Genetics of Hypogonadotropic

1
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
2
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
3
Normal H-P-G Axis
HYPOTHALAMUS
GnRH
PITUITARY
LH
FSH
GONAD
Steroids Gametes
4
Normal Pubertal Milestones
5
Delayed Puberty
1) Females
No breast development age 13 No menses age 15
2) Males
No testes development age 14
6
Clinical Evaluation
Hypogonadism Low sex steroids No pubertal
development
Obtain serum gonadotropins
(LH and FSH)
7
H-P-G Axis Dysfunction
HYPO
GnRH
PIT
Hypergonadotropic Hypogonadism
LH
FSH

• High FSH LH
• Low sex steroids

GONAD
Steroids Gametes
8
Idiopathic Hypogonadotropic Hypogonadism (IHH)

• Irreversible, delayed puberty
• Females age 17 Amenorrhea
• Males age 18 Low T (
• Low FSH, LH
• No CNS lesion
• Normal prolactin, thyroid, adrenal function

9
H-P-G Axis Dysfunction
HYPO
Hypogonadotropic Hypogonadism
GnRH
PIT

• Low FSH LH
• Low sex steroids

LH
FSH
GONAD
Steroids Gametes
10
Gonadotropins in IHH

• Gonadotropin responses to exogenous
• GnRH variable
• LH Pulsatility Patternsserial 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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GNRHR
LHB
FSHB
14
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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IHH
1. Hypogonadal mouse Gnrh1 gene deletion
Mason et al. Science 19862341372.
2. Human IHH no GNRH1 gene mutations
16
Kallmann syndrome
X-linked recessive males
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Kallmann syndrome

• GnRH olfactory neurons migrate
• from olfactory placode to hypothalamus

• KAL1 gene protein directs migration, so
• if mutations

Franco et al. Nat 1991353529.
Legouis et al. Cell 199167423.
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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 19932373)

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

Bick et al. N Eng J Med 19923261752.
Georgopoulos et al. J CEM 199782213.
Layman et al. J Soc Gynecol Invest 1998
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Kallmann syndrome

Expression Phenotype
Olfactory bulb Anosmia Cerebellum Nystagmus
Ataxia Spinal cord (cort/spinal) Synkinesia Ocu
lomotor nucleus Eye movement abnormalities R
etina 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 2001861532-8.
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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 200234675-8.
22
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 20021291283-94.)

• Secreted molecule that binds via heparan sulfate
• proteoglycan to its receptor to induce axon
• branching and misrouting

Bulow et al PNAS 2002996346-51.
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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 2002109217-28.
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Adrenal Hypoplasia Congenita (AHC) Hypogonadotropi
c 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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NROB1 (DAX1) Heterogeneity
Habiby et al. JCI 1996981055.
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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 1999844497-4500.
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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 19993401248-1252.

• 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 199820353-357.
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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 1994372425-432.
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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 199818214-215.
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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 1998392398-401
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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 200333463-465.
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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 genesFGFR1 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 200333463-465.
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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 200333463-465.
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GNRHR Gene Mutations
Partial IHH

• Low LH, low FSH
• Incomplete pubertal development

(deRoux et al. N Engl J Med 19973371597-1602.)
Complete IHH

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

(Layman e al. Nat Genet 19981814-15.)
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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 19973371597.
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GnRH
Receptor binding
GnRH
GnRHR
Membrane
IP3 Production
2nd messenger
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GNRHR Mutations
Compound heterozygotes
Gln106Arg
Arg262Gln
Reduced binding
Reduced IP3
Reduced IP3
de Roux et al. N Engl J Med 19973371597.
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GNRHR Gene Mutations in IHH

• Variable response of FSHLH 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 19981814.
40
GNRHR Gene Mutations
Total IP3
EC50
Layman LC, Cohen DP et al. Nat Genet 19981814.
41
I
1
2
3
4
5
6
7
8
II
Age
17
30
29
Breasts
No
No
--
Testosterone
--
--
75
Basal LH


2.6
Stimulated LH
12.3
6.8
7.5
Basal FSH
3.3
1.6

6.0
Stimulated FSH
5.0

Layman LC, Cohen DP et al. Nat Genet 19981814.
42
GNRHR Gene Mutations in IHH

• Variable response of FSHLH 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 19981814.
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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 19981814.
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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 2001861580-8.
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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 199818147-9.

• 164 males 20 females with IHH
• No mutations identified

Park JL et al. Clin Endocrinol (In press).
48
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
• Rathkes Pouch, pituitary primordium
• Autosomal recessive, dominant

Dattani et al. Nat Genet 199819125-133.
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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 197948279.
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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 197948279.
52
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 1992326179.
53
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 1993583-86.
2) Layman et al. N Engl J Med 1997337607-11.
3) Layman et al. JCEM 2002873702-7.
54
Human FSHB Mutations Female

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

FSH Testosterone
LH
Layman et al. N Engl J Med 1997337607-11.
Barnes et al. N Engl J Med 20003431197-98.
Barnes et al. Hum Reprod 20021788-91.
55
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 199836663-65.
Phillip et al. N Engl J Med 19983381729-32.
Layman et al. JCEM 2002873702-7.
56
Human FSHb Mutations Male

• Low testosterone
• Azoospermia

LH
Sperm
Phillip et al. N Engl J Med 19983381729-32.
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FSH Levels in vitro Cell Lines
100
FSH mIU/mL
50
Val61X
Tyr76X
Media
Untrnsf.
Cys51Gly
WT
Layman et al. JCEM 2002873702-7.
58
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 phenotypeM F 7. LEP
LEPR obesity HH 8. Most causes of inherited
IHH unknown
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GNRHR
LHB
FSHB