Genome-Wide Copy Number Analysis and Systematic Mutation Screening in 58 Patients with Hypogonadotropic Hypogonadism

ORIGINAL ARTICLES: GENETICS Genome-wide copy number analysis and systematic mutation screening in 58 patients with hypogonadotropic hypogonadism

Yoko Izumi, M.D.,a,b Erina Suzuki, M.A.,a Susumu Kanzaki, M.D.,c Shuichi Yatsuga, M.D.,d Saori Kinjo, M.D.,e Maki Igarashi, Ph.D.,a Tetsuo Maruyama, M.D.,b Shinichiro Sano, M.D.,a Reiko Horikawa, M.D.,f Naoko Sato, M.D.,a Kazuhiko Nakabayashi, Ph.D.,g Kenichiro Hata, M.D.,g Akihiro Umezawa, M.D.,h Tsutomu Ogata, M.D.,a,i Yasunori Yoshimura, M.D.,b and Maki Fukami, M.D.a a Department of Molecular Endocrinology, National Research Institute for Child Health and Development; b Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo; c Division of Pediatrics and Perinatology, Tottori University Faculty of Medicine, Tottori; d Department of Pediatrics and Child Health, Kurume University School of Medicine, Fukuoka; e Department of Pediatrics, Okinawa Chubu Hospital, Okinawa; f Division of Endocrinology and Metabolism, National Center for Child Health and Development; g Department of Maternal-Fetal Biology and h Department of Reproductive Biology, National Research Institute for Child Health and Development, Tokyo; and i Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, Japan

Objective: To clarify the molecular basis of hypogonadotropic hypogonadism (HH). Design: Genome-wide copy number analysis by array-based comparative genomic hybridization and systematic mutation screening of 29 known causative genes by next-generation sequencing, followed by in silico functional assessment and messenger RNA/DNA analyses of the mutants/variants. Setting: Research institute. Patient(s): Fifty-eight patients with isolated HH (IHH), combined pituitary hormone deficiency (CPHD), and syndromic HH. Intervention(s): None. Main Outcome Measure(s): Frequency and character of molecular abnormalities. Result(s): Pathogenic defects were identified in 14 patients with various types of HH, although oligogenicity was not evident in this patient group. As rare abnormalities, we identified a submicroscopic deletion involving FGFR1 and an SOX3 polyalanine deletion in patients with IHH, and a WDR11 splice site mutation in a patient with CPHD. No disease-associated polymorphism was detected in the 58 patients. Conclusion(s): The present study provides further evidence that mutations and deletions in the known causative genes play a relatively minor role in the etiology of HH and that submicroscopic rearrangements encompassing FGFR1 can lead to IHH as a sole recognizable clinical feature. Furthermore, the results indicate for the first time that polyalanine deletions in SOX3 and mutations in WDR11 constitute rare genetic causes of IHH and CPHD, respectively. (Fertil SterilÒ 2014;102:1130–6. Ó2014 by American Society for Reproductive Medicine.) Use your smartphone Key Words: FGFR1, genomic rearrangements, gonadotropin deficiency, mutation, SOX3, to scan this QR code WDR11 and connect to the discussion forum for Discuss: You can discuss this article with its authors and with other ASRM members at http:// this article now.* fertstertforum.com/izumiy-hypogonadotropic-hypogonadism-molecular-analysis- * Download a free QR code scanner by searching for “QR screening/ scanner” in your smartphone’s app store or app marketplace.

Received April 4, 2014; revised May 28, 2014; accepted June 11, 2014; published online July 23, 2014. Y.I. has nothing to disclose. E.S. has nothing to disclose. S. Kanzaki has nothing to disclose. S.Y. has nothing to disclose. S. Kinjo has nothing to disclose. M.I. has nothing to disclose. T.M. has nothing to disclose. S.S. has nothing to disclose. R.H. has nothing to disclose. N.S. has nothing to disclose. K.N. has nothing to disclose. K.H. has nothing to disclose. A.U. has nothing to disclose. T.O. has nothing to disclose. Y.Y. has nothing to disclose. M.F. has nothing to disclose. Supported by the Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology; from the Japan Society for the Promotion of Science (grant 22132004-A01, Tokyo, Japan); by the grants from the Ministry of Health, Labor and Welfare, from National Center for Child Health and Development (grant 23A-1, 24-7, Tokyo, Japan); and from Takeda foundation. Reprint requests: Maki Fukami, M.D., Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 2–10–1 Ohkura, Setagaya, Tokyo 157-8535, Japan (E-mail: [email protected]).

