# Presentation: 353718

## 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**

## 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**

## Normal H-P-G Axis

**FSH**

**GnRH**

**HYPOTHALAMUS**

**PITUITARY**

**GONAD**

**Steroids   Gametes**

_**Normal H-P-G Axis**_

## Normal Pubertal Milestones

## Delayed Puberty

**1) **_**Females**_**: **

**No breast development: age 13**
**No menses: age 15**

**2) **_**Males**_**:**

**No testes development: age 14**

## Clinical Evaluation

**	****Low sex steroids**
**	****No pubertal development**

**Obtain serum gonadotropins**

**(LH and FSH)**

**Clinical Evaluation**

## H-P-G Axis Dysfunction

**FSH**

**GnRH**

**HYPO**

**PIT**

**GONAD**

**Steroids   Gametes**

_**H-P-G Axis Dysfunction**_

**Hypergonadotropic**
**Hypogonadism**

** ****High FSH & LH**
** ****Low sex steroids**

## Idiopathic Hypogonadotropic

**	**_**Females**_**: age 17****	**** Amenorrhea**
**	**_**Males**_**: age 18 ****	**** Low T (< 100ng/dL)**
** ****Low FSH, LH **
** ****No CNS lesion**
** ****Normal prolactin, thyroid, adrenal function**

**Idiopathic Hypogonadotropic**
**Hypogonadism (IHH)**

## H-P-G Axis Dysfunction

**FSH**

**GnRH**

**HYPO**

**PIT**

**GONAD**

**Steroids   Gametes**

**Hypogonadotropic**
**Hypogonadism**

_**H-P-G Axis Dysfunction**_

** ****Low FSH & LH**
** ****Low sex steroids**

## Gonadotropins in IHH

**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**

## 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 & 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)**

## GNRHR

**GNRHR**

**LHB**

**FSHB**

## GNRH1 Gene

** ****Pivotal gene in reproduction**

** ****Expressed in:**

**1. Hypothalamus**
**2. Pituitary**
**3. Placenta**
**4. Ovary**
**5. Breast**

** ****Deficiency: hypogonadotropic hypogonadism**

## IHH

**1. Hypogonadal mouse: **
**	****Gnrh1 gene deletion**

**2. Human IHH: no GNRH1 gene**
**	****mutations**

**Mason et al. Science 1986;234:1372.**

## Kallmann syndrome

**X-linked recessive: males**

## 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.**

## 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**

## Kallmann syndrome

** **

**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**

_**Expression**_**	****	****	**_**Phenotype**_

**Kallmann syndrome**

## Prevalence of KAL1 Mutations

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

_**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%)**

## 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.**

## Anosmin-1

**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 sulfate**
**	****proteoglycan to its receptor to induce axon**
**	****branching and misrouting**

## 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.**

## 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**

## NROB1 (DAX1) Heterogeneity

**Habiby et al. JCI 1996;98:1055.**

## 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.**

## 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 NH****2**

**ASHG 2002 meeting 10/02**

** ****Conditional KO: not ovarian determinant, but**
**	****instead important for spermatogenesis**

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

## Leptin Deficiency

** ****Leptin deficient *****ob/ob***** mouse:**

** ****Obesity**
** ****Hyperinsulinemia**
** ****Infertility (2****0**** to HH)**
** ****Hypothermia**
** ****Cold intolerance**
** ****Hypercortisolemia**

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

## Human Leptin Deficiency

** ****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)**

**Autosomal recessive**
** ****2 sibs with similar phenotype**
** ****Mutant not secreted from cell**

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

## Leptin Receptor Gene Mutation

**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) & cranio**
**	****facial-skeletal dysplasia (Jackson-Weiss)**
**	****syndrome**

## FGFR1 Mutations

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

** ****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**

## FGFR1 & KAL1 Relationship

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

** ****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**

## GNRHR 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 Resistance

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

** ****Proposed partial loss of function mutations**
**in GnRHR**

_**Labs**_

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

## GnRH

**GnRH**

**IP****3**** Production**

**GnRHR**

**GnRH**

**Membrane**

**Receptor binding**

**2nd messenger**

## GNRHR Mutations

- <date/time>

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

_**GNRHR Mutations**_

**Compound heterozygotes**

**Gln106Arg**

**Arg262Gln**

**Reduced binding**

**Reduced IP****3**

**Reduced IP****3**

## GNRHR Gene Mutations in IHH

**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)**

## GNRHR Gene Mutations

**GNRHR Gene Mutations**

**  ****Total IP****3**

**   ****EC****50**

## I

**I**

**II**

**1**

**2**

**3**

**4**

**5**

**6**

**7**

**8**

**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**

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

## GNRHR Gene Mutations in IHH

**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)**

## 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 by**
**	****denaturing gradient gel electrophoresis**
**	****with GC-clamps (>95% mutations)**

## 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**

## PROP1 Gene

**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**

## 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.**

## LHB Polymorphisms

** ****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):two**
**	****Abs against LH******

## Immuno- active, Bio- inactive LH 

**Gynecomastia**
**Infantile penis**
**Small descended testes**
**Female distribution pubic hair**

_**Immuno- active, Bio- inactive LH **_

**Axelrod et al. JCEM 1979;48:279.**

**Labs:**

**T= 30-80 ng/dL**
**LH = 30 mIU/mL**
**FSH = 26 mIU/mL**

## Immuno- active, Bio- inactive LH 

**Axelrod et al. JCEM 1979;48:279.**

** ****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 **

## LHB Gene Mutation

**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**

## 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.**

## Human FSHB Mutations: Female

** ****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.**

## Human FSH Mutations: Male

** ****Azoospermia**

**Human FSH******** Mutations: Male**

**Sperm**

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

**+LH**

## FSH Levels in vitro Cell Lines

**Val61X**

**Tyr76X**

**Media**

**Untrnsf.**

**Cys51Gly**

**50**

**100**

**FSH**
**mIU/mL**

**WT**

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

**FSH Levels in vitro Cell Lines**

## 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**

## GNRHR

**GNRHR**

**LHB**

**FSHB**