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Genetics of Hypogonadotropic Hypogonadism

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

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  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 FSH LH GONAD Steroids Gametes

  4. 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

  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 FSH LH • 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 (< 100ng/dL) • 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 FSH LH GONAD Steroids Gametes

  10. 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

  11. 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)

  12. 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)

  13. FGFR1 KAL1 HYPOTHALAMUS GnRH LEP/LEPR NROB1 PITUITARY PROP1 HESX1 FSH LH GONAD Steroids Gametes GNRHR FSHB LHB

  14. GNRH1 Gene • Pivotal gene in reproduction • Expressed in: 1. Hypothalamus 2. Pituitary 3. Placenta 4. Ovary 5. Breast • Deficiency: hypogonadotropic hypogonadism

  15. 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

  16. IHH • Anosmia • Neurologic abnormalities: synkinesia visual abnormalities • Renal anomalies • Midfacial defects Kallmann syndrome X-linked recessive: males

  17. 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.

  18. 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

  19. 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

  20. 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.

  21. 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.

  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 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.

  23. 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.

  24. 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

  25. 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.

  26. 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.

  27. 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.

  28. 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.

  29. 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.) Human Leptin Deficiency • Normally: + correlation of BMI & leptin • Leptin deficiency rare in obesity

  30. C T (Arg105Trp) 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.

  31. 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

  32. 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.

  33. 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.

  34. 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.

  35. 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.)

  36. 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.

  37. GnRH Receptor binding GnRH GnRHR Membrane IP3 Production 2nd messenger

  38. 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.

  39. 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.

  40. 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.

  41. 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.

  42. 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.

  43. 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.

  44. 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.

  45. 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

  46. 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

  47. 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).

  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 • Rathke’s Pouch, pituitary primordium • Autosomal recessive, dominant Dattani et al. Nat Genet 1998;19:125-133.

  49. 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

  50. 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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