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

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 Center

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

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  1. 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 Center Director of Clinical Research, Mass General Hospital

  2. The People Who Did the Work ! Others Jim Gusella Sue Slaughenhaupt Larry Jameson Cricket & John Seidman Eric Lander David Altshuler Female Team Jan Hall Corinne Welt Kathy Martin Judy Adams Yari Jimenez Genetics Team Stephanie Seminara Nelly Pitteloud James Acierno Yousef Bo-Abbas Astrid Meysing Jenna Sagourny Pat Sluss Carl Pallais Paradigm Therapeutics Sam Aparicio Stephen O’Rahilly William Colledge Sophia Messager Emmanouella Chatzidaki U of Pittsburgh: Tony Plant U of Washington: Bob Steiner Oregon Primate Center: Sergio Ojeda Male Team Frances Hayes Nellie Pitteloud Maria Yialamas Andrew Dwyer

  3. 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

  4. ???? Hypothalamus GnRH FSH Pituitary LH Inhibin B T/E2 + Leydig cells T/E2 + + Testes Sperm

  5. 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 Role Links 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 Specific E.g. Alpha adrenergic modulation of GnRH

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

  7. ? Controlling Genes Neonatal Years Childhood Puberty Neuroendocrine Activity of the Reproductive Axis Across Life in Humans GnRH +mRNA GnRH +Immunostaining Neuroendocrine Activity (GnRH,LH,FSH,FAS)

  8. Rule #1 - Start with a Patient with a Disease: = Important Biologic Problem History (1943) • good general health • small genitals; absence of puberty • absent sense of smell Exam • short stature • arm span > height • no axillary or pubic hair • microphallus • small L testis; no R testis palpable

  9. PITUITARY GONADS LH FSH LH FSH LH FSH Gonadotropins Normal Gonadal Fxn Normal Hyper  1o Failure Hypo  2o Failure Rule #2 – Measure Something!- First Classification of Gonadal Function -

  10. GnRH Deficient Normal HYPOTHALAMUS GnRH Pulsatile GnRH Rx PITUITARY FSH LH FSH LH GONADS Hypogonadotropic Hypogonadism? Hypothalamic GnRH DeficiencyAn Opportunity for Therapy based on Physiology

  11. 50 T = 500ng% Normal Adult Male 40 30 Normal Adult Male Range Normal Adult Male Range LH (IU/L) 20 10 0 0 240 480 720 960 1200 1440 50 IHH Male: Baseline T = 20ng% 40 LH (IU/L) 30 20 10 0 0 240 480 720 960 1200 1440 GnRH IV q 2hr + Generates Opportunity for Dose-Response Curves (Spratt et al, JCEM) 80 T = 500ng% 60 (LH (IU/L) 40 20 0 120 240 360 480 600 720 0 TIME (minutes) Pulsatile GnRH Rx:Re-Constitutes NormalHPG Axis in IHH(Crowley et al, JCEM, 1980,Hoffman et, NEJM, 1980)

  12. Mother 46XX shared terminal deletion Xp22.31 Son 46XY Bick et al. Am J Med Gen 1989 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

  13. splice frameshift nonsense missense deletion * * * * 1 2 3 4 5 7 8 9 10 11 12 13 14 6 Novel Mutation C172R R191X R457X 1951delC Anosmin 181 285 402 540 680 FN III FN III FN III FN III His-rich WAP H Localization of the KAL Gene on Xp 21.3 *MGH (Georgopoulos 1997)

  14. Migration of GnRH Neurons in Mouse Brain vno vomeronasal organ gt ganglion terminale ob olfactory bulb poa preoptic area Schwanzel-Fukuda & Pfaff, Nature 1989

  15. Olfactory bulb Olfactory Tract Cribriform Plate Olfactory Placode • Olfactory neurons send processes  hypothalamus • GnRH neurons migrate along olfactory tract • Anosmin in extracellular matrix NORMAL • Kallmann’s Syndrome -olfactory bulb & tract absent in Kallmann’s pts  anosmia • Migration defect in KAL-1 deletion/mutation  GnRH deficiency KALLMANN’S Embryology of GnRH Neuronal Migration

  16. Genotype-phenotype: Males with ConfirmedKAL mutations Inherit TV Crypt Micro LH pulse Other 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. Oliviera et al: JCEM 2002

  17. GnRH Deficiency Anosmia Delayed Puberty 5 +/- DP 64% 25% 11% Waldstreicher et al, JCEM 1996 The Genetics of GnRH Deficiency: Role of the Autosome Fam. HistoryAutosmal Dom.Autosmal Rec.X-Linked IHH/KS alone 32% 47%21% +/-anosmia 50% 32% 18%

  18. 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!!!

