Molecular Genetics of Rare Puberty Disorders in Finland and Denmark

Doctoral Programme in Biomedicine Medicum/Physiology Faculty of Medicine University of Helsinki

MOLECULAR GENETICS OF RARE PUBERTY DISORDERS IN FINLAND AND DENMARK

Johanna Känsäkoski

ACADEMIC DISSERTATION

To be publicly discussed, with the permission of the Faculty of Medicine of the University of Helsinki, in Auditorium XIV, Main Building of the University of Helsinki, Unioninkatu 34, on August 18th 2017, at 12 o’clock noon

Helsinki 2017 Supervised by Professor Taneli Raivio, MD, PhD Physiology, Faculty of Medicine University of Helsinki and Children's Hospital Helsinki University Hospital Helsinki, Finland and Docent Johanna Tommiska, PhD Physiology, Faculty of Medicine University of Helsinki and Children's Hospital Helsinki University Hospital Helsinki, Finland Reviewed by Docent Hannele Laivuori, MD, PhD Department of Medical and Clinical Genetics University of Helsinki and Helsinki University Hospital and Institute for Molecular Medicine Finland / HiLIFE University of Helsinki Helsinki, Finland and Docent Kirsti Näntö-Salonen, MD, PhD Department of Pediatrics Turku University Hospital and University of Turku Turku, Finland Official opponent Professor Helena Kääriäinen, MD, PhD National Institute for Health and Welfare and Department of Clinical Genetics Helsinki University Hospital Helsinki, Finland

ISBN 978-951-51-3532-2 (paperback) ISBN 978-951-51-3533-9 (PDF) http://ethesis.helsinki.fi Unigrafia Oy Helsinki 2017

Table of Contents LIST OF ORIGINAL PUBLICATIONS ...... 7 ABBREVIATIONS AND DEFINITIONS ...... 8 ABSTRACT ...... 12 TIIVISTELMÄ ...... 14 1. INTRODUCTION ...... 16 2. REVIEW OF THE LITERATURE ...... 19 2.1 Sex determination and sex differentiation...... 19 2.1.1 Sex determination ...... 19 2.1.2 Sex differentiation ...... 21 2.2 Disorders of sex development ...... 23 2.2.1 46,XY and 46,XX disorders of sex development ...... 23 2.2.2 Androgen insensitivity syndrome (AIS) ...... 26 2.3 The hypothalamic-pituitary-gonadal axis ...... 27 2.4 The development, migration and control of GnRH neurons ...... 29 2.5 Current understanding on the factors controlling the onset of puberty ...... 32 2.6 Gonadotropin-dependent precocious puberty (GDPP) ...... 34 2.6.1 Clinical manifestation of GDPP ...... 34 2.6.2 Molecular genetics of GDPP ...... 35 2.7 Delayed puberty: congenital hypogonadotropic hypogonadism and Kallmann syndrome ...... 38 2.7.1 Clinical presentation ...... 38 2.7.2 Molecular genetics of CHH ...... 40 2.7.3 Genetic defects underlying Kallmann syndrome ...... 48 2.7.4 Genetic defects underlying normosmic CHH ...... 52 2.7.5 Other genes implicated in CHH ...... 53 2.7.6 Semaphorins ...... 59 3. AIMS OF THE STUDY ...... 65 4. SUBJECTS AND METHODS ...... 66 4.1 Subjects...... 66 4.1.1 Subjects with CAIS ...... 66 4.1.2 Subjects with GDPP ...... 66 4.1.3 Danish subjects with KS or normosmic CHH ...... 67 4.1.4 Finnish subjects with KS or normosmic CHH ...... 67 4.1.5 Controls ...... 67 4.1.6 Ethical issues ...... 68 4.2 Sanger-sequencing ...... 68 4.3 Whole-genome resequencing ...... 69 4.4 Bioinformatic analysis of mutations ...... 69 4.5 RNA extraction and cDNA synthesis ...... 69 4.6 AR cDNA sequencing ...... 69 4.7 RT-qPCR analysis ...... 70 4.8 Cell culture ...... 70 4.9 Immunoprecipitation and western blotting ...... 71 4.10 Amplification of MKRN3 from a hypothalamic cDNA library ...... 72 5. RESULTS ...... 73 5.1 Deep intronic AR mutation leading to CAIS ...... 73 5.1.1 Genomic sequencing and cDNA analysis of AR ...... 73 5.1.2 In silico analysis with Human Splicing Finder ...... 74 5.1.3 Sequencing of the AR cDNA ...... 75 5.1.4 AR protein analysis and quantification of AR cDNA ...... 75 5.2 MKRN3 ...... 79 5.2.1 Results of MKRN3 screening in Danish GDPP patients ...... 79 5.2.2 Expression of MKRN3 in an adult human hypothalamic cDNA library ...... 79 5.3 Phenotypic and genotypic features of Danish patients with CHH ...... 80 5.4 SEMA3A and SEMA7A mutations identified in Finnish CHH patients ...... 86 6. DISCUSSION ...... 89 6.1 New mechanisms underlying complete androgen insensitivity syndrome ... 89 6.2 MKRN3 mutations: a significant cause of GDPP in different populations ... 91 6.3 The genetic features of CHH in Denmark ...... 94 6.4 Current knowledge on the involvement of SEMA3A and SEMA7A mutations in CHH pathogenesis ...... 98 6.4.1 The role of SEMA3A mutations in CHH ...... 98 6.4.2 The role of SEMA7A mutations in CHH ...... 100 7. CONCLUSIONS AND FUTURE PERSPECTIVES ...... 102 8. ACKNOWLEDGEMENTS ...... 105 9. REFERENCES ...... 107

LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following publications, which are referred to in the text by the Roman numerals I-IV:

I. Johanna Känsäkoski, Jarmo Jääskeläinen, Tiina Jääskeläinen, Johanna Tommiska, Lilli Saarinen, Rainer Lehtonen, Sampsa Hautaniemi, Mikko J. Frilander, Jorma J. Palvimo, Jorma Toppari, Taneli Raivio. Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene. Sci Rep. 2016:6:32819. doi: 10.1038/srep32819. II. Johanna Känsäkoski, Taneli Raivio, Anders Juul, Johanna Tommiska. A missense mutation in MKRN3 in a Danish girl with central precocious puberty and her brother with early puberty. Pediatr Res. 2015:78:709-711. doi: 10.1038/pr.2015.159. III. Johanna Tommiska, Johanna Känsäkoski, Peter Christiansen, Niels Jørgensen, Jacob Lawaetz, Anders Juul, Taneli Raivio. Genetics of Congenital Hypogonadotropic Hypogonadism in Denmark. Eur J Med Genet. 2014:57:345-348. doi: 10.1016/j.ejmg.2014.04.002. IV. Johanna Känsäkoski, Rainer Fagerholm, Eeva-Maria Laitinen, Kirsi Vaaralahti, Peter Hackman, Nelly Pitteloud, Taneli Raivio, Johanna Tommiska. Mutation screening of SEMA3A and SEMA7A in patients with congenital hypogonadotropic hypogonadism. Pediatr Res. 2014:75:641–644. doi:10.1038/pr.2014.23

These publications are reproduced with the kind permission of their copyright holders.

ABBREVIATIONS AND DEFINITIONS 17β-HSDIII 17-β hydroxysteroid dehydrogenase 3 AIS Androgen insensitivity syndrome AMH Antimüllerian hormone AMHR Antimüllerian hormone receptor ANOS1 Anosmin-1 AR Androgen receptor AVPV Anteroventral periventricular nucleus AXL AXL receptor tyrosine kinase BMP5 Bone morphogenetic protein 5 BSA Bovine serum albumin BWA Burrows-Wheeler Aligner CAH Congenital adrenal hyperplasia CAIS Complete androgen insensitivity syndrome CBX2 Chromobox 2 CCDC141 Coiled-coil domain containing 141 CCKBR Cholecystokinin B receptor CDGP Constitutional delay of growth and puberty cDNA Complementary DNA CHARGE CHARGE syndrome (Coloboma, Heart defect, Atresia choanae, Retarded growth and development, Genital hypoplasia, Ear anomalies/deafness) CHD7 Chromodomain helicase DNA-binding protein-7 CHH Congenital hypogonadotropic hypogonadism CPHD Combined pituitary hormone deficiency CV Consensus value CYP Cytochrome P450 enzyme DAX1 Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 dbSNP Single Nucleotide Polymorphism database DHH Desert hedgehog DHT Dihydrotestosterone DLK1 Delta like non-canonical Notch ligand 1 DMRT1 Doublesex and mab-3 related transcription factor 1 DMXL2 Dmx like 2 DNA Deoxyribonucleic acid DSD Disorder of sex development DUSP6 Dual specificity phosphatase 6 EMX1 Empty spiracles homeobox 1 8

ESE Exonic splicing enhancer EVS Exome Variant Server FAK Focal adhesion kinase FCS Fetal calf serum FEZF1 FEZ family zinc finger 1 FGF Fibroblast growth factor FGFR1 Fibroblast growth factor receptor 1 FKBP5 FK506 binding protein 5 FLRT3 Fibronectin leucine rich transmembrane protein 3 FOG2 Zinc finger protein, FOG family member 2 FOXL2 Forkhead box L2 FSH Follicle-stimulating hormone GABA γ-aminobutyric acid GABRA1 Gamma-aminobutyric acid type A receptor alpha1 subunit GAPDH Glyceraldehyde-3-phosphate dehydrogenase GATA4 GATA binding protein 4 GDPP Gonadotropin-dependent precocious puberty GnRH Gonadotropin-releasing hormone GNRHR Gonadotropin-releasing hormone receptor GPI Glycosylphosphatidylinositol GPR54 G-protein coupled receptor 54 GWA Genome-wide association HESX1 HESX homeobox 1 HGNC HUGO Gene Nomenclature Committee HPG Hypothalamic-pituitary-gonadal HS6ST1 Heparan sulfate 6-O-sulfotransferase HSD Hydroxysteroid dehydrogenase HSF Human Splicing Finder HSPG Heparan-sulfate proteoglycan IGSF10 Immunoglobulin superfamily member 10 IL17RD Interleukin 17 receptor D ITGB1 Integrin β1 KCNK9 Potassium two pore domain channel subfamily K member 9 KISS1 Kisspeptin 1 KISS1R Kisspeptin 1 receptor KNDy-neuron Kisspeptin-, neurokinin B- and dynorphin A-expressing neurons KO Knockout KS Kallmann syndrome LEP Leptin 9

LEPR Leptin receptor LH Luteinizing hormone LIN28B Lin-28 Homolog B MAF Minor Allele Frequency MAIS Mild androgen insensitivity syndrome MAP3K1 Mitogen-activated protein kinase kinase kinase 1 ME Median eminence MKRN3 Makorin RING finger protein 3 MRI Magnetic resonance imaging mRNA Messenger ribonucleic acid NELF Nasal embryonic LHRH factor NGS Next-generation sequencing nHH Normosmic congenital hypogonadotropic hypogonadism NHLBI National Heart, Lung, and Blood Institute NKB Neurokinin B NMD Nonsense-mediated decay NPY Neuropeptide Y NPY1R Neuropeptide Y receptor Y1 NR0B1 Nuclear receptor subfamily 0 group B member 1 NR5A1 Nuclear receptor subfamily 5 group A member 1 NRP Neuropilin NSMF NMDA receptor synaptonuclear signaling and neuronal migration factor OEC Olfactory ensheathing cell OTUD4 OTU deubiquitinase 4 PAIS Partial androgen insensitivity syndrome PCR Polymerase chain reaction PCSK1 Proprotein convertase subtilisin/kexin type 1

PGD2 Prostaglandin D2 PLXN Plexin PNPLA6 Patatin like phospholipase domain containing 6 PolyPhen-2 Polymorphism Phenotyping v2 POLR3B RNA polymerase III subunit B PROK2 Prokineticin 2 PROKR2 Prokineticin 2 receptor PTC Premature termination codon RNA Ribonucleic acid RNF216 Ring finger protein 216 RSPO1 R-spondin 1 10

RT-qPCR Real-time quantitative PCR SEMA Semaphorin SF-1 Steroidogenic factor 1 SHFM Split-hand/foot malformation SIFT Sorting Tolerant From Intolerant SOD Septo-optic dysplasia SOX SRY-box SPRY4 Sprouty RTK signaling antagonist 4 SRA1 Steroid receptor RNA activator 1 SRD5A2 Steroid 5 alpha-reductase 2 SRSF1 Serine and arginine rich splicing factor 1 SRY Sex determining region Y STAR Steroidogenic acute regulatory protein TAC3 Tachykinin-3 TACR3 Tachykinin-3-receptor U1 snRNP U1 small nuclear ribonucleoprotein UTR Untranslated region WDR11 WD repeat domain 11 WNT4 Wingless-type MMTV integration site family, member 4 WT1 Wilms tumor 1

Genes encoding the proteins are in italics; human genes are in all uppercase letters, mouse genes are in lower case except for the first letter.

ABSTRACT Sexual differentiation and the control of pubertal onset and development are complex processes whose disruption leads to the abnormal development of primary and/or secondary sexual characteristics and usually causes considerable stress for the individual. For example androgens, which signal through the androgen receptor (AR), are required for the correct development of both the internal and the external male sex organs. Mutations in the AR gene cause androgen insensitivity syndrome (AIS), which ranges in severity from complete (CAIS) to partial (PAIS) and to mild (MAIS) forms of androgen resistance. Patients with CAIS are genetically male but phenotypically female, demonstrating the importance of androgen action in sexual differentiation. Other genetic defects cause other variable forms of disorders of sex development (DSD).

During puberty, the ability to reproduce sexually is achieved as sex organs and other sexual characteristics develop further into the mature, adult form. The onset of pubertal development is dependent on the reactivation of the hypothalamic-pituitary-gonadal (HPG) axis by increased secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus. Premature reactivation of this axis results in gonadotropin-dependent precocious puberty (GDPP), defined as the development of secondary sexual characteristics prior to eight years in girls and nine years in boys. On the other hand, in patients with congenital hypogonadotropic hypogonadism (CHH), the HPG axis fails to reactivate properly, leading to delayed, partial, or absent puberty. When a patient with CHH also has absent or defective sense of smell, the condition is called Kallmann syndrome (KS). The KS phenotype reflects the tightly connected development of GnRH neurons and the olfactory system.

Although several genes are implicated in the disorders of puberty and sex development, the majority of the patients remain without a molecular genetic diagnosis. The aim of my thesis work was to identify genetic defects underlying disorders of sex development, specifically CAIS, and disorders of pubertal development, specifically CHH and GDPP, in Finnish and Danish patients.

The genetic cause of CAIS in a family with two affected siblings and without mutations in the AR coding sequence or conserved splice sites was investigated by whole-genome sequencing and cDNA analysis. CAIS was found to be caused by a deep intronic AR mutation, which leads to the formation of two aberrantly spliced mRNA products and a significant reduction in the amount of normal AR mRNA and undetectable AR protein levels. This is the first reported pseudoexon-activating AR mutation that leads to CAIS.

The genetic causes of both extremes of pubertal variation, GDPP and CHH, were investigated in Danish patients. Twenty-nine Danish girls with GDPP were screened for 12

mutations in MKRN3, a maternally imprinted gene recently identified as a regulator of pubertal onset. MKRN3 is expressed only from the paternal allele, and therefore, only paternally inherited MKRN3 mutations can cause precocious puberty. One paternally inherited mutation in MKRN3 was found in one of the screened girls with GDPP, and also in her brother who had early puberty.

Forty-one Danish male patients with CHH were screened for mutations in the known CHH genes ANOS1, FGFR1, FGF8, PROK2, PROKR2, GNRHR, TAC3, TACR3, and KISS1R. Additionally, CDH7 was screened in two Danish CHH patients with hearing loss. In addition, fifty Finnish patients with CHH were screened for mutations in the CHH candidate genes SEMA3A and SEMA7A. Twelve out of forty-one of the Danish CHH patients got a molecular genetic diagnosis; four patients had a mutation in ANOS1, five in FGFR1, one had a homozygous mutation in GNRHR, and the two patients with hearing loss had a mutation in CHD7. Three heterozygous missense variants in SEMA3A were identified in three Finnish KS patients, two of which also had a previously identified mutation in FGFR1. Two heterozygous variants in SEMA7A were identified in one patient with normosmic HH and in one KS patient with a previously identified ANOS1 mutation.

In conclusion, the intronic AR mutation is the first reported case of pseudoexon activation leading to AIS and demonstrates the importance of AR cDNA analysis in AIS patients who still lack a molecular genetic diagnosis. This study also produced new information on the genetic defects underlying the extreme ends of pubertal variation in Finland and Denmark. The results show that mutations in MKRN3 underlie precocious puberty in Danish patients, although the mutations are not very common in sporadic cases. FGFR1, ANOS1, CHD7, and GNRHR mutations were found to underlie CHH in the Danish patients, but the majority of them still remain without a molecular genetic diagnosis. Finally, mutations in SEMA3A and SEMA7A do not seem to contribute significantly to CHH, and it remains to be seen whether mutations in these two genes cause CHH in humans.

TIIVISTELMÄ Sukupuolten erilaistuminen ja murrosikäkehityksen käynnistyminen ja eteneminen ovat monimutkaisia tapahtumasarjoja, joiden häiriöt johtavat primaaristen ja/tai sekundaaristen sukupuoliominaisuuksien epätavalliseen kehitykseen aiheuttaen yleensä paljon huolta niistä kärsiville. Esimerkiksi androgeenireseptorin kautta signaloivat androgeenit ovat välttämättömiä miehen niin ulkoisten kuin sisäistenkin lisääntymiselinten kehitykselle. Mutaatiot androgeenireseptoria koodaavassa AR- geenissä aiheuttavat androgeeniresistenssiä (androgen insensitivity syndrome, AIS), joka vaihtelee vakavuudeltaan täydellisestä (complete AIS eli CAIS) osittaiseen (partial AIS eli PAIS) ja edelleen lievään (mild AIS eli MAIS) androgeeniresistenssiin. CAIS- potilaat ovat geneettisesti miehiä mutta ilmiasultaan naisia, mikä osoittaa androgeenien tärkeän roolin sukupuolten erilaistumisessa. Virheet muissa geeneissä voivat aiheuttaa muita vaihtelevia sukupuolisen kehityksen häiriöitä.

Sukukypsyys saavutetaan murrosiässä sukupuolielimien ja toissijaisten sukupuoliominaisuuksien kehittyessä edelleen aikuiseen muotoonsa. Murrosikäkehityksen käynnistyminen on riippuvainen hypotalamus-aivolisäke- sukupuolirauhanen-akselin (hypothalamic-pituitary-gonadal axis, HPG axis) uudelleenaktivoitumisesta. Uudelleenaktivoituminen tapahtuu gonadotropiineja vapauttavan hormonin (GnRH) erityksen lisääntyessä hypotalamuksesta. Akselin ennenaikainen aktivoituminen johtaa hypotalamuksen toiminnasta johtuvaan ennenaikaiseen murrosikäkehitykseen (gonadotropin-dependent precocious puberty, GDPP), mikä tarkoittaa sekundaaristen sukupuoliominaisuuksien kehittymistä tytöillä ennen kahdeksan ja pojilla ennen yhdeksän vuoden ikää. Toisaalta potilailla, joilla on synnynnäinen hypogonadotrooppinen hypogonadismi (CHH), HPG-akseli ei aktivoidu kunnolla, minkä seurauksena on viivästynyt, puuttuva tai osittainen murrosikäkehitys. Jos potilaalla on CHH:n lisäksi puuttuva tai vajavainen hajuaisti, hänellä sanotaan olevan Kallmannin oireyhtymä (KS). Tämä yhdistelmä on seurausta GnRH:ta tuottavien neuronien ja hajujärjestelmän tiivisti yhteen kytkeytyneestä kehityksestä.

Vaikka sukupuolisen kehityksen ja murrosikäkehityksen häiriöihin on liitetty lukuisia geenejä, valtaosa näistä häiriöistä kärsivistä potilaista on edelleen vailla molekyyligeneettistä diagnoosia. Tämän väitöskirjatyön tavoitteena oli löytää uusia sukupuolen kehityksen ja murrosikäkehityksen häiriöitä aiheuttavia geenivirheitä. Työssä keskityttiin erityisesti androgeeniresistenssin, ennenaikaisen murrosiän ja synnynnäisen hypogonadotrooppisen hypogonadismin taustalla oleviin geenivirheisiin.

Täydellisen androgeeniresistenssin syytä tutkittiin perheestä, jossa oli kaksi androgeeniresistenttiä sisarusta, joilla ei ollut mutaatioita AR-geenin koodaavalla alueella eikä geenin konservoituneissa silmukointikohdissa. Geenivirheen selvityksessä

käytettiin avuksi kokogenomisekvensointia ja AR-geenin cDNA-analyysia. Androgeeniresistenssin aiheuttajaksi paljastui syvällä AR-geenin intronissa sijaitseva virhe, joka johti kahden epätavallisen lähetti-RNA:n syntyyn ja normaalin lähetti-RNA:n määrän huomattavaan vähenemiseen, minkä seurauksena myös AR-proteiinin määrä oli havaitsemattoman alhainen. Tämä geenivirhe on ensimmäinen CAIS-potilailla raportoitu pseudoeksonin aktivoitumiseen johtava mutaatio AR-geenissä.

Tanskalaisista potilaista tutkittiin murrosiän ajoituksen kahden ääripään taustalla olevia geneettisiä syitä. Kahdeltakymmeneltäyhdeksältä tanskalaiselta tytöltä, joilla oli ennenaikainen murrosikä, seulottiin MKRN3-geeni, joka on hiljattain tunnistettu murrosiän alkua säätelevä geeni. MKRN3-geenin äidiltä peritty kopio on hiljennetty, eli vain isältä perityt MKRN3-mutaatiot ilmentyvät ja voivat aiheuttaa ennenaikaisen murrosiän. Yhdeltä seulotuista tytöistä löytyi isältä peritty geenivirhe MKRN3-geenistä. Sama virhe löytyi myös tytön veljeltä, jolla oli aikainen murrosikä.

Neljältäkymmeneltäyhdeltä tanskalaiselta CHH-potilaalta seulottiin tunnetut CHH- geenit ANOS1, FGFR1, FGF8, PROK2, PROKR2, GNRHR, TAC3, TACR3 ja KISS1R. CHD7-geeni seulottiin kahdelta potilaalta, joilla oli kuulonalenema. Lisäksi viideltäkymmeneltä suomalaiselta CHH-potilaalta seulottiin CHH:n kandidaattigeenit SEMA3A ja SEMA7A. Kaksitoista tanskalaista potilasta sai molekyyligeneettisen diagnoosin; neljällä oli mutaatio ANOS1-geenissä, viidellä FGFR1-geenissä, yhdellä oli homotsygoottinen mutaatio GNRHR-geenissä, ja molemmilla kuulonalenemasta kärsivillä potilailla oli mutaatio CHD7-geenissä. SEMA3A-geenistä löytyi kolme heterotsygoottista geenivirhettä kolmelta suomalaiselta KS-potilaalta, joista kahdella oli myös aiemmin löydetty mutaatio FGFR1-geenissä. Kaksi SEMA7A-geenin virhettä löydettiin yhdeltä CHH-potilaalta, jolla oli normaali hajuaisti sekä yhdeltä KS-potilaalta, jolla oli jo aiemmin tunnistettu mutaatio ANOS1-geenissä.

Yhteenvetona totean, että tässä työssä löytynyt ensimmäinen raportoitu AR-geenin pseudoeksonin aktivoitumiseen johtava introninen mutaatio on osoitus siitä, että AR:n cDNA-analyysi voi johtaa taudin molekyyligeneettisen syyn löytymiseen ilman geneettistä diagnoosia jääneillä AIS-potilailla. Lisäksi murrosiän häiriöiden geenivirheistä Suomessa ja Tanskassa saatiin uutta tietoa. Tulokset osoittavat, että virheet MKRN3-geenissä aiheuttavat ennenaikaista murrosikää tanskalaisissa potilaissa, joskin mutaatiot eivät ole kovin yleisiä sporadisissa tapauksissa. Tanskalaisilta CHH- potilailta löytyi mutaatioita FGFR1-, ANOS1-, CHD7 -, ja GNRHR-geeneistä, mutta valtaosa jäi vaille molekyyligeneettistä diagnoosia. Lopuksi totean, että mutaatiot SEMA3A- ja SEMA7A-geeneissä eivät näytä olevan merkittävä CHH:n aiheuttaja, ja nähtäväksi jää, aiheuttavatko mutaatiot näissä kahdessa geenissä ylipäätään CHH:ta ihmisillä.

1. INTRODUCTION The continuation of human reproduction is dependent on the correct formation of two distinct sexes and the subsequent full maturation of the reproductive system in each generation. The phenotypic differences between the two sexes are formed through processes called sex determination and sex differentiation during embryonic development. In mammals, the presence of the SRY locus in the genome sets in motion a series of events that leads to the development of the testes, which in turn secrete hormones that guide the differentiation of the internal and external sex organs towards the male phenotype (Biason-Lauber 2010). The capacity to reproduce sexually is achieved during puberty when the sex organs and secondary sexual characteristics develop into the mature form. The onset of pubertal development is dependent on the reactivation of the hypothalamic-pituitary-gonadal (HPG) axis. The hypothalamic gonadotropin-releasing hormone (GnRH)-secreting neurons begin to secrete GnRH with increased frequency at the onset of puberty. The increased pulsatile secretion of GnRH leads to the secretion of the gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary, which in turn stimulates the formation of gametes and the secretion of sex steroids from the gonads.

Disorders of sex development (DSD) is a broad term for conditions in which sex differentiation or development is atypical. DSDs are usually detected in early childhood due to ambiguous genitalia in the infant, but may also escape detection until adolescence or early adulthood (Hiort et al. 2014), when the first presenting sign may be absent pubertal development, or primary amenorrhea in a female adolescent who has already commenced breast development and has a pubertal growth spurt (Hughes et al. 2012).One example of the latter case is complete androgen insensitivity syndrome (CAIS), in which a loss-of-function mutation in the androgen receptor gene, AR, leads to a female appearance in a genetically male (46,XY) individual (Hughes et al. 2012). Not all patients with CAIS are found to have a mutation in AR (Jääskeläinen 2012), suggesting some mutations in regulatory and other non-coding regions of AR may go undetected in these patients.

Another reason for atypical pubertal development are disorders where the normal timing of puberty is disrupted, such as in gonadotropin-dependent precocious puberty (GDPP) or congenital hypogonadotropic hypogonadism (CHH). GDPP results from the premature reactivation of the HPG axis when GnRH secretion is stimulated or inhibition of GnRH secretion is terminated prematurely, leading to the development of secondary sexual characteristics prior to the age of 8 years in girls or 9 years in boys (Parent et al. 2003). CHH is a rare disorder characterized by absent, delayed or partial puberty that is caused by defects in the development of the hypothalamic GnRH neuron population or defective GnRH secretion or signaling (Kim 2015). CHH presents sometimes as a part of a syndrome, such as Kallmann syndrome (KS), where patients with CHH also have an absent or defective sense of smell. The combination of absent puberty and defective sense of smell is explained by the tightly connected development of the GnRH neurons and the olfactory system (Forni & Wray 2015).

It is estimated that, in the general population, 50-80% of the variation in the timing of puberty is dependent on genetic factors (Gajdos et al. 2010). However, the genetic basis for the control of pubertal onset remains largely unresolved. The development of the GnRH-secreting neurons is also still not fully understood. The identification of genetic defects underlying the extreme ends of pubertal timing reveals new factors that control these critical processes. The molecular genetic causes of GDPP, however, have remained largely a mystery until the recent discovery of mutations in MKRN3, which seems to have an important function in preventing the premature reactivation of the HPG axis in the prepubertal period (Abreu et al. 2013). Additionally, the rarity and the clinical and genetic heterogeneity of CHH make it difficult to study with traditional genetics methods (Cadman et al. 2007). Several genes have already been connected with the disorder; however, in about 50% of the cases, the molecular genetic cause remains unresolved (Kim 2015). The clinical and molecular genetic features of Finnish patients with KS or normosmic HH have been described previously (Laitinen et al. 2011, Laitinen et al. 2012), with 44% of KS patients getting a molecular genetic diagnosis. The high proportion of patients still lacking an identified genetic cause implies that there are more genes whose mutations underlie the disorder in Finland and in other populations. 17

The aim of this thesis work was to identify genetic causes of disorders where the normal development of sex and puberty is disrupted, focusing on CAIS, GDPP, KS, and normosmic CHH.

2. REVIEW OF THE LITERATURE 2.1 Sex determination and sex differentiation 2.1.1 Sex determination During the first weeks of life, embryos of the different sexes are indistinguishable but for their genetic makeup. Development of the different sexes can be divided into two phases; the first phase is sex determination, during which the bipotential gonads develop into testes or ovaries; the second phase is sex differentiation, during which the gonads guide the development of the phenotypical sex by secreting hormones (Biason-Lauber 2010). The bipotential gonads develop around gestational week 6 in humans, and differentiate between weeks 6 and 10 guided by the genetic architecture. In mammals, sex is determined by the sex chromosomes X and Y. The default developmental program is the female developmental pattern, which is suppressed when the embryo carries a Y chromosome, or more specifically, the SRY (sex-determining region on the Y chromosome) locus (Biason-Lauber 2010). Steroidogenic factor 1 (SF-1) is expressed in the bipotential gonad, where it activates the key testis-development-promoting gene SOX9 (SRY-box 9) expression and, if present, SRY expression (Figure 1) (Knarston et al. 2016). SRY and SF-1 act together to upregulate the expression of SOX9, which in turn guides the development of Sertoli cells and thus testis differentiation. Other factors that regulate SOX9 expression include FGF9, prostaglandin D2, DAX1, CBX2, WT1, GATA4, and FOG2 (Knarston et al. 2016). In the absence of SRY, the expression of SOX9 remains low and the gonad develops into an ovary. The main signaling pathway directing ovarian development is the WNT/E-catenin pathway. Wingless-type MMTV integration site family member 4 (WNT4) and R-spondin family member 1 (RSPO1) are positive effectors of E-catenin that are both essential for ovarian development (Knarston et al. 2016). The WNT/E-catenin and SRY/SOX9 signaling pathways repress each other during development, but the repression of the wrong signaling pathway is important even in adulthood to maintain the gonadal identity (Knarston et al. 2016). FOXL2 helps maintain ovarian identity by suppressing SOX9, DMRT1, and other male-specific factors, whereas DMRT1 maintains the identity of the testis by suppressing FOXL2 and retinoic acid signaling (Knarston et al. 2016).

Figure 1. Factors guiding sex determination. WT1 (Wilms tumor 1) and SF1 are expressed in the bipotential gonads, where they induce the expression of SOX9, and SRY, if present. CBX2 promotes the expression of SF1 directly and possibly through the inhibition of DAX1. SF1 and SRY act together to increase SOX9 expression, which is then maintained through a positive autoregulatory loop. SOX9 expression also initiates feedforward loops through FGF9 and prostaglandin D2 (PGD2) signaling. SOX9 promotes Sertoli cell development and represses the β-catenin pathway. When SOX9 expression is low, β-catenin levels rise in response to RSPO1 and WNT4, which shuts down SOX9 expression. FOXL2 helps maintain ovarian identity by repressing male-specific factors. Reprinted from Best Practice & Research Clinical Endocrinology & Metabolism, 24, Anna Biason-Lauber, Control of sex development, 163-186, Copyright (2010), with permission from Elsevier.

2.1.2 Sex differentiation In human development, the reproductive ducts are undifferentiated at weeks 6-7 of gestation, and both sexes have both mesonephric (Wolffian) and paramesonephric (Müllerian) ducts. The differentiation of these ducts is guided by the hormones secreted by the differentiated gonads from around 7 weeks of gestation onwards (Biason-Lauber 2010). In males, the Wolffian ducts develop into epididymis, vas deferens, and the prostate and the Müllerian ducts regress, whereas in females, the Müllerian ducts develop into the fallopian tubes, the uterus, and the upper third of the vagina, and the Wolffian ducts regress (Figure 2) (Biason-Lauber 2010).

In Sertoli cells of the testis, SF-1 activates the expression of antimüllerian hormone (AMH), which causes the regression of the Müllerian ducts. In Leydig cells, SF-1 promotes the expression of androgens, which promote the development of the Wolffian ducts into the male reproductive ducts and the development of the external male genitalia (Figure 2). The testes synthesize testosterone, some of which is metabolized by 5D- reductase type 2 into the more potent dihydrotestosterone (DHT). DHT is required for labioscrotal fusion between the 8th and 12th gestational weeks, and for the growth of the phallus especially in the third trimester; in other words, it is necessary for the development of the external male genitalia, although the internal male genitalia develop normally even in the absence of DHT (Auchus & Chang 2010, Mendonca et al. 2010). In the absence of androgens or androgen action, the Wolffian ducts regress and the external genitalia become female.