Fertility and Sterility® Vol. 102, No. 4, October 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.06.017

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ypogonadotropic hypogonadism (HH) is a clinically DNA was extracted from peripheral leukocytes of the patients. H and genetically heterogeneous condition that occurs We examined copy number alterations in the entire genome as an isolated hormonal defect (isolated HH, IHH) or using an array-based catalog comparative genomic hybridi- in combination with other pituitary hormone deficiencies zation (4 180 k format, catalog number G4449A; Agilent (combined pituitary hormone deficiency, CPHD) (1). Hypogo- Technologies). In the present study, we focused on copy num- nadotropic hypogonadism can also take place as a component ber changes involving the known causative genes and those of congenital syndromes such as Kallmann syndrome (KS), involving genomic intervals more than 1.5 Mb, which have septo-optic dysplasia, and CHARGE syndrome (1–4). a higher probability of being associated with disease pheno- Hypogonadotropic hypogonadism affects men and women, types (27). To exclude benign copy number variants, we although the prevalence is significantly higher in men than referred to the database of genomic variants (http://projects. in women (5). Monoallelic, biallelic, and oligogenic tcag.ca/variation/). mutations in more than 15 genes including FGFR1, PROKR2, KAL1, and CHD7 have been identified in patients Mutation Analysis with HH (1, 6–11). In addition, missense mutations in WDR11 and polyalanine deletions in SOX3 have recently Mutation analysis was carried out for the coding regions of 29 been described as rare causes of IHH/KS and CPHD, known HH causative genes, namely, CHD7, FGF8, FGFR1, respectively (12–16). Because of the highly heterogeneous FSHB, GNRH1, GNRHR, HESX1, HS6ST1, KAL1, KISS1, genetic basis of HH, there have been few reports of KISS1R, LEP, LEPR, LHB, LHX3, LHX4, NELF, NR0B1, systematic mutation analyses in patients with this condition OTX2, POU1F1, PROK2, PROKR2, PROP1, SEMA3A, SOX2, (9–11, 17). Furthermore, genome-wide copy number analyses SOX3, TAC3, TACR3, and WDR11 (8, 28–30). Sequences have rarely been performed for patients with HH, although were determined by the Haloplex system (Agilent submicroscopic deletions involving KAL1, FGFR1, GNRH1, Technologies) on a MiSeq sequencer (Illumina). The average and KISS1R have been identified in a small number of read depth in target regions was 322.9. Nucleotide changes patients (17–25). Thus, the current understanding of the were identified using the Surecall system (Agilent etiology of HH remains fragmentary. Technologies) and SAMtools v0.1.17 software (http:// Recent advances in molecular technology including samtools.sourceforge.net/). In the present study, we focused array-based comparative genomic hybridization and next- on nonsynonymous substitutions and splice site mutations. generation sequencing have enabled researchers to perform Single nucleotide polymorphisms (SNPs) of allele frequency genome-wide copy number analysis and high-throughput >1.0% in the Japanese general population (dbSNP, http:// mutation screening for multiple samples. Here, using compar- www.ncbi.nlm.nih.gov/) and nucleotide changes identified ative genomic hybridization and next-generation sequencing, in our control samples (unaffected Japanese individuals, we analyzed samples from 58 patients with various types of n ¼ 16) were excluded from further study. Nucleotide HH. Our results provide novel information about the molecular substitutions identified by next-generation sequencing were abnormalities underlying this condition. confirmed by Sanger sequencing. Primer sequences are available upon request. MATERIALS AND METHODS The functional consequences of missense substitutions were predicted by in silico analysis using PolyPhen-2 (http:// Patients genetics.bwh.harvard.edu/pph2/). We classified frameshift We studied 58 unrelated Japanese patients with HH (34 male and nonsense mutations as damaging mutations. We referred and 24 female subjects, aged 0–56 years). The patient group to previous articles and the human gene mutation database included patients 1–10 and 35–45 with normosmic IHH (http://www.hgmd.cf.ac.uk/ac/index.php) to identify muta- (nIHH), patients 11–25 and 46–51 with KS, patients 26–28 tions that have been associated with HH. For one patient and 52 with CPHD, patients 29 and 53–55 with CHARGE syn- with a nucleotide change in the consensus sequence at a drome, and patients 30 and 31 with septo-optic dysplasia. splice acceptor site of WDR11, we performed reverse Patients 32–34 and 56–58 were IHH patients who did not un- transcription-polymerase chain reaction (PCR) analysis using dergo quantitative assessment of olfactory function; these pa- lymphoblastoid messenger RNA and a primer pair, 50- tients should have either nIHH or KS. Clinical data of the GGGCTGGCAAGGTTTAATTG-30 and 50-GCTTGGATGGGGA patients are shown in Supplemental Table 1 (available online). GAAGTCT-30. The protein structure of the mutant WDR11 One female subject (patient 45) mothered a child by IVF after was predicted using the WD40 repeat protein structure predic- treatment with hMG, whereas fertility was unknown for the tor (http://wu.scbb.pkusz.edu.cn/wdsp/index.php) (31). remaining 57 patients. Patients with cytogenetically detect- The nucleotide alterations identified in this study were able chromosomal abnormalities were excluded from the categorized into three groups: pathogenic mutations (muta- study. Patient 13 has been reported previously (26). tions previously associated with HH or hitherto unreported mutations that were assessed as ‘‘probably damaging’’ by in silico analyses), apparently benign variants (hitherto unre- Copy Number Analysis ported substitutions assessed as ‘‘benign’’), and rare polymor- This study was approved by the Institutional Review Board phisms (variants of allele frequency <1.0% in general Committee at the National Center for Child Health and Devel- population). To determine whether the rare polymorphisms opment. After obtaining written informed consent, genomic are associated with disease risk, allele frequencies in the