  19. FGFR1 Mutations: Genotype-Phenotype Correlations A. # 1,3,4,9 Absent Puberty T = 45 ng/dL (LH (IU/L) Tyr339Cys 10 Glu274Gly Leu340Ser 5 Gln680X 0 IgI IgII IgIII 0 2 4 6 8 10 12 hr TK TK # 12 Partial Puberty E2<20 pg/mL B. 10 LH (IU/L) 5 0 0 2 4 6 8 10 12 hr #10Reversal of KS T = 368 ng/dL C. (LH (IU/L) 10 5 0 0 2 4 6 8 10 12 hr Arg250Gln Arg622X

  20. 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 Normalcleft 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

  21. FGFR1 Mutation and normosmic IHH IHH Anosmia Gln678X 19yr IHH? GHD? Gln678X Gln678X 19 yr IHH (TV 1mL) 28yr IHH (TV 1mL) Cleft palate Missing teeth Color blindness R/G colorblind 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

  22. FGFR1 Families Delayed Puberty IHH Anosmia Menarche 15 Arg620X Menarche 16 Glu272Gly Arg620X Glu272Gly Arg620X Pedigree 2 Pedigree 1 12 11 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 6 7 10 9 8 3 2 r 4 1 5

  23. Evidence from the Fgfr1 Knock-out Mouse: • 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 Herbert Development 2003

  24. FGF, FGFR, and HS are required for receptor dimerization (Bernfield, 1994) Anos. Anos. Anos. Anos. FGFR1/HS binding domain in the Ig II domain Pellegrini 2001

  25. Biology of FGFR1 • KAL-1 & FGFR1 co-expressed developmentally • Brain & kidney • Conditional K/O of FGFR1 in the telencephalon • No olfactory bulb; no pathway for GnRH neurons • FGFR1-FGF2 interaction with HS • Role for co-receptor (e.g. TGF beta family)

  26. Hypothesis: Kallmann’s Syndrome Due to FGFR1 Mutations is a Digenic Disorder? • KAL1/Ansomin binds HSPG “Syndecans” • Anosmin = ligand or co-receptor for FGFR1 (vs FGF2) • KAL1 = Non-Lyonized X gene • i.e. Females have 2 copies; Males only 1 • ? 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

  27. IHH Anosmia I:1 I:2 II:1 II:2 II:3 II:4 6 III:1III:2 III:3 III:4 S/L S/L S/L 3 III:5 III:6 III:7 III:8 S/L S/L S/L 3 IV:9 IV:10 IV:11 IV:12 IV:13 IV:14 IV:15 IV:16 IV:17 L/L S/S L/S L/L L/L S/S S/L IV:1 IV:2 IV:3 IV:4 IV:5 IV:6 IV:7 IV:8 L/L S/S L/S S/S S/S S/S S/L L/L

  28. Chromosome 19 rs7815 rs731804 rs668447 rs10390 REU-1902 rs1006473 rs1006474 rs1006475 rs757331 REU-1903 rs736926 D19S886 REU-1905 Seminara et al, JCEM, June, 2003

  29. Protein Phosphorylation  Gq/11  • GPCR • Rhodopsin-like • Galanin & Somato- • Statin Receptors • (~35-38%) • Cognition • Nociception • Feeding/Nutrition • Reproduction GTP DAG Phospholipase C PIP2 IP3 Ca 2+ release GPR54

  30. L148S WT Pro Leu Arg Pro Ser Arg Human GPR54 Mouse Gpr54 Rat Gpr54 Human GALR1 Human GALR2 Human GALR3 Human SSTR1 Human SSTR2 Human SSTR3 Human SSTR4 Human SSTR5 7tm consensus SVDRWYVTVFPLRALHRRTPRL SVDRWYVTVFPLRALHRRTPRL SVDRWYVTVFPLRALHRRTPRL SVDRYVAIVHSRRSSSLRVSRN SVDRYLAIRYPLHSRELRTPRN SVDRYLAVRHPLRSRALRTPRN SVDRYVAVVHPIKAARYRRPTV SIDRYLAVVHPIKSAKWRRPRT SVDRYLAVVHPTRSARWRTAPV SVDRYVAVVHPLRAATYRRPSV SVDRYLAVVHPLSSARWRRPRV SIDRYLAIVHPLRYRRIRTPRR Galanin Family Somatostatin Family Genomic Screening of GPR54 • 443T>C [L148S] • Segregates properly • Absent in control pops. • Highly conserved • Nonpolar to polar

