3985s2_02_fda-layman

5/22/2018 3985s2_02_fda-layman

1/59

Genetics of Hypogonadotropic

Hypogonadism

Lawrence C. Layman, M.D.

Professor

Chief, Section of Reproductive Endocrinology,

Infertility, & Genetics

Department of Obstetrics & Gynecology

Neurobiology ProgramThe Institute of Molecular Medicine & Genetics

The Medical College of Georgia

Augusta, GA

5/22/2018 3985s2_02_fda-layman

2/59

Genetics of IHH

1. Normal pubertal milestones

2. Idiopathic hypogonadotropic

hypogonadism (IHH)3. Mutations/phenotype

A.Hypothalamic:

KAL1, NROBI, FGFR1, LEP, LEPRB. Pituitary:

GNRHR, PROP1, HESX1, FSHB, LHB

5/22/2018 3985s2_02_fda-layman

3/59

LHFSH

GnRH

HYPOTHALAMUS

PITUITARY

GONAD

Steroids Gametes

Normal H-P-G Axis

5/22/2018 3985s2_02_fda-layman

4/59

Normal Pubertal Milestones

Females:

Breasts: age 9-11

Pubic hair: 8-9Growth spurt: 12

Menses: age 12

Males:

Testes: age 10-11

Pubic hair: 10-11Penile growth: 13

Growth spurt: 14

5/22/2018 3985s2_02_fda-layman

5/59

Delayed Puberty

1) Females:

No breast development: age 13

No menses: age 15

2) Males:

No testes development: age 14

5/22/2018 3985s2_02_fda-layman

6/59

Hypogonadism:

Low sex steroids

No pubertal development

Obtain serum gonadotropins

(LH and FSH)

Clinical Evaluation

5/22/2018 3985s2_02_fda-layman

7/59

LHFSH

GnRH

HYPO

PIT

GONAD

Steroids Gametes

H-P-G Axis Dysfunction

Hypergonadotropic

Hypogonadism

High FSH & LHLow sex steroids

5/22/2018 3985s2_02_fda-layman

8/59

Irreversible, delayed puberty

Females: age 17 AmenorrheaMales: age 18 Low T (< 100ng/dL)

Low FSH, LHNo CNS lesion

Normal prolactin, thyroid, adrenal function

Idiopathic Hypogonadotropic

Hypogonadism (IHH)

5/22/2018 3985s2_02_fda-layman

9/59

LHFSH

GnRH

HYPO

PIT

GONAD

Steroids Gametes

Hypogonadotropic

Hypogonadism

H-P-G Axis Dysfunction

Low FSH & LH

Low sex steroids

5/22/2018 3985s2_02_fda-layman

10/59

Gonadotropins in IHH

Gonadotropin responses to exogenous

GnRH variable

LH Pulsatility Patternsserial samples

(every 10-20 minutes)

1) Apulsatile

2) Decreased frequency

3) Decreased amplitude

4) Nocturnal prepubertal pattern

5/22/2018 3985s2_02_fda-layman

11/59

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 GnRHGood prognosis depending upon age

(20%/cycle)

5/22/2018 3985s2_02_fda-layman

12/59

OMIM Entries with IHH (>40)

215470 Chorioretinal dystrophy, spinocerebellar

ataxia & HH

253320 Multicore myopathy with mental retardation,

short stature, & HH

212840 Cerebellar ataxia & HH176270 Prader-Willi syndrome

176270 Fertile eunich syndrome

235200 Hemochromatosis (HFE)

602390 Hemochromatosis type (HFE2)157900 Moebius syndrome

209900 Bardet-Biedl syndrome (BBS1-6)

5/22/2018 3985s2_02_fda-layman

13/59

GNRHRNROB1LEP/LEPR

LHFSH

HYPOTHALAMUS

PITUITARY

GONADSteroids

Gametes

GnRH

LHBFSHBPROP1HESX1

FGFR1KAL1

5/22/2018 3985s2_02_fda-layman

14/59

GNRH1 Gene

Pivotal gene in reproduction

Expressed in: 1. Hypothalamus

2. Pituitary3. Placenta

4. Ovary

5. Breast

Deficiency: hypogonadotropic hypogonadism

5/22/2018 3985s2_02_fda-layman

15/59

IHH

1. Hypogonadal mouse:

Gnrh1 gene deletion

2. Human IHH: no GNRH1 gene

mutations

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.