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TABLE 1

Copy number alterations and nucleotide changes identified in the present study. Case Sex Clinical diagnosis Pathogenic mutation Apparently benign variant Rare polymorphismb 1 M nIHH FGFR1 deletion NR0B1 p.A127Ta 2 M nIHH SOX3 p.A234_242del9Aa 3 M nIHH PROKR2 p.D42insED PROKR2 p.W178S 4 M nIHH CHD7 p.C2187Y 5 M nIHH LHB p.Y57H 11 M KS KAL1 g.IVS3þ2T>Ca 12 M KS CHD7 p.G998E CHD7 p.T2227S 13 M KS FGF8 p.S192fsX204 LHX4 p.P17S 14 M KS LHB p.R88W 15 M KS CHD7 p.F1352S 16 M KS LHX3 p.R208C 19 M KS FGFR1 p.V102I 20 M KS CHD7 p.R2065C 26 M CPHD WDR11 g.IVS3-2 A>G 30 M SOD WDR11 p.A1076T 32 M IHH CHD7 p.A2714V 35 F nIHH PROKR2 p.Y113H 36 F nIHH NR0B1 p.A127T 37 F nIHH LHB p.Y57H 38 F nIHH LHB p.Y57H 45 F nIHH FGFR1 p.S107T 46 F KS FGFR1 p.R570W 53 F CHARGE CHD7 p.K645fsX711 LHX3 p.A322T 54 F CHARGE CHD7 p.R987X Note: Deletions and nucleotide changes were identified in 24 of 58 patients. CHARGE ¼ CHARGE syndrome; CPHD ¼ combined pituitary hormone deficiency; F ¼ female; nIHH ¼ normosmic isolated hypogonadotropic hypogonadism; IHH ¼ isolated hypogonadotropic hypogonadism with unknown olfactory function; KS ¼ Kallmann syndrome; M ¼ male; SOD ¼ septo-optic dysplasia. a Mutations were identified in a homozygous or hemizygous state, while other mutations/variants/polymorphisms were detected in a heterozygous state. b Polymorphisms with an allele frequency of less than 1.0% are shown. Izumi. Genetic basis of gonadotropin deficiency. Fertil Steril 2014. patient group and in the general population were analyzed by (p.A234_242del9A). Patient 26 carried a hitherto unreported Fisher's exact probability test. P values of less than .05 were nucleotide changes at a splice acceptor site of WDR11 considered significant. (g.IVS3-2A>G). Reverse transcription-PCR analysis of patient 26 revealed that the mutation led to a deletion of the RESULTS entire exon 4. The Dexon 4 mutant (c.353_526del174) was predicted to cause a 58-amino-acid deletion and 1-amino- Copy Number Analysis acid insertion (p.D118_L175delinsV), thereby disrupting the A submicroscopic genomic rearrangement at 8p11.22–12 was functionally important bladed b-propeller structure of the identified in male case 1 (Table 1, Fig. 1A). This rearrangement WD40 protein (32). Parental samples of patient 2 were not comprised a 5.1-Mb deletion and a 153-kb amplification. available. The mother of patient 26 carried the same mutation. The deletion encompassed all exons of FGFR1, in addition to Six apparently benign substitutions and 7 rare polymor- the exons of 29 other genes and pseudogenes that have not phisms were identified in 15 patients (Table 1). The allele been associated with HH (Supplemental Table 2, available on- frequencies of the seven polymorphisms did not differ be- line). The amplification included two pseudogenes (ADAM5 tween the patient group and the general population, although and ADAM3A). This amplification has been registered in the frequency of the LHB rs371722800 polymorphism in the the SNP database as a frequent copy number variant. Parental general population was unknown (Supplemental Table 3, samples of patient 1 were not available for genetic testing. No available online). pathogenic copy number alterations were identified in the remaining 57 patients. Phenotypes of Three Patients with Rare Genetic Defects Mutation Analysis The clinical and hormonal findings of patients 1, 2, and 26 are A total of 13 mutations were identified in 9 of 34 male summarized in Table 2. The three patients were men and pre- patients and 4 of 24 female patients (Table 1). Mutations in sented with genital abnormalities and/or lack of pubertal FGFR1 and CHD7 accounted for most of the molecular development. Physical examination revealed no additional defects. Oligogenicity was not evident in the 58 patients. clinical features in patient 1, short stature in patient 2, and Rare monogenic mutations were detected in male patients 2 obesity, short stature, and mental retardation in patient 26. and 26 (Fig. 1B and C). Patient 2 had a hemizygous 27-bp Olfactory dysfunction was not apparent in any of the three deletion in SOX3 (c.700_726del27), which resulted in the patients. Brain magnetic resonance imaging revealed normal loss of 9 alanine residues from the first polyalanine tract pituitary in patient 1, and pituitary malformation in patients