Figure 2. The differentiation of the male and female reproductive ducts. Antimüllerian hormone (AMH), which is secreted from the testis, causes the regression of the Müllerian ducts, and testosterone (T) promotes the development of the Wolffian ducts into epididymis, vas deferens, and the prostate. In the absence of AMH and testosterone, the Müllerian ducts develop into the fallopian tubes, the uterus, and the upper third of the vagina, and the Wolffian ducts regress. Reprinted by permission from Macmillan Publishers Ltd: [Nature Medicine] (Martin M Matzuk & Dolores J Lamb. The biology of infertility: research advances and clinical challenges. 14:1197-213), copyright (2008).

2.2 Disorders of sex development 2.2.1 46,XY and 46,XX disorders of sex development Disorders of sex development (DSDs) are defined as ”congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical” (Hughes et al. 2006). DSDs can be either disorders of sex determination or sex differentiation; the former are typically caused by defects in transcription factors that guide gonadal development and the latter by defects in hormones and hormone receptors that are involved in the development of the phenotypical sex (Biason-Lauber 2010). The biological classification of different forms of DSDs is presented in table 1 (Hiort et al. 2014).

The identification of gene defects underlying different forms of DSDs has been invaluable for the identification of the different factors involved in sex determination and differentiation. For example, mutations in SRY, SOX9, NR5A1 (encoding SF-1), GATA4, DMRT1, DHH, CBX2, and MAP3K1 have been identified in individuals with 46,XY disorders of primary sex determination (46,XY complete or partial gonadal dysgenesis) (Bashamboo & McElreavey 2015). 46,XX testicular DSDs and ovotesticular DSDs, in which the gonads are either completely or partially comprised of testicular tissue in an 46,XX individual, are caused by gain-of-function mutations affecting testis- determining genes or loss-of-function mutations in the female pathway genes (Knarston et al. 2016). 46,XX testicular DSD is in 90% of the cases caused by the translocation of the SRY locus into one of the X chromosomes (Knarston et al. 2016). The second most common cause is the duplication of the SOX9 gene or its promoter region, with additional rare cases caused by the duplication of SOX3, SOX10, or FGF9, or a loss-of-function mutation in RSPO1 or WNT4 (Knarston et al. 2016). The majority of the 46,XX ovotesticular DSD cases have an unknown molecular genetic cause, although similar SRY, SOX9, RSPO1, and WNT4 mutations as in 46,XX testicular DSD have been identified in a subset of patients (Knarston et al. 2016).

Table 1. The biological classification of DSDs (table based on Hiort et al. 2014).

Classification based on karyotype Examples of mutated genes Ovotesticular DSD Disorders of Translocation of SRY; gonadal Testicular DSD duplication of FGF9 or SOX9 development Syndromic forms Congenital adrenal CYP21A2, CYP11B1, HSD3B2 hyperplasia (CAH) 46,XX Disorders of Aromatase deficiency CYP19A1 androgen excess Luteoma Latrogenic Mayer-Rokitansky-Küster- LHX1, TBX6 Unclassified Hauser syndrome disoders Complex syndromic disorders Ovotesticular DSD Disorders of Monogenic complete or gonadal SRY, WT1, MAP3K1, NR5A1 partial gonadal dysgenesis development Syndromic forms Syndromic (e.g. Smith- DHCR7 Lemli-Opitz syndrome) Disorders of CAH and early androgen CYP11A1, STAR, androgen biosynthesis defects HSD3B1, CYP17A1 synthesis Androgen biosynthesis 46,XY SRD5A2, HSD17B3 defects Endocrine disruption Disorders of Complete and partial AR androgen action androgen insensitivity Hypospadias with unknown genetic cause Unclassified Epispadias disorders Complex syndromic disorders 45,X (Turner syndrome and variants) Chromosomal 45X/46,XY (mixed gonadal dysgenesis) DSDs 47,XXY (Klinefelter syndrome and variants) Other complex chromosomal rearrengements CYP21A2 encodes 21-hydroxylase; CYP11B1 encodes 11-beta-hydroxylase; HSD3B3 encodes hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (3β-HSDII); CYP19A1 encodes aromatase; DHCR7 encodes 7- dehydrocholesterol reductase; CYP11A1 encodes the cholesterol side-chain cleavage enzyme P450scc; STAR encodes the steroidogenic acute regulatory protein; HSD3B1 encodes Hydroxy-Delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (3β-HSDI); SRD5A2 encodes steroid 5 alpha-reductase 1; HSD17B3 encodes 17-beta hydroxysteroid dehydrogenase 3.

Virilization of the female fetus is a form of 46,XX disorders of sex differentiation caused by excess DHT or foreign androgens during the critical period of gestation when sex differentiation occurs, whereas 46,XY disorders of sex differentiation are commonly caused by defects in androgen production, metabolism, or action (Auchus & Chang 2010, Mendonca et al. 2010). Excess androgens in female fetuses are most commonly derived from the adrenal glands, where a mutation in one of the enzymes involved in steroidogenesis can lead to the diversion of cortisol precursors to other steroid hormones. This disorder is a form of congenital adrenal hyperplasia (CAH), which may lead to early death due to dehydration if left untreated when mineralocorticoid biosynthesis is also affected (Auchus & Chang 2010). Rarely, excess androgens may be the result of aromatase deficiency, i.e. the aromatase enzyme that converts the high levels of fetal adrenal androgens to estrogens does not function properly, which also leads to impaired estrogen production by the ovaries and peripheral tissues and the failed development of female secondary sexual characteristics later in life (Auchus & Chang 2010). The excess androgens may also be of maternal origin, usually due to androgen-producing tumors (Auchus & Chang 2010). 46,XY disorders of sex differentiation may also present as a part of CAH if an enzyme participating in both adrenal and testicular steroidogenesis is affected, or it may be caused by mutations in enzymes involved only in testicular steroidogenesis, such as in 17E-hydroxysteroid dehydrogenase (HSD) type III deficiency (Mendonca et al. 2010). 5D-reductase type 2 deficiency causes a defect in testosterone metabolism, so that sufficient DHT for the development of the external male genitalia is lacking, which results in ambiguous, female-like external genitalia (Mendonca et al. 2010). Defects in androgen signaling are caused by mutations in the androgen receptor gene, leading to androgen resistance, which is discussed in more detail in the next section (2.1.4). Mutations in the genes encoding AMH and its specific receptor, AMHR-II, in turn cause persistent Müllerian duct syndrome, in which 46,XY individuals are otherwise normally virilized and clearly identified as males at birth, but have Müllerian derivatives (uterus and fallopian tubes) that the testes are tightly attached to, causing cryptorchidism and low fertility (Josso et al. 2005).

2.2.2 Androgen insensitivity syndrome (AIS) The most common known cause of 46,XY DSDs is androgen insensitivity syndrome (AIS), which is caused by mutations in AR, the X-linked gene encoding the androgen receptor. There are over 500 CAIS-causing mutations in the Androgen Receptor Gene Mutations Database (Gottlieb et al. 2012) ranging in severity from completely abolished to mildly impaired AR function, which is reflected in the severity of the phenotype. Patients with complete AIS (CAIS), although genetically male (46,XY), are phenotypically female; they have estrogen-dependent secondary sexual characteristics because of peripheral aromatization of androgens, little or no pubic axillary hair and a blind-ending vagina (Hughes et al. 2012). The gonads are testes, but the reproductive ducts fail to develop normally because of the lack of androgen action. The structures that develop from the Müllerian ducts are also missing because of the action of antimüllerian hormone. The typical phenotype in partial AIS (PAIS) is a micropenis, perineoscrotal hypospadias, and a bifid scrotum, although the phenotype can range from mostly male to mostly female (Hughes et al. 2012). Mild AIS presents as infertility without associated genital anomalies. An AR mutation has been identified in 80 to 100% of CAIS patients (Jääskeläinen 2012), but the percentage is much lower, 28-73% (Jääskeläinen 2012), or even as low as 16% (Audi et al. 2010), in PAIS patients. The milder forms of androgen resistance have phenotypic overlap with some other 46,XY DSDs, such as 5D-reductase and 17E-HSD3 deficiencies (Mendonca et al. 2010), and therefore, the identification of the causal mutation is important for a confirmed diagnosis of the disorder (Hughes et al. 2012). The failure to identify an underlying AR mutation in some CAIS patients suggests that mutations in some obligate AR co-factors may cause the disorder in some cases (Adachi et al. 2000), although such mutations have not yet been reported (Jääskeläinen 2012). Alternatively, AIS may be caused by mutations in the introns or regulatory regions of AR that are more difficult to recognize.

2.3 The hypothalamic-pituitary-gonadal axis The hypothalamic-pituitary-gonadal (HPG) axis is responsible for the acquirement and maintenance of reproductive capacity. The hypothalamic gonadotropin-releasing hormone (GnRH) -secreting neurons have a key role in the function of the HPG axis. Mammals have 1000-3000 GnRH neurons that are located as a dispersed continuum from the olfactory bulbs to the medial septal nuclei, preoptic area, anterior hypothalamic area, and mediobasal hypothalamus (Herbison 2014). At least in mice, most GnRH neurons have long projections called dendrons (a combination of an axon and a dendrite) that project into the median eminence (ME), where they release GnRH into the pituitary portal blood vessels (Herde et al. 2013). GnRH then reaches the anterior pituitary, where it stimulates the gonadotropes to secrete the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The gonadotropins are transported in the circulation and target cells in the testes and the ovaries. In males, LH targets the Leydig cells and promotes testosterone production, whereas FSH targets the Sertoli cells where it promotes the synthesis of growth factors that promote spermatogenesis and other factors important for the function of the testis (Smith & Walker 2014). FSH also promotes the synthesis of inhibins in the testes, where they act locally as growth factors, and, through endocrine signaling, exert a negative feedback on FSH secretion at the level of the anterior pituitary (De Kretser et al. 2000). Androgens, which are aromatized into estrogens in the brain, exert a negative feedback on the secretion of GnRH, LH, and FSH at the level of the hypothalamus and the anterior pituitary (Clarke et al. 2012). In females, FSH promotes the maturation of ovarian follicles to the preovulatory stage, and LH promotes ovulation (Hunzicker-Dunn & Mayo 2015). After ovulation, LH acts on the cells that form the corpus luteum and promotes progesterone production. FSH also stimulates inhibin synthesis in the granulosa cells (Hunzicker-Dunn & Mayo 2015). The ovarian cells produce estrogens and progestins, which exert either a positive or a negative feedback on the hypothalamus and the pituitary; during most of the menstrual cycle, the feedback is negative, but it changes to positive just before ovulation and

induces the ovulatory LH surge (Clarke et al. 2012). The HPG axis is depicted in figure 3.

Figure 3. The hypothalamic-pituitary-gonadal (HPG) axis. GnRH is released from the hypothalamus into the pituitary portal vessels. In the pituitary, GnRH stimulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which are transported in the circulation to their target cells in the testes and ovaries, where they promote the synthesis of sex steroids. The sex steroids in return exert either a negative or a positive feedback on the hypothalamus and the pituitary (Clarke et al. 2012).

2.4 The development, migration and control of GnRH neurons GnRH neurons originate outside the brain. GnRH expression is first detected in the nose, in the olfactory placode, around gestational day 10 in the mouse (Schwanzel-Fukuda & Pfaff 1989, Wray et al. 1989b). From there the GnRH neurons migrate into the brain during prenatal development along the axons of the olfactory system (Figure 4) (Schwanzel-Fukuda & Pfaff 1989, Wray et al. 1989a, Schwanzel-Fukuda et al. 1996). The origin of GnRH neurons is still under debate, with some evidence suggesting that some of the cells might actually be derived from the neural crest at least in mice, which could partly explain the heterogeneity of the cells (Forni et al. 2011). In mouse and chicken embryos, neural crest was also identified to be the source of the olfactory ensheathing cells (OECs), which are glial cells that sheath olfactory axons in the same way as nonmyelinating Schwann cells surround other axons of the peripheral nervous system (Barraud et al. 2010). OECs leave the olfactory placode around gestational day 10 in mice, a little before GnRH neurons begin their migratory journey toward the brain (Geller et al. 2013). They form a microenvironment for migrating GnRH neurons, and appear to be crucial for successful migration (Barraud et al. 2013, Pingault et al. 2013, Geller et al. 2013). The correct development and migration of GnRH neurons are dependent on the interplay of multiple factors that regulate craniofacial development, development of the neural crest and the OECs, growth and guidance of the olfactory axons, and neurogenesis, adhesion and migration of the GnRH neurons themselves (Forni & Wray 2015). Defects in any of these processes can lead to impaired reproductive capacity in combination with other developmental abnormalities. The recently developed protocol for GnRH-neuron differentiation from stem cells will potentially reveal previously unknown processes involved in GnRH neuron development and function (Lund et al. 2016).

Figure 4. Schematic drawing of the migratory journey of GnRH neurons. GnRH neurons migrate from the olfactory placode to the hypothalamus along the axons of the olfactory system.

GnRH neurons release GnRH in a pulsatile fashion, which is critical for the secretion of the gonadotropins, since continuous GnRH release has an opposite, inhibitory effect on their secretion from the pituitary (Belchetz et al. 1978). The neurons receive inputs from several parts of the central nervous system to regulate the secretion of GnRH. The long dendrons of GnRH neurons receive synaptic inputs along their entire length, including in the proximity of the ME, which suggests the capability to fine tune neurosecretion (Herde et al. 2013). The dendrons of different neurons also come into close proximity to one another and even share synapses from afferent axon terminals at the sites where they bundle together, which may be a means of synchronization of the dispersed GnRH neuron population (Campbell et al. 2009). There are also dynamic changes in the GnRH dendron trajectories to the ME, for example during the different phases of the estrous cycle, and there appears to be a dynamic interaction between the nerve terminals,

neuroglial cells, and endothelial cells (Prevot et al. 2010). Special ependymoglial cells, called tanycytes, play a major role in controlling the accessibility of the GnRH neuron terminals to the ME, thus controlling the release of GnRH into the pituitary portal vessels, for example during the different phases of the estrous cycle (Prevot et al. 1999). Vascular endothelial cells may be involved in relaying signals, such as hormones, from the circulation to the GnRH neurons and the other cells surrounding them to ensure GnRH is synthesized and secreted effectively at the correct time (De Seranno et al. 2010).

Several neurotransmitters, such as GABA and glutamate, are known to regulate GnRH secretion, but the most potent activator of GnRH neurons identified to date is kisspeptin, which is mainly secreted by neurons located in two different brain regions, namely the preoptic area and the mediobasal hypothalamus (Herbison 2014). At least in rodents, the kisspeptin neuron population located in the preoptic area is bigger in females than in males and is thought to be involved in the activation of the GnRH neurons during the preovulatory LH surge (Kauffman et al. 2007). In humans, however, the effect of estradiol on the pituitary rather than on the kisspeptin neurons may be more important for creating the preovulatory surge (Herbison 2016). The other kisspeptin-producing neurons, located in the arcuate nucleus in the mediobasal hypothalamus, are called KNDy neurons because, in addition to kisspeptin, they also express neurokinin B (NKB) and dynorphin A, which are thought to autoregulate the KNDy neurons in a stimulatory (NKB) or an inhibitory (dynorphin A) manner (Navarro & Tena-Sempere 2012). The importance of NKB as a stimulator of kisspeptin release is seen in cases where loss-of- function mutations either in the gene encoding NKB (TAC3) or its receptor (TACR3) cause CHH, as described later. The KNDy neurons are suggested to be involved in generating the pulsatility of GnRH secretion, in the negative feedback of estrogen on LH secretion, and in the onset of puberty (Herbison 2014). They may also be involved in relaying information on the metabolic status of the body to the GnRH neurons to ensure reproduction is only allowed when sufficient energy is available, although persistent overweight may also reduce Kiss1 expression (Navarro & Tena-Sempere 2012).

2.5 Current understanding on the factors controlling the onset of puberty The ability to reproduce sexually is achieved during puberty, when sex organs and other sexual characteristics develop into the adult form. Usually, the first sign of central puberty in girls is the onset of breast development from Tanner stage B1 (pre-adolescent) into B2 (breast bud stage) (Marshall & Tanner 1969), and in boys the enlargement of testis volume to at least 3 ml (Tanner stage G2) (Marshall & Tanner 1970, Ankarberg- Lindgren & Norjavaara 2004). The onset of puberty is dependent on the reactivation of the HPG axis with the increased pulsatile secretion of GnRH from the hypothalamus, which occurs first during sleep and eventually also during the waking hours as puberty progresses. The HPG axis is also active during the first few months of life (known as minipuberty), when the activity seems to be important for the development of the Sertoli cells in boys (Waldhauser et al. 1981, Andersson et al. 1998), after which it remains quiescent until the onset of puberty.

There is considerable variation, approximately 5 years, in the timing of puberty even among healthy individuals, owing to both genetic and environmental factors (Parent et al. 2003, Gajdos et al. 2010). According to the Copenhagen Puberty study, puberty onset occurs between the ages of 8 and 13 years in most girls, and between the ages of 9 and 14 years in most boys (Aksglaede et al. 2009, Sørensen et al. 2010a). It is estimated that up to 50-80% of the variation in the timing of pubertal onset is explained by genetic factors, which is evidenced by the high correlation of the pubertal timing within ethnic groups, families, and monozygotic twins (Gajdos et al. 2010). Several genome-wide association studies have found that variation at an intergenic locus at 9q31.2 and at the 6q21 locus at or near the LIN28B gene associate with age at menarche (Sulem et al. 2009, He et al. 2009, Ong et al. 2009, Perry et al. 2009), but they are estimated to explain only 0.6% of the variation (He et al. 2009). Because of the complex interplay of multiple factors involved in puberty onset it is most likely impossible to pinpoint the direct influence of only one gene on the timing in the general population. Indeed, a more recent study suggested the involvement of at least hundreds of common variants in pubertal timing (Perry et al. 2014). Interestingly, the authors also found parent-of-origin-specific associations between variation at three imprinted loci, including the genes MKRN3, 32

DLK1, and KCNK9 and age at menarche, and implicated retinoic acid and GABAB receptor II signaling as novel regulators of pubertal timing (Perry et al. 2014).

Epigenetic changes are also involved in the regulation of pubertal timing at least in female rats, where the epigenetic repression of two transcriptional silencers, Eed and Cbx7, is required for increased Kiss1 expression at the onset of puberty (Lomniczi et al. 2013). In addition, microRNAs – miR-200 and miR155 in particular – were recently shown to be involved in the regulation of puberty onset and fertility in mice, where they negatively regulate factors that repress Gnrh expression either directly or indirectly (Messina et al. 2016). From these results it is apparent that the regulation of puberty onset involves a complex system including repressive and activating factors, epigenetic modulators and microRNAs, whose interplay finally leads to the increased pulsatility of GnRH secretion. A model has been proposed where an intrinsic developmental clock measures the overall growth, development, and composition of the body, and ensures that puberty is not initiated until sufficient maturity and the right body composition is reached, after which other factors are able to fine-tune the secretion of GnRH in response to environmental and peripheral signals (Sisk & Foster 2004). One factor that signals information on the body composition is leptin, a hormone produced by adipocytes, which provides the hypothalamus with information on fat mass and energy status of the body and is involved in the control of eating behavior. The importance of leptin signaling for puberty onset is especially seen in patients who have mutations in the genes encoding leptin, LEP, or its receptor, LEPR; these patients are morbidly obese and suffer from hypogonadotropic hypogonadism (Strobel et al. 1998, Clément et al. 1998). The influence of peripheral and environmental factors on puberty is also seen as the secular trend towards earlier puberty onset that occurred most prominently from the mid-1800s to the 1960s in the developed countries, most likely reflecting the influence of both improved nutrition and health care (De Muinck Keizer-Schrama & Mul 2001, Parent et al. 2003). In addition, endocrine-disrupting chemicals in the environment, acute and chronic illnesses, physical and psychological stressors, as well as the climate and the light-dark cycle are all known or suggested to modulate the reproductive axis and pubertal timing (Parent et al. 2003). 33

2.6 Gonadotropin-dependent precocious puberty (GDPP) 2.6.1 Clinical manifestation of GDPP Precocious puberty is defined as the onset of breast development (Tanner B2 stage) before the age of 8 years in girls and as the onset of testicular development (Tanner stage G2) before the age of 9 years in boys (Parent et al. 2003). The recent trend towards earlier puberty onset has raised the question whether the age limits for precocious puberty should be lowered, but no consensus has been reached on the matter (Latronico et al. 2016). Additionally, ethnicity needs to be taken into consideration, as for example African-American girls seem to have earlier puberty onset than white American girls (Herman-Giddens et al. 1997).

Precocious puberty is usually caused by the premature increased pulsatile secretion of GnRH, and it is called gonadotropin-dependent precocious puberty (GDPP) or central precocious puberty, as opposed to peripheral (or gonadotropin-independent) precocious puberty (Latronico et al. 2016). GDPP can be caused by lesions in the central nervous system, such as a hypothalamic hamartoma, hydrocephalus, or a tumor, or it may be caused by endocrine disruptors, changes in the environment (e.g. international adoption), or certain genetic changes. GDPP is much more common in girls than in boys; there are 15-20 times more girls with PP, and the most common causes of PP are also different between the sexes: 50-70% of boys with GDPP have a central nervous system lesion, whereas in girls, 90% of the cases are idiopathic (Latronico et al. 2016). The estimated incidence of GDPP is 1:5 000-10 000 in American girls (Partsch et al. 2002), and the prevalence is 1:500 in Danish girls (Teilmann et al. 2005). Most cases are sporadic, although familial cases also exist, and the mode of inheritance seems to be autosomal dominant with sex-dependent penetrance (de Vries et al. 2004). Few genetic causes of GDPP are known; for example some chromosomal abnormalities resulting in complex syndromes, and mutations in MKRN3, KISS1, and KISS1R, have been reported in GDPP patients (see section 2.6.2) (Latronico et al. 2016).

Individuals with GDPP have progressive pubertal development, and they usually have an advanced bone age and accelerated growth velocity. The follow-up of puberty progression helps to distinguish individuals with GDPP from those who have common 34

variants of premature puberty, for example isolated premature breast development without other pubertal signs, or premature pubic or axillary hair development caused by increased levels of adrenal-derived androgens (Latronico et al. 2016). MRI of the central nervous system is important to determine whether the precocious puberty is due to a central nervous system lesion, which may have no other presenting symptoms (Partsch et al. 2002). Precocious puberty can have several health implications, such as shorter adult height, increased obesity risk, risk of estrogen-dependent cancer, cardiovascular disease, and type 2 diabetes, as well as adverse psychosocial outcomes (Latronico et al. 2016). GDPP can be treated with GnRH agonist treatment, which leads to the desensitization of the pituitary to GnRH and the eventual inhibition of gonadotropin release after a short stimulation period (Partsch et al. 2002). The treatment should result in the regression or stabilization of pubertal symptoms, in lower growth velocity, and in slower advancement of bone age (Latronico et al. 2016).

2.6.2 Molecular genetics of GDPP As mentioned above, the genetic basis of precocious puberty is poorly understood, although the existence of familial cases indicates a genetic cause in a subset of patients (de Vries et al. 2004). Candidate genes have been identified based on genetic linkage and/or function of the gene product; these candidates include KISS1, KISS1R, TAC3, TACR3, GNRHR, LIN28B, LIN28A, GABRA1, and NPY1R (Teles et al. 2011).

LINA28B (HGNC ID: 32207) LIN28B encodes a human homolog of the C. elegans lin-28 gene. lin-28 is a heterochronic gene that controls the developmental timing of the C. elegans larvae; loss- of function mutations of lin-28 lead to precocious development, whereas increased lin- 28 expression leads to a retarded phenotype (Moss et al. 1997). Lin-28 is an RNA- binding protein that is expressed in human embryonic stem cells, and it specifically blocks the processing of the let-7 family miRNAs that control developmental timing (Viswanathan et al. 2008, Winter et al. 2009). Several GWA studies have found an association between variation at or near the LIN28B gene locus and the timing of puberty (Sulem et al. 2009, He et al. 2009, Ong et al. 2009, Perry et al. 2009) or height (Lettre et

al. 2008), but no causal LIN28B mutations have so far been reported in GDPP patients (Silveira-Neto et al. 2012, Tommiska et al. 2011) or in patients with constitutional delay of growth and puberty (Tommiska et al. 2010). Additionally, both Lin28b and Lin28a, a functionally redundant homolog of Lin28b, affect growth and pubertal timing in mice in a sex-dependent manner (Corre et al. 2016). As LIN28A is also another candidate gene whose mutations may be involved in defective pubertal timing, it was screened in Danish girls with GDPP, but no mutations were found in this gene either (Tommiska et al. 2011). Thus, it may be that genetic variation in LIN28B, and possibly LIN28A, affects pubertal timing in the general population but is not at least a common cause of more extreme variants of pubertal timing (Corre et al. 2016).

KISS1 (HGNC ID: 6341) and KISS1R (HGNC ID: 4510) As kisspeptin is an important regulator of GnRH secretion and inactivating mutations in KISS1 and KISS1R cause CHH, it seems reasonable that activating mutations in the same genes could cause precocious puberty. Indeed, a heterozygous activating KISS1R mutation that leads to a prolonged intracellular response to kisspeptin has been reported in a girl with GDPP (Teles et al. 2008). Another heterozygous mutation in KISS1 was reported in 2010 in a boy with GDPP; the mutant protein seems to be more resistant to degradation than the wild-type protein, although it has the same capacity to induce signaling through KISS1R as wild-type kisspeptin (Silveira et al. 2010). Only one other KISS1 variant was identified in the 83 GDPP patients screened in the same study; a homozygous missense variant that was found in two unrelated girls and which did not show any difference to the wild-type protein in the functional assays (Silveira et al. 2010). Other polymorphisms have also been reported in KISS1 in girls and boys with GDPP, but their functional significance is not known (Rhie et al. 2014, Mazaheri et al. 2015). Additionally, some KISS1R and KISS1 polymorphisms may be associated with timing of puberty in Chinese girls, although functional evidence on the effect of the polymorphisms is still lacking (Luan et al. 2007a, Luan et al. 2007b). Overall, KISS1 and KISS1R mutations seem to be a very rare cause of GDPP.

Other candidate genes In the same vein as activating mutations in KISS1 and KISS1R may cause GDPP, same kind of mutations in GNRHR, TAC3, or TACR3 might be expected to underlie the disorder. Other candidate genes based on the function of the gene product include GABRA1 and NPY1R (Teles et al. 2011). GABRA1 encodes the gamma-aminobutyric acid type A receptor alpha1 subunit, which is the main mediator of the inhibitory effects of GABA on GnRH neurons (Terasawa & Fernandez 2001). NPY1R encodes a receptor for neuropeptide Y, which inhibits GnRH release in prepubertal primates (Plant & Barker-Gibb 2004). Since both GABA and NPY exert inhibitory effects on GnRH release during the prepubertal period, loss-of-function mutations in their receptors might be expected to underlie GDPP (Plant & Parker-Gibb 2004, Teles et al. 2011). Conclusive mutations in any of these candidate genes, however, have not been reported in GDPP patients so far, implying these kinds of mutations, if they exist, are very rare (Teles et al. 2011).

MKRN3 (HGNC ID:7114) MKRN3, a maternally imprinted gene that is expressed only from the paternal allele, encodes Makorin RING finger 3 protein, which is a ubiquitously expressed, putative E3 ubiquitin ligase (Jong et al. 1999). MKRN3 is located in the Prader–Willi syndrome critical region on chromosome 15, although its disruption does not seem to be required for the syndrome to develop (Kanber et al. 2009). In 2013, Abreu and colleagues identified paternally inherited mutations in MKRN3 to underlie GDPP (Abreu et al. 2013), and these findings have since been supported by several studies in diverse populations, making MKRN3 mutations the most common identified genetic cause of GDPP to date (Macedo et al. 2014, Settas et al. 2014, Schreiner et al. 2014, de Vries et al. 2014, Lee et al. 2015, Simsek et al. 2016, Dimitrova-Mladenova et al. 2016). Based on the finding that the expression of MKRN3 decreases simultaneously with increased expression of Tac2 and Kiss1 in the mouse arcuate nucleus, and that most of the mutations –being ones that lead to a premature stop-codon– were predicted to be loss- of-function, Abreu et al. suggested that MKRN3 could act as a pubertal break (Abreu et al. 2013). The mechanism of action, however, still remains elusive. The median age of 37

puberty onset in girls with MKRN3 mutations is 6.0 and in boys 8.25, which suggests that the suppression of GnRH release occurs normally after mini-puberty in these patients, but is lost prematurely (Abreu et al. 2015). Therefore, the function of MKRN3 as a GnRH suppressor seems to be critical especially right before puberty onset (Macedo et al. 2014). Recently, Hagen et al. (2015) found that circulating levels of MKRN3 decreased prior to pubertal onset in healthy Danish girls, and that MKRN3 levels were lower in girls who matured early than in the healthy controls. Of note, some of the girls with GDPP had very low or undetectable MKRN3 levels (Hagen et al. 2015). A similar decrease in circulating MKRN3 levels at the onset of puberty has later been reported also in boys (Varimo et al. 2016a, Busch et al. 2016), but the levels do not differ between healthy men and men with CHH (Varimo et al. 2016b). The declining MKRN3 levels at the onset of puberty are in accordance with the current hypothesis that MKRN3 acts as a pubertal break.

2.7 Delayed puberty: congenital hypogonadotropic hypogonadism and Kallmann syndrome 2.7.1 Clinical presentation Delayed puberty is traditionally defined as the absence of breast development by the age of 13 years in girls and the absence of testicular development by the age of 14 years in boys (Sedlmeyer et al. 2002). In most cases, there is no underlying pathological condition, so the delay reflects a part of the normal, wide spectrum of pubertal timing, and puberty will start spontaneously in due course. This condition is called constitutional delay of growth and puberty (CDGP). Adolescents with CDGP tend to be short for their age and have delayed skeletal maturation (Boehm et al. 2015). In contrast, individuals with congenital hypogonadotropic hypogonadism (CHH) typically will not undergo spontaneous pubertal development even by the age of 18 years, although some may have partial pubertal development, and they grow steadily but lack a growth spurt (Boehm et al. 2015).

CHH is caused by defective or absent GnRH secretion or action, which can result from the absence or reduced amount of hypothalamic GnRH neurons, insufficient secretion of GnRH, or the inability of GnRH to stimulate gonadotropin secretion from the pituitary 38

(Bianco & Kaiser 2009). Patients with CHH have low serum levels of testosterone (men) or estradiol (women) and low or normal serum levels of gonadotropins, whereas other pituitary functions are normal (Boehm et al. 2015). When making a CHH diagnosis it is important to confirm that the GnRH deficiency is permanent and not functional, i.e. caused by for example medication, another illness, or excessive stress, weight loss or exercise such as in the case of hypothalamic amenorrhea in women (Seminara et al. 1998). Structural lesions of the hypothalamus or pituitary that can disturb hormone synthesis and secretion must also be ruled out by MRI (Seminara et al. 1998).

CHH is most often diagnosed when puberty fails to occur, although sometimes, when the GnRH deficiency is severe, it can present already in infancy as a micropenis and/or as uni- or bilateral cryptorchidism. Female infants lack such specific clinical signs of CHH, but mini-puberty offers an opportunity to investigate reproductive hormone levels in both sexes if there is a family history of CHH, or if CHH is suspected based on the presence of micropenis or cryptorchidism (Boehm et al. 2015). On the other hand, in some cases, CHH is diagnosed in men who have undergone normal pubertal development but who later develop GnRH deficiency (adult-onset hypogonadotropic hypogonadism) (Nachtigall et al. 1997). Although the GnRH deficiency is usually permanent, approximately 10-15% of patients with CHH have a reversal of the phenotype after discontinuing hormone therapy, so that the patient’s own HPG axis is activated and the levels of reproductive hormones stay normal even after discontinuation of the hormone therapy (Raivio et al. 2007, Laitinen et al. 2012, Sidhoum et al. 2014, Dwyer et al. 2016). This may be due to the plasticity and maturation of the neuronal system responsible for GnRH secretion that occurs in response to the sex steroids given during therapy (Raivio et al. 2007). Some patients carrying milder and more common mutations may have a greater tendency for reversal, although reversal of the phenotype cannot be predicted solely based on the underlying genetic defect (Laitinen et al. 2012, Sidhoum et al. 2014).

