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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