The Role of the Clinical Investigator in Discovering how the Brain Controls

Reproduction?

Evidence from Genetic Approaches in the Human

William F. Crowley, Jr., M.D.Professor of Medicine, Harvard Medical School

Director, Harvard Reproductive Endocrine Sciences CenterDirector of Clinical Research, Mass General Hospital

The People Who Did the Work !

Genetics TeamStephanie Seminara

Nelly PitteloudJames Acierno

Yousef Bo-Abbas Astrid Meysing

Jenna SagournyPat Sluss

Carl Pallais

Male TeamFrances HayesNellie PitteloudMaria YialamasAndrew Dwyer

Female TeamJan Hall

Corinne WeltKathy MartinJudy AdamsYari Jimenez

OthersJim Gusella

Sue SlaughenhauptLarry Jameson

Cricket & John SeidmanEric Lander

David Altshuler

Paradigm TherapeuticsSam AparicioStephen O’RahillyWilliam ColledgeSophia MessagerEmmanouella Chatzidaki

U of Pittsburgh: Tony PlantU of Washington: Bob SteinerOregon Primate Center: Sergio Ojeda

Today’s Goals

• Use human disease model to address fundamental problem

• Idiopathic hypogonadotropic hypogonadism & Kallmann’s Syndrome

• Genotype/phenotypes of 2 new genes that control GnRH

• We propose to be “gatekeepers” of GnRH secretion, sexual

maturation, & puberty

• Put them in some context

• Biology, Development, Evolution & Opportunities

Pituitary

HypothalamusGnRH

TestesSperm

Leydig cells T/E2

Inhibin BT/E2

FSH

LH

????

Why Study the GnRH Neuronal System?

GnRH is Central, Critical, & Highly Conserved in Reproduction‘Pilot Light’ of mammalian reproduction

• Initiates all reproductive activity

• ‘On’ During neonatal and pubertal periods

• ‘Off’ during the childhood period or anestrus

• Initiates puberty/sexual maturation

• Rest of system - passively regulated

“The Boss”

Key Evolutionary RoleLinks external environment and internal endocrine milieu

Synchronizes nutrition, light/dark cycles, stress, olfaction, & predators & reproduction

Key to evolutionary success (‘fitness’)

Genetic Control is Highly Species SpecificE.g. Alpha adrenergic modulation of GnRH

Links Between Reproduction & Environment: Role of Predators, Nutrition & Genes for Selection

Neu

roen

do

crin

e A

ctiv

ity

(Gn

RH

,LH

,FS

H,F

AS

)

GnRH +mRNAGnRH +Immunostaining

NeonatalYears

Childhood Puberty

Neuroendocrine Activity of the Reproductive Axis Across Life in Humans

? Controlling Genes

Rule #1 - Start with a Patient with a Disease: = Important Biologic Problem

Exam• short stature• arm span > height• no axillary or pubic hair• microphallus• small L testis; no R testis palpable

History (1943)• good general health• small genitals; absence of puberty• absent sense of smell

Rule #2 – Measure Something!- First Classification of Gonadal Function -

FSHLH

Hypo 2o Failure

FSHLH

PITUITARY

GONADS

Gonadotropins Normal

Gonadal Fxn Normal

FSHLH

Hyper 1o Failure

Hypogonadotropic Hypogonadism? Hypothalamic GnRH Deficiency

An Opportunity for Therapy based on Physiology

FSHLH

GnRH Deficient

GONADS

FSHLH

Normal

HYPOTHALAMUS

GnRHPITUITARY

PulsatileGnRH Rx

Pulsatile GnRH Rx:

Re-Constitutes NormalHPG Axis in IHH

(Crowley et al, JCEM, 1980,

Hoffman et, NEJM, 1980)

Normal Adult Male Range

0 240 480 720 960 1200 14400

10

20

30

40

50

Normal Adult Male T = 500ng%L

H (

IU/L

)

Normal Adult Male Range

0 240 480 720 960 1200 14400

10

20

30

40

50

IHH Male: Baseline T = 20ng%

LH

(IU

/L)

TIME (minutes)0 120 240 360 480 600 720

0

20

40

60

80

T = 500ng%

GnRH IV q 2hr+ Generates

Opportunity forDose-Response

Curves(Spratt et al, JCEM)

(LH

(IU

/L)