CHH can present as isolated or syndromic with variable non-reproductive anomalies. About 50% of CHH patients have a defective sense of smell (anosmia or hyposmia,

meaning completely or partially lacking sense of smell, respectively) with usually – although not always– hypoplastic/aplastic olfactory bulbs (Boehm et al. 2015). These patients are said to have Kallmann syndrome (KS), named after Franz Josef Kallmann, a psychiatrist and geneticist who first suggested a genetic basis for this disorder (Kallmann et al. 1944) that had already been described in 1856 by Maestre de San Juan (Maestre de San Juan, A. 1856). As a distinction from patients with KS, those who have a normal sense of smell are said to have normosmic HH (nHH). In addition to anosmia/hyposmia, patients with CHH can also have other developmental abnormalities that reflect the underlying genetic defect, such as a cleft lip or palate, renal agenesis, missing teeth, hearing loss, or bimanual synkinesia. CHH can also be a part of other syndromes, such as combined pituitary hormone deficiency (CPHD), septo-optic dysplasia (SOD), CHARGE syndrome, Waardenburg syndrome, and Bardet-Biedl syndrome (Boehm et al. 2015), but this work is mainly focused on nonsyndromic HH and Kallmann syndrome.

The incidence of CHH in different populations is estimated to be 1-10 per every 100 000 births, and it is approximately five times more common in men than in women (Bianco & Kaiser 2009). The estimated minimal incidence of Kallmann syndrome in Finland is 1:48 000; 1:30 000 in males and 1:125 000 in females (Laitinen et al. 2011). Possible explanations for the male predominance include sex-dependent penetrance of the underlying mutations due to higher susceptibility of the HPG axis to disturbances in boys and underdiagnosis of CHH in females (Laitinen et al. 2011).

2.7.2 Molecular genetics of CHH CHH is genetically a very heterogeneous disorder with X-linked recessive, autosomal dominant, and autosomal recessive modes of inheritance, although most of the cases are sporadic (Quinton et al. 2001). Although originally considered a monogenic disorder, di- and oligogenic inheritance of CHH have also been suggested, which could partly explain the very variable penetrance of some CHH-causing mutations (Pitteloud et al. 2007a, Sykiotis et al. 2010). Environmental and peripheral factors may also modulate the phenotype together with the genetic background (Mitchell et al. 2011). There are

already over 25 genes, mutations in which have been implicated in CHH, but still about 50% of the patients remain without a molecular genetic diagnosis (Kim 2015), which suggests the involvement of as-yet-unidentified genes whose defects underlie the disorder. It may be that mutations in non-coding regulatory regions of the already identified genes also underlie CHH in some patients. The identification of new CHH genes has been complicated by the rarity and heterogeneity of CHH, the lack of large pedigrees that would permit linkage analysis, and also the possibility of variable penetrance of the mutations. Modern high-throughput sequencing techniques provide a valuable tool that may help to overcome some of these issues.

Normosmic HH and KS are for the most part caused by mutations in different genes, although there is also some genetic overlap. nHH is typically caused by mutations that affect the regulation of GnRH secretion, GnRH signaling in the pituitary, or GnRH itself, whereas KS is caused by mutations that disrupt the development of the olfactory system along with the development and/or migration of the GnRH neurons (Figure 5) (Kim 2015). Genes that have so far been implicated in CHH are listed in table 2.

Figure 5. Genes, whose mutations have been implicated in CHH, grouped according to their known/hypothesized function (see main text for references). Note that CHH can also present as a part of combined pituitary hormone deficiency due to defects in other genes involved in pituitary development, which are not presented here.

Table 2. Genes implicated in congenital hypogonadotropic hypogonadism. The genes are grouped according to their known/suggested function. In cases where the gene may be involved either in GnRH secretion or pituitary development/function, the gene has been grouped under the heading of GnRH secretion.

Typical Mutations first HGNC Chromosomal Mode of additional reported Also Gene Full name nHH/KS ID location inheritance phenotypic in KS/nHH implicated in features patients Defective GnRH neuron development/function X-linked Synkinesia, Franco et al. 1991, ANOS1 Anosmin 1 6211 Xp22.31 KS recessive renal agenesis Legouis et al. 1991 SOD, Cleft lip/palate, CPHD, Fibroblast growth Autosomal dental agenesis, FGFR1 3688 8p11.23 nHH/KS Dodé et al., 2003 Hartsfield factor receptor 1 dominant ear anomalies, syndrome, limb anomalies SHFM Cleft lip/palate, Fibroblast growth Autosomal Falardeau et al., FGF8 3686 10q24.32 nHH/KS hearing CPHD factor 8 dominant 2008 impairment Autosomal PROK2 Prokineticin 2 18455 3p13 nHH/KS Dodé et al., 2006 recessive Prokineticin Autosomal PROKR2 15836 20p12.3 nHH/KS Dodé et al., 2006 receptor 2 recessive Chromodomain Hearing Kim et al. 2008, helicase Autosomal CHARGE CHD7 20626 8q12.2 nHH/KS impairment, Jongmans et al. DNA binding dominant syndrome ear anomalies 2009 protein 7 Autosomal Waardenburg SOX10 SRY-box 10 11190 22q13.1 KS Hearing loss Pingault et al. 2013 dominant syndrome 43

WD repeat domain Autosomal WDR11 13831 10q26.12 nHH/KS Kim et al. 2010 CPHD 11 dominant FEZ family zinc Autosomal FEZF1 22788 7q31.32 KS Kotan et al. 2014 finger 1 recessive Kotan et al. 2014, Coiled-coil domain CCDC141 26821 2q31.2 Digenic? KS? Hutchins et al. containing 141 2016 NMDA receptor synaptonuclear NSMF signaling and 29843 9q34.3 Oligogenic? nHH/KS Miura et al. 2004 neuronal migration factor Heparan sulfate Tornberg et al. HS6ST1 6-O- 5201 2q14.3 Oligogenic? nHH/KS 2011 sulfotransferase 1 Autosomal Young et al. 2012, SEMA3A Semaphorin 3A 10723 7q21.11 dominant/ KS? Hanchate et al. oligogenic? 2012 Interleukin 17 IL17RD 17616 3p14.3 Oligogenic? KS Hearing loss Miraoui et al. 2013 receptor D Dual specificity DUSP6 3072 12q21.33 Oligogenic? nHH/KS Miraoui et al. 2013 phosphatase 6 Sprouty RTK SPRY4 signaling 15533 5q31.3 Oligogenic? nHH/KS Miraoui et al. 2013 antagonist 4 Fibronectin leucine rich FLRT3 3762 20p12.1 Oligogenic? KS Miraoui et al. 2013 transmembrane protein 3

Fibroblast growth FGF17 3673 8p21.3 Oligogenic? nHH/KS Miraoui et al. 2013 factor 17 Immunoglobulin IGSF10 superfamily 26384 3q25.1 Oligogenic? nHH Howard et al. 2016 member 10 AXL receptor Salian-Mehta et AXL 905 19q13.2 Oligogenic? nHH/KS tyrosine kinase al.2014 Defective GnRH secretion/Defective GnRH Autosomal Topaloglu et al., TAC3 Tachykinin 3 11521 12q13.3 nHH recessive 2009 Tachykinin Autosomal Topaloglu et al., TACR3 11528 4q24 nHH receptor 3 recessive 2009 KiSS-1 metastasis- Autosomal Topaloglu et al. KISS1 6341 1q32.1 nHH suppressor recessive 2012 Seminara et al. Autosomal 2003, KISS1R KISS1 receptor 4510 19p13.3 nHH recessive De Roux et al. 2003 Proprotein convertase Autosomal PCSK1 8743 5q15 nHH Obesity Jackson et al. 1997 subtilisin/kexin recessive type 1 Autosomal LEP Leptin 6553 7q32.1 nHH Morbid obesity Strobel et al. 1998 recessive Autosomal Clément et al. LEPR Leptin receptor 6554 1p31.3 nHH Morbid obesity recessive 1998 Ring finger protein Ataxia, Margolin et al. RNF216 21698 7p22.1 Oligogenic? nHH 216 dementia 2013 45

OTU Ataxia, Margolin et al. OTUD4 24949 4q31.21 Oligogenic? nHH deubiquitinase 4 dementia 2013 Central hypothyroidism, peripheral Autosomal polyneuropathy, DMXL2 Dmx like 2 2938 15q21.2 nHH Tata et al., 2014 recessive mental retardation, profound hypoglycemia Nuclear receptor Adrenal X-linked Muscatelli et al. NR0B1 subfamily 0 7960 Xp21.2 nHH hypoplasia recessive 1994 group B member 1 congenita Gordon Patatin like nHH (as Ataxia, Holmes phospholipase Autosomal part chorioretinal Synofzik et al. syndrome, PNPLA6 16268 19p13.2 domain containing recessive of dystrophy, 2014 Boucher- 6 syndromes) brisk reflexes Neuhäuser syndrome Gonadotropin Bouligand et al. Autosomal GNRH1 releasing 4419 8p21.2 nHH 2009, recessive hormone 1 Chan et al. 2009 Defective pituitary development, response, or function Gonadotropin Autosomal De Roux et al. GNRHR releasing 4421 4q13.2 nHH recessive 1997 hormone receptor SOD, Newbern et al. HESX1 HESX homeobox 1 4877 3p14.3 Unknown KS? IGHD, 2013 CPHD

RNA polymerase Autosomal Richards et al. 4H leuco- POLR3B III 30348 12q23.3 nHH recessive 2016 dystrophy subunit B Autosomal Steroid receptor SRA1 11281 5q31.3 recessive/ nHH Kotan et al. 2016 RNA activator 1 digenic? HGNC: HUGO gene nomenclature committee; KS:Kallmann syndrome; nHH: normosmic hypogonadotropic hypogonadism; SOD: septo- optic dysplasia; IGHD: isolated growth hormone deficiency; CPHD: combined pituitary hormone deficiency; SHFM: split-hand/foot malformation

2.7.3 Genetic defects underlying Kallmann syndrome ANOS1 (previously known as KAL1, HGNC ID: 6211) ANOS1 (previously known as KAL1) is an X-linked gene that encodes anosmin-1, which is an extracellular matrix protein that binds to the cell membrane via heparan-sulfate proteoglycans (HSPG) (Soussi-Yanicostas et al. 1996). Anosmin-1 stimulates axonal growth and branching and acts as an axonal guidance molecule (Bülow et al. 2002, Soussi-Yanicostas et al. 2002). It is expressed in the developing olfactory bulbs and kidneys (Hardelln et al. 1999), and has the ability to exert a chemotactic effect on immortalized immature GnRH neurons (Cariboni et al. 2004). Findings in two fetuses, one of which had a deletion of ANOS1 and the other a mutation causing a premature stop-codon in the gene, demonstrated the importance of anosmin-1 in the development of the olfactory system and the migration of GnRH neurons; in both, the olfactory axons and GnRH neurons left the olfactory placode but failed to reach the brain and instead accumulated over the cribriform plate (Schwanzel-Fukuda et al. 1989, Teixeira et al. 2010). Therefore, it seems likely that anosmin-1 helps guide the olfactory axons and possibly also directly promotes GnRH neuron migration during embryonic development, especially around the nasal-forebrain junction (Hardelln et al. 1999, Cariboni et al. 2004). Additionally, studies in chicken have provided some evidence for the involvement of anosmin-1 in the development of the cranial neural crest through the modulation of FGF8, BMP5, and WNT3a signaling (Endo et al. 2012). In adult rodents, new neurons arise in the subventricular zone and migrate to the olfactory bulb via the rostral migratory stream, and anosmin-1 overexpression increases the cell proliferation in the subventricular zone and the migratory activity of the neuroblasts, apparently through FGFR1 signaling (García-González et al. 2016).

ANOS1 was the first gene whose mutations were identified to cause KS (Franco et al. 1991, Legouis et al. 1991). ANOS1 mutations are identified in 5-10% of KS patients (Bianco & Kaiser 2009), they cause severe GnRH deficiency and seem to affect the sense of smell invariably (Hu & Bouloux 2011, Costa-Barbosa et al. 2013). Common additional findings in patients with ANOS1 mutations are bimanual synkinesia, unilateral renal agenesis, and cryptorchidism (Hu & Bouloux 2011, Costa-Barbosa et al. 2013). 48

FGFR1 (HGNC ID: 3688) and FGF8 (HGNC ID: 3686) FGF8 encodes fibroblast growth factor 8, and FGFR1 encodes fibroblast growth factor receptor 1. FGFs regulate morphogenesis during embryonic development through the regulation of cell proliferation, differentiation, and migration, and correct FGF/FGFR signaling is especially important for cranial, digital, and skeletal development (Eswarakumar et al. 2005). FGFs require HSPGs to bind to and activate the FGF receptors, and thus, HSPGs form a potential link between anosmin-1 and FGF signaling (González-Martínez et al. 2004). FGF8 signaling is crucial for correct midline facial integration, the formation of the nasal region, and the development of GnRH neurons (Kawauchi et al. 2005, Forni et al. 2013). Mice with hypomorphic Fgfr1 or Fgf8 mutations either lack or have a significantly reduced amount of GnRH neurons, which seems to be the result of a defect in the early development of the neurons (Falardeau et al. 2008, Chung et al. 2008). The disrupted olfactory and GnRH neuron development may be secondary to defective craniofacial development in Fgf8 hypomorphic mice rather than due to a direct effect of FGF8 on GnRH neuron specification (Forni et al. 2013).

The first mutations in FGFR1 in KS patients were identified in 2003 (Dodé et al. 2003), and they are found in 10% of KS patients and in 6% of all CHH patients (Bianco & Kaiser 2009). In Finnish KS patients, FGFR1 mutations are the most frequently identified molecular genetic cause, especially in females (Laitinen et al. 2011). The first mutations in FGF8 were identified in 2008 (Falardeau et al. 2008) and are rarer than FGFR1 mutations; an FGF8 mutation is identified in less than 5% of KS patients and in less than 2% of all CHH patients (Bianco & Kaiser 2009). Both FGFR1 and FGF8 mutations seem to cause autosomal dominantly inherited CHH. They can cause highly variable phenotypes even within the same family, ranging from apparently completely unaffected individuals, isolated anosmia, or delayed puberty to severe GnRH deficiency with variable non-reproductive anomalies (Dodé et al. 2003, Pitteloud et al. 2006a, Pitteloud et al. 2006b). Additionally, some patients with an FGFR1 mutation undergo reversal of CHH (Pitteloud et al. 2005, Raivio et al. 2007, Laitinen et al. 2012), and an FGF8 mutation has been found to underlie a case of adult-onset HH (Falardeau et al. 49

2008). Common associated anomalies in patients with FGFR1 or FGF8 mutations are a cleft lip or palate, dental agenesis, and ear and limb anomalies (Costa-Barbosa et al. 2013). FGFR1 mutations have also been found to underlie various disorders of craniofacial and skeletal development, such as Pfeiffer syndrome, Hartsfield syndrome, and osteoglophonic dysplasia (Moosa & Wollnik 2016).

PROK2 (HGNC ID: 18455) and PROKR2 (HGNC ID: 15836) PROK2 encodes prokineticin 2, and PROKR2 its receptor. PROK2 is a secreted protein that is involved in various biological processes such as circadian rhythms, pain perception, hematopoiesis, and immune response (Martin et al. 2011). The phenotype of homozygous Prok2- and Prokr2-knockout (KO) mice resembles Kallmann syndrome; they have hypoplasia of the olfactory bulbs and of the reproductive system, and a significantly reduced number of GnRH neurons in the hypothalamus (Ng et al. 2005, Matsumoto et al. 2006, Pitteloud et al. 2007b). Prok2-KO mice also have defective migration of neuroblasts through the rostral migratory stream to the olfactory bulbs (Ng et al. 2005). The most important mechanism by which PROK2/PROKR2 deficiency leads to GnRH deficiency seems to be the disrupted development of the olfactory system, although, as PROK2 and PROKR2 mutations have also been found in nHH patients, an additional direct effect on GnRH neurons cannot be ruled out (Martin et al. 2011). PROK2 and PROKR2 are also expressed in the gonads, so a direct effect on gonadal development/function may contribute to the phenotype (Matsumoto et al. 2006, Martin et al. 2011). Heterozygous mice are apparently unaffected, indicating that at least in mice, one functional Prok2/Prokr2 allele is sufficient for the normal development of the olfactory system and GnRH neurons (Ng et al. 2005, Matsumoto et al. 2006, Pitteloud et al. 2007b).

PROK2 and PROKR2 mutations were first reported in KS patients in 2006 (Dodé et al. 2006), and later also in nHH patients (Pitteloud et al. 2007b, Cole et al. 2008). Mutations in PROK2 and PROKR2 account for 5-10% of KS cases and 3-6% of all CHH cases (Bianco & Kaiser 2009). The mode of inheritance of PROK2 and PROKR2 mutations seems to be autosomal recessive, although heterozygous mutations have also been

reported in CHH patients (Dodé et al. 2006, Pitteloud et al. 2007b, Abreu et al. 2008, Tommiska et al. 2013a). Of note, PROKR2 mutations reported in nHH patients seem to be invariably heterozygous (Cole et al. 2008, Abreu et al. 2008, Martin et al. 2011), whereas one homozygous PROK2 mutation has been reported also in an nHH patient (Pitteloud et al. 2007b), which is in keeping with the fact that Prok2-KO mice seem to have a milder OB phenotype than Prokr2-KO mice (Matsumoto et al. 2006). No additional anomalies have been found to be significantly enriched in KS patients with PROK2 or PROKR2 mutations (Martin et al. 2011, Costa-Barbosa et al. 2013). Interestingly, no PROK2 or PROKR2 mutations have thus far been identified in Finnish CHH patients (Laitinen et al. 2011), but certain PROKR2 mutations are enriched in the Maghrebian population compared to Europeans, suggesting that the population may have undergone a bottleneck or a founder event, or that there are different selection pressures affecting PROKR2 allele frequencies in the Maghrebian population than in the European population (Sarfati et al. 2013).

CHD7 (HGNC ID: 20626) CHD7 encodes the chromodomain helicase DNA-binding protein 7, which participates in gene expression regulation through chromatin remodeling (Basson & van Ravenswaaij-Arts 2015). CHD7 is expressed in the developing olfactory epithelium and olfactory bulbs, the hypothalamus, and several other neural and non-neural tissues during development in mice and humans (Layman et al. 2009). Chd7-deficient mice have olfactory defects, small olfactory bulbs, and a reduced number of olfactory sensory neurons (Layman et al. 2009), as well as a 30-35% reduction in the number of GnRH neurons in embryos and adult mice, and as a result, reproductive dysfunction (Layman et al. 2011). The reduced number of GnRH neurons may in part be explained by the finding that CHD7 is required for the formation of multipotent migratory neural crest cells (Bajpai et al. 2010). In adult mice, CHD7 is required for neurogenesis in the subventricular zone and the subgranular zone (Feng et al. 2013), as well as for neuronal regeneration in the olfactory epithelium (Layman et al. 2009).

Mutations in CHD7 were originally implicated in CHARGE syndrome (coloboma of the eye, heart defects, atresia of the choanae, severe retardation of growth/development, genital abnormalities and ear abnormalities), and the majority of patients with typical CHARGE syndrome are found to have a CHD7 mutation (Janssen et al. 2012). Some CHARGE patients also have hypogonadotropic hypogonadism and anosmia, which led Kim et al. (2008) to search for CHD7 mutations in patients with CHH and KS. Mutations were found in 6% of the patients, both in KS and CHH patients, which led the authors to conclude that CHH caused by CHD7 mutations is a milder allelic form of CHARGE syndrome (Kim et al. 2008). Jongmans et al. (2009) found CHD7 mutations in three out of 56 patients initially diagnosed with CHH. All three patients had anosmia and some phenotypic features of CHARGE syndrome, and two of them were later diagnosed as having CHARGE. The authors concluded that screening of CHD7 is useful especially in those CHH patients who have hearing impairment or other ear anomalies (Jongmans et al. 2009).

SOX10 (HGNC ID: 11190) SOX10 (SRY (Sex Determining Region Y)-Box 10) encodes a transcription factor that is crucial for the differentiation of olfactory ensheathing cells, and thus for axonal targeting of olfactory axons and for the migration of GnRH neurons (Barraud et al. 2013, Pingault et al. 2013). SOX10 mutations have been traditionally associated with Waardenburg syndrome, which has some phenotypic overlap with Kallmann syndrome (Pingault et al. 1998, Bondurand et al. 2007). In fact, in 2013, mutations in SOX10 were also identified to underlie KS with hearing loss (Pingault et al. 2013), and they seem to contribute to a significant number of these cases (Pingault et al. 2013, Vaaralahti et al. 2014).

2.7.4 Genetic defects underlying normosmic CHH GNRH1 (HGNC ID: 4419) and GHRNR (HGNC ID: 4421) GNRH1 encodes the 92-amino acid preprohormone that is cleaved into the biologically active GnRH decapeptide, and GNRHR encodes the GnRH receptor. Homozygous or compound heterozygous mutations in these genes cause normosmic HH. The first

GNRHR mutations were reported in 1997 (De Roux et al. 1997), and 3.5-16% of sporadic nHH cases and up to 40% of familial cases are explained by GNRHR mutations (Bianco & Kaiser 2009). Some patients especially with milder GNRHR mutations undergo reversal of the phenotype (Raivio et al. 2007, Laitinen et al. 2012b). GNRH1 mutations are rare with only a few reported cases to date (Bouligand et al. 2009, Chan et al. 2009, Mengen et al. 2015).

KISS1 and KISS1R KISS1 encodes a 145 amino acid protein that is cleaved into the 54, 14, 13 and 10 amino acid kisspeptins found in human (Kotani et al. 2001). The only KISS1 mutations causing nHH to date were reported in 2012 (Topaloglu et al. 2012). KISS1R, also known as GPR54, encodes the kisspeptin receptor, and the first mutations in this gene were identified in 2003 (Seminara et al. 2003, De Roux et al. 2003). KISS1 and KISS1R mutations are recessive, and KISS1R mutations are found to underlie nHH in about 5% of cases (Bianco & Kaiser 2009).

TAC3 (HGNC ID: 11521) and TACR3 (HGNC ID: 11528) TAC3 and TACR3 encode neurokinin B and the neurokinin B receptor, respectively. The first mutations in TAC3 and TACR3 were reported in 2009 in four consanguineous families, where homozygous mutations in either gene were identified to underlie the phenotype (Topaloglu et al. 2009). The normal phenotype of heterozygous TAC3 and TACR3 mutation carriers points to recessive inheritance of the mutations (Guran et al. 2009, Young et al. 2010, Francou et al. 2011), although mutations in these genes have been found in CHH patients also in the heterozygous state (Gianetti et al. 2010).

2.7.5 Other genes implicated in CHH NSMF (HGNC ID: 29843, previously known as NELF; nasal embryonic LHRH factor) encodes NMDA receptor synaptonuclear signaling and neuronal migration factor. A heterozygous possibly pathogenic NSMF variant was first reported in an nHH patient in 2004 (Miura et al. 2004). Since then, only a few NSMF variants have been reported, most of them heterozygous and present in combination with a variant in another CHH gene, suggesting NSMF variants may contribute to di/oligogenic inheritance of CHH but

heterozygous variants alone are probably not sufficient to cause CHH (Pitteloud et al. 2007a, Xu et al. 2011). Female Nsmf-knockout mice have been reported to have reduced fertility and number of GnRH neurons in the hypothalamus (Quaynor et al. 2015). However, the biological role of NSMF is still under debate, as another report described Nsmf-KO mice with hippocampal dysplasia and no KS-associated features (Spilker et al. 2016).

WDR11 (HGNC ID: 13831) encodes WD repeat-containing protein 11, which is a possible interaction partner of the homeobox protein EMX1 that is involved in olfactory neuron development (Kim et al. 2010). WDR11 was identified as a CHH gene based on a balanced translocation on 10q26, located near WDR11, in a KS patient, and the subsequent screening of WDR11 in 201 CHH patients, revealing heterozygous, probably pathogenic variants in six patients (Kim et al. 2010). WDR11 variants were found both in KS and nHH patients. Few reports on WDR11 mutations in CHH patients exist, and WDR11 mutations have so far not been identified in Finnish KS patients (Laitinen et al. 2011).

FEZF1 (HGNC ID: 22788) encodes a zinc-finger protein that acts as a transcriptional repressor and is expressed in the olfactory epithelium, the amygdala, and the hypothalamus during embryonic development (Watanabe et al. 2009, Damla Kotan et al. 2014). In mice, Fezf1 seems to be required for the olfactory axons to be able to cross the central nervous system basal lamina, and deficiency of Fezf1 leads to the inability of olfactory axons to reach the olfactory bulb and to the disrupted migration of GnRH neurons (Watanabe et al. 2009). Consistently, homozygous recessive FEZF1 mutations have been identified in two consaguineous families, in which the affected individuals had severe HH and anosmia without any additional anomalies, indicating the importance of FEZF1 function during the development of the olfactory system also in human (Damla Kotan et al. 2014). In one of the families, another homozygous mutation in CCDC141 was also identified, suggesting an additional genetic defect underlying the phenotype (Damla Kotan et al. 2014, Hutchins et al. 2016). CCDC141 encodes a coiled-coil domain-containing protein that is expressed in GnRH neurons and olfactory fibers

during the migration of GnRH neurons in mice. The knockdown of Ccdc141 in mouse nasal explants reduces the migration of GnRH neurons without affecting olfactory axons or GnRH neuron adhesion, suggesting a cell motility defect as the cause of the reduced migration and possible contribution of CCDC141 mutations to the CHH phenotype (Hutchins et al. 2016).

Heparan sulfates (HS) are extracellular glycosaminoglycans that are important for the development of the nervous system (Inatani et al. 2003). 6-O-sulfated heparin sulfates seem to be especially important for the function of anosmin 1 in C. elegans, and also for the function of FGFR1 and FGF8 (Eak & Salmivirta 2002, Bülow et al. 2002, Tornberg et al. 2011). HS6ST1, encoding HS 6-O-sulfotransferase 1, was therefore screened in 338 individuals with GnRH deficiency, of which 2% –both KS and nHH patients– were found to have either homozygous or heterozygous loss-of-function mutations in the gene, and some mutations were found in several individuals with widely varying phenotypes (Tornberg et al. 2011). The varying phenotypes and the presence of FGFR1 and NSMF mutations in two families led the authors to suggest that HS6ST1 mutations may not alone be sufficient to cause full-blown GnRH deficiency (Tornberg et al. 2011).

AXL encodes a member of the Tyro3-Axl-Mer receptor tyrosine kinase subfamily that transduces signals from the outside of the cell into the cytoplasm by binding GAS6 (Hafizi & Dahlbäck 2006). Axl/Tyro3 double-knockout mice have a reduced number of GnRH neurons, delayed first estrus, and abnormal estrus cyclicity (Pierce et al. 2008), whereas Axl-knockout mice have a delayed first estrus and prolonged interval between vaginal opening and first estrus. This led Salian-Mehta et al. (Salian-Mehta et al. 2014) to screen 104 subjects with CHH for mutations in AXL. Four heterozygous variants were found, both in individuals with nHH and KS, and some were also reported in databases of genetic variation (Salian-Mehta et al. 2014). Although two of the variants showed partial loss of function in functional assays, none of them is most likely sufficient to cause the phenotype alone (Salian-Mehta et al. 2014).

FGF8 synexpression group FGF17 (HGNC ID: 3673), IL17RD (HGNC ID: 17616), DUSP6 (HGNC ID: 3072), SPRY4 (HGNC ID: 15533), and FLRT3 (HGNC ID: 3762) are all members of the so- called ‘FGF8 synexpression group’, i.e. a group of genes whose expression pattern during development is similar to FGF8, and which are also able to modulate FGF8 signaling through FGFR1 (Miraoui et al. 2013). Variants in these genes were recently identified in CHH patients, and FGF17 and IL17RD were deemed as the best CHH candidate genes based on their interactions with previously identified CHH genes. Mutations especially in the FGF8-signaling inhibitor IL17RD seem to be enriched in KS patients with hearing loss (Miraoui et al. 2013). 19% of the patients had variants in more than one gene of the FGF8 synexpression group or other known CHH genes, suggesting oligogenic inheritance; additionally, control subjects were found to carry some of the variants found in CHH patients, but a significantly higher proportion of the CHH patients compared to the controls carried two or more variants (Miraoui et al. 2013).

CHH as part of other syndromes PCSK1 (HGNC ID: 8743) encodes proprotein convertase-1 that processes precursor proteins, such as prohormones, into smaller, bioactive products. Recessive mutations in PCSK1 cause the deficiency of several neuroendocrine hormones and insulin, leading to severe obesity, HH, hypocortisolism, elevated pro-opiomelanocortin (POMC) levels, and abnormal glucose homeostasis coupled with low levels of insulin and elevated levels of proinsulin as a result of impaired proprotein processing (Jackson et al. 1997).

NR0B1, previously known as DAX1, is located on the X chromosome and encodes an orphan nuclear receptor that acts as a transcriptional regulator. Mutations in NR0B1 cause X-linked adrenal hypoplasia congenita with hypogonadotropic hypogonadism (Muscatelli et al. 1994). Although the exact function of NR0B1 is still unclear, it seems to have a role in the development of the testes and the adrenal glands. NR0B1 mutations seem to cause a combined hypothalamic and pituitary defect leading to impaired gonadotropin secretion from the pituitary (Suntharalingham et al. 2015). Interestingly, some NR0B1 mutations result in early puberty, and one frameshift mutation in NR0B1

has also been identified in a boy with central precocious puberty and no signs of adrenal insufficiency (Shima et al. 2016).

A homozygous recessive mutation in DMXL2 (HGNC ID: 2938), encoding rabconnectin-3D, was identified to underlie a new syndromic form of CHH in three brothers of a consanguineous family (Tata et al. 2014). The brothers had gonadotropin deficiency, central hypothyroidism, peripheral neuropathy, mental retardation, and diabetes (Tata et al. 2014). Rabconnectin-3D is a putative scaffold protein for the regulators of Rab3a, Rab3-GAP and Rab3-GEP, and it is expressed in exocytotic vesicles in GnRH neuron axonal extremities and in gonadotropes in the pituitary (Tata et al. 2014). Rabconnectin-3D is involved in regulated insulin secretion and most likely also in neurosecretion, and conditional Dmxl2-KO mice have a reduced number of GnRH neurons in the hypothalamus, possibly due to a mechanism where the defective neurosecretion leads to the loss of GnRH neurons in the hypothalamus (Tata et al. 2014).

HESX1 (homeobox gene expressed in embryonic stem cells 1) is expressed in Rathke’s pouch during early development of the anterior pituitary, and it is important for the development of the pituitary, hypothalamus, optic nerve, and the forebrain (Dattani et al. 1998). HESX1 mutations have been identified in individuals with SOD, isolated growth hormone deficiency, and combined pituitary hormone deficiency, and more recently, also in some individuals with KS and no other associated anomalies (Newbern et al. 2013). Notably, all the KS patients had heterozygous mutations; HESX1 mutations have been identified both in homozygous and heterozygous states, and homozygous mutations seem to lead to a more severe phenotype such as SOD or panhypopituitarism (Newbern et al. 2013).

Recessive mutations in PNPLA6 are implicated in the Gordon Holmes and Boucher- Neuhäuser syndromes, which are characterized by ataxia, nHH, and either brisk reflexes (in Gordon Holmes syndrome) or chorioretinal dystrophy (in Boucher-Neuhäuser syndrome) (Synofzik et al. 2014). PNPLA6 encodes neuropathy target esterase, which de-esterifies phosphatidylcholine into fatty acids and glycerophosphocholine, which is required for the biosynthesis of acetylcholine. PNPLA6 mutations seem to disrupt the 57

ability of the pituitary gland to secrete gonadotropins in response to GnRH stimulus, although a contributing hypothalamic effect may also be present (Topaloglu et al. 2014). Digenic homozygous mutations in RNF216 and OTUD4 have also been identified in a consanguineous family with nHH, ataxia, and dementia (Margolin et al. 2013). Compound heterozygous and heterozygous RNF216 mutations were subsequently also identified in unrelated individuals with a similar phenotype, suggesting a potential di-or oligogenic inheritance (Margolin et al. 2013). RNF216 encodes a ubiquitin E3 ligase, and OTUD4 encodes a deubiquitinase, which, when knocked down simultaneously in zebrafish, exhibit a more severe phenotype than when either one is knocked down individually, suggesting an epistatic relationship between the two genes (Margolin et al. 2013). This phenotype includes cerebellar hypoplasia, microphthalmia, and small optic tecta, indicating that loss-of-function mutations in the two genes are involved in neurodegeneration. The patients seemed to have defects at both the hypothalamic and pituitary levels, but the exact mechanism behind the reproductive phenotype remains unknown (Margolin et al. 2013).

Recent advancements in the molecular genetics of CHH Heterozygous rare variants in IGSF10 were recently identified in several individuals with self-limited delayed puberty, CHH, or functional hypogonadism (Howard et al. 2016). The function of IGSF10 is unknown, but it is expressed in the nasal mesenchyme of mice and human embryos at the time of GnRH neuron migration. Its knockdown reduces the migration of immature GnRH neurons in vitro as well as the migration and neurite outgrowth of GnRH3 neurons in zebrafish (Howard et al. 2016). The authors hypothesized that the pathogenic variants may lead to reduced GnRH neuron numbers in the hypothalamus or an impaired GnRH neuronal network, which may predispose the individuals carrying them to delayed puberty (Howard et al. 2016). It may be that other predisposing factors, such as mutations in other CHH genes, are needed for the phenotype to manifest, as most of the rare variants identified in delayed puberty/CHH patients are also reported in genetic variation databases (Howard et al. 2016).