5/22/2018 3985s2_02_fda-layman

16/59

Kallmann syndrome

IHHAnosmia

Neurologic abnormalities:

synkinesiavisual abnormalities

Renal anomalies

Midfacial defects

X-linked recessive: males

5/22/2018 3985s2_02_fda-layman

17/59

Kallmann syndrome

GnRH & olfactory neurons migrate

from olfactory placode to hypothalamus

KAL1 gene: protein directs migration, soif mutations 1. Anosmia

2. GnRH deficiency

Franco et al. Nat 1991;353:529.

Legouis et al. Cell 1991;67:423.

5/22/2018 3985s2_02_fda-layman

18/59

Kallmann Syndrome

KAL1 gene mutations in ~50% X-linkedfamilies

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

5/22/2018 3985s2_02_fda-layman

19/59

Olfactory bulb AnosmiaCerebellum 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

5/22/2018 3985s2_02_fda-layman

20/59

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%)

5/22/2018 3985s2_02_fda-layman

21/59

Kallmann syndrome

KAL1 on pseudoautosomal XpInactive pseudogene on Yq

Encodes anosmin-1, a protein with

neural cell adhesion properties

Orthologs in chicks, zebrafish, C.

elegans, DrosophilaNot cloned in murine species yet, but

human 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.

5/22/2018 3985s2_02_fda-layman

22/59

(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 & maleray (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

5/22/2018 3985s2_02_fda-layman

23/59

Kallmann syndrome

1) Absent LOT branches causes anosmia2) 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.

5/22/2018 3985s2_02_fda-layman

24/59

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.

5/22/2018 3985s2_02_fda-layman

25/59

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 418Normal FAS, no response to GnRH

(suggesting pituitary)

5/22/2018 3985s2_02_fda-layman

26/59

NROB1 (DAX1) in IHH

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

No mutations

Conclusion: NROB1 mutations uncommonin IHH patients without AHC

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

5/22/2018 3985s2_02_fda-layman

27/59

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 NH2ASHG 2002 meeting 10/02

Conditional KO: not ovarian determinant, but

instead important for spermatogenesis

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

5/22/2018 3985s2_02_fda-layman

28/59

Leptin Deficiency

Leptin deficient ob/obmouse:

Obesity

Hyperinsulinemia

Infertility (20to HH)

Hypothermia

Cold intoleranceHypercortisolemia

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

5/22/2018 3985s2_02_fda-layman

29/59

Human 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

5/22/2018 3985s2_02_fda-layman

30/59

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 phenotypeMutant not secreted from cell

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

5/22/2018 3985s2_02_fda-layman

31/59

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)

5/22/2018 3985s2_02_fda-layman

32/59

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 M t ti

5/22/2018 3985s2_02_fda-layman

33/59

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 expressivitySome patients with cleft palate/lip,

dentogenesis, synkinesis

FGFR1 & KAL1 R l ti hi

5/22/2018 3985s2_02_fda-layman

34/59

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 isexpressed in rostral forebrain & required

for olfactory bulb evagination in mouse

5/22/2018 3985s2_02_fda-layman

35/59

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.)