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FIGURE 1

Rare monogenic defects identified in the present study. (A) Genomic rearrangement in patient 1. The black, red, and green dots denote signals in comparative genomic hybridization analyses that indicate the normal, increased (>þ0.5), and decreased (<0.8) copy numbers, respectively. The deletion included all exons of FGFR1 and exons of an additional 29 genes and pseudogenes. The amplification affected two pseudogenes including ADAM5. Genomic positions refer to the human genome database (hg19, build 37). The position of genes around the breakpoints is shown. (B) SOX3 mutation in patient 2. Top, genomic position of the mutation. The black boxes indicate polyalanine tracts. Middle and lower, chromatographs of the mutation. The mutation resulted in a deletion of nine amino acids from the first polyalanine tract. The asterisk indicates a common polymorphism. WT ¼ wild type. (C) WDR11 mutation in patient 26. Top, genomic position of the mutation. Left, chromatographs of the mutation. The mutation (arrowheads) affected the splice donor site of exon 4. Right, complementary DNA (cDNA) analysis. The mutation resulted in exon skipping. C ¼ control; P ¼ patient. Izumi. Genetic basis of gonadotropin deficiency. Fertil Steril 2014.

2 and 26. Endocrine evaluation indicated grossly normal they were present in the patients at frequencies similar to pituitary function except for HH in patients 1 and 2, and com- those of the Japanese general population. However, the allele bined deficiencies of LH and GH in patient 26. The mother of frequency of LHB rs371722800 in the general population patient 26 with the same WDR11 mutation as the proband needs to be determined to clarify whether this SNP is a suscep- allegedly had menstrual irregularity with oligomenorrhea tibility allele. Our data suggest a relatively minor role of (one cycle of 6 months), although she had no history of known gene mutations in the development of HH and demon- delayed puberty (menarche at age 12 years). strate the rarity of oligogenic mutations in Japanese patients. Three of the 58 patients harbored rare molecular defects. Patient 1 carried a small genomic rearrangement at 8p11.22– DISCUSSION 12. Chromosomal rearrangements at 8p11.22–12 have been We identified pathogenic molecular defects in 14 of 58 cases identified in a small number of patients with HH with various with HH. The mutation-positive rate was higher in male than complications (19, 21–23). Our findings imply that in female patients, reflecting the presence of X-linked muta- submicroscopic chromosomal deletions involving FGFR1 tions in KAL1 and SOX3. Although we identified several other can cause nIHH as a sole recognizable clinical feature. nucleotide alterations that have not been submitted to the Because the short arm of chromosome 8 is known as a SNP database, in silico analyses indicated that most of these hotspot for chromosomal rearrangements (19, 21–23, 33), substitutions are functionally neutral. Likewise, although such submicroscopic rearrangements may be hidden in a rare polymorphisms were detected in nine patients, these certain fraction of patients with HH. Patient 2 harbored a SNPs were unlikely to associate with the disease risk, because hemizygous deletion affecting nine alanine residues of the