Recessive mutations in POLR3B, encoding a subunit of RNA polymerase III, are known to cause 4H leucodystrophy, a syndrome consisting of ataxia, hypogonadotropic hypogonadism, and hypodontia (Tétreault et al. 2011). Recently, however, potentially pathogenic mutations in this gene were also found in individuals with CHH but without any signs of neurodegeneration or dental anomalies, expanding the phenotypic range caused by POLR3B mutations (Richards et al. 2016). One of these patients also showed resistance to GnRH treatment, suggesting a pituitary rather than a hypothalamic defect (Richards et al. 2016).

A targeted next-generation sequencing approach of 261 genes known or suggested to be involved in CHH pathogenesis or in the development or function of GnRH neurons, the hypothalamus, the pituitary, or the olfactory system, revealed promising variants in 18 novel candidate genes in 48 CHH patients (Quaynor et al. 2016). The authors suggested CCKBR, which encodes the cholecystokinin B receptor, to be one of the most promising candidate genes based on the cosegregation of a CCKBR variant with the phenotype in a large family (Quaynor et al. 2016). While these results are promising, extensive functional studies and larger patient cohorts are needed before anything conclusive can be said about the pathogenicity of the variants.

Very recently, Kotan et al. (2016) reported inactivating mutations in the SRA1 gene, which functions both through a protein and a non-coding RNA product, in three families with nHH. The mutations appear to be recessive because the parents, who were heterozygous carriers, had normal pubertal development. In one of the families, the patients had a heterozygous mutation in SRA1 in combination with another heterozygous mutation in PNPLA6, suggesting digenic inheritance (Kotan et al. 2016). The authors suggested that SRA1 may regulate the expression of SF-1 target genes –including GNRHR and the gonadotropins—in the pituitary together with DAX1, thus in part regulating gonadotropin release from the pituitary (Kotan et al. 2016).

2.7.6 Semaphorins The semaphorin family is a group of developmental guidance molecules, all 27 members containing a conserved sema domain responsible for the specificity of receptor binding

(Tamagnone & Comoglio 2000). The semaphorin family is divided into eight classes, of which classes 3-7 are found in vertebrates; class 3 semaphorins are secreted molecules, class 4-6 semaphorins have a transmembrane domain, and the one member of class 7 is glycosylphosphatidylinositol (GPI)-anchored to the cell membrane (Pasterkamp 2012). Semaphorins were first identified as having repulsive, growth-cone collapsing properties towards growing axons, but it is now known that they have diverse functions in the development of the nervous system, in vascular patterning, in the formation of different tissues, and in the immune system (Mann et al. 2007). Semaphorins bind to receptors located in the growth cones of growing axons, which induces changes in the cytoskeleton and cell adhesion to guide the axon to the appropriate target. They regulate, among other things, the targeting and branching of axons, axon bundling, the migration of neurons, apoptosis, the targeting and branching of dendrites, axonal pruning, and synaptic signaling (Mann et al. 2007). Semaphorins signal in neurons predominantly through receptor complexes containing plexins, nine of which have been identified in vertebrates (PLXNA1-4, PLXNB1-3, PLXNC1, and PLXND1) (Pasterkamp 2012). In addition, SEMA3 family members require neuropilins (either NRP1 or NRP2) in order to bind to the receptor complex (Pasterkamp 2012). Plexins are large transmembrane proteins that all have an extracellular sema domain and a conserved intracellular domain which can be phosphorylated and acts as an R-Ras guanosine triphosphatase –activating protein (Perälä et al. 2012). Several signaling molecules bind to the intracellular domains of plexins, thus allowing the activation of multiple intracellular signaling pathways in response to semaphorin binding (Franco & Tamagnone 2008). In addition, some membrane-spanning semaphorins are able to function as receptors for plexins and other semaphorins, thus allowing for even more diversity in semaphorin/plexin signaling (Pasterkamp 2012).

SEMA3A (HGNC ID: 10723) SEMA3A is one of the most studied members of the semaphorin family, and it is the first identified member of the secreted semaphorin class (Sharma et al. 2012). SEMA3A signals mainly through receptors containing neuropilin 1 (NRP1), but in some cells it can also signal through NRP2-containing receptors (Cariboni et al. 2011). SEMA3A is 60

also able to signal through receptors containing any of the plexin A family members, plexin B1 or plexin D1, so SEMA3A signaling is not dependent on any one single plexin family member (Sharma et al. 2012). The role of SEMA3A in development has been studied extensively both in vitro and in mouse models, and it has been shown that SEMA3A can, among other things, regulate the formation of dendrites and the migration of neurons, induce neuronal apoptosis, promote the formation of axon bundles in the peripheral nervous system, and prevent the premature growth of axons during development as well as guide the migration of neural crest cells (Taniguchi et al. 1997, Huber et al. 2005, Mann et al. 2007, Osborne et al. 2005, Schwarz et al. 2009). Despite the diverse roles of SEMA3A during development, most Sema3A-KO mice do not seem to have as extensive developmental defects as might be expected, which may be due to the redundant roles of different guidance molecules during neuronal development (Taniguchi et al. 1997, Catalano et al. 1998). Behar et al. (1996), however, reported severe developmental defects in Sema3A-KO mice including skeletal, heart, and central nervous system defects leading to early postnatal death. These differences probably reflect the different genetic backgrounds of different laboratory mouse strains, which makes the interpretation of the results quite challenging (Taniguchi et al. 1997). Interestingly, the most severe axonal defects of the central nervous system in other Sema3A-KO mouse strains seem to affect the axons of the olfactory system and indicate that SEMA3A is involved in organizing the axons of the olfactory sensory neurons that project to the olfactory bulbs, thus participating in the formation of the topographic map of the olfactory system (Schwarting et al. 2000, Imai et al. 2009). Mice lacking either SEMA3A or both NRP1 and NRP2 also have a phenotype resembling Kallmann syndrome caused by ANOS1 mutations; their vomeronasal axons accumulate above the cribriform plate together with GnRH neurons and the mice have smaller testes, making SEMA3A a strong KS candidate gene (Cariboni et al. 2011).

Mutations in SEMA3A have also been found in Kallmann syndrome patients: Young et al. (2012) reported a family, in which a heterozygous 213-kb deletion in SEMA3A, deleting the last 11 exons of the gene, was present in patients with KS with no other associated anomalies. KS seemed to be transmitted with an autosomal dominant mode 61

of inheritance with reduced penetrance in the family, as one family member had only isolated hyposmia and normal fertility (Young et al. 2012). Hanchate et al. (2012) screened 386 KS patients for SEMA3A mutations and reported a frameshift mutation and seven missense mutations, all in the heterozygous state, in 6% of the studied patients. All changes, excluding the frameshift mutations, had been previously reported in databases, and some of the variants were also present in healthy controls. Additionally, five patients had a mutation in another gene known to be involved in CHH (ANOS1, FGFR1, PROKR2, or PROK2), leading the authors to conclude that the identified SEMA3A mutations are most likely not sufficient to cause the phenotype on their own but most likely contribute to the pathogenesis of KS together with mutations in other genes (Hanchate et al. 2012).

SEMA7A (HGNC ID: 10741) Semaphorin 7A was originally detected in the immune system and is the closest relative of viral semaphorins (Lange et al. 1998). SEMA7A can be present both as a GPI- anchored membrane protein and as a soluble protein, and it signals through both plexin C1 and β1-integrins (Pasterkamp et al. 2003, Pasterkamp 2012). SEMA7A stimulates the production of cytokines in monocytes and monocyte movement as well as their differentiation into dendritic cells (Holmes et al. 2002), and Sema7A-knockout mice have a deficient T-cell-mediated inflammatory response (Suzuki et al. 2007). SEMA7A also has a potential role in the differentiation of osteoblasts and osteoclasts (Delorme et al. 2005), and some SEMA7A polymorphisms are associated with bone density (Koh et al. 2006). SEMA7A also carries the John Milton Hagen blood group antigens (Seltsam et al. 2007).

Among its many functions, SEMA7A is also involved in the development of the nervous system. During development, SEMA7A is expressed in the neural crest in chick (Bao & Jin 2006), and in the median eminence and other parts of the hypothalamus, Rathke’s pouch and the later developing adenohypophysis, olfactory epithelium, olfactory bulbs, and other parts of the developing nervous system in rat (Pasterkamp et al. 2007). Both the GPI-anchored and the soluble form of SEMA7A promote axon growth, and

SEMA7A signaling through β1-integrins promotes axon outgrowth from the olfactory bulbs to the olfactory cortex (Pasterkamp et al. 2003).

SEMA7A is expressed along the axons of olfactory and vomeronasal nerves during mouse development and promotes the migration of GnRH neurons through β1-integrin- dependent signaling (Messina et al. 2011). Sema7A-KO mice have approximately a 30% reduction in the number of GnRH neurons in their brains, the number of GnRH fibers in the ME is reduced, and their fertility is also significantly reduced compared to wild-type littermates (Messina et al. 2011). The olfactory system, however, develops normally in these mice, suggesting SEMA7A promotes the migration of GnRH neurons directly but is not crucial for the correct growth and targeting of the nerves of the olfactory system (Messina et al. 2011). Targeted disruption of Itgb1 in mouse GnRH neurons leads to a 30% reduction of GnRH-neuron cell bodies in the adult mouse brain, and innervation of the ME by GnRH fibers is reduced by over 70% in females and 33% in males: accordingly, female GnRH-Itgb1-KO mice also have significantly reduced fertility compared to wild-type mice (Parkash et al. 2012). Based on the phenotype of Sema7A- KO mice, SEMA7A is a promising candidate gene for normosmic HH, but the role of SEMA7A mutations in the pathogenesis of CHH is still unclear.

SEMA3E (HGNC ID: 10727) Recently, a heterozygous missense mutation in another secreted semaphorin, SEMA3E, previously identified to be involved in vascular patterning, axonal guidance, and synapse formation, was identified in two brothers with KS (Cariboni et al. 2015). Unexpectedly, SEMA3E seems to protect maturing GnRH neurons in the brain rather than promote their migration. In mice where either Sema3E or its receptor, PlxnD1 had been knocket out, there was increased apoptosis of GnRH neurons in the developing brain, and innervations to the ME were reduced (Cariboni et al. 2015). The brothers also had a heterozygous mutation in CHD7, and it is unclear to what extent the different mutations are responsible for the phenotype (Cariboni et al. 2015). A heterozygous de novo mutation in SEMA3E has also been identified in a patient with CHARGE syndrome (Lalani et al. 2004). As no other reports on SEMA3E mutations in CHARGE patients

exist, the role of SEMA3E mutations in the pathogenesis of CHARGE syndrome remains unresolved (Sanlaville & Verloes 2007).

Semaphorin signaling in the postnatal brain Recent studies have provided evidence that, in addition to guiding the migration of GnRH neurons during embryonic development, semaphorin signaling may also be involved in the morphological changes occurring throughout the estrous cycle in response to changing levels of estrogen and progesterone in rodents (Giacobini et al. 2014, Parkash et al. 2015). Recently, Parkash et al. (2015) showed that Semaphorin 7A functions also in the adult rodent brain to modulate the connections of GnRH neuron terminals to the ME. SEMA7A is secreted by tanycytes, the ependymal cells surrounding GnRH neuron nerve terminals, in response to circulating ovarian steroids, and the expression is highest on the day of the diestrus. GnRH neurons express PlexinC1, and when SEMA7A signals through this receptor, it causes the nerve terminals to retract. At the same time, mediated by SEMA7A signaling through ITGB1, tanycyte processes expand to further eliminate contact between GnRH neurons and the ME (Parkash et al. 2015). This mechanism could provide one link between changes in the circulating sex steroids and changes in GnRH secretion from the hypothalamus. The physiological relevance of this mechanism is seen in female PlexinC1-KO mice, which lack normal estrous cyclicity and have reduced fertility as a result (Parkash et al. 2015). In contrast, SEMA3A, which is expressed by vascular endothelial cells in the ME, has an opposite effect on the GnRH nerve terminals, which also express the SEMA3A receptor NRP1 (Giacobini et al. 2014). The 65 kDa isoform of SEMA3A reaches a peak in expression on the day of proestrus in the ME of female rats in response to circulating estradiol, and promotes the growth of the GnRH nerve terminals towards the vascular walls. The disruption of SEMA3A/NRP1 signaling in rats leads to abnormalities in the ovarian cycle, demonstrating the importance of SEMA3A as a regulator of GnRH neuron access to the ME during the different phases of the estrous cycle (Giacobini et al. 2014).

3. AIMS OF THE STUDY To identify genetic causes of disorders where the normal timing of pubertal development or normal development of sex is disrupted. Specifically, to investigate:

1) the cause of complete androgen insensitivity syndrome in two patients without mutations in the coding region or conserved splice sites of the androgen receptor gene (I) 2) if mutations in MKRN3 contribute to gonadotropin-dependent precocious puberty in Danish girls (II) 3) the genetics of congenital hypogonadotropic hypogonadism in Denmark (III) 4) whether mutations in the candidate CHH genes SEMA3A and SEMA7A are present in Finnish patients with congenital hypogonadotropic hypogonadism (IV)

4. SUBJECTS AND METHODS 4.1 Subjects 4.1.1 Subjects with CAIS The two subjects with CAIS were born to a non-consanguineous couple after normal pregnancy and birth. They are the first (A) and the third (B) child in the family; the second child is a 46,XX girl. The first child was tested for her karyotype after an operation to treat an inguinal hernia revealed a gonad in the hernia sac. Her karyotype was found to be 46,XY, and ultrasound and MRI revealed completely feminine external genitalia and abdominal gonads, which were confirmed to be functional testicular tissue based on serum testosterone and inhibin levels (2.5 nmol/l and inhibin-B 668 pg/ml, respectively, at 3 months 3 weeks age). The vagina was only 0.7 cm. MRI revealed no uterine structures. Her serum hormone levels at the age of 12.9 years were: LH 19 U/L, FSH 4.1 U/L, testosterone 29 nmol/L, estradiol 0.149 nmol/L, and inhibin B 426 ng/L, which verifies functional testicular tissue.

Child B was karyotyped from the amniotic fluid and the result (46,XY) was confirmed after birth. Her phenotype was identical to that of the first child, with no uterine structures found in the MRI. At the age of 8.9 years, her serum hormone levels were: LH 3.2 U/L, FSH 4.4 U/L, testosterone 1.1 nmol/L, estradiol <0.025 nmol/L, and inhibin B 222 ng/L, which is consistent with the presence of functional testicular tissue and commencing puberty.

4.1.2 Subjects with GDPP 29 girls with GDPP were recruited from the outpatient clinic at the Department of Growth and Reproduction, Copenhagen University Hospital. Details of this study have been published previously (Sørensen et al. 2010b). In short, the girls were diagnosed with GDPP if their age at onset of breast development was <8 years, peak-LH level in response to rapid-acting GnRHa was >5 IU/l, and their brain MRI was normal. The girls had previously been screened for mutations in KISS1, KISS1R, LIN28A, and LIN28B, but no causative mutations had been found (Tommiska et al. 2011).

4.1.3 Danish subjects with KS or normosmic CHH All patients registered with an ICD10 code corresponding to CHH (DE23.0D) were identified through the register of the Department of Growth and Reproduction, Copenhagen University Hospital, and the diagnosis was validated by carefully evaluating the patient records. All clinical and biomedical data were reordered and results from dual-energy X-ray absorptiometry, brain MRI, and sense of smell (tested or self-reported) were included when available. DNA from 41 male patients with CHH was available through the biobank.

4.1.4 Finnish subjects with KS or normosmic CHH 50 patients were previously diagnosed as having CHH based on i) clinical signs and symptoms of hypogonadism (absent puberty, infertility, decreased libido), ii) low sex steroid levels in association with inappropriately low or normal gonadotropin levels, and subnormal/normal response to GnRH stimulation test, iii) otherwise normal anterior pituitary function, iv) normal radiological imaging of the hypothalamic and pituitary areas, and in case of KS, v) defective sense of smell detected by formal testing (UPSIT score <5th percentile of age (University of Pennsylvania Smell Identification Test, Sensonic, Haddon Heights, NJ, USA)) and/or absent or rudimentary olfactory bulbs in MRI. The patients were enrolled from all five university hospitals in Finland. All patients had previously been screened for mutations in the known CHH genes (FGFR1, FGF8, PROK2, PROKR2, CHD7, WDR11 (all patients), ANOS1 (KS patients only), GNRHR, GNRH1, KISS1R, KISS1, TAC3, and TACR3 (nHH patients only)). Of the 50 patients, 20 had been found to have mutation(s) in ANOS1 (3 patients), FGFR1 (12 patients), GNRHR (4 patients), or CHD7 (1 patient). Thirty of the patients had not been found to have a conclusive mutation in any of the known CHH genes. Of the mutation-negative patients, 19 (18 men, 1 woman) had KS, 9 (7 men, 2 women) had nHH, and two men had adult-onset HH.

4.1.5 Controls Controls were from the same geographical region as the subjects. The controls used in study III were 95 healthy prepubertal girls from the Copenhagen Puberty study (Aksglaede et al. 2009). Controls used in study IV were DNA samples from 200 healthy

anonymous blood donors obtained from the Finnish Red Cross blood service. Additionally, the presence and the frequency of the identified variants were checked from the dbSNP (https://www.ncbi.nlm.nih.gov/SNP/) (studies I and III), NHLBI Exome Sequencing Project (Exome Variant Server, http://evs.gs.washington.edu/EVS/) (studies II, III and IV), and 1000 Genomes (www.1000genomes.org; phase 3, all variants) (study I) databases.

4.1.6 Ethical issues All participants and the guardians of participants less than 16 years old gave their written informed consent. Studies I and IV were approved by the Ethics Committee of the Helsinki University Central Hospital, study II was approved by the scientific ethics committee for Copenhagen and Frederiksberg (KF 01 282214 and KF 11 2006-2033) and the Danish Data Protection Agency, and study III was approved by the regional ethics committee (KF01328087) and the Danish Data Protection Agency (2006-41- 7251). All studies adhered to the approved guidelines.

4.2 Sanger-sequencing The Sanger-sequencing method was used to screen the AR gene (RefSeq ID: NM_000044) in the two CAIS patients (study I); MKRN3 (NM_005664.3) in the Danish girls with GDPP (study II); ANOS1 (NM_000216.3), FGFR1 (NM_023110.2), FGF8 (NM_033163.3), PROK2 (NM_001126128.1), PROKR2 (NM_144773.2), GNRHR (NM_000406.2), TAC3 (NM_013251.3), TACR3 (NM_001059.2), and KISS1R (NM_032551.4) in the Danish HH patients, as well as CHD7 (NM_017780.3) in the two patients with hearing loss (study III); and SEMA3A (NM_006080.2) and SEMA7A (NM_003612.4) in the Finnish patients with CHH (study IV).

Genomic DNA samples had been previously obtained from all patients by extraction from peripheral blood leukocytes. Coding exons and exon-intron boundaries of the genes were PCR-amplified from the genomic DNA samples and purified with ExoSAP-IT (Amersham Biosciences, Piscataway, NJ, USA) or illustra ExoProStar 1-step (GE Healthcare Life Sciences, Chicago, IL, USA) treatment. The purified products were sequenced bi-directionally using the ABI BigDyeTerminator Cycle Sequencing Kit

(v3.1) and ABI Prism 3730xl DNA Analyzer automated sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were aligned and read with the Sequencher software (v4.9 or v5.4.1) (Gene Codes Corporation, AnnArbor, MI, USA).

4.3 Whole-genome resequencing Whole-genome resequencing of the two siblings with CAIS and of their healthy father was performed at the Beijing Genomics Institute (BGI, Shenzhen, China) by using Illumina’s HiSeq 2000 technology.

4.4 Bioinformatic analysis of mutations The effects of the identified missense mutations were predicted with the web-based analysis tools PolyPhen2 (Adzhubei et al. 2013) (studies II, III, and IV), SIFT (Kumar et al. 2009) (study II), and Mutation Taster (Schwarz et al. 2014) (study II). The effects of identified splice-site mutations were predicted with the web-based analysis tool Human Splicing Finder v.2.4.1 or v.3.0 (Desmet et al. 2009) (studies I and III). All analyses were performed with the default settings.

4.5 RNA extraction and cDNA synthesis TriPure Isolation Reagent (Roche) was used to extract total RNA from cultured cells according to the manufacturer’s recommendations, and RNA pellets were suspended in

30 μl of sterile H2O. Complementary DNA (cDNA) synthesis was done with Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics) using 1 μg of RNA in a 20 μl volume, and the cDNA samples were further diluted into 200 μl with sterile

H2O.

4.6 AR cDNA sequencing The entire coding region of AR cDNA was PCR-amplified in overlapping fragments from the cDNA samples obtained from the patients’ genital skin fibroblasts, control fibroblasts, and LNCaP cells. The PCR products were visualized on a 1.5% agarose gel. The different-sized bands produced from the patient samples were extracted from the gel with QIAquick gel extraction kit (Qiagen, Hilden, Germany) and were sequenced as described above (section 4.2).

4.7 RT-qPCR analysis A 4 μl aliquot of cDNA was used in each qPCR reaction, which contained 0.4 μM each primer and 1 x LightCycler® 480 SYBR Green I Master (Roche) in a 10 μl reaction volume. The PCR program was as follows: denaturation at 95 ˚C for 10 min, 40 cycles of 95 ˚C, 20 sec; 58 ˚C, 20 sec and 72 ˚C, 20 sec, and final extension 72 ˚C for 5 min. The primers used are listed in table 3. Quantification of the normally spliced AR mRNA was performed with primers targeting specifically the normal variant with the reverse primer located at the junction of exons 6 and 7. The results were calculated for each gene of interest in relation to GAPDH expression. Further, the results of all samples were expressed in relation to control sample XX31B vehicle (0.1% ethanol) treatment (value

-(ΔΔCt) set to 1) using the formula 2 , where ΔΔCt is ΔCt(sample)-ΔCt(XX31B vehicle), ΔCt is Ct(gene of interest)-Ct(GAPDH) and Ct is the cycle where the threshold value is crossed.

Table 3. Sequences of the primers used in the RT-qPCR reactions

Forward 5’- TTGGAGACTGCCAGGGAC-3’ Total AR mRNA Reverse 5’- TCAGGGGCGAAGTAGAGC-3’ Forward 5’- AAAAGGCCAAGGAGCACAAC-3’ FKBP5 Reverse 5’-TTGAGGAGGGGCCGAGTTC-3’ Forward 5’-TGGGGAAGGTGAAGGTCGG -3’ GAPDH Reverse 5’-TCTCAGCCTTGACGGTGCC-3’ Forward 5’- CAGTGTGTCCGAATGAGGCA-3’ Normally spliced AR Reverse 5’- CCCATCCACTGGAATAATGCTGA-3’

4.8 Cell culture Patient fibroblasts (obtained from the labia majora) and human control fibroblasts were grown in Dulbecco's Modified Eagle Medium (Gibco, Life Technologies, 41965, Paisley, Scotland) supplemented with 1% (vol/vol) penicillin and streptomycin and 10% fetal calf serum (FCS; HyClone, Thermo Scientific SV30160-03, Gramlington, UK). LNCaP prostatic cancer cells were grown in RPMI (Gibco, Life Technologies, A10491, Grand Island, NY, USA) supplemented with 1% (vol/vol) penicillin and streptomycin and 10% FCS. For the experiments, the cells were split onto 6-well plates (250 000

cells/well) and allowed to grow for 24 h, after which the cells were incubated in steroid- depleted medium (fibroblasts in Dulbecco's Modified Eagle Medium supplemented with 2.5% stripped serum and LNCaP cells in RPMI supplemented with 10% stripped serum) for 6 h. Subsequently, vehicle (0.1% EtOH) was added to a half and 1 nM AR agonist methyltrienolone (R1881) (Perkin Elmer, NCP-005, Boston, MA, USA) to a half of the wells. Cells were collected for immunoprecipitation or RNA extraction 18 hours after the addition of vehicle or R1881.

4.9 Immunoprecipitation and western blotting Cells were collected by using phosphate-buffered saline containing 10 mM N- ethylmaleimide, and cells from three wells were pooled into one sample yielding approximately 2 million cells. The cells were resuspended in lysis buffer (50 mM Tris- HCl, pH 8.0, 140 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% (vol/vol) Triton X-100, 10% glycerol, 10 mM Na-phosphate, 50 mM NaF, 10 mM N-ethylmaleimide, 1x protease inhibitor cocktail (cOmplete Protease Inhibitor Cocktail tablets, Roche Diagnostics GmbH, Mannheim, Germany)) and incubated on ice for 20 min, after which they were sonicated for 10 sec, centrifuged at 13000 x g for 20 min at +4 ˚C, and the supernatants were transferred into new tubes. The supernatants were precleared in rotation at +4 ˚C for 1h with magnetic beads that had been equilibrated in the lysis buffer containing 0.5% bovine serum albumin (BSA). A magnet was used to separate the beads from the supernatant from which an aliquot was taken to serve as an input sample.

The immunoprecipitation was performed with Magna ChIP™ Protein A magnetic beads (Millipore, Temecula, CA, USA) by using a polyclonal antibody (rabbit α-AR (Karvonen et al. 1997)) raised against the full-length AR coupled to beads in lysis buffer which contained 0.5% BSA in rotation over night at +4 ˚C. The supernatants were immunoprecipitated with beads coupled to the antibody in rotation at +4 ˚C overnight, after which the beads were washed thrice with the lysis buffer. 1 x sodium dodecyl sulfate sample buffer containing 10 mM N-ethylmaleimide and 1 x protease inhibitor cocktail was used to elute the immunoprecipitated proteins from the beads, and the input and the immunoprecipitated samples (corresponding to 40% of each immunoprecipitate)

were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels followed by transfer onto nitrocellulose membranes. A mouse monoclonal α-AR antibody 441 targeting amino acids 299-315 of the N-terminal domain (sc-7305, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and a horseradish-peroxidase-conjucated anti-mouse secondary antibody (Life Technologies, Eugene, OR, USA) were used to detect AR on the membrane. To determine the loading of the input samples, a rabbit polyclonal α-GAPDH antibody (sc-25778; Santa Cruz Biotechnology Inc.) was used.

4.10 Amplification of MKRN3 from a hypothalamic cDNA library Expression of MKRN3 in adult human hypothalamus was investigated by amplifying a 598-bp-long fragment of the MKRN3 cDNA from a commercial human hypothalamic cDNA library (Clontech Alboratories Inc., Mountain View, CA, USA). The hypothalami of altogether 30 male and female Caucasians aged 15-68 years had been collected after sudden death to construct the cDNA library. Cyclophilin G served as a positive control gene for the PCR. Following amplification, the PCR products were visualized on a 2.0% agarose gel.

5. RESULTS 5.1 Deep intronic AR mutation leading to CAIS 5.1.1 Genomic sequencing and cDNA analysis of AR Sanger sequencing of the coding region and the exon-intron boundaries of AR revealed no rare potentially pathogenic variants. Both CAIS siblings had only one synonymous polymorphism (c.639G>A p.(Glu213=), rs6152, minor allele frequency 0.24 in the 1000 Genomes (www.1000genomes.org; phase 3, all variants) database) in the coding region of AR.

The entire coding region of AR cDNA was PCR amplified and the products were visualized on a 1.5% agarose gel. Amplification with primers situated on exon 5 and the 3’untranslated region (UTR) produced two abnormally long products from the patients’ cDNA samples, and a significantly reduced amount of the normal-sized product as compared to the control fibroblasts (Figure 6).

Figure 6. PCR amplification of AR cDNA with primers situated on exon 5 and the 3’UTR from the cDNA samples of patient (T1, T2) and control (XX31B, XY31A) fibroblasts and LNCaP cells.

Whole-genome sequencing of the two CAIS patients and their father yielded sequencing data with an average sequencing depth of ≥35.14 and ≥99.68% coverage for each sample.

The AR gene region (GRCh37 genomic position chrX:66,764,465-66,950,461) had an average sequencing depth of ≥19.04 and at least 99.4% bases had ≥4x coverage in each sample. The data revealed a point mutation, c.2450-118A>G (GRCh37 genomic location chrX:66,942,551), in intron 6 of AR, consistent with the observed abnormal splicing of the cDNA. The mutation was not reported in the dbSNP or the 1000 Genomes database, and it was confirmed by Sanger sequencing to be present as hemizygous in both CAIS patients, heterozygous in their mother, and absent in the father and the healthy sister (Figure 7). The whole-genome sequencing data revealed no other potentially pathogenic variants in the AR gene region.

Figure 7. Sanger sequencing of the deep intronic mutation in intron 6 of AR. The mutated base (c.2450-118A>G) is marked with black. The mutation was hemizygous in both affected siblings with CAIS, heterozygous in their mother, and absent in the father and the healthy sister.

5.1.2 In silico analysis with Human Splicing Finder According to the in silico analysis, the intronic mutation may create either a new acceptor splice site [+61.08% increase in the motif score from 47.4 (wild type), to 76.35 (mut) with the HSF prediction algorithm (consensus value (CV) threshold 65, variation threshold +/-10%)] or a new donor splice site [+15.34% increase of the score from 68.92 (wild type) to 79.49 (mut) with the HSF prediction algorithm (CV threshold 65, variation threshold +/-10%), and +1053.95% increase from 0.76 (wild type) to 7.25 (mut) with

MaxEntScan algorithm (CV threshold 3, variation threshold +/-30%)]. In addition to a new splice site, HSF predicted that the mutation may also create a new exonic splicing enhancer (ESE) motif for the serine/arginine-rich splicing factor SRSF1 (SF2/ASF) [mutant motif value 75.19 with ESE Finder algorithm (threshold value 72.98)].

5.1.3 Sequencing of the AR cDNA Consistent with the in silico analysis, the sequencing of the abnormal products revealed the addition of either 85 bp of intronic sequence which comprised the mutated base and the 84 nucleotides upstream of the mutation, or 202 bp of intronic sequence comprising the aforementioned 85 nucleotides and all of the intronic sequence downstream of the mutation site leading up to exon 7 (Figure 8). Thus, the mutation led to the formation of two abnormally spliced mRNAs that contained either a new cryptic 85-bp exon between exons 6 and 7, or an abnormally long exon 7 containing 202 bp of the upstream intronic sequence.

5.1.4 AR protein analysis and quantification of AR cDNA Both abnormal splice variants code for 12 additional amino acids (NRIQLSFPLRSP) followed by a premature termination codon (PTC) after amino acid 816. Both aberrant AR mRNA isoforms are predicted to undergo nonsense-mediated decay due to the presence of an intron downstream of the PTC (Isken & Maquat 2007). RT-qPCR analysis revealed that the patient fibroblasts expressed overall similar amounts of AR mRNA when compared to control fibroblasts (Figure 9a), but the amount of the normally spliced AR mRNA in patient fibroblasts was only approximately 10% of the expression levels in the XX31B control fibroblasts (T1: 9.0-10.9% expression, T2: 11.6-12.9% expression) (Figure 9b). Consistent with the qPCR result, AR protein was undetectable in the patient samples in the western blot performed after immunoprecipitation with a polyclonal anti-AR antibody, although it was detected in the control fibroblast samples (Figure 9c). The expression of both the AR mRNA and the AR protein were significantly higher in the LNCaP prostate cancer cell line in comparison to the sample and control fibroblasts. Androgen-receptor signaling was examined in patient and control cells by adding the AR agonist R1881 to the cells and quantitating the expression of the

androgen-inducible gene FKBP5, but the amount of AR was sufficient for any induction only in the LNCaP cells (Figure 10).

Figure 8. The schematic representation of the two aberrant mRNAs (A), sequence chromatograms showing the borders of the normal and cryptic exons of the two aberrant splice variants (B), and the nucleotide sequences of exon6 and 7, intron 6 and the cryptic exons (C). A) In splice variant 1, there is a new 5’splice site (ss) motif where the U1 snRNP binds; in splice variant 2, there is a new exonic splicing enhancer (ESE) motif where the splicing factor SRSF1 binds. In both scenarios, the cryptic exonic sequences are marked with Ψ. B) Sequence chromatograms showing the borders of the normal exons and the cryptic sequences of splice variant 1 (top two chromatograms) and 2 (bottom two chromatograms). C) The nucleotide sequences of the cryptic exons and the flanking exons and introns. The identified mutation is in bold. The cryptic exonic sequences are highlighted in yellow (and red/green) and correspond to the boxes marked with Ψ in panel A. The new splicing motifs created by the mutation are highlighted either in red (5’ splice (donor) site) or in green (ESE motif).