Infant Male with Terminal Deletion at Xp22.31 Karyotypic Abnormality

• Dysmorphic features

• Cryptorchism & microphallus

• Low LH, FSH, & T levels

• Absent olfactory bulbs/tracks

• Icthyiosis & Calcium Disorder

Bick et al. Am J Med Gen 1989

Mother 46XX

shared terminal deletion Xp22.31

Son 46XY

Localization of the KAL Gene on Xp 21.3

680Anosmin 181 285 402 540

His-rich WAP HFN III FN III FN III FN III

141 2 3 4 5 6 7 8 9 10 11 12 13

1951delCR457X

R191XC172R

** *

*

Novel

Mutation

splice

frameshift

nonsense

missense

deletion

*MGH (Georgopoulos 1997)

Migration of GnRH Neurons in Mouse Brain

Schwanzel-Fukuda & Pfaff, Nature 1989

vno vomeronasal organgt ganglion terminaleob olfactory bulbpoa preoptic area

Embryology of GnRH Neuronal Migration

• Olfactory neurons send processes hypothalamus

• GnRH neurons migrate along olfactory tract

• Anosmin in extracellular matrix

Olfactory Tract

CribriformPlateOlfactoryPlacode

Olfactory

bulb

NORMAL

KALLMANN’S

• Kallmann’s Syndrome -olfactory bulb & tract absent in Kallmann’s pts anosmia

• Migration defect in KAL-1 deletion/mutation GnRH deficiency

Genotype-phenotype: Males with Confirmed KAL mutations

Cys172Arg 3 Brothers 1-2 mL - - No Synkinesia

Arg 191X 1 sporadic 1-2 mL 1 - No

1 sporadic 1-2 mL 2 +

Tyr328X X-linked 1-2 mL - - No Hi-Arched

palate color

blindness

11 base del X-linked 1-2 mL 2 - No

14 base del Sporadic 1-2 ml - - No color

blindness

Arg457X X-linked 4.5 mL* 1 - No

Del. Sporadic 1-2 mL 1 + No Renal

agen.

Inherit TV Crypt Micro LH pulse Other

Oliviera et al: JCEM 2002

The Genetics of GnRH Deficiency: Role of the Autosome

5

GnRH Deficiency

Anosmia

Delayed Puberty

Fam. History Autosmal Dom. Autosmal Rec. X-Linked

IHH/KS alone 32% 47% 21%

+/-anosmia 50% 32% 18%

+/- DP 64% 25% 11%

Waldstreicher et al, JCEM 1996

Mutations in FGFR1 Gene (8p11.2) cause Kallmann’s Syndrome

• Hardelin et al, Nature Genetics, 2003• 2 Patients with deletions of 8p11.2 & 12 region (GnRH)• 1 with hereditary spherocytosis (ANK1) + KS• Multiple congential anomalies (shortened 5th finger, micrognathia)• 12% incidence of heterozygous mutations in FGFR1 in 129 KS (91M/28F)• Autosomal Dominant

• Dode et al, Nature Genetics, 2003• 9% incidence of FGFR1 coding sequence mutations in KS patients (12/129)• High frequency of cleft lip/palate• Asymptomatic carriers (female)

• Sato et al, JCEM, 2004• 2 heterozygous mutations of FGFR1 in KS patients• 11% incidence

• No Reproductive Phenotypes!!!

FGFR1 Mutations: Genotype-Phenotype Correlations

IgIII TK TKIgIIIgI

Gln680XLeu340Ser

Tyr339Cys

Glu274Gly

Arg250Gln Arg622X

A.

(LH

(IU

/L)

0

5

10

# 1,3,4,9 Absent PubertyT = 45 ng/dL

0 2 4 6 8 10 12 hr

#10 Reversal of KST = 368 ng/dL

0 2 4 6 8 10 12 hr

(LH

(IU

/L)

0

5

10

C.

# 12 Partial PubertyE2<20 pg/mL

0 2 4 6 8 10 12 hr0

5

10

LH

(IU

/L)

B.

Genotype-phenotype - FGFR1 mutations(MGH Pitteloud, unpublished)

Sex FH TV Crypt. Micro. LH Ansomia Other Gly696Ser M - 8 mL - N U +

Tyr337Cys M - 3 mL - N U +

Arg620X M + 10mL(S+) - N Reversal + Ser344Cys M+ 4 mL - N LH3.5/FSH6 +

Arg252Glyn M - No puberty - N ? +

Gly235Ser M+ 2 ml* Bilat Y U + Glu272Gly M + 2 mL Bilat Y U + cleft palate

Val271Met M + 2 mL - Y U + Gly678X M + 1 ml - N U Normal cleft palate

Gly701Ser M - 3 mL* Uni Y U+ + Hirschspr.

