Genetics of HypogonadotropicHypogonadism

Lawrence C. Layman, M.D.ProfessorChief, Section of Reproductive Endocrinology,

Infertility, & GeneticsDepartment of Obstetrics & GynecologyNeurobiology ProgramThe Institute of Molecular Medicine & GeneticsThe Medical College of GeorgiaAugusta, GA

Genetics of IHHGenetics of IHH

1. Normal pubertal milestones2. Idiopathic hypogonadotropic

hypogonadism (IHH)3. Mutations/phenotype

A.Hypothalamic:KAL1, NROBI, FGFR1, LEP, LEPR

B. Pituitary: GNRHR, PROP1, HESX1, FSHB, LHB

LHFSH

GnRH

HYPOTHALAMUS

PITUITARY

GONAD

Steroids Gametes

Normal H-P-G AxisNormal H-P-G Axis

Normal Pubertal Milestones

Females: Breasts: age 9-11Pubic hair: 8-9Growth spurt: 12Menses: age 12

Males:Testes: age 10-11Pubic hair: 10-11Penile growth: 13Growth spurt: 14

Delayed Puberty

1) Females:

No breast development: age 13No menses: age 15

2) Males:

No testes development: age 14

Hypogonadism:Low sex steroidsNo pubertal development

Obtain serum gonadotropins(LH and FSH)

Clinical Evaluation

LHFSH

GnRH

HYPO

PIT

GONAD

Steroids Gametes

H-P-G Axis DysfunctionH-P-G Axis Dysfunction

HypergonadotropicHypogonadism

• High FSH & LH• Low sex steroids

• Irreversible, delayed puberty Females: age 17 AmenorrheaMales: age 18 Low T (< 100ng/dL)

• Low FSH, LH • No CNS lesion• Normal prolactin, thyroid, adrenal function

Idiopathic HypogonadotropicHypogonadism (IHH)

LHFSH

GnRH

HYPO

PIT

GONAD

Steroids Gametes

HypogonadotropicHypogonadism

H-P-G Axis DysfunctionH-P-G Axis Dysfunction

• Low FSH & LH• Low sex steroids

Gonadotropins in IHH

• Gonadotropin responses to exogenous GnRH variable

• LH Pulsatility Patterns—serial samples(every 10-20 minutes)

1) Apulsatile2) Decreased frequency3) Decreased amplitude4) Nocturnal prepubertal pattern

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)

OMIM Entries with IHH (>40)215470 Chorioretinal dystrophy, spinocerebellar

ataxia & HH253320 Multicore myopathy with mental retardation,

short stature, & HH212840 Cerebellar ataxia & HH176270 Prader-Willi syndrome176270 Fertile eunich syndrome235200 Hemochromatosis (HFE)602390 Hemochromatosis type (HFE2)157900 Moebius syndrome209900 Bardet-Biedl syndrome (BBS1-6)

GNRHR

NROB1LEP/LEPR

LHFSH

HYPOTHALAMUS

PITUITARY

GONAD

SteroidsGametes

GnRH

LHBFSHB

PROP1

HESX1

FGFR1

KAL1

GNRH1 Gene

• Pivotal gene in reproduction

• Expressed in: 1. Hypothalamus2. Pituitary3. Placenta4. Ovary5. Breast

• Deficiency: hypogonadotropic hypogonadism

IHH

1. Hypogonadal mouse: Gnrh1 gene deletion

2. Human IHH: no GNRH1 genemutations

Mason et al. Science 1986;234:1372.

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.

Kallmann syndrome• IHH• Anosmia• Neurologic abnormalities:

synkinesiavisual abnormalities

• Renal anomalies• Midfacial defects

X-linked recessive: males

Kallmann syndrome

• GnRH & olfactory neurons migrate from olfactory placode to hypothalamus

• KAL1 gene: protein directs migration, so if mutations 1. Anosmia

2. GnRH deficiency

Franco et al. Nat 1991;353:529.Legouis et al. Cell 1991;67:423.

Kallmann Syndrome• KAL1 gene mutations in ~50% X-linked

families• Half of males with KAL1 mutations have

unilateral renal agenesis

• About 5% or less of unselected K.S. males have KAL1 gene mutations

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

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

Olfactory bulb AnosmiaCerebellum Nystagmus

AtaxiaSpinal cord (cort/spinal) SynkinesiaOculomotor nucleus Eye movement

abnormalitiesRetina Visual defectsMeso- & meta- nephros Renal agenesisFacial mesenchyme Cleft palateCartilage & Limb bud Club foot

Expression PhenotypeKallmann syndromeKallmann syndrome

Prevalence of KAL1 Mutations

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

KAL1 mutations

1) Familial KS: 3/21 (14%)2) Sporadic KS: 4/38 (11%)3) Normosmic IHH: 0/42

Total KS: 7/59 (12%)Total IHH patients: 7/101 (7%)

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, buthuman Abs detect its presence

Franco et al. Nat 1991;353:529.Legouis et al. Cell 1991;67:423.