Figure 9. The quantification of the total AR mRNA (A), the normally-spliced AR mRNA (B), and the AR protein (C) expression. Control (XX31B, XY31A, XX54A) or patient (T1, T2) fibroblasts and LNCaP cells were seeded on 6-well plates and after 24h, the medium was changed to steroid-depleted medium for 6h. The cells were treated with 1nM R1881 (+) or vehicle (-) (0.1% EtOH) for 18h before RNA extraction (A, B) or immunoprecipitation (C). In A and B, the expression of total AR mRNA (A) or the normally spliced AR mRNA (B) was quantitated with RT-qPCR. GAPDH served as the reference gene. Expression in all samples was normalized to the expression in vehicle-treated XX31B cells. Normally spliced AR mRNA was quantitated from vehicle-treated samples. In C, AR was immunoprecipitated with a rabbit polyclonal α-AR antibody and detected in western blot with a mouse monoclonal α-AR antibody. GAPDH served as a reference to control for the loading of the input samples. 77

Figure 10. Quantification of the androgen-inducible FKBP5 gene mRNA expression in response to the AR agonist R1881. Control (XX31B, XY31A, XX54A) or patient (T1, T2) fibroblasts and LNCaP cells were seeded on 6-well plates. After 24h, the medium was changed to steroid-depleted medium for 6h, after which the cells were treated for 18h with 1nM R1881 (+) or vehicle (-) (0.1% EtOH). Expression of FKBP5 was quantitated with RT-qPCR. GAPDH served as the reference gene, and the expression in all samples was normalized to the expression in vehicle-treated XX31B cells. Bars represent mean ± SD of 3-5 independent samples.

5.2 MKRN3 5.2.1 Results of MKRN3 screening in Danish GDPP patients Screening of MKRN3 revealed one heterozygous missense mutation, c.1034G>A p.(Arg345His), in one Danish girl with GDPP, who was born from non-consanguineous Caucasian parents. The girl had experienced periodic breast enlargement starting at 6 years of age and rapid breast and pubic hair development starting at 7 years. At 7.5 years, she had breast stage 4 and pubic hair stage 3, accelerated linear growth (height SDS +2.0) and advanced bone age (10 years Greulich Pyle). She had pubertal LH response (from 1.28 to 32.2 IU/l) to a GnRH test (0.1 mg Relefact), basal estradiol 68 pmol/l, inhibin B 124 pg/ml, and IGF-1 469 ng/ml (2.82 SDS). MRI of the brain was normal. The girl was treated with GnRH analogue for 2 years, after which it was stopped, and subsequently, her puberty resumed and she had menarche at 11.5 years of age. She had final height of 164.2 cm (target height was 169.2 cm).

The same mutation was also identified in the girl’s brother, who had a very early voice break at 10.5 years, indicating early pubertal development. His puberty, however, was not formally assessed. He also had cystic fibrosis and was therefore seen at regular visits. The siblings had inherited the mutation from their father, since the mother did not have it. The father’s puberty was described as average, and the mother had normal timing of menarche at 11 years old.

The mutation has been reported with a frequency of 1/8,600 chromosomes in the European American population in the EVS database. The mutation is situated in the C3HC4 RING domain of MKRN3, and is predicted to be deleterious by PolyPhen-2 (“probably damaging”, score 0.01), Mutation Taster (“disease causing”, probability 0.999999998), and SIFT (“affects protein function”, score 0.01).

No other mutations in MKRN3 were identified in the 29 Danish girls with GDPP.

5.2.2 Expression of MKRN3 in an adult human hypothalamic cDNA library The PCR amplification of the MKRN3 cDNA, visualized by agarose gel electrophoresis, revealed strong expression in the human hypothalamic cDNA library (Figure 11).

Figure 11. PCR amplification of MKRN3 cDNA from the human hypothalamic cDNA library. MKRN3 expression in adult human hypothalamus was investigated by PCR- amplification of a 598-bp fragment of the MKRN3 cDNA from a commercial hypothalamic cDNA library. Cyclophilin G was used as a positive control for the PCR. The PCR products were visualized on a 2.0% agarose gel. NT: no template (negative control).

5.3 Phenotypic and genotypic features of Danish patients with CHH Of the 41 patients, 16 had anosmia/hyposmia (as tested clinically or reported by the patient), 13 had normal sense of smell, and for 12 patients the data on olfaction was not available. Twenty-three (56%) of the patients had a history of microphallus, which is consistent with severe neonatal GnRH deficiency.

Twelve (29%) of the patients were identified to have a conclusive mutation in one of the screened genes. The most commonly mutated gene was FGFR1, where 5 (12%) patients were found to have a mutation; only one of the mutations (c.289G>A p.(Gly97Ser)) had been previously reported in a KS patient, but the rest (c.625C>T p.(Arg209Cys), c.1535C>T p.(Ala512Val), c.1936C>T p.(Arg646Trp), and c.1614C>T p.(Ile538=)) were previously unreported. All of the missense mutations were predicted by PolyPhen- 2 to be probably damaging with a score of 1.000 (sensitivity 0.00; specificity 1.00), and

all were absent from the dbSNP and the NHLBI Exome Sequencing Project (EVS) database. The synonymous mutation c.1614C>T p.(Ile538=)) was predicted by Human Splicing Finder to create an exonic silencer motif and to probably affect splicing. All the patients with an FGFR1 mutation had a history of cryptorchidism and/or micropenis. Additionally, two had a cleft lip or palate.

The second most mutations were identified in ANOS1, where three patients harbored a nonsense mutation (c.[309C>A;310delT] p.[(Ser103Arg);(Cys105ValfsTer13)], c.154_157dupAGGT p.(Cys53Ter), and c.769C>T p.(Arg257Ter)), and one patient had a deletion of exons 5-8 as determined by the failed amplification of just those exons in only that patient. Three of the patients had severe GnRH deficiency as indicated by a history of micropenis and/or cryptorchidism; for one patient this information was not available. Additionally, three patients had anosmia, and information on olfaction was unavailable for one patient.

One homozygous GNRHR mutation (c.785G>A p.(Arg262Gln)) was identified in an nHH patient who had reversal of CHH before the age of 32 years but presented with late- onset hypogonadism at the age of 60 years. Details of this case have been previously reported (Tommiska et al. 2013b). In addition, the same p.(Arg262Gln) mutation was identified as heterozygous in three patients, and another mutation (c.317A>G p.(Gln106Arg)) was found as heterozygous in one patient.

Two patients with CHARGE-like phenotypic features were also screened for mutations in CHD7, and both were found to harbor a mutation in this gene. One patient had an esophagotracheal fistula, a bilateral cleft lip and palate, anosmia, deafness in the left ear, and partial hearing loss in the right ear, and he was found to have the frameshift mutation c.7832_7841del p.(Lys2611MetfsTer25). The other patient had syndactyly and hearing loss, and a mutation (c.2443-2A>C) in a conserved acceptor splice site, predicted by Human Splicing Finder to disrupt the normal splicing of the CHD7 mRNA. Neither of these mutations has been reported in the CHD7 mutation database (https://molgenis51.gcc.rug.nl/menu/main/home). Both patients had a history of micropenis and cryptorchidism. 81

One heterozygous PROK2 mutation, c.163delA p.(Ile55fsTer1), was identified in a KS patient, and one heterozygous TAC3 variant, c.1A>T, which changes the start codon ATG to TTG (coding for leucine), was identified in the KS patient with the p.(Gly97Ser) FGFR1 mutation. The TAC3 variant may result in deletion of the first three amino acids of the protein, as the next codon for methionine is found at the fourth amino acid position. No biallelic mutations were found in PROK2 or TAC3. No mutations in FGF8, PROKR2, TACR3, or KISS1R were found in the Danish CHH patients.

The mutations identified in the Danish HH patients and their phenotypic features are summarized in table 4.

Table 4. Conclusive mutations identified in the Danish male patients with congenital hypogonadotropic hypogonadism (CHH), and their phenotypic features. Genes ANOS1, FGFR1, FGF8, PROK2, PROKR2, GNRHR, TAC3, TACR3, and KISS1R were screened in all patients. Additionally, CHD7 was screened in two patients with CHARGE-like phenotypic features.

Olfaction Predicted effect on function Mutation Other Family Self- Clinically Micro- Crypt- Proband Mutation (PolyPhen-2a or Human previously Puberty associated history reported examined penis orchidism Splicing Finder) reported in phenotypes of CHH Probably damaging (score FGFR1 1.000) Anosmic 1 c.625C>T anosmia Yes Yes Nob (sensitivity: 0.00; specificity: father p.(Arg209Cys) 1.00) Probably damaging (score Cleft FGFR1 1.000) lip/palate; 2 c.1936C>T hyposmia Yes Yes No No (sensitivity: 0.00; specificity: tooth p.(Arg646Trp) 1.00) agenesis Brother with Probably damaging (score Cleft FGFR1 cleft lip and 1.000) lip/palate; 3 c.1535C>T hyposmia Yes Yes No palate, and (sensitivity: 0.00; specificity: normal p.(Ala512Val) tooth 1.00) MRI agenesis Creation of an exonic FGFR1 silencer motif, 4 c.1614C>T normosmia Yes No No potential alteration of p.(Ile538=) splicing Probably damaging (score FGFR1 1.000) Jarzabek et c.289G>A (sensitivity: 0.00; specificity: al., 2012 Two anosmic 5 p.(Gly97Ser) anosmia Yes No Partialc 1.00) sons TAC3 c.1A>T N/A p.(Met1Leu)

ANOS1 c.[309C>A;310d elT] Normal 6 N/A anosmia No No p.[(Ser103Arg); MRI (Cys105ValfsTer 13)] Hypoplasti c olfatory groove and sulci ANOS1 (MRI); c.154_157dupA Brother 7 N/A anosmia Yes No olfactory GGT with KS bulbs not p.(Cys53Ter) visible; normal renal ultrasound Hearing loss; rheumatoid 8 ANOS1 delex5-8 N/A Yes Yes No No arthritis; normal MRI Hardelin et ANOS1 al., 1993, 9 c.769C>T N/A anosmia Yes No No Bhagavath p.(Arg257Ter) et al., 2007

Delayed; GNRHR N/A reversal; c.785G>A de Roux et 10 (reported to partially normosmia late-onset No (homozygous) al., 1997 impair receptor function) hypogona p.(Arg262Gln) -dism

Cleft lip and CHD7 palate; c.7832_7841del esophagotr 11 N/A anosmia Yes Yes No No p.(Lys2611Metfs acheal Ter25) fistula; hearing loss Alteration of wild-type Syndactyly CHD7 c.2443- acceptor splice-site, ; 12 normosmia Yes Yes No No 2A>C most probably affects hearing splicing loss KS: Kallmann syndrome; MRI: magnetic resonance imaging aHumDiv-trained PolyPhen-2 model bTesticular volume less than 4 ml cTesticular volume 6 ml at 32 years of age

5.4 SEMA3A and SEMA7A mutations identified in Finnish CHH patients Mutation screening of SEMA3A revealed three heterozygous missense mutations (c.1303G>A p.(Val435Ile), c.458A>G p.(Asn153Ser), and c.1253A>G p.(Asn418Ser)) in three KS patients (table 5). The patient with the p.(Asn418Ser) mutation had a previously identified mutation in FGFR1 (c.1305_1306dupAT p.(Ser436Tyrfs*3), as did the patient with the p.(Asn153Ser) mutation, who had the nonsense mutation c.1825C>T p.(Arg609*) in FGFR1. The third patient was not found to have any mutations in the previously screened KS genes (ANOS1, FGFR1, FGF8, PROK2, PROKR2, CHD7, WDR11, or NELF). All of the identified SEMA3A mutations were reported in the Exome Variant Server database. Minor allele frequencies in the European American population were 0.03% for the Asn418Ser mutation, 0.5% for the Asn153Ser mutation, and 1.4% for the Val435Ile mutation. All three mutations are located in the sema domain of SEMA3A protein (Figure 12a).

Mutation screening of SEMA7A revealed two heterozygous missense variants in one patient with nHH (c.442C>T p.(Arg148Trp) and one patient with KS (c.1421G>A p.(Arg474Gln). Both patients also had a previously identified heterozygous mutation in a known CHH gene; the patient with nHH had a heterozygous nonsense mutation in KISS1R (c.1167C>A p.(Cys398*) and the KS patient had a splice-site-disrupting ANOS1 mutation (g.2357_2360delAgta) at the exon 8-intron 8 boundary. The p.(Arg148Trp) mutation was reported once in the EVS database with a frequency of 1/12986 and was not present in the screened 200 healthy controls. The p.(Arg474Gln) variant was not reported in the EVS database, but several other amino acid substitutions had been reported in the same position, indicating that at least some variation is tolerated in that position. Both mutations are located in the conserved sema domain of the protein (Figure 12b).

The variants identified in the Finnish CHH patients and the patients’ clinical characteristics are summarized in table 5.

Figure 12. The SEMA3A (A) and SEMA7A (B) variants identified in the Finnish CHH patients. The protein domains are marked according to the UniProtKB database. Aa: amino acid position; Ig domain: immunoglobulin-like domain; Arg/Lys-rich region: basic region that is rich in arginine and lysine; RGD motif: arginine-glycine-aspartate motif recognized by some integrin receptors.

Table 5. The nonsynonymous SEMA3A and SEMA7A variants identified in the Finnish CHH patients and the patients’ clinical charateristics

Predicted effect on Variant Family Variant (rs MAF Other phenotypic Mutations in other Proband Diagnosis Sex function previously history of number) (%)a features CHH genesc (PolyPhen-2b) reported in CHH SEMA3A Benign (score 0.113) c.1303G>A Hanchate et 1 KS M 1.4 (sensitivity: 0.93; No No not detected p.(Val435Ile) al., 2012 specificity: 0.86) (rs147436181) Sister, SEMA3A Benign (score 0.002) father, and FGFR1 c.1825C>T c.458A>G Hanchate et Cleft lip; dental 2 KS F 0.5 (sensitivity: 0.99; paternal p.(Arg609Ter) p.(Asn153Ser) al., 2012 agenesis specificity: 0.3) aunt with (heterozygous) (rs139295139) KS SEMA3A FGFR1 Benign (score 0.000) Son and c.1253A>G c.1305_1306dupAT 3 KS F 0.03 (sensitivity: 1.00; not reported No daughter p.(Asn418Ser) p.(Ser436TyrfsTer3) specificity: 0.00) with KS (rs142967103) (heterozygous) SEMA7A Probably damaging KISS1R c.1167C>A c.442C>T (score 1.00) 4 nHH M 0.01 not reported No No p.(Cys389Ter) p.(Arg148Trp) (sensitivity: 0.00; (heterozygous) (rs200895370) specificity: 1.00) SEMA7A Probably damaging Micropenis; Maternal KAL1 c.1421G>A (score 0.999) 5 KS M N/A not reported cryptorchidism; uncle with g.2357_2360delAgta p.(Arg474Gln) (sensitivity: 0.14; synkinesia KS (hemizygous) (N/A) specificity: 0.99) KS: Kallmann syndrome; nHH: normosmic CHH; M: male; F: female, MAF: minor allele frequency aMinor allele frequency in the European American population in the NHLBI Exome Sequencing Project database bHumDiv-trained PolyPhen-2 model cScreened genes: FGFR1, FGF8, PROK2, PROKR2, CHD7, WDR11 (in all patients); ANOS1 (only in KS patients); GNRHR, GNRH1, KSS1R, KISS1, TAC3, TACR3 (only in nHH patients) 88

6. DISCUSSION Disorders of puberty and sex development cause considerable psychosocial stress for the affected individual and can have severe long-term health outcomes. Identifying the genetic defects behind these disorders not only increases our understanding on the genetic regulation of sex development and puberty but is also valuable for the treatment and genetic counseling of the individual patient and his/her family members. Unfortunately, although several genes have already been implicated in the different disorders of puberty and sex development, only in about 13-43% of DSD cases (Bashamboo & McElreavey 2015, Eggers et al. 2016) and in about 50% of CHH cases is a genetic cause identified (Kim 2015). Not even all patients with a clear phenotype that has an established monogenic cause, such as in CAIS, are found to have a causal mutation. The aim of this study was to identify genetic causes of defective development of sex and puberty. A new mechanism of CAIS was detected, as a deep intronic, pseudoexon-activating mutation was found in the androgen receptor gene.

The genetic defects underlying both precocious and absent puberty were investigated in Danish patients. Mutations in MKRN3, a gene with a putative role in the inhibition of premature GnRH secretion, were confirmed to underlie GDPP in the Danish population, supporting the important role of mutations in this gene in precocious puberty. Additionally, conclusive mutations were found in 29% of the Danish CHH patients in the genes FGFR1, ANOS1, CHD7, and GNRHR. Finally, mutations in SEMA3A and SEMA7A, two promising CHH candidate genes, were found to be rare in Finnish CHH patients. These results are discussed in more detail in the following sections.

6.1 New mechanisms underlying complete androgen insensitivity syndrome Most, but not all, CAIS patients are found to have a mutation in AR, indicating some cases may be caused by mutations in obligate AR cofactors or that some AR mutations are missed in routine genetic screenings. In this study, a deep intronic mutation in AR was identified to underlie CAIS in two siblings who had no mutations in the AR coding region or in the conserved splice sites. The mutation lies 118 bp upstream of exon 7 and was thus missed in a routine genetic screening focused on the coding exons and exon-

intron boundaries of the gene. Analysis of the AR cDNA and whole-genome sequencing of the two patients, however, enabled the identification of the causal mutation with high confidence. Only one other deep intronic AR mutation, located 44 bp from exon 6, has been previously reported in a CAIS patient; the mutation caused reduced DHT binding, but the effect on mRNA splicing was not reported (Audi et al. 2010).

AR cDNA analysis showed that the c.2450-118A>G mutation leads to the formation of two aberrantly spliced mRNA products at the expense of the normally spliced AR mRNA. Computational analysis and sequencing of the abnormal products revealed that the mutation creates both a novel 5’splice site (ss), where the U1 small nuclear ribonucleoprotein (snRNP) binds, and a new exonic splicing enhancer (ESE) motif for the SRSF1 protein, which is a member of the splicing activator family (Graveley 2000). Both the binding of the U1 snRNP and the SRSF1 allow the splicing machinery to recognize the sequence 84 bp upstream of the mutation as a new 3’ ss (de Conti et al. 2012), thus leading to a situation where either an 85-bp-long pseudoexon located in intron 6, normally unrecognized by the splicing machinery, is activated (Sun & Chasin 2000), or the entire intronic 202-nt long fragment between the new 3’ss and exon 7 is spliced into the mRNA. U1 snRNP and SRSF1 compete for binding to the overlapping sequence motifs, so that the two aberrant products are made in approximately equal amounts, while the amount of the normally spliced mRNA is significantly reduced.

Both abnormal AR mRNAs are predicted to lead to a premature termination codon (PTC) after the addition of 12 novel amino acids. As the PTC is located more than 50-55 nt upstream of the last exon-exon boundary, it is predicted to lead to nonsense-mediated decay (NMD) of the mRNA (Isken & Maquat 2007). The overall amount of AR mRNA, however, was not lower in the patient fibroblasts than in the controls, although the amount of the normally spliced mRNA was significantly reduced. Notably, it has been shown that the half-life of some PTC-containing mRNAs can be similar to normal mRNAs, while some are destroyed rapidly in the cell (Trcek et al. 2013). Therefore, it is possible that the aberrant AR mRNAs are targeted by the NMD pathway even though they are detected in the patient fibroblasts. Additionally, even if the PTC-containing

mRNAs are translated into protein, the lack of detectable AR protein in the patient fibroblasts indicates that the resulting protein product is unstable, and that the low amount of normal AR mRNA is not sufficient to yield significant AR protein levels.

Some DSD patients have an increased risk to develop gonadal tumors; for example, PAIS patients with non-scrotal gonads have a high malignancy risk and early gonadectomy is recommended (Hughes et al. 2006, Wünsch et al. 2012). On the other hand, patients with 17β-HSD3 deficiency, a similar phenotype, have an intermediate risk, in which case careful monitoring of the situation rather than gonadectomy is recommended to avoid overtreatment and to preserve endogenous hormone production (Wünsch et al. 2012). In this case, the molecular genetic diagnosis is an important factor in deciding whether gonadectomy should be performed or not (Wünsch et al. 2012). AR splice-site mutations have been reported in patients with PAIS (Brüggenwirth et al. 1997, Sammarco et al. 2000), so it seems plausible that deep intronic mutations may also underlie these cases. Notably, a mutation in the AR 5’UTR was recently identified as a novel mechanism underlying CAIS in two unrelated patients (Hornig et al. 2016), which further demonstrates the importance of mutations outside of the coding region to the pathogenesis of AIS. Therefore, AR cDNA analysis should be routinely performed in cases when no mutation is identified with sequencing of the genomic AR coding regions.

6.2 MKRN3 mutations: a significant cause of GDPP in different populations The molecular genetic causes of GDPP were largely unknown until the discovery of MKRN3 loss-of-function mutations in several GDPP patients in 2013 (Abreu et al. 2013). In this study, one heterozygous missense mutation, p.(Arg345His), was identified in one Danish girl with GDPP, and subsequently also in her brother who had early puberty. This is a similar frequency (1/29=3.4%) to that reported previously for 207 girls with apparently sporadic GDPP (8/207=3.9%) from three different university medical centers (Macedo et al. 2014), although in familial cases, MKRN3 mutations may be identified in up to 46% of the cases (Simon et al. 2016). The mother did not carry the mutation, indicating the siblings had inherited it from their father, which is consistent with the maternal imprinting of the gene; only mutations that are inherited from the father can 91

cause the phenotype. Unfortunately, detailed information on the timing of the father’s puberty or DNA samples of the paternal grandparents were unavailable, but since the father’s puberty was described as average, he probably inherited the mutation from his mother. The mutation is also reported in the EVS database with low frequency (1/8600), which may be expected for this type of variant whose effects are only revealed if it is inherited from the father. Additionally, the database does not include information on the pubertal timing of the person carrying the mutation in the database.

The girl’s age at puberty onset (around 6 years of age) was similar to that reported for other girls with MKRN3 mutations (median 6.0, range 3.0 to 7.5 years) (Abreu et al. 2015). Although MKRN3 mutations are found in both girls and boys, they seem to affect girls more strongly, and the median age of puberty onset for boys carrying MKRN3 mutations is 8.25 (range 5.9 to 9.0 years) (Abreu et al. 2015). In fact, some boys carrying an MKRN3 mutation are apparently unaffected (Dimitrova-Mladenova et al. 2016). The puberty of the boy in our family was not formally assessed, but he seems to be affected by the mutation as he did have very early voice break at 10.5 years of age; voice break usually occurs between Tanner stages G3 and G4 (Harries et al. 1997) at the median age of 14.0 in Danish boys (Juul et al. 2007).

The mutation is predicted to be pathogenic by three prediction tools. It is located in the C3HC4 RING domain, predicted to have E3 ubiquitin ligase activity, where at least 5 other missense mutations have been identified so far in GDPP patients (Abreu et al. 2013, Settas et al. 2014, Simon et al. 2016, Neocleous et al. 2016). Interestingly, the majority of the patients with an identified MKRN3 mutation have either a nonsense mutation or a frameshift mutation leading to a premature stop codon, and the majority of the identified missense mutations are located in the C3HC4 domain or one of the C3H domains (Figure 13) (Abreu et al. 2015, Simsek et al. 2016). Several of the frameshift mutations are located in a poly-C stretch (codons for amino acids Pro160 and Pro161), indicating a mutational hotspot (Macedo et al. 2014, Simon et al. 2016, Dimitrova- Mladenova et al. 2016). Due to the lack of an available bioassay, the functional effects of the mutations have not been assessed in vitro, but in silico modeling has predicted a

disrupted tertiary protein structure for some of the missense mutations (Settas et al. 2014, Neocleous et al. 2016).

Figure 13. The MKRN3 mutation identified in this study along with other reported MKRN3 mutations. The mutation (p.Arg345His) identified in this study is drawn inside a box with bold font. In addition, mutations reported in Abreu et al. 2013, Macedo et al. 2014, Schreiner et al. 2014, Settas et al. 2014, de Vries et al. 2014, Grandone et al. 2015, Simon et al. 2016, Neocleous et al. 2016, and Simsek et al. 2016, are displayed in the figure.

The exact function of MKRN3 is still unknown, but it is presumed to act as a suppressor of puberty onset based on two findings: the expression of Mkrn3 declines in the mouse arcuate nucleus at the onset of puberty, and the majority of the identified GDPP-causing mutations lead to a premature stop codon, indicating a loss of protein function (Abreu et al. 2013). Although MKRN3 is widely expressed in adult and fetal tissues (Jong et al. 1999), few GDPP patients with an MKRN3 mutation have been reported to have additional anomalies, such as esotropia, high-arched palate, or dental abnormalities (Abreu et al. 2013, Macedo et al. 2014), suggesting MKRN3 function is not indispensable outside the HPG axis. In this study, MKRN3 was also found to be expressed in an adult human hypothalamic cDNA collection, suggesting that the hypothalamic MKRN3 function is not limited to the prepubertal inhibition of GnRH release. Unfortunately, as human hypothalamic samples are poorly available, the 93

potential temporal changes in MKRN3 expression at puberty could not be addressed in this study. MKRN3 is a zinc-finger protein that contains a C3HC4 RING domain that is found in most E3 ubiquitin ligases, three C3H zinc fingers which are typically found in ribonucleoproteins, and a Cys-His-rich motif which may bind DNA (Böhne et al. 2010, Abreu et al. 2015). MKRN3 is an intronless retrocopy of MKRN1, which has several identified functions mainly as an E3 ubiquitin ligase, for example in defense against virus infection, cell cycle arrest, and apoptosis, and possibly, independent of E3-ligase activity, as a transcriptional regulator (Böhne et al. 2010). The zinc-finger domain structure of MKRN3 and its similarity with MKRN1 suggest that it can participate in multiple cellular processes such as marking proteins for proteasomal degradation, modulation of protein function or localization, and transcriptional regulation (Böhne et al. 2010, Abreu et al. 2015). The elucidation of the exact mechanism by which MKRN3 inhibits the HPG axis will be highly interesting and may reveal new players in the control of puberty onset.

6.3 The genetic features of CHH in Denmark Although several genes have already been implicated in CHH, the majority of the patients still lack a conclusive molecular genetic diagnosis. This was also the case in Denmark, where a conclusive mutation underlying CHH was identified in 12 of the 41 (29%) patients. FGFR1 mutations were the most frequent genetic cause: five (12%) patients had a heterozygous mutation in this gene. One of the FGFR1 mutations, p.(Gly97Ser), identified in a KS patient, has been reported previously in another KS patient who had scoliosis and hearing loss (Jarzabek et al. 2012) and more recently, in another patient with CHH and split-hand/foot malformation (Ohtaka et al. 2017); no such additional features had been reported for the Danish patient. The patient was also found to carry a heterozygous start-codon-disrupting mutation in TAC3, c.1A>T. The next possible start codon in the TAC3 gene sequence is in the fourth amino acid position, so this mutation may lead to the absence of the first three amino acids of the polypeptide. TAC3 mutations are implicated in autosomal recessice nHH (Topaloglu et al. 2009), and since the patient and his two sons all have KS, the FGFR1 mutation is most likely enough to explain the phenotype, although a contribution of the TAC3 mutation cannot be ruled 94

out. None of the other FGFR1 mutations [p.(Arg209Cys), p.(Ala512Val), p.(Arg646Trp), and p.(Ile538=)] have been reported previously, although a mutation affecting the same codon as Arg209Cys, c.626G>A p.(Arg209His), has been reported in a Finnish KS patient (Laitinen et al. 2011). Notably, neither the Danish nor the Finnish patient had additional phenotypic features. Although the functional effects of the mutations were not tested in vitro, the in silico predictions, the rarity of the mutations, and the fact that FGFR1 is an established CHH gene, indicate that they are highly probable causal mutations in these patients. Of note, all of the patients with an FGFR1 mutation had a history of micropenis and/or cryptorchidism, indicating severe GnRH deficiency. Additionally, two had a cleft lip or palate, which is a typical finding associated with FGFR1 mutations. All had hyposmia or anosmia, except the patient with the p.(Ile538=) mutation, who had self-reported normal sense of smell. This demonstrates the phenotypic variation in CHH caused by FGFR1 mutations.

The second most often mutated gene in Danish patients was ANOS1. Four patients (a10%) had a mutation in this gene, and all mutations are predicted to affect the protein product severely; three patients had a mutation leading to a premature stop codon [p.[(Ser103Arg);(Cys105ValfsTer13)], p.(Cys53Ter), and p.(Arg257Ter)], and one patient had a deletion of exons 5 to 8, as they could not be PCR-amplified from the patient’s genomic DNA even though the amplification from other DNA samples was successful at the same time. Failed amplification due to DNA degradation is unlikely because the other ANOS1 exons were successfully amplified from the same DNA sample after the amplification of exons 5-8 had failed. Consistent with the common finding of a severe reproductive phenotype in patients harboring ANOS1 mutations (Hu & Bouloux 2011), three of the patients had a history of micropenis and/or cryptorchidism; for one patient this information was not available. In addition, three patients had anosmia as expected. Information on olfaction was unavailable for one patient, but his sense of smell is probably also affected, as invariably seems to be the case for patients with CHH caused by an ANOS1 mutation (Hu & Bouloux 2011). Unexpectedly, none of the patients were reported to have synkinesia or renal agenesis, which are typical findings in patients with ANOS1 mutations (Hu & Bouloux 2011, Costa-Barbosa et al. 2013). 95

Only one patient was found to have a biallelic mutation in GNRHR, the homozygous p.(Arg262Gln) mutation, which has been shown to partially impair the GnRH receptor function due to decreased signaling activity (de Roux et al. 1997). This patient has been reported previously (Tommiska et al. 2013b); he had presented with delayed puberty at 16 years of age, had reversal of HH after testosterone treatment around the age of 30, and at 61, presented again with signs of hypogonadism. Additionally, the Arg262Gln mutation was identified in the heterozygous state in three patients, and a heterozygous p.(Gln106Arg) mutation, previously shown to cause reduced GnRH binding and impaired intracellular signaling, was identified in one patient. The p.(Arg262Gln) mutation is, together with the p.(Gln106Arg) mutation, the most commonly identified GNRHR defect underlying nHH, and homozygous Arg262Gln/Arg262Gln and Gln106Arg/Gln106Arg mutations, as well as compound heterozygous Arg262Gln/Gln106Arg mutations, tend to cause a mild phenotype (Brioude et al. 2010). Both mutations were also detected with a frequency of 1/95 in control individuals from The Copenhagen Puberty Study, a frequency similar to that previously reported in the Finnish population (Vaaralahti et al. 2011). Considering the relatively high carrier frequency of these mutations in the Danish population and the fact that mutations in GNRHR are the most common known genetic defect underlying nHH (Bianco & Kaiser 2009), it is somewhat surprising that no other biallelic GNRHR mutations were identified. It is possible, however, that the phenotype caused by the Arg262Gln and Gln106Arg mutations is so mild that it goes undetected in some individuals who may only present with delayed puberty, or in some females may be brushed off as primary or secondary amenorrhea caused by excessive exercise or low body fat. In addition, it is unlikely that a second mutation in GNRHR went undetected in the four patients with a heterozygous mutation, as three of them had anosmia, indicating a different genetic cause. No other genetic causes of nHH were identified in the Danish patients, as there were no biallelic mutations in TAC3, TACR3, or KISS1R. GNRH1 and KISS1 were not screened because mutations in them are an extremely rare cause of nHH, so it is possible, although unlikely, that some mutations in these genes may have gone undetected.

CHD7 was screened in two patients who had CHARGE-like phenotypic features, and both were found to have a previously unreported mutation in this gene. One patient had a heterozygous frameshift mutation (p.(Lys2611MetfsTer25)) and several nonreproductive phenotypic features such as anosmia, a bilateral cleft lip and palate, an esophagotracheal fistula, deafness in the left ear, and partial hearing loss in the right ear. The other patient had a mutation in the conserved acceptor splice site of exon 7 (c.2443- 2A>C), which most probably disrupts the splice site and leads to defective splicing of the CHD7 mRNA; he had syndactyly and hearing loss in addition to severe CHH. CHD7 mutations cause highly variable phenotypic features (Husu et al. 2013), and although they are not a very common cause of CHH, they have been shown to be enriched especially in patients with hearing loss or other ear anomalies (Jongmans et al. 2009, Costa-Barbosa et al. 2013). CHD7 screening should thus be prioritized in these kinds of patients to allow genetic counseling of the family.