L>S (exon8) M - 1mL Bilat N U Hypo. cleft palate

RforQ (ex7) F + Partial pub. ? Y cleft palate

FGFR1 Mutation and normosmic IHH

colorblind

28yr IHH (TV 1mL) Cleft palate Missing teethColor blindness R/G

Gln678X

19 yrIHH (TV 1mL)

Gln678X

IHHAnosmia

19yrIHH? GHD?

Gln678X

Clues/Lessons re FGFR1 Mutations:

1. AD that Mimics X-linked (? Females)

2. Causes not just KS but IHH s anosmia

3. Confirms cleft palate = part of spectrum

FGFR1 Families

Arg620X

Pedigree 2

Arg620X

Menarche 16

Glu272Gly1

23

4 5r

6 7

8

910

1112

Pedigree 1

Menarche 15

Glu272Gly

IHHAnosmia

Delayed Puberty

Arg620X

Clues re FGFR1 Mutations:

1. Reproductive phenotype quite variable

2. Cause delayed puberty = first gene

3. Females are attenuated to no

phenotype

4. ? Why are males more severely affected

Evidence from the Fgfr1 Knock-out Mouse:

Herbert Development 2003

FGFR1 and KAL1 Genes• both expressed in the

Olfactory Bulb during development

FGFR1 Knock-out• Telencephalon does not

develop normally• Interfers with Olfactory

Bulb development

FGF, FGFR, and HS are required for receptor dimerization (Bernfield, 1994)

FGFR1/HS binding domainin the Ig II domain

Pellegrini 2001

Anos.Anos.

Anos. Anos.

KAL-1 & FGFR1 co-expressed developmentallyBrain & kidney

Conditional K/O of FGFR1 in the telencephalonNo olfactory bulb; no pathway for GnRH neurons

FGFR1-FGF2 interaction with HS Role for co-receptor (e.g. TGF beta family)

Biology of FGFR1

Hypothesis: Kallmann’s Syndrome Due to FGFR1 Mutations is a Digenic Disorder?

1. KAL1/Ansomin binds HSPG “Syndecans”• Anosmin = ligand or co-receptor for FGFR1 (vs FGF2)

2. KAL1 = Non-Lyonized X gene• i.e. Females have 2 copies; Males only 1

3. ? FGFR1 Defects Digenic in nature• Females have 2 copies; males only 1 ligand • i.e. males are ‘haploinsufficient’ for anosmin• Therefore males get the more severe phenotypes

I:1 I:2

II:1 II:2 II:3 II:4

6

III:1 III:2 III:3 III:4

III:5 III:6 III:7 III:8

3

IV:1 IV:2 IV:3 IV:4 IV:5 IV:6 IV:7 IV:8

IV:9 IV:10 IV:11 IV:12 IV:13 IV:14 IV:15 IV:16 IV:17

3

S/S L/S L/LS/S S/S S/S

S/S S/S

S/LL/L

L/L L/S L/L L/L S/L

S/L S/LS/L

S/L S/L S/L

IHH

Anosmia

rs7815rs731804rs668447

rs10390REU-1902rs1006473rs1006474rs1006475

rs757331REU-1903rs736926D19S886

REU-1905

Chromosome 19

Seminara et al, JCEM, June, 2003

GTP

Gq/11

Phospholipase CPIP2

IP3

DAG

Protein Phosphorylation

Ca 2+ release

GPR54

• GPCR• Rhodopsin-like• Galanin & Somato- Statin Receptors (~35-38%)

CognitionNociceptionFeeding/NutritionReproduction

WTLeuPro Arg

L148SSerPro Arg

443T>C [L148S]•Segregates properly•Absent in control pops.•Highly conserved• Nonpolar to polar

Genomic Screening of GPR54

SVDRWYVTVFPLRALHRRTPRLSVDRWYVTVFPLRALHRRTPRLSVDRWYVTVFPLRALHRRTPRL

SVDRYVAIVHSRRSSSLRVSRNSVDRYLAIRYPLHSRELRTPRNSVDRYLAVRHPLRSRALRTPRNSVDRYVAVVHPIKAARYRRPTVSIDRYLAVVHPIKSAKWRRPRTSVDRYLAVVHPTRSARWRTAPVSVDRYVAVVHPLRAATYRRPSVSVDRYLAVVHPLSSARWRRPRVSIDRYLAIVHPLRYRRIRTPRR