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TABLE 2

Clinical ﬁndings of patients 1, 2, and 26 with rare molecular defects. Patient 1 Patient 2 Patient 26 Molecular defects Mutation FGFR1 deletion SOX3 mutation WDR11 mutation Parental origin of mutation Unknown Unknown Mother Clinical features Diagnosis nIHH nIHH CPHD Genital abnormality at birth Cryptorchidism (left)a None None Defective sex development Micropenis/small Small testis, no pubertal Micropenis (at 7.0 y) testis (at 13.2 y) development (at 16 y) Other clinical feature None Short stature Short stature,b obesity, MR Olfactory function Normal Normal Normal Brain MRI Normal Hypoplastic anterior pituitary Pituitary malformationc

Endocrine data (age at exam.) Stimulantd 13.2 10.9 16.9, 18.3e 1.7, 2.3, 3.5f 7.0 LH (IU/L) GnRH 0.1i/ 1.7i <0.1i/ 6.4 <0.1i/ 3.6i <0.2i/ 0.2i FSH (IU/L) GnRH 0.3/ 3.2 2.4/ 16.2 0.5/ 4.8 <0.5/ 2.0 T (nmol/L) hCG 0.1/ 0.1 0.6 GH (ng/mL) Insulin 1.16/ 10.10 0.60/ 1.50i GH (ng/mL) Arginine / 1.42g,i GH (ng/mL) Clonidine 1.30/ 9.81 / 0.62g,i GH (ng/mL) L-dopa 0.30/ 18.78 IGF-I (ng/mL) 151 178 222 / <4g,i TSH (mIU/L) TRH 1.2/ 12.7 1.2 3.2 2.3/ 10.4 Free T4 (pmol/L) 0.18 0.17 0.17 0.15 ACTH (pg/mL) Insulin/CRHh 13.1/ 393.0 67.8 20.1 / 57.3 11.0/ 28.7 Cortisol (mg/dL) Insulin/CRHh 3.1/ 29.1 23.6 10.0/ 20.9 7.0/ 20.8 PRL (ng/mL) TRH 3.5/ 55.2 6.1 4.0/ 4.9i Note: Hormone levels are shown as (basal values)/ (peak values). CPHD ¼ combined pituitary hormone deﬁciency; CRH ¼ corticotropin- releasing hormone; fT4 ¼ free thyroxine; IGF1 ¼ Insulin- like growth factor 1; MR ¼ mental retardation; T ¼ testosterone; TRH ¼ thyrotropin releasing hormone; nIHH ¼ normosmic isolated hypogonadotropic hypogonadism. a Subjected to orchiopexy at 2 years of age. b Treated with GH from 1.9 years of age. c Stalk interruption and ectopic posterior lobe. d GnRH (100 mg/m2), insulin (0.1 U/kg), arginine (0.5 g/kg), TRH (10 mg/kg), and CRH (1.5 mg/kg) IV; clonidine (0.1 mg/m2) and L-DOPA (10 mg/kg) orally; blood sampling at 0, 30, 60, 90, and 120 minutes. In CRH stimulation test, additional blood sampling was performed at 15 minutes. hCG (3,000 U). IM for 3 consecutive days; blood sampling on days 1 and 4. O.12N.4/OTBR2014 OCTOBER / 4 NO. 102 VOL. e LH and FSH were measured at 16.9 years of age and IGF-I was measured at 18.3 of age. f GH and IGF-I were measured at 1.7 years of age, ACTH and cortisol were measured at 2.3 years of age, and other hormones were measured at 3.5 years of age. g Basal values were not available. h Insulin and CRH were used for patients 1 and 26, respectively. i Hormone values below the age- and sex-matched reference range. Izumi. Genetic basis of gonadotropin deﬁciency. Fertil Steril 2014. Fertility and Sterility®