One patient with KS and a history of micropenis and cryptorchidism had a heterozygous PROK2 mutation, p.(Ile55fsTer1), which has been identified previously both in KS and nHH patients (Leroy et al. 2008, Pitteloud et al. 2007b). The mutation has been shown to cause loss-of-function in vitro, but heterozygous carriers of this mutation seem to be unaffected (Pitteloud et al. 2007b), so another mutation in PROK2 or PROKR2 would be needed to explain the phenotype. Of note, no biallelic PROK2 or PROKR2 mutations were found in the Danish patients, similarly as previously in Finnish patients (Laitinen et al. 2011). Interestingly, some PROKR2 mutations are enriched in the Maghrebian population: in a recent study, Sarfati et al. (2013) found PROKR2 mutations in 23.3% of Maghrebian KS patients but in only 5.1% of the European patients, and PROK2/PROKR2 mutations have been found to underlie 5-10% of KS cases in other studies (Bianco & Kaiser 2009, Martin et al. 2011). It may be that PROK2/PROKR2 mutations are rarer in some populations, such as in the Danes and the Finns, although genetically the Danes are more closely related to other Northern Europeans than to the Finns (Beekman et al. 2013). It should be noted, however, that another, non-coding, PROK2 mutation may have gone undetected in the Danish patient, whose phenotype of

severe GnRH deficiency would fit with a biallelic mutation in this gene (Martin et al. 2011).

Mutations in the genes currently implicated in CHH can be found in approximately 50% of the patients (Kim 2015). It should be noted, however, that this figure also includes genes and variants whose contribution to the disease pathogenesis has not been shown conclusively. In this study, we focused on the genes that are the most relevant in a clinical setting, and identified a conclusive mutation in 29% of the patients. Not all genes implicated in CHH, such as HS6ST1, SEMA3A, WDR11, or NSMF, were screened, but as the contribution of variants in these genes to the pathogenesis of CHH is still unclear, they would not be of much value for example in the genetic counseling of the patients. Notably, although di -or oligogenicity has been suggested in CHH, (Pitteloud et al. 2007a, Sykiotis et al. 2010), no evidence of oligogenicity was found in this patient series, which may largely be explained by the fact that the genes whose relevance to CHH is still uncertain were left unscreened.

6.4 Current knowledge on the involvement of SEMA3A and SEMA7A mutations in CHH pathogenesis SEMA3A and SEMA7A are very promising candidate genes for Kallmann syndrome and normosmic HH, respectively. To elucidate the contribution of mutations in these genes to the pathogenesis of CHH in Finland, they were screened in 50 patients with CHH. All in all, only few variants were found, discussed in more detail in the following sections.

6.4.1 The role of SEMA3A mutations in CHH In SEMA3A, three heterozygous missense variants (p.(Asn418Ser), p.(Asn153Ser), and p.(Val435Ile)) were identified in three KS patients. All of the variants have been reported in the Exome Variant Server database, and the p.(Val435Ile) and p.(Asn153Ser) variants have also been previously reported in KS patients by Hanchate et al. (2012). Both Val435Ile and Asn153Ser variants have been shown to cause loss-of-function in vitro; Val435Ile resulted in impaired SEMA3A secretion, whereas Asn153Ser caused reduced signaling activity in GN11 cells (Hanchate et al. 2012), although both are predicted to

be benign by PolyPhen-2. The p.(Asn418Ser) variant was not reported and thus not tested by Hanchate et al., but it is likewise predicted to be benign by PolyPhen-2.

Although the functional studies showed impaired function of the p.(Val435Ile) and p.(Asn153Ser) variants indicating a pathogenic effect, the two variants are not very rare, as seen from their frequencies in the European American population in the EVS database; 1.4% for Val435Ile and 0.5% for Asn153Ser. The p.(Asn418Ser) variant is somewhat rarer with a frequency of 0.03% in the EVS database, but it seems to be enriched in the Finnish population where the frequency is 1.16% according to the SISu (Sequencing Initiative Suomi) Project database. Therefore, if the identified variants do have an effect on the development of the olfactory system or on the development or function of GnRH neurons, the effect must be very mild and the variants alone are not sufficient to cause the phenotype. These results are in accordance with those obtained by Hanchate et al. (2012). Based on the existence of the variants in databases and in healthy controls, and the fact that some patients had mutations in additional CHH genes, they suggested that monoallelic SEMA3A mutations are not sufficient to cause the KS phenotype. This is in contrast to the findings of Young et al. (2012), who proposed that the heterozygous deletion mutation in SEMA3A is sufficient to cause KS in a family with autosomal dominant mode of inheritance of the disorder. It may of course be argued that a deletion has a more drastic effect than a missense variant. It is also possible that the KS patients harboring the SEMA3A deletion have another, as-yet-unidentified mutation contributing to the phenotype. In this study, both of the patients carrying the p.(Asn153Ser) and p.(Asn418Ser) variants also had a previously identified truncating mutation in FGFR1, which in the light of current knowledge is sufficient to explain the patients’ phenotypes. As di-/oligogenicity has been repeatedly suggested in CHH patients (Pitteloud et al. 2007a, Sykiotis et al. 2010), however, it is possible that the identified SEMA3A variants contribute to the phenotype. The patient carrying the p.(Val435Ile) variant had no identified defects in the other known CHH genes, indicating that he most probably carriers at least one unidentified mutation underlying the disorder.

Interestingly, Hofmann et al (2013) reported biallelic SEMA3A mutations in a patient with short stature, skeletal abnormalities, a congenital heart defect, and camptodactyly. He had a deletion affecting the first exon of SEMA3A, inherited from the father, and a mutation disrupting the normal splicing of SEMA3A, inherited from the mother, together leading to significantly reduced expression of the gene in the patient. The patient was prepubertal but he had a normal sense of smell, and, inconsistent with the findings of Young et al (2012), the parents did not have KS, although the father had had a delayed pubertal growth spurt which may indicate delayed puberty (Hofmann et al. 2013). Therefore, the role of SEMA3A in human development still remains unclear. Of note, although most Sema3A-KO mouse models do not have severe developmental defects, the mouse phenotype reported by Behar et al. (1996) resembles that of the patient described by Hofmann et al., although the patient’s phenotype was less severe than observed in the mice (Hofmann et al. 2013). Taken together, the function of SEMA3A in human development requires further study.

6.4.2 The role of SEMA7A mutations in CHH Only two heterozygous missense variants were identified in SEMA7A, one (p.(Arg148Trp)) in an nHH patient and one (p.(Arg474Gln) in a KS patient. The p.(Arg148Trp) variant has been reported in the EVS database with a frequency of 0.01% and 38/120024 chromosomes in the ExAC database, and the p.(Arg474Gln) variant is absent from the EVS database but is found in the ExAC database with a frequency of 0.0075%. In addition, several other substitutions (p.(Arg474Leu), p.(Arg474Pro), and p.(Arg474Trp)) have been reported in the same amino acid position, suggesting that some variation is tolerated in that position. The frequency of the SEMA7A variants suggests that although they are predicted to be probably pathogenic by PolyPhen-2, they are most likely not alone sufficient to cause a CHH phenotype. The nHH patient carrying the p.(Arg148Trp) variant also has a previously identified heterozygous nonsense variant in KISS1R (c.(Cys389Ter)), a gene implicated in autosomal recessive nHH. The KISS1R variant is reported in the ExAC database with a frequency of 0.2% and has unknown functional significance. It is located in the last exon of the gene close to the normal termination codon. As the variant is located in the last exon, it is not predicted to lead to 100

nonsense-mediated decay of the mRNA but may result in a protein product that lacks the ten last amino acids, which may have an effect on receptor internalization and degradation (Pampillo et al. 2009). Whether SEMA7A and KISS1R are involved in a same cellular process that could be affected by the identified variants is currently unknown. The KS patient carrying the p.(Arg474Gln) variant has a previously identified splice-site disrupting mutation (g.2357_2360delAgta) in ANOS1, and his phenotypic features (severe HH and synkinesia) and family history (maternal uncle with KS) are consistent with KS caused by an ANOS1 mutation. Taken together, it seems unlikely that the identified SEMA7A variants have a significant contribution to the patients’ phenotypes.

101

7. CONCLUSIONS AND FUTURE PERSPECTIVES The existence of patients for whom no molecular genetic cause can be found in the already identified disease-associated genes, such as in some patients with CAIS or KS, suggests there are still new genes whose mutations underlie the phenotype in some of the unsolved cases. This has led to great efforts to identify new genetic defects underlying for example CHH by utilizing high-throughput sequencing methods, which hold great promise for the elucidation of the genetic basis of genetically heterogeneous disorders. As demonstrated in this study by the identification of a deep intronic mutation underlying CAIS in an already established gene, however, it is clear that sometimes the answers are found by taking a closer look at the non-coding regions of already well- known disease-associated genes. The importance of mutations outside the coding regions of genes is being recognized more and more as our understanding of the regulation of gene expression increases. Whole-genome resequencing will potentially reveal new mutations in intronic and regulatory regions of genes already implicated in CHH, GDPP, and the different DSDs. Importantly, as seen in this study, older methods, such as cDNA analysis, should not be forgotten as they can invaluably complement the newer techniques.

MKRN3 mutations are currently the most frequent known genetic cause of GDPP, and the identification of MKRN3 mutations both in boys and in girls in different populations around the world indicates MKRN3 should be screened in GDPP patients regardless of ethnic background or sex. Additionally, although MKRN3 mutations are more frequent in familial cases, they are also identified in approximately 4% of apparently sporadic cases. This study showed that MKRN3 mutations also underlie GDPP in Danish patients, although the frequency of mutations was quite low, possibly due to lack of other familial cases. The majority of GDPP patients still remain without an identified genetic cause, and the identification of the mechanism by which MKRN3 regulates pubertal onset will potentially reveal new candidate genes involved in GDPP. In addition, precocious puberty has so far not been studied systematically in Finland, and it will be interesting to study the genetic causes of this condition also in Finnish patients.

102

In this study, a conclusive mutation was identified in only 29% of Danish CHH patients with most mutations found in FGFR1 and ANOS1. One homozygous GNRHR mutation was associated with temporal variation in the endocrine phenotype and supports the notion that some, more common, GNRHR mutations may cause a milder phenotype. Expectedly, a CHD7 mutation was identified in two patients with CHARGE-like phenotypic characteristics. No biallelic PROK2 or PROKR2 mutations or evidence of oligogenicity were identified. These results can be utilized in prioritizing genetic testing of Danish CHH patients, although the more effective targeted gene panels are increasingly replacing the traditional Sanger-sequencing-based genetic screenings. Of note, causal mutations in the non-coding regions of some of the screened genes may have gone undetected in some patients, but mutations in genes not yet implicated in CHH probably underlie some of these cases. The identification of new genetic defects underlying CHH remains a huge challenge due to the clinical and genetic heterogeneity of the disorder. The potential variable penetrance of the mutations, which has already been reported for established genes such as FGFR1, further increases the difficulty. The increased availability and employment of high-throughput sequencing techniques has led to the discovery of several new candidate genes involved in the pathogenesis of CHH, but the true challenge will be the identification of the real culprits behind the phenotype among all the candidates. Major efforts in the form of in vitro studies and in vivo modeling is still required before mutations found any one of those genes can be used for example in the genetic counseling of the patients carrying the mutations.

Genes are often considered as good candidate disease genes based on mouse models that show phenotypic similarity to the disorder of interest. The role of SEMA3A and SEMA7A in the development and postnatal function of GnRH neurons has recently gained interest as, based on rodent models, they seem to have important functions in the guidance of olfactory system development, GnRH neuron migration, and relaying hormonal signals from the circulation to the GnRH neuron nerve terminals. Conflicting results from previous studies, however, have left the role of SEMA3A in human unclear. Of note, several other genetic-KO mice have shown a phenotype resembling KS or CHH, but mutations in the same genes have not been reported to cause CHH in humans. This 103

study showed that mutations in SEMA3A and SEMA7A are not a significant cause of CHH in Finland. Additionally, based on previous studies, SEMA3A mutations may not cause KS but an entirely different syndrome in humans. Therefore, the potential role of SEMA3A and SEMA7A in human reproduction still requires further study.

104

8. ACKNOWLEDGEMENTS This work was carried out at the Department of Physiology, Medicum, at the Faculty of Medicine, University of Helsinki, and supported by the Academy of Finland, the Foundation for Pediatric Research, the Biomedicum Helsinki Foundation, the Sigrid Jusélius Foundation, the Finnish Concordia Fund, the Finnish Cultural Foundation, and the Alfred Kordelin Foundation, which are all gratefully acknowledged.

All subjects are sincerely thanked for their participation in this study.

I am deeply grateful to my supervisors, Professor Taneli Raivio and Docent Johanna Tommiska, for their excellent guidance throughout the years. Taneli, thank you for letting me join your group to do my master’s thesis and to continue all the way to this PhD thesis. I admire your enthusiasm, drive, and never-ending ability to come up with new ideas. Thank you for your encouragement and faith in my projects. Johanna, thank you for giving me invaluable insight into the complexities of disease genetics and for teaching me critical thinking, and thank you for your friendship and support throughout this work.

My sincerest gratitude goes to the pre-examiners of this thesis, Docent Hannele Laivuori and Docent Kirsti Näntö-Salonen. Thank you both for our discussions, your words of encouragement, and your invaluable input on how to improve this work. Thank you also to the members of my thesis committee, Docent Katarina Pelin and Docent Minna Pöyhönen, for your kind and encouraging words.

Thank you to all the co-authors for fruitful collaboration: Professor Jarmo Jääskeläinen, Docent Tiina Jääskeläinen, Lilli Saarinen, Docent Rainer Lehtonen, Professor Sampsa Hautaniemi, Dr. Mikko Frilander, Professor Jorma Palvimo, Professor Jorma Toppari, Professor Anders Juul, Dr. Peter Christiansen, Dr. Niels Jørgensen, Jacob Gerner Lawaetz, Rainer Fagerholm, Dr. Eeva-Maria Laitinen, Dr. Kirsi Vaaralahti, Docent Peter Hackman, and Professor Nelly Pitteloud. I also want to thank Professor Nelly Pitteloud for hosting my visit in Lausanne, Switzerland, and all her group members, especially Dr. Yisrael Sidis, for welcoming me and taking the time to supervise me during my visit.

Big Thank You’s go to fellow lab members Kirsi, Carina, Kristiina, Ram, Ida, and Ansku. Thank you for the good times in- and outside the lab, and for much-needed peer support during the not-so-good times that are an inevitable part of the PhD project. Especially big thanks to Kirsi; you always have the best tips and words of advice on all aspects of the PhD process. Also, thank you for keeping our lab running so smoothly even in the middle of moving and renovations. Your organizational skills are truly inspiring! I also thank Kristiina for sharing the challenging times in the mouse facility. Our former lab technician Lennu is thanked for her skillful help and advice during my 105

first years in the lab. Thanks also to other co-workers and researchers; Annika, Tero, Matti, Päivi, Johanna H, Heta, and Ella, for your hard work and for the times shared in- and outside of work and on conference trips.

Thanks to all the former group members and visitors I have had the pleasure of knowing and working with: Eve, Elina, Sanna, Ken, Michaela, Pat, and Elisa. Thanks also to current and former members from other groups for sharing thoughts and ideas on both scientific and everyday topics, especially Laura, Linda, Pauli, Giorgio, Swaroop, Ulla- Kaisa, Tommy, and Jarno. I also thank my friends outside the lab; times spent with you have provided a refreshing break from the world of science and given some much-needed perspective. Special thanks to Jenny: you are the sister I never had, and you probably know too much about me but have nevertheless always believed in me.

Last but not least, I thank my family for their love and support. My mom Helena, who has always been very supportive of my academic endeavors, and my late grandparents Heljä and Pentti, who set a great example of life-long-learning and always staying curious. To my two older brothers Antti and Lauri, for teaching me how to be resilient and stand up for myself, and to all the rest of my family for all your support. Finally to Tommi, my other half and best friend, thank you for your love and companionship, and for always believing in me. Thank you for bearing with me throughout all these years and the stressful projects starting with the bachelor’s thesis and leading up to, but hopefully not ending, here. Your support has been invaluable, I love you!

Helsinki, June 2017

106

9. REFERENCES Abreu AP, Dauber A, Macedo DB, Noel SD, Brito Audi L, Fernández-Cancio M, Carrascosa A, Andaluz VN, Gill JC, Cukier P, Thompson IR, Navarro VM, P, Torán N, Piró C, Vilaró E, Vicens-Calvet E, Gagliardi PC, Rodrigues T, Kochi C, Longui CA, Gussinyé M, Albisu MA, Yeste D, Clemente M, Beckers D, De Zegher F, Montenegro LR, Mendonca Hernández De La Calle I, Del Campo M, Vendrell T, BB, Carroll RS, Hirschhorn JN, Latronico AC, Kaiser Blanco A, Martínez-Mora J, Granada ML, Salinas I, UB. Central precocious puberty caused by mutations Forn J, Calaf J, Angerri O, Martínez-Sopena MJ, Del in the imprinted gene MKRN3. New Engl J Med Valle J, García E, Gracia-Bouthelier R, Lapunzina P, 2013, 368: 2467-2475. Mayayo E, Labarta JI, Lledó G, Sánchez Del Pozo J, Arroyo J, Pérez-Aytes A, Beneyto M, Segura A, Borrás V, Gabau E, Caimarí M, Rodríguez A, Abreu AP, Trarbach EB, De Castro M, Costa EMF, Martínez-Aedo MJ, Carrera M, Castaño L, Andrade Versiani B, Baptista MTM, Garmes HM, Mendonca M, Bermúdez De La Vega JA. Novel (60%) and BB, Latronico AC. Loss-of-function mutations in the recurrent (40%) androgen receptor gene mutations in genes encoding prokineticin-2 or prokineticin a series of 59 patients with a 46,XY disorder of sex receptor-2 cause autosomal recessive Kallmann development. J Clin Endocrinol Metab 2010, 95: syndrome. J Clin Endocrinol Metab 2008, 93: 4113- 1876-1888. 4118.

Bajpai R, Chen DA, Rada-Iglesias A, Zhang J, Xiong Abreu AP, Macedo DB, Brito VN, Kaiser UB, Y, Helms J, Chang C-P, Zhao Y, Swigut T, Wysocka Latronico AC. A new pathway in the control of the J. CHD7 cooperates with PBAF to control multipotent initiation of puberty: The MKRN3 gene. J Mol neural crest formation. Nature 2010, 463: 958-962. Endocrinol 2015, 54: R131-R139.

Bao Z-Z, Jin Z. Sema3D and Sema7A have distinct Adachi M, Takayanagi R, Tomura A, Imasaki K, Kato expression patterns in chick embryonic development. S, Goto K, Yanase T, Ikuyama S, Nawata H. Developmental Dynamics 2006, 235: 2282-2289. Androgen-insensitivity syndrome as a possible coactivator disease. New Engl J Med 2000, 343: 856- 862. Barraud P, St John JA, Stolt CC, Wegner M, Baker CV. Olfactory ensheathing glia are required for embryonic olfactory axon targeting and the migration Adzhubei I, Jordan DM, Sunyaev SR. Predicting of gonadotropin-releasing hormone neurons. Biology functional effect of human missense mutations using Open 2013, 2: 750-759. PolyPhen-2. Current Protocols in Human Genetics 2013, Chapter 7:Unit7.20. Barraud P, Seferiadis AA, Tyson LD, Zwart MF, Szabo-Rogers HL, Ruhrberg C, Liu KJ, Baker CVH. Aksglaede L, Sørensen K, Petersen JH, Skakkebæk Neural crest origin of olfactory ensheathing glia. Proc NE, Juul A. Recent decline in age at breast Natl Acad Sci U S A 2010, 107: 21040-21045. development: The Copenhagen puberty study. Pediatrics 2009, 123: e932-e939. Bashamboo A, McElreavey K. Human sex- determination and disorders of sex-development Andersson A-M, Toppari J, Haavisto A-, Petersen JH, (DSD). Semin Cell Dev Biol 2015, 45: 77-83. Simell T, Simell O, Skakkebæk NE. Longitudinal reproductive hormone profiles in infants: Peak of inhibin B levels in infant boys exceeds levels in adult Basson MA, van Ravenswaaij-Arts C. Functional men. J Clin Endocrinol Metab 1998, 83: 675-681. Insights into Chromatin Remodelling from Studies on CHARGE Syndrome. Trends Genet 2015, 31: 600- 611. Ankarberg-Lindgren C, Norjavaara E. Changes of diurnal rhythm and levels of total and free testosterone secretion from pre to late puberty in boys: Beekman M, Blanché H, Perola M, Hervonen A, Testis size of 3 ml is a transition stage to puberty. Eur Bezrukov V, Sikora E, Flachsbart F, Christiansen L, J Endocrinol 2004, 151: 747-757. De Craen AJM, Kirkwood TBL, Rea IM, Poulain M, Robine J-, Valensin S, Stazi MA, Passarino G, Deiana L, Gonos ES, Paternoster L, Sørensen TIA, Tan Q, Auchus RJ, Chang AY. 46,XX DSD: The Helmer Q, Van Den Akker EB, Deelen J, Martella F, masculinised female. Best Pract Res Clin Endocrinol Cordell HJ, Ayers KL, Vaupel JW, Törnwall O, Metab 2010, 24: 219-242. Johnson TE, Schreiber S, Lathrop M, Skytthe A, Westendorp RGJ, Christensen K, Gampe J, Nebel A, Houwing-Duistermaat JJ, Slagboom PE, Franceschi 107

C. Genome-wide linkage analysis for human unusual type of intronic mutation. Am J Hum Genet longevity: Genetics of healthy aging study. Aging 1997, 61: 1067-1077. Cell 2013, 12: 184-193. Bülow HE, Berry KL, Topper LH, Peles E, Hobert O. Behar O, Golden JA, Mashimo H, Schoen FJ, Heparan sulfate proteoglycan-dependent induction of Fishman MC. Semaphorin III is needed for normal axon branching and axon misrouting by the Kallmann patterning and growth of nerves, bones and heart. syndrome gene kal-1. Proc Natl Acad Sci U S A 2002, Nature 1996, 383: 525-528. 99: 6346-6351.

Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil Busch AS, Hagen CP, Almstrup K, Juul A. E. Hypophysial responses to continuous and Circulating MKRN3 levels decline during puberty in intermittent delivery of hypothalamic gonadotropin healthy boys. J Clin Endocrinol Metab 2016, 101: releasing hormone. Science 1978, 202: 631-633. 2588-2593.

Bianco SDC, Kaiser UB. The genetic and molecular Böhne A, Darras A, D'Cotta H, Baroiller J-F, Galiana- basis of idiopathic hypogonadotropic hypogonadism. Arnoux D, Volff J-N. The vertebrate makorin Nat Rev Endocrionol 2009, 5: 569-576. ubiquitin ligase gene family has been shaped by large- scale duplication and retroposition from an ancestral gonad-specific, maternal-effect gene. BMC Genomics Biason-Lauber A. Control of sex development. Best 2010, 11: 721. Practice and Research: Clinical Endocrinology and Metabolism 2010, 24: 163-186. Cadman SM, Kim S-H, Hu Y, González-Martínez D, Bouloux P-M. Molecular pathogenesis of Kallmann's Boehm U, Bouloux P-M, Dattani MT, De Roux N, syndrome. Horm Res 2007, 67: 231-242. Dodé C, Dunkel L, Dwyer AA, Giacobini P, Hardelin J-P, Juul A, Maghnie M, Pitteloud N, Prevot V, Raivio T, Tena-Sempere M, Quinton R, Young J. Campbell RE, Gaidamaka G, Han S-K, Herbison AE. Expert consensus document: European Consensus Dendro-dendritic bundling and shared synapses Statement on congenital hypogonadotropic between gonadotropin- releasing hormone neurons. hypogonadism-pathogenesis, diagnosis and treatment. Proc Natl Acad Sci U S A 2009, 106: 10835-10840. Nat Rev Endocrionol 2015, 11: 547-564. Cariboni A, Pimpinelli F, Colamarino S, Zaninetti R, Bondurand N, Dastot-Le Moal F, Stanchina L, Collot Piccolella M, Rumio C, Piva F, Rugarli EI, Maggi R. N, Baral V, Marlin S, Attie-Bitach T, Giurgea I, The product of X-linked Kallmann's syndrome gene Skopinski L, Reardon W, Toutain A, Sarda P, Echaieb (KAL1) affects the migratory activity of A, Lackmy-Port-Lis M, Touraine R, Amiel J, gonadotropin-releasing hormone (GnRH)-producing Goossens M, Pingault V. Deletions at the SOX10 neurons. Hum Mol Genet 2004, 13: 2781-2791. gene locus cause Waardenburg syndrome types 2 and 4. Am J Hum Genet 2007, 81: 1169-1185. Cariboni A, Davidson K, Rakic S, Maggi R, Parnavelas JG, Ruhrberg C. Defective gonadotropin- Bouligand J, Ghervan C, Tello JA, Brailly-Tabard S, releasing hormone neuron migration in mice lacking Salenave S, Chanson P, Lombès M, Millar RP, SEMA3A signalling through NRP1 and NRP2: Guiochon-Mantel A, Young J. Isolated familial Implications for the aetiology of hypogonadotropic hypogonadotropic hypogonadism and a GNRH1 hypogonadism. Hum Mol Genet 2011, 20: 336-344. mutation. N Engl J Med 2009, 360: 2742-2748. Cariboni A, André V, Chauvet S, Cassatella D, Brioude F, Bouligand J, Trabado S, Francou B, Davidson K, Caramello A, Fantin A, Bouloux P, Salenave S, Kamenicky P, Brailly-Tabard S, Chanson Mann F, Ruhrberg C. Dysfunctional SEMA3E P, Guiochon-Mantel A, Young J. Non-syndromic signaling underlies gonadotropin-releasing hormone congenital hypogonadotropic hypogonadism: Clinical neuron deficiency in Kallmann syndrome. J Clin presentation and genotype-phenotype relationships. Invest 2015, 125: 2413-2428. Eur J Endocrinol 2010, 162: 835-851. Catalano SM, Messersmith EK, Goodman CS, Shatz Brüggenwirth HT, Boehmer ALM, Ramnarain S, CJ, Chédotal A. Many major CNS axon projections Verleun-Mooijman MCT, Satijn DPE, Trapman J, develop normally in the absence of semaphorin III. Grootegoed JA, Brinkmann AO. Molecular analysis Mol Cell Neurosci 1998, 11: 173-182. of the androgen-receptor gene in a family with receptor-positive partial androgen insensitivity: An 108

Chan Y-M, De Guillebon A, Lang-Muritano M, homeobox gene HESX1/Hesx1 associated with septo- Plummer L, Cerrato F, Tsiaras S, Gaspert A, Lavoie optic dysplasia in human and mouse. Nat Genet 1998, HB, Wu C-H, Crowley Jr. WF, Amory JK, Pitteloud 19: 125-133. N, Seminara SB. GNRH1 mutations in patients with idiopathic hypogonadotropic hypogonadism. Proc De Conti L, Skoko N, Buratti E, Baralle M. Natl Acad Sci U S A 2009, 106: 11703-11708. Complexities of 5' splice site definition: Implications in clinical analyses. RNA Biology 2012, 9: 911-923. Chung WCJ, Moyle SS, Tsai P-S. Fibroblast growth factor 8 signaling through fibroblast growth factor De Kretser DM, Meinhardt A, Meehan T, Phillips DJ, receptor 1 is required for the emergence of O'Bryan MK, Loveland KA. The roles of inhibin and gonadotropin-releasing hormone neurons. related peptides in gonadal function. Mol Cell Endocrinology 2008, 149: 4997-5003. Endocrinol 2000, 161: 43-46.

Clarke IJ, Campbell R, Smith JT, Prevot V, Wray S. De Muinck Keizer-Schrama SMPF, Mul D. Trends in Neuroendocrine control of reproduction. In: pubertal development in Europe. Hum Reprod Update Handbook of Neuroendocrinology. 2012: 197-235. 2001, 7: 287-291.

Clément K, Vaisse C, Lahlou N, Cabrol S, Pelloux V, De Roux N, Genin E, Carel J-C, Matsuda F, Cassuto D, Gourmelen M, Dina C, Chambaz J, Chaussain J-L, Milgrom E. Hypogonadotropic Lacorte J-, Basdevant A, Bougnères P, Lebouc Y, hypogonadism due to loss of function of the KiSS1- Froguel P, Guy-Grand B. A mutation in the human derived peptide receptor GPR54. Proc Natl Acad Sci leptin receptor gene causes obesity and pituitary U S A 2003, 100: 10972-10976. dysfunction. Nature 1998, 392: 398-401.

De Roux N, Young J, Misrahi M, Genet R, Chanson Cole LW, Sidis Y, Zhang C, Quinton R, Plummer L, P, Schaison G, Milgrom E. A family with Pignatelli D, Hughes VA, Dwyer AA, Raivio T, hypogonadotropic hypogonadism and mutations in the Hayes FJ, Seminara SB, Huot C, Alos N, Speiser P, gonadotropin-releasing hormone receptor. N Engl J Takeshita A, Van Vliet G, Pearce S, Crowley Jr. WF, Med 1997, 337: 1597-1602. Zhou Q-Y, Pitteloud N. Mutations in prokineticin 2 and prokineticin receptor 2 genes in human gonadotrophin-releasing hormone deficiency: De Seranno S, D'Anglemont De Tassigny X, Estrella Molecular genetics and clinical spectrum. J Clin C, Loyens A, Kasparov S, Leroy D, Ojeda SR, Endocrinol Metab 2008, 93: 3551-3559. Beauvillain J-C, Prevot V. Role of Estradiol in the Dynamic Control of Tanycyte Plasticity Mediated by Vascular Endothelial Cells in the Median Eminence. Corre C, Shinoda G, Zhu H, Cousminer DL, Endocrinology 2010, 151: 1760-1772. Crossman C, Bellissimo C, Goldenberg A, Daley GQ, Palmert MR. Sex-specific regulation of weight and puberty by the Lin28/let-7 axis. J Endocrinol 2016, De Vries L, Kauschansky A, Shohat M, Phillip M. 228: 179-191. Familial central precocious puberty suggests autosomal dominant inheritance. J Clin Endocrinol Metab 2004, 89: 1794-1800. Costa-Barbosa FA, Balasubramanian R, Keefe KW, Shaw ND, Al-Tassan N, Plummer L, Dwyer AA, Buck CL, Choi J-H, Seminara SB, Quinton R, Monies De Vries L, Gat-Yablonski G, Dror N, Singer A, D, Meyer B, Hall JE, Pitteloud N, Crowley Jr WF. Phillip M. A novel MKRN3 missense mutation Prioritizing genetic testing in patients with kallmann causing familial precocious puberty. Hum Reprod syndrome using clinical phenotypes. J Clin 2014, 29: 2838-2843. Endocrinol Metab 2013, 98: E943-953. Delorme G, Saltel F, Bonnelye E, Jurdic P, Machuca- Damla Kotan L, Ian Hutchins B, Ozkan Y, Demirel F, Gayet I. Expression and function of semaphorin 7A in Stoner H, Cheng PJ, Esen I, Gurbuz F, Kenan Bicakci bone cells. Biology of the Cell 2005, 97: 589-597. Y, Mengen E, Yuksel B, Wray S, Kemal Topaloglu A. Mutations in FEZF1 cause kallmann syndrome. Am J Hum Genet 2014, 95: 326-331. Desmet F-O, Hamroun D, Lalande M, Collod-Bëroud G, Claustres M, Béroud C. Human Splicing Finder: An online bioinformatics tool to predict splicing Dattani MT, Martinez-Barbera J-P, Thomas PQ, signals. Nucleic Acids Res 2009, 37: e67. Brickman JM, Gupta R, Mårtensson I-L, Toresson H, Fox M, Wales JKH, Hindmarsh PC, Krauss S, Beddington RSP, Robinson ICAF. Mutations in the 109

Dimitrova-Mladenova MS, Stefanova EM, Glushkova Eswarakumar VP, Lax I, Schlessinger J. Cellular M, Todorova AP, Todorov T, Konstantinova MM, signaling by fibroblast growth factor receptors. Kazakova K, Tincheva RS. Males with Paternally Cytokine Growth Factor Rev 2005, 16: 139-149. Inherited MKRN3 Mutations May Be Asymptomatic. J Pediatr 2016, 179: 263-265. Falardeau J, Chung WCJ, Beenken A, Raivio T, Plummer L, Sidis Y, Jacobson-Dickman EE, Dodé C, Levilliers J, Dupont J-M, De Paepe A, Le Dû Eliseenkova AV, Ma J, Dwyer A, Quinton R, Na S, N, Soussi-Yanicostas N, Coimbra RS, Delmaghani S, Hall JE, Huot C, Alois N, Pearce SHS, Cole LW, Compain-Nouaille S, Baverel F, Pêcheux C, Le Hughes V, Mohammadi M, Tsai P, Pitteloud N. Tessier D, Cruaud C, Delpech M, Speleman F, Decreased FGF8 signaling causes deficiency of Vermeulen S, Amalfitano A, Bachelot Y, Bouchard P, gonadotropin-releasing hormone in humans and mice. Cabrol S, Carel J-C, Van de Waal HD, Goulet-Salmon J Clin Invest 2008, 118: 2822-2831. B, Kottler M-L, Richard O, Sanchez-Franco F, Saura R, Young J, Petit C, Hardelin J-P. Loss-of-function Feng W, Khan MA, Bellvis P, Zhu Z, Bernhardt O, mutations in FGFR1 cause autosomal dominant Herold-Mende C, Liu H-K. The chromatin remodeler Kallmann syndrome. Nat Genet 2003, 33: 463-465. CHD7 regulates adult neurogenesis via activation of soxc transcription factors. Cell Stem Cell 2013, 13: Dodé C, Teixeira L, Levilliers J, Fouveaut C, 62-72. Bouchard P, Kottler M-L, Lespinasse J, Lienhardt- Roussie A, Mathieu M, Moerman A, Morgan G, Forni PE, Bharti K, Flannery EM, Shimogori T, Wray Murat A, Toublanc J-E, Wolczynski S, Delpech M, S. The indirect role of fibroblast growth factor-8 in Petit C, Young J, Hardelin J-P. Kallmann syndrome: defining neurogenic niches of the olfactory/GnRH Mutations in the genes encoding prokineticin-2 and systems. J Neurosci 2013, 33: 19620-19634. prokineticin receptor-2. PLoS Genetics 2006, 2: 1648- 1652. Forni PE, Taylor-Burds C, Melvin VS, Williams T, Wray S. Neural crest and ectodermal cells intermix in Dwyer AA, Raivio T, Pitteloud N. Reversible the nasal placode to give rise to GnRH-1 neurons, hypogonadotropic hypogonadism. Eur J Endocrinol sensory neurons, and olfactory ensheathing cells. J 2016, 174: R267-R274. Neurosci 2011, 31: 6915-6927.