Human GPR54Mouse Gpr54Rat Gpr54

Human GALR1Human GALR2Human GALR3Human SSTR1Human SSTR2Human SSTR3Human SSTR4Human SSTR57tm consensus

Galanin Family

Somatostatin Family

TIME (minutes)0 240 480 720 960 1200 1440

0

2

4

6

8

10

+ Low AmplitudeLH Pulses

IHH Patient: +GPR54 Mutations

Normal Adult Male Range

0 240 480 720 960 1200 14400

10

20

30

40

50

Normal Adult Male T = 500ng%L

H (

IU/L

)

Normal Adult Male Range

0 240 480 720 960 1200 14400

10

20

30

40

50

GnRH Deficient Male: BaselineT = 20ng%

LH

(IU

/L)

GPR54 Patient: Baseline Studies

LOG (10) GnRH0 1 2 3

0

20

40

60

LH

Am

pli

tud

e

+ Left ShiftedDose ResponseCurve Compared

To 6 IHH Men Without GPR54

Mutations

LH

Am

pli

tud

e

0 1 2 3

0

20

40

60

LH

(IU

/L)

0 2 6 8

0

20

40

60

TIME (hr)

Dose ResponseStudies

25 ng/kg 75ng/kg 2.5 ng/kg 250ng/kg

Seminara SB et al, NEJM, 2003

B-/-

+/++/+ -/-

+/+ -/- +/+ -/-

-/-

GPR54 -/-Paradigm Therapetuics

“Harry Potter”

-/-

0

1

2

3

4

5

6

7Te

stoste

rone

pg/

ml

Genotype

P<0.001a)

0

10

20

30

40

50

FSH

ng/m

l

Genotype

c) P<0.01 P<0.01

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

LH

ng

/ml

Genotype

d)

0

1

2

3

4

5

6

7

8

LH

ng/

ml

Genotype/Treatment

P<0.01 P<0.01f)

0

100

200

300

400

500

600

Pitu

itary

LH

ng/m

g tis

sue

Genotype/Treatment

g)

P<0.01

P<0.05

0

10

20

30

40

50

60

70

80

90

100

110

120

Oestr

adiol

pg/m

l

Genotype/cycle stage

P<0.05b)

0

10

20

30

40

GnR

H p

g/m

g hy

poth

alam

us

Genotype

e)

Phenotype of GPR54 -/- Mouse (“Harry Potter”)

• Faithful Recapitulation of Human IHH

• Normal to GnRH Levels in Hypothalamus- ? Means GPCR 54 maybe responsible for GnRH

processing

- ? Like prepubertal monkey and rat hypothalamus

• If mutation IHH, Could Antagonists HH- ? Good candidate for small ligand screening

If GPR54 is the Receptor,What is the ligand?

Kisspeptin-1/Metastin

M G E RK RLSRR GTS GLRF GKR Q

1 19 20 67 68 121 122 145

GTS GLRF Metastin NH2

1 GTSLSPPPESSGSRQ 1516 QPGLSAPHSRQIPAP 3031 QGAVLVQREKDLPNY 4546 NWNSFGLRF-NH2 54

Signal Peptide

Dibasic cleavage sites

Structure of Metastin

Kisspeptin-1

GPR54 & KiSS1 mRNA in over-

lapping cell populations

of female monkeys (Ojeda & Plant)

KiSS-1 mRNA transcripts

in neurons of arcuate nucleus

in the rodent (Steiner Lab)

GPR54 Expression in Female and

Agonadal Male Monkeys Across

the Pubertal Transition

Expression of GPR54 does not increase in the agonadal male

monkey

KISS1 Expression in Female and

Agonadal Male Monkeys Across

the Pubertal Transition

Expression of KISS1 increases in both animal

models

0.0

4.0

8.0

12.0

16.0

-30 0 30 60 90 120 150 180 210 240

Time (min)

Pla

sma

LH

(n

g/m

l)

Metastin

Vehicle

30 ug Metastin

100 ug Metastin

Acyl+100 ug Metastin

Central Administration of Metastin in Juvenile Orchidectomized Monkeys

Metastin ellicits a brisk LH response in juvenile,

agonadal male monkeys

Physiology of Metastin/Kisspeptin

• GPR54 is on nearly all GnRH neurons + median eminence

• Metastin & GPR 54 mRNA across sexual maturation in monkey,

rat, and mouse

• (Ojeda, Plant, Steiner, Manuel-Tempore)

• This appears to be regulated by sex steroids

• Kisspeptin neurons overlay GPR54 & GnRH neurons

• Steiner; Ojeda

• Metastin administration induces c-fos in GnRH neurons

• Metastin administration induces LH pulses

• (rat, mice & monkey)

• Metastin administration ‘trumphs’ leptin def.