first polyalanine tract in SOX3. SOX3 encodes a transcription normalities in the known causative genes accounts for a factor involved in pituitary morphogenesis (13). Duplications relatively small percentage of the genetic causes of HH and and deletions of alanine residues in SOX3 have been shown to that submicroscopic rearrangements encompassing FGFR1 cause various types of pituitary dysfunction in male and, less can lead to IHH as a sole clinical feature. In addition, this frequently, in female subjects (14, 16). Our data broaden the study indicates for the first time that polyalanine deletions phenotypic variation of polyalanine deletions in SOX3 to in SOX3 and mutations in WDR11 constitute rare genetic include nIHH. Patient 26 carried a hitherto unreported causes of IHH and CPHD, respectively. Further studies, mutation of WDR11 that probably disrupts the protein including molecular analysis of large patient groups of structure of WDR11 by causing exon skipping. WDR11 has various ethnicities and identification of novel causative recently been shown to play a role in the development of genes/factors of HH, will serve to advance our understanding hypothalamic neurons (34). Five missense mutations in of the etiology of HH. WDR11 have been identified in six patients with nIHH/KS (12). Our results indicate that WDR11 mutations can Acknowledgments: The authors appreciate patients and underlie not only nIHH/KS but also CPHD. Because previous families for participating in the study. The authors thank studies revealed that nIHH, KS, and CPHD are genetically the following clinicians for providing the samples and infor- related conditions resulting from defective formation of the mation of the patients: Drs. Katsuya Aizu (Saitama Children's anterior midline in the forebrain (2), CPHD may be a Medical Center), Hiroshi Arimura (Kagoshima University), manifestation of a severely truncated WDR11 protein. In Katsunori Asai (Kobe City Medical Center General Hospital), this context, the menstrual irregularity in the mother of Hiroyuki Iuchi (Jikei University), Tomohiro Ishii (Keio Univer- patient 26 may also be ascribed to pituitary dysfunction due sity), Yoshiki Ueno (Komatsu Municipal Hospital), Akira to the WDR11 mutation, although endocrine evaluation Ohtake (Saitama Medical University), Kenichi Kashimada was not performed for the mother. (Tokyo Medical and Dental University), Reina Kawarabayashi The present study provides a model of molecular diagnosis (Osaka City University), Junko Kanno (Tohoku University), of genetically heterogeneous disorders. Comparative genomic Yoshitomo Kobori (Koshigaya Hospital, Dokkyo Medical hybridization and next-generation sequencing identified University), Yume Suzuki (Jichi Medical University), Sumito several pathogenic mutations/deletions in our patients. Identifi- Dateki (Nagasaki University), Keisuke Nagasaki (Niigata Uni- cation of the genetic factors underlying diseases is beneficial in versity), Akie Nakamura (Hokkaido University), Atsushi Nish- the prediction of disease outcomes and possible complications. iyama (Kakogawa West City Hospital), Chikahiko Numakura Furthermore, the molecular diagnosis of each patient serves to (Yamagata University), Reiji Hirano (Yamaguchi-ken Saisei- improve the accuracy of genetic counseling for the family. kai Shimonoseki General Hospital), Hiroyo Mabe (Kumamoto For example, mutations in SOX3 are known to lead to familial University), Junko Nishioka (Kurume University), Yoko HH in an X-linked manner, whereas deletions involving FGFR1 Miyoshi (Osaka University), Masaaki Yamamoto (Nippon are inherited in an autosomal dominant manner. 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Cooper GM, Coe BP, Girirajan S, Rosenfeld JA, Vu TH, Baker C, et al. A copy 15. Laumonnier F, Ronce N, Hamel BC, Thomas P, Lespinasse J, Raynaud M, number variation morbidity map of developmental delay. Nat Genet 2011; et al. Transcription factor SOX3 is involved in X-linked mental retardation 43:838–46. with growth hormone deficiency. Am J Hum Genet 2002;71:1450–5. 28. Gorbenko Del Blanco D, Romero CJ, Diaczok D, de Graaff LC, Radovick S, 16. Takagi M, Ishii T, Torii C, Kosaki K, Hasegawa T. A novel mutation in SOX3 Hokken-Koelega AC. A novel OTX2 mutation in a patient with combined polyalanine tract: a case of kabuki syndrome with combined pituitary pituitary hormone deficiency, pituitary malformation, and an underdevel- hormone deficiency harboring double mutations in MLL2 and SOX3. Pitui- oped left optic nerve. Eur J Endocrinol 2012;167:441–52. tary. 2013 Dec 18. [Epub ahead of print.] 29. Turton JP, Strom M, Langham S, Dattani MT, Le Tissier P. Two novel muta- 17. Pedersen-White JR, Chorich LP, Bick DP, Sherins RJ, Layman LC. The tions in the POU1F1 gene generate null alleles through different mecha- prevalence of intragenic deletions in patients with idiopathic hypogonado- nisms leading to combined pituitary hormone deficiency. Clin Endocrinol tropic hypogonadism and Kallmann syndrome. Mol Hum Reprod 2008;14: (Oxf) 2012;76:387–93. 367–70. 30. Kelberman D, Dattani MT. Hypopituitarism oddities: congenital causes. 18. De Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E. Hypogo- Horm Res 2007;68(Suppl 5):138–44. nadotropic hypogonadism due to loss of function of the KiSS1-derived 31. Wang Y, Jiang F, Zhuo Z, Wu XH, Wu YD. A method for WD40 repeat detec- peptide receptor GPR54. Proc Natl Acad Sci U S A 2003;100:10972–6. tion and secondary structure prediction. PLoS One 2013;8:e65705. 19. Klopocki E, Fiebig B, Robinson P, Tonnies H, Erdogan F, Ropers HH, et al. A 32. Wu XH, Wang Y, Zhuo Z, Jiang F, Wu YD. Identifying the hotspots on the top novel 8 Mb interstitial deletion of chromosome 8p12-p21.2. Am J Med faces of WD40-repeat proteins from their primary sequences by beta-bulges Genet A 2006;140:873–7. and DHSW tetrads. PLoS One 2012;7:e43005. 20. Wincent J, Schulze A, Schoumans J. Detection of CHD7 deletions by MLPA in 33. Yu S, Fiedler S, Stegner A, Graf WD. Genomic profile of copy number vari- CHARGE syndrome patients with a less typical phenotype. Eur J Med Genet ants on the short arm of human chromosome 8. Eur J Hum Genet 2010;18: 2009;52:271–2. 1114–20. 21. Trarbach EB, Teles MG, Costa EM, Abreu AP, Garmes HM, Guerra G Jr, et al. 34. Schwarting GA, Wierman ME, Tobet SA. Gonadotropin-releasing hormone Screening of autosomal gene deletions in patients with hypogonadotropic neuronal migration. Semin Reprod Med 2007;25:305–12. hypogonadism using multiplex ligation-dependent probe amplification: 35. Miraoui H, Dwyer AA, Sykiotis GP, Plummer L, Chung W, Feng B, et al. 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SUPPLEMENTAL TABLE 1