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

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

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

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

• Secreted molecule that binds via heparan sulfateproteoglycan to its receptor to induce axonbranching and misrouting

Kallmann syndrome

1) Absent LOT branches causes anosmia2) Lack of GnRH neurons to forebrain causes

IHH3) May be anosmia also because of lack of

primary contacts between olfactory axons& OB anlage

Hypothesis: anosmin-1 in OB area exertsattractive effect of olfactory receptor neuronsto create contact

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

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

Zanaria et al. Nat 1994;372:635.Muscatelli et al. Nat 1994;372:672.

NROB1 (DAX1) Heterogeneity

• Normal response to GnRH (suggesting hypothalamic defect)

• Minimal LH response during GnRHpriming (suggesting pituitary)

Del1219nt & Gly329Glu

Habiby et al. JCI 1996;98:1055.

GGAT duplication codon 418• Normal FAS, no response to GnRH

(suggesting pituitary)

NROB1 (DAX1) in IHH

• 106 IHH males (85 sporadic; 21 familial)• DNA sequencing of the coding region

No mutations

ConclusionConclusion: NROB1 mutations uncommonin IHH patients without AHC

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

NROB1 (DAX1) in Females

• Mutation in female with HH (no AHC), whohad with skewed X-inactivation

• Variabile expression within the family (bothmales had HH/AHC)Merke et al. NEJM 1999;340:1248-1252.

• Female with HH & missense mutation? in NH2ASHG 2002 meeting 10/02

• Conditional KO: not ovarian determinant, butinstead important for spermatogenesis

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

Leptin DeficiencyLeptin Deficiency Leptin deficient ob/ob mouse:

• Obesity• Hyperinsulinemia• Infertility (20 to HH)• Hypothermia• Cold intolerance• Hypercortisolemia

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

Human Leptin DeficiencyHuman Leptin Deficiency

• LEP deficiency causes early onset obesity(Montague et al. Nat 1997;387:903-908)

• Causes obesity & HH(Strobel et al. Nat Genet 1998;18:214-215.)

• Normally: + correlation of BMI & leptin

• Leptin deficiency rare in obesity

LEP Gene Mutations & HH

Obese Male:• BMI = 55.8 kg/m2• Low serum leptin (0.9ng/mL)

• C T (Arg105Trp)•Autosomal recessive• 2 sibs with similar phenotype• Mutant not secreted from cell

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

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

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)

FGFR1 Mutations

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

• 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) & craniofacial-skeletal dysplasia (Jackson-Weiss)syndrome

FGFR1 Mutations

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

• Identified 10-11Mb region on 8p11.2-p12 via2 patients with contiguous gene deletionsyndromes, 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

FGFR1 & KAL1 Relationship

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

• Could anosmin-1 (KAL1 protein) be the ligandfor FGFR1?

• FGF interacts with the FGFR1 and heparansulfate proteoglycans (HSPGs)—necessaryfor receptor dimerization & autophosphorylation

• Anosmin-1 binds to HSPGs

• KAL1 expressed in olfactory bulbs & Fgfr1 isexpressed in rostral forebrain & requiredfor olfactory bulb evagination in mouse

GNRHR Gene MutationsGNRHR Gene Mutations

Partial IHH

Complete IHH

• Low LH, low FSH• Incomplete pubertal development

• Low LH, low FSH• Absent pubertal development• No response to GnRH

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

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

GnRH ResistanceGnRH Resistance

• 22 yr. old male with delayed puberty at 18,decreased libido, small (8 cc) testes, small penis

• Proposed partial loss of function mutationsin GnRHR

Labs

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

Testosterone = 80 ng/dL (260-690)Low FSH, LH LH pulses: Nl frequency, amplitudeSemen analysis: 39 million/mL; 5% motile

GnRH

IP3 Production

GnRHR

GnRH

Membrane

Receptor binding

2nd messenger

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

GNRHR MutationsCompound heterozygotes

Gln106Arg Arg262Gln

Gln(CAA) (CGA)

Arg Gln(CAG)(CGG)

Arg

Reduced binding Reduced IP3Reduced IP3

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

GNRHR Gene Mutations in IHHGNRHR Gene Mutations in IHH

• Variable response of FSH&LH to GnRHsuggested GNRHR mutations possible

• Screened 46 IHH (32 males; 14 females)for mutations using DGGE

• 1 of 46 with GNRHR mutations (compoundheterozygote)

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

GNRHR Gene Mutations

Total IP3

EC50

Tyr284Cys

Cys(TGT)(TAT)

Tyr

20% WT

75%

20X

Arg262Gln

Gln(CAG)(CGG)