Eak B-L, Salmivirta M. Heparin/heparan sulfate Forni PE, Wray S. GnRH, anosmia and domains in binding and signaling of fibroblast growth hypogonadotropic hypogonadism - Where are we? factor 8b. J Biol Chem 2002, 277: 32616-32623. Front Neuroendocrinol 2015, 36: 165-177.

Eggers S, Sadedin S, van den Bergen JA, Robevska G, Ohnesorg Franco B, Guioli S, Pragliola A, Incerti B, Bardoni B, T, Hewitt J, Lambeth L, Bouty A, Knarston IM, Tan TY, Tonlorenzi R, Carrozzo R, Maestrini E, Pieretti M, Cameron F, Werther G, Hutson J, O'Connell M, Grover SR, Taillon-Miller P, Brown CJ, Willard HF, Lawrence C, Heloury Y, Zacharin M, Bergman P, Kimber C, Brown J, Webb Persico MG, Camerino G, Ballabio A. A gene deleted N, Hunter MF, Srinivasan S, Titmuss A, Verge CF, Mowat D, in Kallmann's syndrome shares homology with neural Smith G, Smith J, Ewans L, Shalhoub C, Crock P, Cowell C, cell adhesion and axonal path-finding molecules. Leong GM, Ono M, Lafferty AR, Huynh T, Visser U, Choong Nature 1991, 353: 529-536. CS, McKenzie F, Pachter N, Thompson EM, Couper J, Baxendale A, Gecz J, Wheeler BJ, Jefferies C, MacKenzie K, Hofman P, Carter P, King RI, Krausz C, van Ravenswaaij-Arts Franco M, Tamagnone L. Tyrosine phosphorylation in CMA, Looijenga L, Drop S, Riedl S, Cools M, Dawson A, semaphorin signalling: Shifting into overdrive. Juniarto AZ, Khadilkar V, Khadilkar A, Bhatia V, Dung VC, EMBO Rep 2008, 9: 865-871. Atta I, Raza J, thi Diem Chi N, Hao TK, Harley V, Koopman P, Warne G, Faradz S, Oshlack A, Ayers KL, Sinclair AH. Francou B, Bouligand J, Voican A, Amazit L, Disorders of sex development: Insights from targeted gene sequencing of a large international patient cohort. Genome Biol Trabado S, Fagart J, Meduri G, Brailly-Tabard S, 2016, 17: Chanson P, Lecomte P, Guiochon-Mantel A, Young J. Normosmic congenital hypogonadotropic hypogonadism due to TAC3/TACR3 mutations: Endo Y, Ishiwata-Endo H, Yamada K. Extracellular Characterization of neuroendocrine phenotypes and Matrix Protein Anosmin Promotes Neural Crest novel mutations. PLoS ONE 2011, 6: e25614. Formation and Regulates FGF, BMP, and WNT Activities. Dev Cell 2012, 23: 305-316. Gajdos ZKZ, Henderson KD, Hirschhorn JN, Palmert MR. Genetic determinants of pubertal timing in the

110

general population. Mol Cell Endocrinol 2010, 324: Graveley BR. Sorting out the complexity of SR 21-29. protein functions. RNA 2000, 6: 1197-1211.

García-González D, Murcia-Belmonte V, Esteban PF, Guran T, Tolhurst G, Bereket A, Rocha N, Porter K, Ortega F, Díaz D, Sánchez-Vera I, Lebrón-Galán R, Turan S, Gribble FM, Kotan LD, Akcay T, Atay Z, Escobar-Castañondo L, Martínez-Millán L, Weruaga Canan H, Serin A, O'Rahilly S, Reimann F, Semple E, García-Verdugo JM, Berninger B, de Castro F. RK, Topaloglu AK. Hypogonadotropic hypogonadism Anosmin-1 over-expression increases adult due to a novel missense mutation in the first neurogenesis in the subventricular zone and extracellular loop of the neurokinin B receptor. J Clin neuroblast migration to the olfactory bulb. Brain Endocrinol Metab 2009, 94: 3633-3639. Struct Funct 2016, 221: 239-260. Hafizi S, Dahlbäck B. Gas6 and protein S: Vitamin K- Geller S, Kolasa E, Tillet Y, Duittoz A, Vaudin P. dependent ligands for the Axl receptor tyrosine kinase Olfactory ensheathing cells form the subfamily. FEBS J 2006, 273: 5231-5244. microenvironment of migrating GnRH-1 neurons during mouse development. Glia 2013, 61: 550-566. Hagen CP, Sørensen K, Mieritz MG, Johannsen TH, Almstrup K, Juul A. Circulating MKRN3 levels Giacobini P, Parkash J, Campagne C, Messina A, decline prior to pubertal onset and through puberty: A Casoni F, Vanacker C, Langlet F, Hobo B, Cagnoni longitudinal study of healthy girls. J Clin Endocrinol G, Gallet S, Hanchate NK, Mazur D, Taniguchi M, Metab 2015, 100: 1920-1926. Mazzone M, Verhaagen J, Ciofi P, Bouret SG, Tamagnone L, Prevot V. Brain Endothelial Cells Hanchate NK, Giacobini P, Lhuillier P, Parkash J, Control Fertility through Ovarian-Steroid-Dependent Espy C, Fouveaut C, Leroy C, Baron S, Campagne C, Release of Semaphorin 3A. PLoS Biology 2014, 12: Vanacker C, Collier F, Cruaud C, Meyer V, García- e1001808. Piñero A, Dewailly D, Cortet-Rudelli C, Gersak K, Metz C, Chabrier G, Pugeat M, Young J, Hardelin J- Gianetti E, Tusset C, Noel SD, Au MG, Dwyer AA, P, Prevot V, Dodé C. SEMA3A, a Gene Involved in Hughes VA, Abreu AP, Carroll J, Trarbach E, Silveira Axonal Pathfinding, Is Mutated in Patients with LFG, Costa EMF, De Mendonça BB, De Castro M, Kallmann Syndrome. PLoS Genetics 2012, 8: Lofrano A, Hall JE, Bolu E, Ozata M, Quinton R, e1002896. Amory JK, Stewart SE, Arlt W, Cole TR, Crowley WF, Kaiser UB, Latronico AC, Seminara SB. Hardelin J-P, Julliard AK, Moniot B, Soussi- TAC3/TACR3 mutations reveal preferential Yanicostas N, Verney C, Schwanzel-Fukuda M, activation of gonadotropin- releasing hormone release Lievre CA, Petit C. Anosmin-1 is a regionally by neurokinin B in neonatal life followed by reversal restricted component of basement membranes and in adulthood. J Clin Endocrinol Metab 2010, 95: interstitial matrices during organogenesis: 2857-2867. Implications for the developmental anomalies of X chromosome-linked Kallmann syndrome. Dev Dyn González-Martínez D, Kim S-H, Hu Y, Guimond S, 1999, 215: 26-44. Schofield J, Winyard P, Vannelli GB, Turnbull J, Bouloux P-M. Anosmin-1 modulates fibroblast Harries MLL, Walker JM, Williams DM, Hawkins S, growth factor receptor 1 signaling in human Hughes IA. Changes in the male voice at puberty. gonadotropin-releasing hormone olfactory neuroblasts Arch Dis Child 1997, 77: 445-447. through a heparan sulfate-dependent mechanism. J Neurosci 2004, 24: 10384-10392. He C, Kraft P, Chen C, Buring JE, Paré G, Hankinson SE, Chanock SJ, Ridker PM, Hunter DJ, Chasman DI. Gottlieb B, Beitel LK, Nadarajah A, Paliouras M, Genome-wide association studies identify loci Trifiro M. The androgen receptor gene mutations associated with age at menarche and age at natural database: 2012 update. Hum Mutat 2012, 33: 887- menopause. Nat Genet 2009, 41: 724-728. 894.

Herbison AE. Physiology of the Adult Gonadotropin- Grandone A, Cantelmi G, Cirillo G, Marzuillo P, Releasing Hormone Neuronal Network. In: Knobil Luongo C, Miraglia del Giudice E, Perrone L. A case and Neill's Physiology of Reproduction: Two-Volume of familial central precocious puberty caused by a Set. 2014: 399-467. novel mutation in the makorin RING finger protein 3 gene. BMC Endocr Disord 2015, 15: 60.

111

Herbison AE. Control of puberty onset and fertility by semaphorin signaling in spinal motor axon guidance. gonadotropin-releasing hormone neurons. Nat Rev Neuron 2005, 48: 949-964. Endocrionol 2016, 12: 452-466. Hughes IA, Davies JD, Bunch TI, Pasterski V, Herde MK, Iremonger KJ, Constantin S, Herbison Mastroyannopoulou K, Macdougall J. Androgen AE. GnRH neurons elaborate a long-range projection insensitivity syndrome. The Lancet 2012, 380: 1419- with shared axonal and dendritic functions. J Neurosci 1428. 2013, 33: 12689-12697. Hughes IA, Houk C, Ahmed SF, Lee PA. Consensus Herman-Giddens ME, Slora EJ, Wasserman RC, statement on management of intersex disorders. Arch Bourdony CJ, Bhapkar MV, Koch GG, Hasemeier Dis Child 2006, 91: 554-563. CM. Secondary sexual characteristics and menses in young girls seen in office practice: A study from the Hunzicker-Dunn M, Mayo K. Chapter 20 - pediatric research in office settings network. Gonadotropin Signaling in the Ovary. In: Plant,TM, , Pediatrics 1997, 99: 505-512. Zeleznik,AJ (eds.) Knobil and Neill's Physiology of Reproduction (Fourth Edition). Academic Press, San Hiort O, Birnbaum W, Marshall L, Wünsch L, Werner Diego 2015: 895-945. R, Schröder T, Döhnert U, Holterhus P-M. Management of disorders of sex development. Nat Husu E, Hove HD, Farholt S, Bille M, Tranebjærg L, Rev Endocrionol 2014, 10: 520-529. Vogel I, Kreiborg S. Phenotype in 18 Danish subjects with genetically verified CHARGE syndrome. Clin Hofmann K, Zweier M, Sticht H, Zweier C, Wittmann Genet 2013, 83: 125-134. W, Hoyer J, Uebe S, van Haeringen A, Thiel CT, Ekici AB, Reis A, Rauch A. Biallelic SEMA3A Hutchins BI, Kotan LD, Taylor-Burds C, Ozkan Y, defects cause a novel type of syndromic short stature. Cheng PJ, Gurbuz F, Tiong JDR, Mengen E, Yuksel Am J Med Genet Part A 2013, 161: 2880-2889. B, Topaloglu AK, Wray S. CCDC141 mutation identified in anosmic hypogonadotropic Holmes S, Downs A-M, Fosberry A, Hayes PD, hypogonadism (kallmann syndrome) alters GnRH Michalovich D, Murdoch P, Moores K, Fox J, Deen neuronal migration. Endocrinology 2016, 157: 1956- K, Pettman G, Wattam T, Lewis C. Sema7A is a 1966. potent monocyte stimulator. Scand J Immunol 2002, 56: 270-275. Imai T, Yamazaki T, Kobayakawa R, Kobayakawa K, Abe T, Suzuki M, Sakano H. Pre-Target axon sorting Hornig NC, De Beaufort C, Denzer F, Cools M, establishes the neural map topography. Science 2009, Wabitsch M, Ukat M, Kulle AE, Schweikert H-U, 325: 585-590. Werner R, Hiort O, Audi L, Siebert R, Ammerpohl O, Holterhus P-M. A recurrent germline mutation in the Inatani M, Irie F, Plump AS, Tessier-Lavigne M, 5'UTR of the androgen receptor causes complete Yamaguchi Y. Mammalian Brain Morphogenesis and androgen insensitivity by activating aberrant uORF Midline Axon Guidance Require Heparan Sulfate. translation. PLoS ONE 2016, 11: e0154158. Science 2003, 302: 1044-1046.

Howard SR, Guasti L, Ruiz-Babot G, Mancini A, Isken O, Maquat LE. Quality control of eukaryotic David A, Storr H, Metherell LA, Sternberg MJ, mRNA: Safeguarding cells from abnormal mRNA Cabrera CP, Warren HR, Barnes MR, Quinton R, de function. Genes Dev 2007, 21: 1833-1856. Roux N, Young J, Guiochon-Mantel A, Wehkalampi K, André V, Gothilf Y, Cariboni A, Dunkel L. IGSF10 mutations dysregulate gonadotropin-releasing Josso N, Belville C, di Clemente N, Picard J-Y. AMH hormone neuronal migration resulting in delayed and AMH receptor defects in persistent Müllerian puberty. EMBO Mol Med 2016, 8: 626-642. duct syndrome. Hum Reprod Update 2005, 11: 351- 356. Hu Y, Bouloux P-M. X-linked GnRH deficiency: Role of KAL-1 mutations in GnRH deficiency. Mol Jääskeläinen J. Molecular biology of androgen Cell Endocrinol 2011, 346: 13-20. insensitivity. Mol Cell Endocrinol 2012, 352: 4-12.

Huber AB, Kania A, Tran TS, Gu C, De Marco Jackson RS, Creemers JWM, Ohagi S, Raffin-Sanson Garcia N, Lieberam I, Johnson D, Jessell TM, Ginty M-, Sanders L, Montague CT, Hutton JC, O'Rahilly S. DD, Kolodkin AL. Distinct roles for secreted Obesity and impaired prohormone processing 112

associated with mutations in the human prohormone Kawauchi S, Shou J, Santos R, Hébert JM, convertase 1 gene. Nat Genet 1997, 16: 303-306. McConnell SK, Mason I, Calof AL. Fgf8 expression defines a morphogenetic center required for olfactory neurogenesis and nasal cavity development in the Janssen N, Bergman JEH, Swertz MA, Tranebjaerg L, mouse. Development (Cambridge) 2005, 132: 5211- Lodahl M, Schoots J, Hofstra RMW, Van 5223. Ravenswaaij-Arts CMA, Hoefsloot LH. Mutation update on the CHD7 gene involved in CHARGE syndrome. Hum Mutat 2012, 33: 1149-1160. Kim H-G, Ahn J-W, Kurth I, Ullmann R, Kim H-T, Kulharya A, Ha K-S, Itokawa Y, Meliciani I, Wenzel W, Lee D, Rosenberger G, Ozata M, Bick DP, Sherins Jarzabek K, Wolczynski S, Lesniewicz R, Plessis G, RJ, Nagase T, Tekin M, Kim S-H, Kim C-H, Ropers Kottler.M.L. Evidence that FGFR1 loss-of-function H-H, Gusella JF, Kalscheuer V, Choi CY, Layman mutations may cause variable skeletal malformations LC. WDR11, a WD protein that interacts with in patients with Kallmann syndrome. Adv Med Sci transcription factor EMX1, is mutated in idiopathic 2012, 57: 314-321. hypogonadotropic hypogonadism and Kallmann syndrome. Am J Hum Genet 2010, 87: 465-479. Jong MTC, Gray TA, Ji Y, Glenn CC, Saitoh S, Driscoll DJ, Nicholls RD. A novel imprinted gene, Kim H-G, Kurth I, Lan F, Meliciani I, Wenzel W, encoding a RING zinc-finger protein, and overlapping Eom SH, Kang GB, Rosenberger G, Tekin M, Ozata antisense transcript in the Prader-Willi syndrome M, Bick DP, Sherins RJ, Walker SL, Shi Y, Gusella critical region. Hum Mol Genet 1999, 8: 783-793. JF, Layman LC. Mutations in CHD7, Encoding a Chromatin-Remodeling Protein, Cause Idiopathic Jongmans MCJ, van Ravenswaaij-Arts CMA, Hypogonadotropic Hypogonadism and Kallmann Pitteloud N, Ogata T, Sato N, Claahsen-van der Syndrome. Am J Hum Genet 2008, 83: 511-519. Grinten HL, van der Donk K, Seminara S, Bergman JEH, Brunner HG, Crowley Jr. WF, Hoefsloot LH. Kim S-H. Congenital hypogonadotropic CHD7 mutations in patients initially diagnosed with hypogonadism and Kallmann syndrome: Past, present, Kallmann syndrome - The clinical overlap with and future. Endocrinol Metab 2015, 30: 456-466. CHARGE syndrome. Clin Genet 2009, 75: 65-71.

Knarston I, Ayers K, Sinclair A. Molecular Juul A, Magnusdottir S, Scheike T, Prytz S, mechanisms associated with 46,XX disorders of sex Skakkebæk NE. Age at voice break in Danish boys: development. Clin Sci 2016, 130: 421-432. Effects of pre-pubertal body mass index and secular trend. Int J Androl 2007, 30: 537-542. Koh J-M, Oh B, Lee JY, Lee J-K, Kimm K, Kim GS, Park BL, Cheong HS, Shin HD, Hong JM, Kim T-H, Kallmann FJ, Schoenfeld WA, Barrera SE. The Park EK, Kim S-Y. Association study of semaphorin genetic aspects of primary eunuchoidism. Am J Ment 7a (sema7a) polymorphisms with bone mineral Defic 1944, 158: 203-236. density and fracture risk in postmenopausal Korean women. J Hum Genet 2006, 51: 112-117. Kanber D, Giltay J, Wieczorek D, Zogel C, Hochstenbach R, Caliebe A, Kuechler A, Horsthemke Kotan LD, Cooper C, Darcan S, Carr IM, Özen S, B, Buiting K. A paternal deletion of MKRN3, Yan Y, Hamedani MK, Gürbüz F, Mengen E, Turan I, MAGEL2 and NDN does not result in Prader-Willi Ulubay A, Akkus G, Yüksel B, Topaloglu AK, syndrome. Eur J Hum Genet 2009, 17: 582-590. Leygue E. Idiopathic hypogonadotropic hypogonadism caused by inactivating mutations in Karvonen U, Kallio PJ, Jänne OA, Palvimo JJ. SRA1. JCRPE J Clin Res Pediatr Endocrinol 2016, 8: Interaction of androgen receptors with androgen 125-134. response element in intact cells. Roles of amino- and carboxyl-terminal regions and the ligand. J Biol Chem Kotani M, Detheux M, Vandenbogaerde A, Communi 1997, 272: 15973-15979. D, Vanderwinden J-, Le Poul E, Brézillon S, Tyldesley R, Suarez-Huerta N, Vandeput F, Blanpain Kauffman AS, Gottsch ML, Roa J, Byquist AC, C, Schiffmann SN, Vassart G, Parmentier M. The Crown A, Clifton DK, Hoffman GE, Steiner RA, Metastasis Suppressor Gene KiSS-1 Encodes Tena-Sempere M. Sexual differentiation of Kiss1 Kisspeptins, the Natural Ligands of the Orphan G gene expression in the brain of the rat. Endocrinology Protein-coupled Receptor GPR54. J Biol Chem 2001, 2007, 148: 1774-1783. 276: 34631-34636.

113

Kumar P, Henikoff S, Ng PC. Predicting the effects of encodes a protein related to adhesion molecules. Cell coding non-synonymous variants on protein function 1991, 67: 423-433. using the SIFT algorithm. Nature Protocols 2009, 4: 1073-1082. Leroy C, Fouveaut C, Leclercq S, Jacquemont S, Boullay HD, Lespinasse J, Delpech M, Dupont J-, Laitinen E-M, Tommiska J, Sane T, Vaaralahti K, Hardelin J-P, Dodé C. Biallelic mutations in the Toppari J, Raivio T. Reversible congenital prokineticin-2 gene in two sporadic cases of Kallmann hypogonadotropic hypogonadism in patients with syndrome. Eur J Hum Genet 2008, 16: 865-868. CHD7, FGFR1 or GNRHR mutations. PLoS ONE 2012, 7: e39450. Lettre G, Jackson AU, Gieger C, Schumacher FR, Berndt SI, Sanna S, Eyheramendy S, Voight BF, Laitinen E-M, Vaaralahti K, Tommiska J, Eklund E, Butler JL, Guiducci C, Illig T, Hackett R, Heid IM, Tervaniemi M, Valanne L, Raivio T. Incidence, Jacobs KB, Lyssenko V, Uda M, Boehnke M, phenotypic features and molecular genetics of Chanock SJ, Groop LC, Hu FB, Isomaa B, Kraft P, Kallmann syndrome in Finland. Orphanet Journal of Peltonen L, Salomaa V, Schlessinger D, Hunter DJ, Rare Diseases 2011, 6: 41. Hayes RB, Abecasis GR, Wichmann H-E, Mohlke KL, Hirschhorn JN. Identification of ten loci associated with height highlights new biological Lalani SR, Safiullah AM, Molinari LM, Fernbach SD, pathways in human growth. Nat Genet 2008, 40: 584- Martin DM, Belmont JW. SEMA3E mutation in a 591. patient with CHARGE syndrome. J Med Genet 2004, 41: e94. Lomniczi A, Loche A, Castellano JM, Ronnekleiv OK, Bosch M, Kaidar G, Knoll JG, Wright H, Pfeifer Lange C, Liehr T, Goen M, Gebhart E, Fleckenstein GP, Ojeda SR. Epigenetic control of female puberty. B, Ensser A. New eukaryotic semaphorins with close Nat Neurosci 2013, 16: 281-289. homology to semaphorins of DNA viruses. Genomics 1998, 51: 340-350. Luan X, Yu H, Wei X, Zhou Y, Wang W, Li P, Gan X, Wei D, Xiao J. GPR54 polymorphisms in Chinese Latronico AC, Brito VN, Carel J-C. Causes, girls with central precocious puberty. diagnosis, and treatment of central precocious Neuroendocrinology 2007a, 86: 77-83. puberty. Lancet Diabetes Endocrinol 2016, 4: 265- 274. Luan X, Zhou Y, Wang W, Yu H, Li P, Gan X, Wei D, Xiao J. Association study of the polymorphisms in Layman WS, Hurd EA, Martin DM. Reproductive the KISS1 gene with central precocious puberty in dysfunction and decreased GnRH neurogenesis in a Chinese girls. European Journal of Endocrinology mouse model of CHARGE syndrome. Hum Mol 2007b, 157: 113-118. Genet 2011, 20: 3138-3150.

Lund C, Pulli K, Yellapragada V, Giacobini P, Lundin Layman WS, McEwen DP, Beyer LA, Lalani SR, K, Vuoristo S, Tuuri T, Noisa P, Raivio T. Fernbach SD, Oh E, Swaroop A, Hegg CC, Raphael Development of Gonadotropin-Releasing Hormone- Y, Martens JR, Martin DM. Defects in neural stem Secreting Neurons from Human Pluripotent Stem cell proliferation and olfaction in Chd7 deficient mice Cells. Stem Cell Rep 2016, 7: 149-157. indicate a mechanism for hyposmia in human CHARGE syndrome. Hum Mol Genet 2009, 18: 1909-1923. Macedo DB, Abreu AP, Reis ACS, Montenegro LR, Dauber A, Beneduzzi D, Cukier P, Silveira LFG, Teles MG, Carroll RS, Junior GG, Filho GG, Gucev Lee HS, Jin H-S, Shim YS, Jeong HR, Kwon E, Choi Z, Arnhold IJP, De Castro M, Moreira AC, Martinelli V, Kim M-C, Chung I-C, Jeong S-Y, Hwang JS. Low Jr. CE, Hirschhorn JN, Mendonca BB, Brito VN, Frequency of MKRN3 Mutations in Central Antonini SR, Kaiser UB, Latronico AC. Central Precocious Puberty among Korean Girls. Horm Metab precocious puberty that appears to be sporadic caused Res 2015, 48: 118-122. by paternally inherited mutations in the imprinted gene makorin ring finger 3. J Clin Endocrinol Metab Legouis R, Hardelin J-P, Leyilliers J, Claverle J-M, 2014, 99: E1097-E1103. Compain S, Wunderle V, Millasseau P, Paslier DL, Cohen D, Caterina D, Bougueleret L, Delemarre-Van Maestre de San Juan, A. Falta total de los de Waal H, Lutfalla G, Weissenbach J, Petit C. The nerviosolfactorios con anosmia en un individuo en candidate gene for the X-linked Kallmann syndrome quien existia una atrofia congenita de los testiculos y miembro viril. Siglo Med 1856, 131: 211. 114

Mann F, Chauvet S, Rougon G. Semaphorins in in the mutational hot spots in the region encoding the development and adult brain: Implication for decapeptide. Horm Res Paediatr 2015, 85: 107-111. neurological diseases. Prog Neurobiol 2007, 82: 57- 79. Messina A, Ferraris N, Wray S, Cagnoni G, Donohue DE, Casoni F, Kramer PR, Derijck AA, Adolfs Y, Margolin DH, Kousi M, Chan Y-M, Lim ET, Fasolo A, Pasterkamp RJ, Giacobini P. Dysregulation Schmahmann JD, Hadjivassiliou M, Hall JE, Adam I, of semaphorin7A/ß1-integrin signaling leads to Dwyer A, Plummer L, Aldrin SV, O'Rourke J, Kirby defective GnRH-1 cell migration, abnormal gonadal A, Lage K, Milunsky A, Milunsky JM, Chan J, development and altered fertility. Hum Mol Genet Hedley-Whyte ET, Daly MJ, Katsanis N, Seminara 2011, 20: 4759-4774. SB. Ataxia, dementia, and hypogonadotropism caused by disordered ubiquitination. New Engl J Med 2013, Messina A, Langlet F, Chachlaki K, Roa J, Rasika S, 368: 1992-2003. Jouy N, Gallet S, Gaytan F, Parkash J, Tena-Sempere M, Giacobini P, Prevot V. A microRNA switch Marshall WA, Tanner JM. Variations in pattern of regulates the rise in hypothalamic GnRH production pubertal changes in girls. Arch Dis Child 1969, 44: before puberty. Nat Neurosci 2016, 19: 835-844. 291-303. Miraoui H, Dwyer AA, Sykiotis GP, Plummer L, Marshall WA, Tanner JM. Variations in the pattern of Chung W, Feng B, Beenken A, Clarke J, Pers TH, pubertal changes in boys. Arch Dis Child 1970, 45: Dworzynski P, Keefe K, Niedziela M, Raivio T, 13-23. Crowley Jr. WF, Seminara SB, Quinton R, Hughes VA, Kumanov P, Young J, Yialamas MA, Hall JE, Van Vliet G, Chanoine J-P, Rubenstein J, Martin C, Balasubramanian R, Dwyer AA, Au MG, Mohammadi M, Tsai P-S, Sidis Y, Lage K, Pitteloud Sidis Y, Kaiser UB, Seminara SB, Pitteloud N, Zhou N. Mutations in FGF17, IL17RD, DUSP6, SPRY4, Q-Y, Crowley Jr. WF. The role of the prokineticin 2 and FLRT3 are identified in individuals with pathway in human reproduction: Evidence from the congenital hypogonadotropic hypogonadism. Am J study of human and murine gene mutations. Endocr Hum Genet 2013, 92: 725-743. Rev 2011, 32: 225-246.

Mitchell AL, Dwyer A, Pitteloud N, Quinton R. Matsumoto S, Yamazaki C, Masumoto K-H, Nagano Genetic basis and variable phenotypic expression of M, Naito M, Soga T, Hiyama H, Matsumoto M, Kallmann syndrome: Towards a unifying theory. Takasaki J, Kamohara M, Matsuo A, Ishii H, Kobori Trends Endocrinol Metab 2011, 22: 249-258. M, Katoh M, Matsushime H, Furuichi K, Shigeyoshi Y. Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin Miura K, Acierno Jr. JS, Seminara SB. receptor PKR2. Proc Natl Acad Sci U S A 2006, 103: Characterization of the human nasal embryonic 4140-4145. LHRH factor gene, NELF, and a mutation screening among 65 patients with idiopathic hypogonadotropic hypogonadism (IHH). J Hum Genet 2004, 49: 265- Matzuk MM, Lamb DJ. The biology of infertility: 268. research advances and clinical challenges. Nat Med 2008, 14: 1197-1213. Moosa S, Wollnik B. Altered FGF signalling in congenital craniofacial and skeletal disorders. Semin Mazaheri A, Hashemipour M, Salehi M, Behnam M, Cell Dev Biol 2016, 53: 115-125. Hovsepian S, Hassanzadeh A. Mutation of kisspeptin 1 gene in children with precocious puberty in Isfahan city. Int J Prev Med 2015, 6:41. Moss EG, Lee RC, Ambros V. The cold shock domain protein LIN-28 controls developmental timing in C. elegans and is regulated by the lin-4 RNA. Cell Mendonca BB, Costa EMF, Belgorosky A, Rivarola 1997, 88: 637-646. MA, Domenice S. 46,XY DSD due to impaired androgen production. Best Pract Res Clin Endocrinol Metab 2010, 24: 243-262. Muscatelli F, Strom TM, Walker AP, Zanaria E, Récan D, Meindl A, Bardoni B, Guioli S, Zehetner G, Rabl W, Peter Schwarz H, Kaplan J-, Camerino G, Mengen E, Tunc S, Kotan LD, Nalbantoglu O, Demir Meitinger T, Monaco AP. Mutations in the DAX-1 K, Gurbuz F, Turan I, Seker G, Yuksel B, Topaloglu gene give rise to both X-linked adrenal hypoplasia AK. Complete idiopathic hypogonadotropic congenita and hypogonadotropic hypogonadism. hypogonadism due to homozygous GNRH1 mutations Nature 1994, 372: 672-676.

115

Nachtigall LB, Boepple PA, Pralong FP, Crowley Jr. Parent A-S, Teilmann G, Juul A, Skakkebaek NE, WF. Adult-onset idiopathic hypogonadotropic Toppari J, Bourguignon J-P. The Timing of Normal hypogonadism - A treatable form of male infertility. Puberty and the Age Limits of Sexual Precocity: NEW ENGL J MED 1997, 336: 410-415. Variations around the World, Secular Trends, and Changes after Migration. Endocr Rev 2003, 24: 668- 693. Navarro VM, Tena-Sempere M. Neuroendocrine control by kisspeptins: Role in metabolic regulation of fertility. Nat Rev Endocrionol 2012, 8: 40-53. Parkash J, Cimino I, Ferraris N, Casoni F, Wray S, Cappy H, Prevot V, Giacobini P. Suppression of ß1- integrin in gonadotropin-releasing hormone cells Neocleous V, Shammas C, Phelan MM, Nicolaou S, disrupts migration and axonal extension resulting in Phylactou LA, Skordis N. In silico analysis of a novel severe reproductive alterations. Journal of MKRN3 missense mutation in familial central Neuroscience 2012, 32: 16992-17002. precocious puberty. Clin Endocrinol 2016, 84: 80-84.