Patients participating in the present study. Age at Clinical Testes Patient no. diagnosis (y) diagnosis Family history Additional clinical feature volume (ml) Male 1 13 nIHH 1.0 2 15 nIHH Father: delayed puberty 1.5 3 17 nIHH No data 4 18 nIHH No data 5 15 nIHH No data 6 17 nIHH No data 7 18 nIHH 2.0 8 18 nIHH 0.5 9 34 nIHH No data 10 No data nIHH No data 11 13 KS Brother: KS Small 12 16 KS Cleft lip and plate, MR No data 13 16 KS Cleft lip and plate, hearing loss, VSD, MR >1.0 14 25 KS No data 15 13 KS 3.0 16 7 KS No data 17 7 KS No data 18 20 KS No data 19 21 KS 2.0 20 21 KS Sister: delayed puberty Cleft lip and plate, ASD Small 21 40 KS Brother: KS No data 22 38 KS Brother: KS No data 23 45 KS Brother: KS Synkinesis, renal agenesis, persistent No data olfactory artery aneurysm, obesity 24 10 KS No data 25 28 KS 3.0 26 2 CPHD MR, obesity 2.0 27 0 CPHD No data 28 5 CPHD No data 29 16 CHARGE Tetralogy of Fallot, hearing loss, No data cleft lip and plate 30 2 SOD Brother: cleft plate, Microphthalmia, optic atrophy No data hypothyroidism 31 13 SOD No data 32 1.2 IHH No data 33 14 IHH No data 34 No data IHH No data Female 35 22 nIHH – 36 22 nIHH – 37 1 nIHH – 38 20 nIHH – 39 14 nIHH – 40 16 nIHH – 41 17 nIHH – 42 27 nIHH – 43 28 nIHH – 44 15 nIHH – 45 27 nIHH – 46 56 KS – 47 9 KS – 48 14 KS – 49 15 KS – 50 29 KS – 51 No data KS – 52 0.2 CPHD Cleft lip and plate, hypoplastic nasal septum – 53 2 CHARGE – 54 12 CHARGE – 55 16 CHARGE Microphthalmia, choroidal coloboma – 56 No data IHH – 57 16 IHH Hearing loss – 58 No data IHH – Note: ASD ¼ atral septal defect; CHARGE ¼ CHARGE syndrome; CPHD ¼ combined pituitary hormone deﬁciency; F ¼ female; nIHH ¼ normosmic isolated hypogonadotropic hypogonadism; IHH ¼ isolated hypogonadotropic hypogonadism with unknown olfactory function; KS ¼ Kallmann syndrome; M ¼ male; MR ¼ mental retardation; SOD ¼ septo-optic dysplasia; VSD ¼ ventricular septal defect. Izumi. Genetic basis of gonadotropin deﬁciency. Fertil Steril 2014.