  31. GPR54 Patient: Baseline Studies 50 T = 500ng% Normal Adult Male 40 30 Normal Adult Male Range Normal Adult Male Range LH (IU/L) 20 10 0 0 240 480 720 960 1200 1440 50 GnRH Deficient Male: Baseline 40 T = 20ng% 30 LH (IU/L) 20 10 0 0 240 480 720 960 1200 1440 10 IHH Patient: +GPR54 Mutations + Low Amplitude LH Pulses 8 6 4 2 0 0 240 480 720 960 1200 1440 TIME (minutes)

  32. 25 ng/kg75ng/kg2.5 ng/kg250ng/kg 60 Dose Response Studies 40 LH (IU/L) 20 0 TIME (hr) 0 2 6 8 60 40 LH Amplitude 20 + Left Shifted Dose Response Curve Compared To 6 IHH Men Without GPR54 Mutations 0 0 1 2 3 60 40 LH Amplitude 20 0 0 1 2 3 LOG (10) GnRH Seminara SB et al, NEJM, 2003

  33. -/- -/- +/+ +/+ -/- -/- +/+ -/- +/+ GPR54 -/- Paradigm Therapetuics “Harry Potter” -/- B

  34. 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

  35. If GPR54 is the Receptor,What is the ligand? Kisspeptin-1 /Metastin

  36. Structure of Metastin Kisspeptin-1 Signal Peptide Dibasic cleavage sites 1 19 20 67 68121 122 145 M G E RK RLSRR GTSGLRF GKR Q Metastin GTS GLRF NH2 1 GTSLSPPPESSGSRQ 15 16 QPGLSAPHSRQIPAP 30 31 QGAVLVQREKDLPNY 45 46 NWNSFGLRF-NH2 54

  37. KiSS-1 mRNA transcripts in neurons of arcuate nucleus in the rodent (Steiner Lab) GPR54 & KiSS1 mRNA in over- lapping cell populations of female monkeys (Ojeda & Plant)

  38. GPR54 Expression in Female and Agonadal Male Monkeys Across the Pubertal Transition Expression of GPR54 does not increase in the agonadal male monkey

  39. KISS1 Expression in Female and Agonadal Male Monkeys Across the Pubertal Transition Expression of KISS1 increases in both animal models

  40. Vehicle Metastin 16.0 30 ug Metastin 100 ug Metastin 12.0 Acyl+100 ug Metastin 8.0 Plasma LH (ng/ml) 4.0 0.0 -30 0 30 60 90 120 150 180 210 240 Time (min) Central Administration of Metastin in Juvenile Orchidectomized Monkeys Metastin ellicits a brisk LH response in juvenile, agonadal male monkeys

  41. 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.

  42. 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

  43. GPR 54 & Metstatin: ? Site of Action within the Hypothalamus M M GPR54 GPR54 Hypophyseal- Portal Blood Supply GnRH

  44. IHH (GPR54/Metastain) Neuroendocrine Activity (GnRH,LH,FSH,FAS) Neonatal Years Childhood Puberty Role of GPR54 & Metastin in IHH

  45. Hypothalamic Amenorrhea (+ stress) (- stress) Neuroendocrine Activity (GnRH,LH,FSH,FAS) Neonatal Years Childhood Puberty ? Role of Metastin/GPR54 in Hypothalamic Amenorrhea?

  46. Adult-Onset IHH (- stress) Neuroendocrine Activity (GnRH,LH,FSH,FAS) Neonatal Years Childhood Puberty ? Role of Metastin/GPR54 in Adult Onset IHH

  47. ? Role of Metastin/GPR54 in Disorders of Pubertal Timing? ? Precocious Puberty/ Constitutional Delay Neuroendocrine Activity (GnRH,LH,FSH,FAS) Neonatal Years Childhood Puberty

  48. + Anosmia ? Developmental Pathways • Anosmia • ? Regulatory GnRH Human Disease Model:Idiopathic Hypogonadotropic Hypogonadism Kallmann’s Syndrome KAL1 (X) FGFR1 (AD) ? FGF 2 ? Others Normosmic IHH GPR 54 ? Kisspeptin FGFR1 ? Others

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