene Mutations

Partial IHH

  • Low LH, low FSH

  • Incomplete pubertal development

(deRoux et al. N Engl J Med 1997;337:1597-1602.)

Complete IHH

  • Low LH, low FSH

  • Absent pubertal development

  • No response to GnRH

(Layman e al. Nat Genet 1998;18:14-15.)


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Testosterone = 80 ng/dL (260-690)

Low FSH, LH

LH pulses: Nl frequency, amplitude

Semen analysis: 39 million/mL; 5% motile

GnRH Resistance

  • Proposed partial loss of function mutations

    • in GnRHR

  • 22 yr. old male with delayed puberty at 18,

    • decreased libido, small (8 cc) testes, small penis

Labs

  • de Roux et al. N Engl J Med 1997;337:1597.


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GnRH

Receptor binding

GnRH

GnRHR

Membrane

IP3 Production

2nd messenger


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Gln

Arg

Arg

Gln

(CAA)

(CGA)

(CGG)

(CAG)

GNRHR Mutations

Compound heterozygotes

Gln106Arg

Arg262Gln

Reduced binding

Reduced IP3

Reduced IP3

  • de Roux et al. N Engl J Med 1997;337:1597.


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GNRHR Gene Mutations in IHH

  • Variable response of FSH&LH to GnRH

  • suggested GNRHR mutations possible

  • Screened 46 IHH (32 males; 14 females)

  • for mutations using DGGE

  • 1 of 46 with GNRHR mutations (compound

    • heterozygote)

Layman LC, Cohen DP et al. Nat Genet 1998;18:14.


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Cys

Tyr

Arg

Gln

(TGT)

(TAT)

(CGG)

(CAG)

Tyr284Cys

Arg262Gln

receptor

20% WT

75% WT

expression

75%

40%

20X

10X

GNRHR Gene Mutations

Total IP3

EC50

Layman LC, Cohen DP et al. Nat Genet 1998;18:14.


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1

2

3

4

5

6

7

8

9

10

11

21

No

--

3.3

12.2

2.3

4.7

I

1

2

3

4

5

6

7

8

II

Age

30

29

17

Breasts

No

--

No

Testosterone

--

75

--

Basal LH

< 2.0

2.6

< 2.0

Stimulated LH

6.8

7.5

12.3

Basal FSH

1.6

< 2.0

3.3

Stimulated FSH

5.0

< 2.0

6.0

Layman LC, Cohen DP et al. Nat Genet 1998;18:14.


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GNRHR Gene Mutations in IHH

  • Variable response of FSH&LH to GnRH

  • suggested GNRHR mutations possible

  • Screened 46 IHH (32 males; 14 females)

  • for mutations using DGGE

  • 1 of 46 with GNRHR mutations (compound

    • heterozygote)

Layman LC, Cohen DP et al. Nat Genet 1998;18:14.


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Prevalence of GNRHR Mutations

Normosmic IHH: 1/46 (2.2%)

Normosmic IHH with female: 1/14 (7%)

Anosmic IHH males: 0/50*

*not included in final paper

Layman LC, Cohen DP et al. Nat Genet 1998;18:14.


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Prevalence of GNRHR Mutations

Normosmic IHH: 5/48 (10%)

a) Sporadic: 3/18 (16.7%)

b) Autosomal recessive: 2/5 (40%)

Anosmic/hyposmic IHH: 0/60

Beranova et al. JCEM 2001;86:1580-8.


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Prevalence of GNRHR Mutations

165 IHH unrelated probands screened by

denaturing gradient gel electrophoresis

with GC-clamps (>95% mutations)

  • 3/165 (1.8%) IHH patients

  • 1/15 (6.7%) if >2 affecteds/family

  • 2/38 (5.3%) if only female probands

Bhagavath et al. Endocr Soc 2003


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GNRHR Mutations

  • ~ 15 different mutations identified

  • Most compound HTZ

  • May affect binding and/or signal transduction

  • Phenotype varies from complete IHH to

  • partial IHH

  • 5) Patients do not have anosmia

  • 6) Gonadotropin response to GnRH is

  • variable (at least 1 pregnancy to GnRH)

  • 7) Prevalence is ~3-10% of normosmic IHH


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PROP1 Gene

  • Autosomal recessive form of combined

  • pituitary deficiency (short stature &

  • delayed puberty)

  • Deficiencies of GH, PRL, TSH, FSH, LH,

  • & ACTH

Wu et al. Nat Genet 1998;18:147-9.