Parkash J, Messina A, Langlet F, Cimino I, Loyens A, Newbern K, Natrajan N, Kim H-G, Chorich LP, Mazur D, Gallet S, Balland E, Malone SA, Pralong F, Halvorson LM, Cameron RS, Layman LC. Cagnoni G, Schellino R, De Marchis S, Mazzone M, Identification of HESX1 mutations in Kallmann Pasterkamp RJ, Tamagnone L, Prevot V, Giacobini P. syndrome. Fertil Steril 2013, 99: 1831-1837. Semaphorin7A regulates neuroglial plasticity in the adult hypothalamic median eminence. Nature Ng KL, Li J-D, Cheng MY, Leslie FM, Lee AC, Zhou Communications 2015, 6: 6385. Q-Y. Neuroscience: Dependence of olfactory bulb neurogenesis on prokineticin 2 signaling. Science Partsch C-J, Heger S, Sippell WG. Management and 2005, 308: 1923-1927. outcome of central precocious puberty. Clin Endocrinol 2002, 56: 129-148. Ohtaka K, Fujisawa Y, Takada F, Hasegawa Y, Miyoshi T, Hasegawa T, Miyoshi H, Kameda H, Pasterkamp RJ, Kolk SM, Hellemons AJCGM, Kurokawa-Seo M, Fukami M, Ogata T. FGFR1 Kolodkin AL. Expression patterns of semaphorin7A Analyses in Four Patients with Hypogonadotropic and plexinC1 during rat neural development suggest Hypogonadism with Split-Hand/Foot Malformation: roles in axon guidance and neuronal migration. BMC Implications for the Promoter Region. Hum Mutat. Developmental Biology 2007, 7: 98. 2017 doi: 10.1002/humu.23178. [Epub ahead of print]. Pasterkamp RJ, Peschon JJ, Spriggs MK, Kolodkin AL. Semaphorin 7A promotes axon outgrowth Ong KK, Elks CE, Li S, Zhao JH, Luan J, Andersen through integrins and MAPKs. Nature 2003, 424: LB, Bingham SA, Brage S, Smith GD, Ekelund U, 398-405. Gillson CJ, Glaser B, Golding J, Hardy R, Khaw K-, Kuh D, Luben R, Marcus M, McGeehin MA, Ness AR, Northstone K, Ring SM, Rubin C, Sims MA, Pasterkamp RJ. Getting neural circuits into shape with Song K, Strachan DP, Vollenweider P, Waeber G, semaphorins. Nat Rev Neurosci 2012, 13: 605-618. Waterworth DM, Wong A, Deloukas P, Barroso I, Mooser V, Loos RJ, Wareham NJ. Genetic variation Perry JRB, Day F, Elks CE, Sulem P, Thompson DJ, in LIN28B is associated with the timing of puberty. Ferreira T, He C, Chasman DI, Esko T, Thorleifsson Nat Genet 2009, 41: 729-733. G, Albrecht E, Ang WQ, Corre T, Cousminer DL, Feenstra B, Franceschini N, Ganna A, Johnson AD, Osborne NJ, Begbie J, Chilton JK, Schmidt H, Kjellqvist S, Lunetta KL, McMahon G, Nolte IM, Eickholt BJ. Semaphorin/neuropilin signaling Paternoster L, Porcu E, Smith AV, Stolk L, Teumer influences the positioning of migratory neural crest A, Tšernikova N, Tikkanen E, Ulivi S, Wagner EK, cells within the hindbrain region of the chick. Dev Amin N, Bierut LJ, Byrne EM, Hottenga J-, Koller Dyn 2005, 232: 939-949. DL, Mangino M, Pers TH, Yerges-Armstrong LM, Zhao JH, Andrulis IL, Anton-Culver H, Atsma F, Bandinelli S, Beckmann MW, Benitez J, Blomqvist C, Pampillo M, Camuso N, Taylor JE, Szereszewski JM, Bojesen SE, Bolla MK, Bonanni B, Brauch H, Ahow MR, Zajac M, Millar RP, Bhattacharya M, Brenner H, Buring JE, Chang-Claude J, Chanock S, Babwah AV. Regulation of GPR54 signaling by Chen J, Chenevix-Trench G, Collée JM, Couch FJ, GRK2 and ß-arrestin. Mol Endocrinol 2009, 23: 2060- Couper D, Coviello AD, Cox A, Czene K, D'adamo 2074. AP, Smith GD, De Vivo I, Demerath EW, Dennis J, Devilee P, Dieffenbach AK, Dunning AM, Eiriksdottir G, Eriksson JG, Fasching PA, Ferrucci L, 116

Flesch-Janys D, Flyger H, Foroud T, Franke L, Garcia Mine O, Francannet C, Dupin-Deguine D, ME, García-Closas M, Geller F, De Geus EEJ, Giles Archambeaud F, Kurtz F-, Young J, Bertherat J, GG, Gudbjartsson DF, Gudnason V, Guénel P, Guo S, Marlin S, Goossens M, Hardelin J-, Dodé C, Hall P, Hamann U, Haring R, Hartman CA, Heath Bondurand N. Loss-of-function mutations in SOX10 AC, Hofman A, Hooning MJ, Hopper JL, Hu FB, cause Kallmann syndrome with deafness. Am J Hum Hunter DJ, Karasik D, Kiel DP, Knight JA, Kosma V- Genet 2013, 92: 707-724. , Kutalik Z, Lai S, Lambrechts D, Lindblom A, Mägi R, Magnusson PK, Mannermaa A, Martin NG, Pingault V, Bondurand N, Kuhlbrodt K, Goerich DE, Masson G, McArdle PF, McArdle WL, Melbye M, Préhu M-, Puliti A, Herbarth B, Hermans-Borgmeyer Michailidou K, Mihailov E, Milani L, Milne RL, I, Legius E, Matthijs G, Amiel J, Lyonnet S, Nevanlinna H, Neven P, Nohr EA, Oldehinkel AJ, Ceccherini I, Romeo G, Smith JC, Read AP, Wegner Oostra BA, Palotie A, Peacock M, Pedersen NL, M, Goossens M. SOX10 mutations in patients with Peterlongo P, Peto J, Pharoah PDP, Postma DS, Pouta Waardenburg-Hirschsprung disease. Nat Genet 1998, A, Pylkäs K, Radice P, Ring S, Rivadeneira F, Robino 18: 171-173. A, Rose LM, Rudolph A, Salomaa V, Sanna S, Schlessinger D, Schmidt MK, Southey MC, Sovio U, Stampfer MJ, Stöckl D, Storniolo AM, Timpson NJ, Pitteloud N, Acierno Jr. JS, Meysing A, Eliseenkova Tyrer J, Visser JA, Vollenweider P, Völzke H, AV, Ma J, Ibrahimi OA, Metzger DL, Hayes FJ, Waeber G, Waldenberger M, Wallaschofski H, Wang Dwyer AA, Hughes VA, Yialamas M, Hall JE, Grant Q, Willemsen G, Winqvist R, Wolffenbuttel BHR, E, Mohammadi M, Crowley Jr. WF. Mutations in Wright MJ, Boomsma DI, Econs MJ, Khaw K-, Loos fibroblast growth factor receptor 1 cause both RJF, McCarthy MI, Montgomery GW, Rice JP, Kallmann syndrome and normosmic idiopathic Streeten EA, Thorsteinsdottir U, Van Duijn CM, hypogonadotropic hypogonadism. Proc Natl Acad Sci Alizadeh BZ, Bergmann S, Boerwinkle E, Boyd HA, U S A 2006a, 103: 6281-6286. Crisponi L, Gasparini P, Gieger C, Harris TB, Ingelsson E, Järvelin M-, Kraft P, Lawlor D, Metspalu Pitteloud N, Acierno Jr. JS, Meysing AU, Dwyer AA, A, Pennell CE, Ridker PM, Snieder H, Sørensen TIA, Hayes FJ, Crowley Jr. WF. Reversible kallmann Spector TD, Strachan DP, Uitterlinden AG, Wareham syndrome, delayed puberty, and isolated anosmia NJ, Widen E, Zygmunt M, Murray A, Easton DF, occurring in a single family with a mutation in the Stefansson K, Murabito JM, Ong KK. Parent-of- fibroblast growth factor receptor 1 gene. J Clin origin-specific allelic associations among 106 Endocrinol Metab 2005, 90: 1317-1322. genomic loci for age at menarche. Nature 2014, 514: 92-97. Pitteloud N, Meysing A, Quinton R, Acierno Jr. JS, Dwyer AA, Plummer L, Fliers E, Boepple P, Hayes F, Perry JRB, Stolk L, Franceschini N, Lunetta KL, Zhai Seminara S, Hughes VA, Ma J, Bouloux P, G, McArdle PF, Smith AV, Aspelund T, Bandinelli S, Mohammadi M, Crowley Jr. WF. Mutations in Boerwinkle E, Cherkas L, Eiriksdottir G, Estrada K, fibroblast growth factor receptor 1 cause Kallmann Ferrucci L, Folsom AR, Garcia M, Gudnason V, syndrome with a wide spectrum of reproductive Hofman A, Karasik D, Kiel DP, Launer LJ, Van phenotypes. Mol Cell Endocrinol 2006b, 254-255: 60- Meurs J, Nalls MA, Rivadeneira F, Shuldiner AR, 69. Singleton A, Soranzo N, Tanaka T, Visser JA, Weedon MN, Wilson SG, Zhuang V, Streeten EA, Harris TB, Murray A, Spector TD, Demerath EW, Pitteloud N, Quinton R, Pearce S, Raivio T, Acierno Uitterlinden AG, Murabito JM. Meta-analysis of J, Dwyer A, Plummer L, Hughes V, Seminara S, genome-wide association data identifies two loci Cheng Y-Z, Li W-P, Maccoll G, Eliseenkova AV, influencing age at menarche. Nat Genet 2009, 41: Olsen SK, Ibrahimi OA, Hayes FJ, Boepple P, Hall 648-650. JE, Bouloux P, Mohammadi M, Crowley Jr. W. Digenic mutations account for variable phenotypes in idiopathic hypogonadotropic hypogonadism. J Clin Perälä N, Sariola H, Immonen T. More than nervous: Invest 2007a, 117: 457-463. The emerging roles of plexins. Differentiation 2012, 83: 77-91. Pitteloud N, Zhang C, Pignatelli D, Li J-D, Raivio T, Cole LW, Plummer L, Jacobson-Dickman EE, Mellon Pierce A, Bliesner B, Xu M, Nielsen-Preiss S, Lemke PL, Zhou Q-Y, Crowley Jr. WF. Loss-of-function G, Tobet S, Wierman ME. Axl and Tyro3 modulate mutation in the prokineticin 2 gene causes Kallmann female reproduction by influencing gonadotropin- syndrome and normosmic idiopathic releasing hormone neuron survival and migration. hypogonadotropic hypogonadism. Proc Natl Acad Sci Mol Endocrinol 2008, 22: 2481-2495. U S A 2007b, 104: 17447-17452.

Pingault V, Bodereau V, Baral V, Marcos S, Watanabe Y, Chaoui A, Fouveaut C, Leroy C, Vérier- 117

Plant TM, Barker-Gibb ML. Neurobiological Salian-Mehta S, Xu M, Knox AJ, Plummer L, Slavov mechanisms of puberty in higher primates. Hum D, Taylor M, Bevers S, Hodges RS, Crowley Jr. WF, Reprod Update 2004, 10: 67-77. Wierman ME. Functional consequences of AXL sequence variants in hypogonadotropic hypogonadism. J Clin Endocrinol Metab 2014, 99: Prevot V, Croix D, Bouret S, Dutoit S, Tramu G, 1452-1460. Stefano GB, Beauvillain JC. Definitive evidence for the existence of morphological plasticity in the external zone of the median eminence during the rat Sammarco I, Grimaldi P, Rossi P, Cappa M, Moretti estrous cycle: Implication of neuro-glio-endothelial C, Frajese G, Geremia R. Novel point mutation in the interactions in gonadotropin-releasing hormone splice donor site of exon-intron junction 6 of the release. Neuroscience 1999, 94: 809-819. androgen receptor gene in a patient with partial androgen insensitivity syndrome. J Clin Endocrinol Metab 2000, 85: 3256-3261. Prevot V, Hanchate NK, Bellefontaine N, Sharif A, Parkash J, Estrella C, Allet C, de Seranno S, Campagne C, d'Anglemont de Tassigny X, Baroncini Sanlaville D, Verloes A. CHARGE syndrome: An M. Function-related structural plasticity of the GnRH update. Eur J Hum Genet 2007, 15: 389-399. system. A role for neuronal-glial-endothelial interactions. Front Neuroendocrinol 2010, 31: 241- Sarfati J, Fouveaut C, Leroy C, Jeanpierre M, 258. Hardelin J-P, Dodé C. Greater prevalence of PROKR2 mutations in Kallmann syndrome patients from the Quaynor SD, Bosley ME, Duckworth CG, Porter KR, Maghreb than in European patients. Eur J Endocrinol Kim S-H, Kim H-G, Chorich LP, Sullivan ME, Choi 2013, 169: 805-809. J-H, Cameron RS, Layman LC. Targeted next generation sequencing approach identifies eighteen Schreiner F, Gohlke B, Hamm M, Korsch E, Woelfle new candidate genes in normosmic hypogonadotropic J. MKRN3 mutations in familial central precocious hypogonadism and Kallmann syndrome. Mol Cell puberty. Horm Res Paediatr 2014, 82: 122-126. Endocrinol 2016, 437: 86-96.

Schwanzel-Fukuda M, Bick D, Pfaff DW. Luteinizing Quaynor SD, Ko EK, Chorich LP, Sullivan ME, hormone-releasing hormone (LHRH)-expressing cells Demir D, Waller JL, Kim H-G, Cameron RS, Layman do not migrate normally in an inherited hypogonadal LC. NELF knockout is associated with impaired (Kallmann) syndrome. Mol Brain Res 1989, 6: 311- pubertal development and subfertility. Mol Cell 326. Endocrinol 2015, 407: 26-36.

Schwanzel-Fukuda M, Crossin KL, Pfaff DW, Quinton R, Duke VM, Robertson A, Kirk JMW, Bouloux PMG, Hardelin J-P, Petit C. Migration of Matfin G, De Zoysa PA, Azcona C, MacColl GS, luteinizing hormone-releasing hormone (LHRH) Jacobs HS, Conway GS, Besser M, Stanhope RG, neurons in early human embryos. J Comp Neurol Bouloux P-G. Idiopathic gonadotrophin deficiency: 1996, 366: 547-557. Genetic questions addressed through phenotypic characterization. Clin Endocrinol 2001, 55: 163-174. Schwanzel-Fukuda M, Pfaff DW. Origin of luteinizing hormone-releasing hormone neurons. Raivio T, Falardeau J, Dwyer A, Quinton R, Hayes Nature 1989, 338: 161-164. FJ, Hughes VA, Cole LW, Pearce SH, Lee H, Boepple P, Crowley Jr. WF, Pitteloud N. Reversal of idiopathic hypogonadotropic hypogonadism. New Schwarting GA, Kostek C, Ahmad N, Dibble C, Pays Engl J Med 2007, 357: 863-873. L, Puschel AW. Semaphorin 3A is required for guidance of olfactory axons in mice. J Neurosci 2000, 20: 7691-7697. Rhie Y-J, Lee K-H, Ko JM, Lee WJ, Kim JH, Kim H- S. KISS1 gene polymorphisms in Korean girls with central precocious puberty. J Korean Med Sci 2014, Schwarz Q, Maden CH, Davidson K, Ruhrberg C. 29: 1120-1125. Neuropilin-mediated neural crest cell guidance is essential to organise sensory neurons into segmented dorsal root ganglia. Development (Cambridge) 2009, Richards MR, Plummer L, Chan Y-M, Lippincott MF, 136: 1785-1789. Quinton R, Kumanov P, Seminara SB. Phenotypic spectrum of POLR3B mutations: Isolated hypogonadotropic hypogonadism without Schwarz JM, Cooper DN, Schuelke M, Seelow D. neurological or dental anomalies. J Med Genet 2016, Mutationtaster2: Mutation prediction for the deep- 54: 19-25. sequencing age. Nature Methods 2014, 11: 361-362. 118

Sedlmeyer IL, Palmert MR, Hospital C. Delayed of the KISS1 gene in disorders of puberty. J Clin puberty: Analysis of a large case series from an Endocrinol Metab 2010, 95: 2276-2280. academic center. J Clin Endocrinol Metab 2002, 87: 1613-1620. Silveira-Neto AP, Leal LF, Emerman AB, Henderson KD, Piskounova E, Henderson BE, Gregory RI, Seltsam A, Strigens S, Levene C, Yahalom V, Moulds Silveira LFG, Hirschhorn JN, Nguyen TT, Beneduzzi M, Moulds JJ, Hustinx H, Weisbach V, Figueroa D, D, Tusset C, Reis ACS, Brito VN, Mendonca BB, Bade-Doeding C, DeLuca DS, Blasczyk R. The Palmert MR, Antonini SR, Latronico AC. Absence of molecular diversity of Sema7A, the semaphorin that functional lin28b mutations in a large cohort of carries the JMH blood group antigens. Transfusion patients with idiopathic central precocious puberty. 2007, 47: 133-146. Horm Res Paediatr 2012, 78: 144-150.

Seminara SB, Hayes FJ, Crowley Jr. WF. Simon D, Ba I, Mekhail N, Ecosse E, Paulsen A, Gonadotropin-releasing hormone deficiency in the Zenaty D, Houang M, Perelroizen MJ, De Filippo G-, human (idiopathic hypogonadotropic hypogonadism Salerno M, Simonin G, Reynaud R, Carel J-C, Léger and Kallmann's syndrome): Pathophysiological and J, De Roux N. Mutations in the maternally imprinted genetic considerations. Endocr Rev 1998, 19: 521- gene MKRN3 are common in familial central 539. precocious puberty. Eur J Endocrinol 2016, 174: 1-8.

Seminara SB, Messager S, Chatzidaki EE, Thresher Simsek E, Demiral M, Ceylaner S, Kirel B. Two RR, Acierno Jr. JS, Shagoury JK, Bo-Abbas Y, Frameshift Mutations in MKRN3 in Turkish Patients Kuohung W, Schwinof KM, Hendrick AG, Zahn D, with Familial Central Precocious Puberty. Horm Res Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, Paediatr 2016, O'Rahilly S, Carlton MBL, Crowley Jr. WF, Aparicio SAJR, Colledge WH. The GPR54 Gene as a Sisk CL, Foster DL. The neural basis of puberty and Regulator of Puberty. N Engl J Med 2003, 349: 1614- adolescence. Nat Neurosci 2004, 7: 1040-1047. 1627.

Smith LB, Walker WH. Hormone Signaling in the Settas N, Dacou-Voutetakis C, Karantza M, Kanaka- Testis. In: Knobil and Neill's Physiology of Gantenbein C, Chrousos GP, Voutetakis A. Central Reproduction: Two-Volume Set. 2014: 637-690. precocious puberty in a girl and early puberty in her brother caused by a novel mutation in the MKRN3 gene. J Clin Endocrinol Metab 2014, 99: E647-E651. Sørensen K, Aksglaede L, Petersen JH, Juul A. Recent changes in pubertal timing in healthy Danish boys: Associations with body mass index. J Clin Sharma A, Verhaagen J, Harvey AR. Receptor Endocrinol Metab 2010a, 95: 263-270. complexes for each of the class 3 Semaphorins. Front Cell Neurosci 2012, 6: 28. Sørensen K, Mouritsen A, Mogensen SS, Aksglaede L, Juul A. Insulin sensitivity and lipid profiles in girls Shima H, Yatsuga S, Nakamura A, Sano S, Sasaki T, with central precocious puberty before and during Katsumata N, Suzuki E, Hata K, Nakabayashi K, gonadal suppression. J Clin Endocrinol Metab 2010b, Momozawa Y, Kubo M, Okamura K, Kure S, 95: 3736-3744. Matsubara Y, Ogata T, Narumi S, Fukami M. NR0B1 Frameshift Mutation in a Boy with Idiopathic Central Precocious Puberty. Sex Dev 2016, 10: 205-209. Soussi-Yanicostas N, De Castro F, Julliard AK, Perfettini I, Chédotal A, Petit C. Anosmin-1, defective in the X-linked form of Kallmann syndrome, Sidhoum VF, Chan Y-M, Lippincott MF, promotes axonal branch formation from olfactory Balasubramanian R, Quinton R, Plummer L, Dwyer bulb output neurons. Cell 2002, 109: 217-228. A, Pitteloud N, Hayes FJ, Hall JE, Martin KA, Boepple PA, Seminara SB. Reversal and relapse of hypogonadotropic hypogonadism: Resilience and Soussi-Yanicostas N, Hardelin J-P, Arroyo-Jimenez fragility of the reproductive neuroendocrine system. J MDM, Ardouin O, Legouis R, Levilliers J, Traincard Clin Endocrinol Metab 2014, 99: 861-870. F, Betton J-M, Cabanié L, Petit C. Initial characterization of anosmin-1, a putative extracellular matrix protein synthesized by definite neuronal cell Silveira LG, Noel SD, Silveira-Neto AP, Abreu AP, populations in the central nervous system. J Cell Sci Brito VN, Santos MG, Bianco SDC, Kuohung W, Xu 1996, 109: 1749-1757. S, Gryngarten M, Escobar ME, Arnhold IJP, Mendonca BB, Kaiser UB, Latronico AC. Mutations

119

Spilker C, Nullmeier S, Grochowska KM, Boucher-Neuhäuser and Gordon Holmes syndromes Schumacher A, Butnaru I, Macharadze T, Gomes as part of a broad neurodegenerative spectrum. Brain GM, Yuanxiang P, Bayraktar G, Rodenstein C, 2014, 137: 69-77. Geiseler C, Kolodziej A, Lopez-Rojas J, Montag D, Angenstein F, Bär J, D’Hanis W, Roskoden T, Tamagnone L, Comoglio PM. Signalling by Mikhaylova M, Budinger E, Ohl FW, Stork O, semaphorin receptors: Cell guidance and beyond. Zenclussen AC, Karpova A, Schwegler H, Kreutz Trends Cell Biol 2000, 10: 377-383. MR. A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis. PLoS Genet 2016, 12: Taniguchi M, Yuasa S, Fujisawa H, Naruse I, Saga S, e1005907. Mishina M, Yagi T. Disruption of semaphorin III/D gene causes severe abnormality in peripheral nerve projection. Neuron 1997, 19: 519-530. Strobel A, Issad T, Camoin L, Ozata M, Strosberg AD. A leptin missense mutation associated with hypogonadism and morbid obesity. Nat Genet 1998, Tata B, Huijbregts L, Jacquier S, Csaba Z, Genin E, 18: 214-215. Meyer V, Leka S, Dupont J, Charles P, Chevenne D, Carel J-C, Léger J, de Roux N. Haploinsufficiency of Dmxl2, Encoding a Synaptic Protein, Causes Sulem P, Gudbjartsson DF, Rafnar T, Holm H, Infertility Associated with a Loss of GnRH Neurons Olafsdottir EJ, Olafsdottir GH, Jonsson T, in Mouse. PloS Biol 2014, 12: e1001952. Alexandersen P, Feenstra B, Boyd HA, Aben KK, Verbeek ALM, Roeleveld N, Jonasdottir A, Styrkarsdottir U, Steinthorsdottir V, Karason A, Teilmann G, Pedersen CB, Jensen TK, Skakkebæk Stacey SN, Gudmundsson J, Jakobsdottir M, NE, Juul A. Prevalence and incidence of precocious Thorleifsson G, Hardarson G, Gulcher J, Kong A, pubertal development in Denmark: An epidemiologic Kiemeney LA, Melbye M, Christiansen C, study based on national registries. Pediatrics 2005, Tryggvadottir L, Thorsteinsdottir U, Stefansson K. 116: 1323-1328. Genome-wide association study identifies sequence variants on 6q21 associated with age at menarche. Nat Genet 2009, 41: 734-738. Teixeira L, Guimiot F, Dodé C, Fallet-Bianco C, Millar RP, Delezoide A-L, Hardelin J-P. Defective migration of neuroendocrine GnRH cells in human Sun H, Chasin LA. Multiple splicing defects in an arrhinencephalic conditions. J Clin Invest 2010, 120: intronic false exon. Mol Cell Biol 2000, 20: 6414- 3668-3672. 6425. Teles MG, Bianco SDC, Brito VN, Trarbach EB, Suntharalingham JP, Buonocore F, Duncan AJ, Kuohung W, Xu S, Seminara SB, Mendonca BB, Achermann JC. DAX-1 (NR0B1) and steroidogenic Kaiser UB, Latronico AC. Brief report: A GPR54- factor-1 (SF-1, NR5A1) in human disease. Best Pract activating mutation in a patient with central Res Clin Endocrinol Metab 2015, 29: 607-619. precocious puberty. N Engl J Med 2008, 358: 709- 715. Suzuki K, Okuno T, Yamamoto M, Pasterkamp RJ, Takegahara N, Takamatsu H, Kitao T, Takagi J, Teles MG, Silveira LFG, Tusset C, Latronico AC. Rennert PD, Kolodkin AL, Kumanogoh A, Kikutani New genetic factors implicated in human GnRH- H. Semaphorin 7A initiates T-cell-mediated dependent precocious puberty: The role of kisspeptin inflammatory responses through a1ß1 integrin. Nature system. Mol Cell Endocrinol 2011, 346: 84-90. 2007, 446: 680-684. Terasawa E, Fernandez DL. Neurobiological Sykiotis GP, Plummer L, Hughes VA, Au M, Durrani mechanisms of the onset of puberty in primates. S, Nayak-Young S, Dwyer AA, Quinton R, Hall JE, Endocr Rev 2001, 22: 111-151. Gusella JF, Seminara SB, Crowley Jr. WF, Pitteloud N. Oligogenic basis of isolated gonadotropin- releasing hormone deficiency. Proc Natl Acad Sci U S Tétreault M, Choquet K, Orcesi S, Tonduti D, Balottin U, Teichmann M, Fribourg S, Schiffmann R, A 2010, 107: 15140-15144. Brais B, Vanderver A, Bernard G. Recessive mutations in POLR3B, encoding the second largest Synofzik M, Gonzalez MA, Lourenco CM, Coutelier subunit of Pol III, cause a rare hypomyelinating M, Haack TB, Rebelo A, Hannequin D, Strom TM, leukodystrophy. Am J Hum Genet 2011, 89: 652-655. Prokisch H, Kernstock C, Durr A, Schöls L, Lima- Martínez MM, Farooq A, Schüle R, Stevanin G, Tommiska J, Wehkalampi K, Vaaralahti K, Laitinen Marques W, Züchner S. PNPLA6 mutations cause E-M, Raivio T, Dunkel L. LIN28B in constitutional 120

delay of growth and puberty. J Clin Endocrinol Metab Vaaralahti K, Wehkalampi K, Tommiska J, Laitinen 2010, 95: 3063-3066. E-, Dunkel L, Raivio T. The role of gene defects underlying isolated hypogonadotropic hypogonadism in patients with constitutional delay of growth and Tommiska J, Sørensen K, Aksglaede L, Koivu R, puberty. Fertil Steril 2011, 95: 2756-2758. Puhakka L, Juul A, Raivio T. LIN28B, LIN28A, KISS1, and KISS1R in idiopathic central precocious puberty. BMC Research Notes 2011, 4: 363. Vaaralahti K, Tommiska J, Tillmann V, Liivak N, Känsäkoski J, Laitinen E-M, Raivio T. De novo SOX10 nonsense mutation in a patient with Kallmann Tommiska J, Toppari J, Vaaralahti K, Känsäkoski J, syndrome and hearing loss. Pediatr Res 2014, 76: Laitinen E-, Noisa P, Kinnala A, Niinikoski H, Raivio 115-116. T. PROKR2 mutations in autosomal recessive Kallmann syndrome. Fertil Steril 2013a, 99: 815-818. Varimo T, Dunkel L, Vaaralahti K, Miettinen PJ, Hero M, Raivio T. Circulating makorin ring finger Tommiska J, Jørgensen N, Christiansen P, Juul A, protein 3 levels decline in boys before the clinical Raivio T. A homozygous R262Q mutation in the onset of puberty. Eur J Endocrinol 2016a, 174: 785- gonadotropin-releasing hormone receptor presenting 790. as reversal of hypogonadotropic hypogonadism and late-onset hypogonadism. Clin Endocrinol 2013b, 78: 316-317. Varimo T, Hero M, Känsäkoski J, Vaaralahti K, Matikainen N, Raivio T. Circulating makorin ring- finger protein-3 (MKRN3) levels in healthy men and Topaloglu AK, Reimann F, Guclu M, Yalin AS, in men with hypogonadotropic hypogonadism. Clin Kotan LD, Porter KM, Serin A, Mungan NO, Cook Endocrinol 2016b, 84: 151-152. JR, Ozbek MN, Imamoglu S, Akalin NS, Yuksel B, O'Rahilly S, Semple RK. TAC3 and TACR3 mutations in familial hypogonadotropic Viswanathan SR, Daley GQ, Gregory RI. Selective hypogonadism reveal a key role for Neurokinin B in blockade of microRNA processing by Lin28. Science the central control of reproduction. Nat Genet 2009, 2008, 320: 97-100. 41: 354-358. Waldhauser F, Weißenbacher G, Frisch H, Pollak A. Topaloglu AK, Lomniczi A, Kretzschmar D, Dissen Pulsatile secretion of gonadotropins in early infancy. GA, Kotan LD, McArdle CA, Koc AF, Hamel BC, Eur J Pediatr 1981, 137: 71-74. Guclu M, Papatya ED, Eren E, Mengen E, Gurbuz F, Cook M, Castellano JM, Kekil MB, Mungan NO, Watanabe Y, Inoue K, Okuyama-Yamamoto A, Nakai Yuksel B, Ojeda SR. Loss-of-function mutations in N, Nakatani J, Nibu K-, Sato N, Iiboshi Y, Yusa K, PNPLA6 encoding neuropathy target esterase underlie Kondoh G, Takeda J, Terashima T, Takumi T. Fezf1 pubertal failure and neurological deficits in Gordon is required for penetration of the basal lamina by Holmes syndrome. J Clin Endocrinol Metab 2014, 99: olfactory axons to promote olfactory development. J E2067-E2075. Comp Neurol 2009, 515: 565-584.

Topaloglu AK, Tello JA, Kotan LD, Ozbek M MN, Winter J, Jung S, Keller S, Gregory RI, Diederichs S. Yilmaz MB, Erdogan S, Gurbuz F, Temiz F, Millar Many roads to maturity: MicroRNA biogenesis RP, Yuksel B. Inactivating KISS1 mutation and pathways and their regulation. Nature Cell Biol 2009, hypogonadotropic hypogonadism. N Engl J Med 11: 228-234. 2012, 366: 629-635.

Wray S, Grant P, Gainer H. Evidence that cells Tornberg J, Sykiotis GP, Keefe K, Plummer L, Hoang expressing luteinizing hormone-releasing hormone X, Hall JE, Quinton R, Seminara SB, Hughes V, Van mRNA in the mouse are derived from progenitor cells Vliet G, Van Uum S, Crowley WF, Habuchi H, in the olfactory placode. Proc Natl Acad Sci U S A Kimata K, Pitteloud N, Bülow HE. Heparan sulfate 6- 1989a, 86: 8132-8136. O-sulfotransferase 1, a gene involved in extracellular sugar modifications, is mutated in patients with idiopathic hypogonadotrophic hypogonadism. Proc Wray S, Nieburgs A, Elkabes S. Spatiotemporal cell Natl Acad Sci U S A 2011, 108: 11524-11529. expression of luteinizing hormone-releasing hormone in the prenatal mouse: evidence for an embryonic origin in the olfactory placode. Dev Brain Res 1989b, Trcek T, Sato H, Singer RH, Maquat LE. Temporal 46: 309-318. and spatial characterization of nonsense-mediated mRNA decay. Genes Dev 2013, 27: 541-551.

121

Wünsch L, Holterhus PM, Wessel L, Hiort O. Patients with disorders of sex development (DSD) at risk of gonadal tumour development: Management based on laparoscopic biopsy and molecular diagnosis. BJU Int 2012, 110: E958-965.

Xu N, Kim H-G, Bhagavath B, Cho S-G, Lee JH, Ha K, Meliciani I, Wenzel W, Podolsky RH, Chorich LP, Stackhouse KA, Grove AMH, Odom LN, Ozata M, Bick DP, Sherins RJ, Kim S-H, Cameron RS, Layman LC. Nasal embryonic LHRH factor (NELF) mutations in patients with normosmic hypogonadotropic hypogonadism and Kallmann syndrome. Fertil Steril 2011, 95: 1613-1620.e1-7.

Young J, Bouligand J, Francou B, Raffin-Sanson M-, Gaillez S, Jeanpierre M, Grynberg M, Kamenicky P, Chanson P, Brailly-Tabard S, Guiochon-Mantel A. TAC3 and TACR3 defects cause hypothalamic congenital hypogonadotropic hypogonadism in humans. J Clin Endocrinol Metab 2010, 95: 2287- 2295.

Young J, Metay C, Bouligand J, Tou B, Francou B, Maione L, Tosca L, Sarfati J, Brioude F, Esteva B, Briand-Suleau A, Brisset S, Goossens M, Tachdjian G, Guiochon-Mantel A. SEMA3A deletion in a family with Kallmann syndrome validates the role of semaphorin 3A in human puberty and olfactory system development. Human Reproduction 2012, 27: 1460-1465.

122