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SUPPLEMENTAL TABLE 2

Genes and pseudogenes involved in the deletion. Gene symbol (Refseq) Accession no. Description UNC5D NM_080872 unc-5 homologue D (C. elegans) KCNU1 NM_001031836 Potassium channel, subfamily U, member 1 ZNF703 NM_025069 Zinc finger protein 703 ERLIN2 NM_007175 ER lipid raft associated 2 LOC728024 NR_003671 chromosome X open reading frame 56 pseudogene PROSC NM_007198 proline synthetase co-transcribed homolog (bacterial) GPR124 NM_032777 G protein-coupled receptor 124 BRF2 NM_018310 BRF2, RNA polymerase III transcription initiation factor 50 kDa subunit RAB11FIP1 NM_025151 RAB11 family interacting protein 1 (class I) GOT1L1 NM_152413 glutamic-oxaloacetic transaminase 1-like 1 ADRB3 NM_000025 adrenoceptor beta 3 EIF4EBP1 NM_004095 eukaryotic translation initiation factor 4E binding protein 1 ASH2L NM_004674 ash2 (absent, small, or homeotic)-like (Drosophila) STAR NM_000349 steroidogenic acute regulatory protein LSM1 NM_014462 LSM1 homologue, U6 small nuclear RNA associated (S. cerevisiae) BAG4 NM_004874 BCL2-associated athanogene 4 DDHD2 NM_001164232 DDHD domain containing 2 PPAPDC1B NM_001102559 phosphatidic acid phosphatase type 2 domain containing 1B WHSC1L1 NM_017778 Wolf-Hirschhorn syndrome candidate 1-like 1 LETM2 NM_001199659 leucine zipper-EF-hand containing transmembrane protein 2 FGFR1 NM_001174063 fibroblast growth factor receptor 1 C8orf86 NM_207412 chromosome 8 open reading frame 86 RNF5P1 NR_003129 ring finger protein 5, E3 ubiquitin protein ligase pseudogene 1 TACC1 NM_001122824 transforming, acidic coiled-coil containing protein 1 PLEKHA2 NM_021623 pleckstrin homology domain containing, family A (phosphoinositide-binding specific) member 2 HTRA4 NM_153692 HtrA serine peptidase 4 TM2D2 NM_001024380 TM2 domain containing 2 ADAM9 NM_003816 ADAM metallopeptidase domain 9 ADAM32 NM_145004 ADAM metallopeptidase domain 32 ADAM5 NM_001040073 ADAM metallopeptidase domain 5, pseudogene Izumi. Genetic basis of gonadotropin deficiency. Fertil Steril 2014.

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SUPPLEMENTAL TABLE 3

Allele frequency of rare polymorphisms. Allele frequency Allele frequency in the Japanese Statistical signiﬁcance Gene Polymorphism dbSNP ID in our patients general populationa (P value) WDR11 p.A1076T rs200126172 1/116 18/2,286 .9294 LHB p.R88W rs146251380 1/116 4/1,434 .2867 LHB p.Y57H rs371722800 3/116 No data No data LHX3 p.R208C rs146251380 1/116 4/1,434 .2867 LHX3 p.A322T rs201356862 1/116 13/2,000 .7842 PROKR2 p.Y113H rs202203360 1/116 4/2,204 .1234 PROKR2 p.W178S rs201835496 1/116 1/76 .762 a Based on the Human Genetic Variation Browser. Izumi. Genetic basis of gonadotropin deﬁciency. Fertil Steril 2014.

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