Arg

75% WT

40%

10X

receptorexpression

I

II1 2 3 4 5 6 7 8

7 81 2 6543 9 10 11

Age

Breasts

Testosterone

Basal LH

Stimulated LH

Basal FSH

6.0

17

No

--

< 2.0

12.3

3.3

Stimulated FSH

30

No

--

< 2.0

6.8

1.6

5.0

29

--

75

2.6

7.5

< 2.0

< 2.0

21

No

--

3.3

12.2

2.3

4.7

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

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

GNRHR Gene Mutations in IHHGNRHR Gene Mutations in IHH

• Variable response of FSH&LH to GnRHsuggested GNRHR mutations possible

• Screened 46 IHH (32 males; 14 females)for mutations using DGGE

• 1 of 46 with GNRHR mutations (compoundheterozygote)

Prevalence of GNRHR Mutations

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

Normosmic IHH: 1/46 (2.2%)Normosmic IHH with female: 1/14 (7%)Anosmic IHH males: 0/50*

*not included in final paper

Prevalence of GNRHR Mutations

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

Normosmic IHH: 5/48 (10%)a) Sporadic: 3/18 (16.7%)b) Autosomal recessive: 2/5 (40%)

Anosmic/hyposmic IHH: 0/60

Prevalence of GNRHR Mutations

Bhagavath et al. Endocr Soc 2003

• 3/165 (1.8%) IHH patients• 1/15 (6.7%) if >2 affecteds/family• 2/38 (5.3%) if only female probands

165 IHH unrelated probands screened bydenaturing gradient gel electrophoresiswith GC-clamps (>95% mutations)

GNRHR Mutations

1) ~ 15 different mutations identified2) Most compound HTZ3) May affect binding and/or signal transduction4) Phenotype varies from complete IHH to

partial IHH5) Patients do not have anosmia6) Gonadotropin response to GnRH is

variable (at least 1 pregnancy to GnRH)7) Prevalence is ~3-10% of normosmic IHH

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

PROP1 Gene

• Autosomal recessive form of combinedpituitary 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

Septo-optic Dysplasia

• Agenesis of corpus callosum, panhypopit,optic nerve hypoplasia, absent septumpellucidum

• 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.

Furui et al. JCEM 1994;78:107.Haavisto et al. JCEM 1995;80:1257.Suganuma et al. Fertil Steril 1995;63:989.

• Two LHB missense mutations same allele(Trp8Arg & Ile15Thr)

• In infertility and control patients• Does interfere with LH assay

LHB Polymorphisms

1. Unmeasurable: IRMA (SPAC-S kit)monoclonal Ab to whole molecule

2. Measurable: IMFMA (DELFIA):twoAbs against LH

Male: delayed puberty at 17 yr.GynecomastiaInfantile penisSmall descended testesFemale distribution pubic hair

Immuno- active, Bio- inactive LH Immuno- active, Bio- inactive LH

Axelrod et al. JCEM 1979;48:279.

Labs: T= 30-80 ng/dLLH = 30 mIU/mLFSH = 26 mIU/mL

Immuno- active, Bio- inactive LHImmuno- active, Bio- inactive LH

Axelrod et al. JCEM 1979;48:279.

• Exogenous T induced secondary sexcharacteristics; 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

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

LHB Gene Mutation• Homozygous LHB gene missense mutation

in exon 3 (Gln54Arg)

1. Detected by dimer-specific IRMA2. Undetectable by RRA

• Mutant LH not capable of receptor binding• Autosomal recessive• Heterozygotes probably normal

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.

• Low testosterone • No clinical effects (no hirsutism)• Clinical studies

Human FSHB Mutations: Female

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

FSH Testosterone

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

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

+LH

Human FSH 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.

• Low testosterone • Azoospermia

Human FSH Mutations: Male

FSH TestosteroneSperm

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

+LH

Immuno- ; Bio- Untrnsf = Untransfected cells.

Val61X Tyr76X MediaUntrnsf.Cys51Gly

50

100

FSHmIU/mL

WT

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

FSH Levels in vitro Cell Lines

Hypogonadotropic HypogonadismHypogonadotropic Hypogonadism

1. No GNRH1 gene mutations, so rare2. KAL1: 10-15% male IHH patients3. KAL1 gene expression explains associated

anomalies4. FGFR1 mutations in 10% male KS? 5. NROB1 affect hypothalamic, pituitary,

adrenal function; M + F6. GNRHR: variable phenotype:M + F 7. LEP & LEPR: obesity & HH8. Most causes of inherited IHH unknown

GNRHR

NROB1LEP/LEPR

LHFSH

HYPOTHALAMUS

PITUITARY

GONAD

SteroidsGametes

GnRH

LHBFSHB

PROP1

HESX1

FGFR1

KAL1