  • 164 males & 20 females with IHH

  • No mutations identified

Park JL et al. Clin Endocrinol (In press).


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Septo-optic Dysplasia

  • Agenesis of corpus callosum, panhypopit,

  • optic nerve hypoplasia, absent septum

  • pellucidum

  • One form due to HESX1 gene mutations

  • HESX1 is homeobox gene expressed in

  • Rathke’s Pouch, pituitary primordium

  • Autosomal recessive, dominant

Dattani et al. Nat Genet 1998;19:125-133.


Slide49 l.jpg

Furui et al. JCEM 1994;78:107.

Haavisto et al. JCEM 1995;80:1257.

Suganuma et al. Fertil Steril 1995;63:989.

LHB Polymorphisms

  • Two LHB missense mutations same allele

  • (Trp8Arg & Ile15Thr)

  • In infertility and control patients

  • Does interfere with LH assay

1. Unmeasurable: IRMA (SPAC-S kit)

monoclonal Ab to whole molecule

2. Measurable: IMFMA (DELFIA):two

Abs against LHb


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Immuno- active, Bio- inactive LH

Male: delayed puberty at 17 yr.

Gynecomastia

Infantile penis

Small descended testes

Female distribution pubic hair

T= 30-80 ng/dL

LH = 30 mIU/mL

FSH = 26 mIU/mL

Labs:

Axelrod et al. JCEM 1979;48:279.


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Immuno- active, Bio- inactive LH

  • Exogenous T induced secondary sex

  • characteristics; then d/c

  • hCG restored adult phenotype &

  • sperm (1 million/cc after 2 mo. &

  • 11 million/cc, 50% motility, 50% nl)

  • T also increased to exogenous LH

  • Testicular Bx: maturation arrest, no Leydig

Axelrod et al. JCEM 1979;48:279.


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LHB Gene Mutation

  • Homozygous LHB gene missense mutation

  • in exon 3 (Gln54Arg)

1. Detected by dimer-specific IRMA

2. Undetectable by RRA

  • Mutant LH not capable of receptor binding

  • Autosomal recessive

  • Heterozygotes probably normal

Weiss et al. N Engl J Med 1992;326:179.


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Human FSHB Mutations: Females

  • No breast development or menses (1,2)

  • Partial breast development (3)

  • Low FSH, High LH

  • Low estradiol

  • Immature ovarian follicles (antral)

  • Infertility

1) Matthews et al. Nat Genet 1993;5:83-86.

2) Layman et al. N Engl J Med 1997;337:607-11.

3) Layman et al. JCEM 2002;87:3702-7.


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Human FSHB Mutations: Female

  • Low testosterone

  • No clinical effects (no hirsutism)

  • Clinical studies

FSH Testosterone

+LH

Layman et al. N Engl J Med 1997;337:607-11.

Barnes et al. N Engl J Med 2000;343:1197-98.

Barnes et al. Hum Reprod 2002;17:88-91.


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Human FSHb Mutations: Males

  • Normal puberty or absent puberty

  • Low FSH, High LH

  • Low or normal testosterone

  • Small testes

  • Azoospermia

  • Infertility

Lindstedt et al. Clin Chem Lab Med 1998;36:663-65.

Phillip et al. N Engl J Med 1998;338:1729-32.

Layman et al. JCEM 2002;87:3702-7.


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FSH Testosterone

Human FSHb Mutations: Male

  • Low testosterone

  • Azoospermia

+LH

Sperm

Phillip et al. N Engl J Med 1998;338:1729-32.


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FSH Levels in vitro Cell Lines

Immuno- ; Bio- Untrnsf = Untransfected cells.

100

FSH

mIU/mL

50

WT

Val61X

Cys51Gly

Tyr76X

Untrnsf.

Media

Layman et al. JCEM 2002;87:3702-7.


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Hypogonadotropic Hypogonadism

1. No GNRH1 gene mutations, so rare

2. KAL1: 10-15% male IHH patients

3. KAL1 gene expression explains associated

anomalies

4. FGFR1 mutations in 10% male KS?

5. NROB1 affect hypothalamic, pituitary,

adrenal function; M + F

6. GNRHR: variable phenotype:M + F

7. LEP & LEPR: obesity & HH

8. Most causes of inherited IHH unknown


Slide59 l.jpg

FGFR1

KAL1

HYPOTHALAMUS

GnRH

LEP/LEPR

NROB1

PITUITARY

PROP1

HESX1

FSH

LH

GONAD

Steroids

Gametes

GNRHR

FSHB

LHB


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