IHH Due to Possible Metastin Mutation?

• IHH male with homozygous duplication at 5’ end of metastin gene (Paris [Deroux] + Boston collaboration [Seminara + Crowley]

• No mRNA present in peripheral lymphocytes

• Some normal controls + for heterozygous changes

• Being evaluated

GPR54

M

GPR54

M

GPR 54 & Metstatin: ? Site of Action within the Hypothalamus

GnRH

Hypophyseal-PortalBloodSupply

Role of GPR54 & Metastin in IHH

Neu

roen

do

crin

e A

ctiv

ity

(Gn

RH

,LH

,FS

H,F

AS

)

NeonatalYears

Childhood Puberty

IHH (GPR54/Metastain)

? Role of Metastin/GPR54 in Hypothalamic Amenorrhea?

Neu

roen

do

crin

e A

ctiv

ity

(Gn

RH

,LH

,FS

H,F

AS

)

NeonatalYears

Childhood Puberty

Hypothalamic Amenorrhea

(+ stress) (- stress)

? Role of Metastin/GPR54 in Adult Onset IHH

Neu

roen

do

crin

e A

ctiv

ity

(Gn

RH

,LH

,FS

H,F

AS

)

NeonatalYears

Childhood Puberty

Adult-Onset IHH(- stress)

? Role of Metastin/GPR54 in Disorders of Pubertal Timing?N

euro

end

ocr

ine

Act

ivit

y(G

nR

H,L

H,F

SH

,FA

S)

NeonatalYears

Childhood Puberty

? Precocious Puberty/Constitutional Delay

Human Disease Model: Idiopathic Hypogonadotropic Hypogonadism

+ Anosmia? Developmental Pathways

-Anosmia-? Regulatory GnRH

Kallmann’s SyndromeKAL1 (X)

FGFR1 (AD)? FGF 2? Others

Normosmic IHHGPR 54

? KisspeptinFGFR1

? Others

KarotypicAbnormality

X-p22.1KAL1

ContiguousGene Syndrome

8p11.2FGFR1

Linkage

19q13.3GPR54

71 2 3 4 5 6 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 X Y

GnRNR4q13.2

FGFR18p12

? GnRH18p 12.2

GPR5419p13.3

KAL1Xp22.31

DAX1Xp21.2

LEPR1p31.2

PROP15q35.3

PIT13p11.2

LEP7q32.1

2003 Human Genome Assembly

Common Features All MonogenicAll PrismaticNone are Polygenic

Trends in Bedside to Bench

Bedside Bench

1930-60s

1960-00s

2000+

ThoughtfulClinicians

ClinicalInvestigators

Basic Investigators

Universities, AHCs, &Professional Societies

1. Take full advantage of UBOs

• patients or families (espeically those with contiguous gene syndromes or chromosomal translocations) afford Unique Biologic Opportunities to locate and identify new genes

SpiralStaircase

ToSuccess

Lessons for the Clinical Investigatorin the Post-Genomic Era

1. Take full advantage of UBOs

2. Partnerships with basic investigators are key for translational investigators to establish & are mutually beneficial

• basic research collaborations will be increasingly bi-directional in the future

• human mutations provide critical information re: structure-activity relationships

• accurate, detailed, & quantitative phenotyping will be key to identifying new genes & their biology

SpiralStaircase

ToSuccess

Lessons for the Clinical Investigatorin the Post-Genomic Era

1. Take full advantage of UBOs

2. Partnerships key & mutually beneficial

3. Strap on the new tool belt

• Human Genome Project provides expanding collection of new tools

SpiralStaircase

ToSuccess

Lessons for the Clinical Investigatorin the Post-Genomic Era

SpiralStaircase

ToSuccess

Lessons for the Clinical Investigatorin the Post-Genomic Era

1. Take full advantage of UBOs

2. Partnerships key & mutually beneficial

3. Strap on the new tool belt

4. Access to the DNA is THE rate-limiting step!

• samples from patients & relatives are a key resource:

• phenotyping of proband

• complete family histories

• relational databases

GPR54

M?

??

?

?

?

GPR54

M

GPR 54 & Metstatin: ? Site of Action within the Hypothalamus