5/22/2018 3985s2_02_fda-layman

36/59

GnRH 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

5/22/2018 3985s2_02_fda-layman

37/59

GnRH

IP3Production

GnRHR

GnRH

Membrane

Receptor binding

2nd messenger

5/22/2018 3985s2_02_fda-layman

38/59

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

GNRHR Mutations

Compound heterozygotes

Gln106Arg Arg262Gln

Gln(CAA) (CGA)

Arg Gln(CAG)(CGG)

Arg

Reduced binding Reduced IP3Reduced IP3

5/22/2018 3985s2_02_fda-layman

39/59

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

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)

5/22/2018 3985s2_02_fda-layman

40/59


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

5/22/2018 3985s2_02_fda-layman

41/59

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


5/22/2018 3985s2_02_fda-layman

42/59


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)

5/22/2018 3985s2_02_fda-layman

43/59

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

5/22/2018 3985s2_02_fda-layman

44/59


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

5/22/2018 3985s2_02_fda-layman

45/59


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)

5/22/2018 3985s2_02_fda-layman

46/59

GNRHR Mutations

1) ~ 15 different mutations identified2) Most compound HTZ

3) May affect binding and/or signal transduction

4) Phenotype varies from complete IHH topartial 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 G

5/22/2018 3985s2_02_fda-layman

47/59

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

5/22/2018 3985s2_02_fda-layman

48/59

Septo-optic Dysplasia

Agenesis of corpus callosum, panhypopit,

optic nerve hypoplasia, absent septum

pellucidum

One form due to HESX1 gene mutationsHESX1 is homeobox gene expressed in

Rathkes Pouch, pituitary primordium

Autosomal recessive, dominant

Dattani et al. Nat Genet 1998;19:125-133.

5/22/2018 3985s2_02_fda-layman

49/59

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):two

Abs against LH

5/22/2018 3985s2_02_fda-layman

50/59

Male: delayed puberty at 17 yr.Gynecomastia

Infantile penis

Small descended testesFemale distribution pubic hair

Immuno- active, Bio- inactive LH

Axelrod et al. JCEM 1979;48:279.

Labs: T= 30-80 ng/dL

LH = 30 mIU/mLFSH = 26 mIU/mL

5/22/2018 3985s2_02_fda-layman

51/59

Immuno- 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

5/22/2018 3985s2_02_fda-layman

52/59

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

LHB Gene Mutation

Homozygous LHB gene missense mutationin exon 3 (Gln54Arg)

1. Detected by dimer-specific IRMA

2. Undetectable by RRA

Mutant LH not capable of receptor binding

Autosomal recessiveHeterozygotes probably normal

5/22/2018 3985s2_02_fda-layman

53/59

Human FSHB Mutations: Females

No breast development or menses (1,2)

Partial breast development (3)

Low FSH, High LHLow estradiol

Immature ovarian follicles (antral)

Infertility1) 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.

5/22/2018 3985s2_02_fda-layman

54/59

Low testosteroneNo 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

5/22/2018 3985s2_02_fda-layman

55/59

Human FSH Mutations: MalesNormal puberty or absent pubertyLow 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.

5/22/2018 3985s2_02_fda-layman

56/59

Low testosterone

Azoospermia

Human FSH Mutations: Male

FSH Testosterone

Sperm

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

+LH

FSH Levels in vitro Cell Lines

5/22/2018 3985s2_02_fda-layman

57/59

Immuno- ; Bio- Untrnsf = Untransfected cells.Val61X Tyr76X MediaUntrnsf.Cys51Gly

50

100

FSH

mIU/mL

WT

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

FSH Levels in vitro Cell Lines

H d t i H di

5/22/2018 3985s2_02_fda-layman

58/59

Hypogonadotropic Hypogonadism

1. No GNRH1 gene mutations, so rare2. KAL1: 10-15% male IHH patients

3. 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 & HH

8. Most causes of inherited IHH unknown

5/22/2018 3985s2_02_fda-layman

59/59

GNRHRNROB1LEP/LEPR

LHFSH

HYPOTHALAMUS

PITUITARY

GONADSteroids

GnRH

LHBFSHBPROP1HESX1

FGFR1KAL1

Documents

3985s2_02_fda-layman