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Mutation profiles and clinical characteristics of Chinese males with isolated hypogonadotropic hypogonadism

  • Chengming Zhou
    Affiliations
    Division of Cardiology, Department of Internal Medicine and Genetic Diagnosis Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China

    Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China
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  • Yonghua Niu
    Affiliations
    Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China

    Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China
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  • Hao Xu
    Affiliations
    Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China

    Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China
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  • Zongzhe Li
    Affiliations
    Division of Cardiology, Department of Internal Medicine and Genetic Diagnosis Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China

    Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China
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  • Tao Wang
    Affiliations
    Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China

    Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China
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  • Weimin Yang
    Affiliations
    Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China

    Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China
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  • Shaogang Wang
    Affiliations
    Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China

    Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China
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  • Dao Wen Wang
    Affiliations
    Division of Cardiology, Department of Internal Medicine and Genetic Diagnosis Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China

    Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China
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  • Jihong Liu
    Correspondence
    Reprint requests: Jihong Liu, M.D., Ph.D., Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030, Hubei, PR China.
    Affiliations
    Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China

    Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, People's Republic of China
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      Objective

      To investigate the mutation profiles and clinical characteristics of Chinese males with isolated hypogonadotropic hypogonadism (IHH) and discover new pathogenic genes that cause IHH.

      Design

      A gene panel, including 31 known IHH genes and 52 candidate genes, was used to perform semiconductor next-generation sequencing.

      Setting

      University hospital.

      Patients

      One hundred thirty-eight sporadic male IHH patients and 10 IHH families; 100 healthy men with normal fertility served as control subjects.

      Interventions(s)

      None.

      Main Outcome Measure(s)

      Targeted next-generation sequencing, polymerase chain reaction and sequencing, pedigree analysis, and bioinformatics analysis.

      Result(s)

      Variants were distributed uniformly throughout 52 genes (52/83, 62.65%), including 16 (16/31, 51.61%) causal genes and 36 (36/52, 69.23%) candidate genes. Six new pathogenic variants and 52 likely pathogenic variants were identified in 16 genes known to cause nIHH/KS (normosmic IHH/Kallmann syndrome). In the 148 probands, PROKR2 (22/148, 14.86%), CHD7, FGFR1, and KAL1 had high mutation rates, and 8.78% (13/148) of the patients carried at least two variants in known genes. In addition, variants were identified in 36 candidate genes, and EGFR, ERBB4, PAX6, IGF1, SEMA4D, and SEMA7A should be prioritized for further research and genetic testing in IHH.

      Conclusion(s)

      The mutation frequency of IHH-causal genes in Chinese HAN males was different from the data reported in white populations. Oligogenic inheritance was a common phenomenon in IHH. Our study expands the mutation profile for IHH, and the new likely pathogenic genes identified in our study warrant further research in GnRH neuronal networks.
      Perfiles de mutaciones y características clínicas de varones chinos con hipogonadismo hipogonadotrópico aislado

      Objetivo

      Investigar los perfiles de mutaciones y las características clínicas de varones chinos con hipogonadismo hipogonadotrópico aislado (IHH) y descubrir nuevos genes patogénicos que causen IHH.

      Diseño

      Se analizó un panel que incluye 31 genes conocidos de IHH y 52 genes candidatos, mediante secuenciación de última generación por semiconductores.

      Entorno

      Hospital universitario.

      Paciente (s)

      Ciento treinta y ocho varones con IHH esporádico y 10 familias con IHH; 100 varones sanos con fertilidad normal sirvieron como control.

      Intervención (s)

      Ninguna.

      Principales medidas de resultado

      resultados de la secuenciación dirigida de última generación, reacción en cadena de polimerasa y secuenciación, análisis de pedigrí y análisis bioinformático.

      Resultado (s)

      Las variantes estuvieron uniformemente distribuidas a lo largo de los 52 genes (52/83, 62.65%), incluyendo 16 genes causales (16/31, 51.61%) y 36 genes candidatos (36/52, 69.23%). Seis nuevas variantes patogénicas y 52 variantes probablemente patogénicas se identificaron en 16 genes conocidos causales de nIHH/KS (IHH normósmico/ Síndrome de Kallmann). En los 148 probandos tuvieron altas tasas de mutaciones en PROKR2 (22/148, 14.86%), CHD7, FGFR1, y KAL1 y el 8.78% (13/148) eran portadores de al menos dos variantes de genes conocidos. Además, se identificaron variantes en 36 genes candidatos, y los genes EGFR, ERBB4, PAX6, IGF1, SEMA4D, y SEMA7A deberían ser priorizados en futuras investigaciones y estudios genéticos en casos de IHH.

      Conclusión (s)

      La frecuencia de mutaciones de genes causales de IHH en varones chinos HAN fue diferente de los datos informados en la población blanca. La herencia oligogénica fue un fenómeno común en IHH. Nuestro estudio amplía el perfil de mutaciones de IHH y el descubrimiento de nuevas variantes probablemente patogénicas en nuestro estudio justifica más investigaciones en las redes neuronales de la GnRH.

      Palabras clave

      Hipogonadismo hipogonadotrópico aislado, nuevas variantes, diagnóstico genético oligogénico, secuenciación dirigida de última generación.

      Key Words

      Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/31872-25384
      Isolated hypogonadotropic hypogonadism (IHH) is a rare genetic disease characterized by hypogonadotropic hypogonadism with or without anosmia or hyposmia (
      • Kallmann F.
      The genetic aspects of primary eunuchoidism.
      ). Kallmann syndrome (KS) is a form of IHH with anosmia or hyposmia and accounts for approximately 60% of IHH cases. Another form of IHH characterized by a normal sense of smell is defined as normosmic IHH (nIHH). An additional rare form of IHH, adult-onset hypogonadotropic hypogonadism (AHH), is observed in patients who go through normal puberty and subsequently develop GnRH deficiency after achieving sexual maturity. IHH mainly affects pubertal development and reproductive functions and differs in prevalence between males and females (1:30,000 and 1:125,000, respectively) (
      • Laitinen E.M.
      • Vaaralahti K.
      • Tommiska J.
      • Eklund E.
      • Tervaniemi M.
      • Valanne L.
      • et al.
      Incidence, phenotypic features and molecular genetics of Kallmann syndrome in Finland.
      ). IHH is a heterogeneous disease that is characterized by varying extents of abnormalities related to puberty in addition to infertility, which is caused by the deficient production, secretion, or action of GnRH. IHH is often accompanied by manifestations in other systems, such as renal agenesis or hypoplasia, cleft lip/palate, hearing loss, and cryptorchidism (
      • Quinton R.
      • Duke V.M.
      • Robertson A.
      • Kirk J.M.
      • Matfin G.
      • de Zoysa P.A.
      • et al.
      Idiopathic gonadotrophin deficiency: genetic questions addressed through phenotypic characterization.
      ). Currently, at least 31 genes have reported associations with IHH. These include ANOS1, FGFR1/FGF8, and PROK2/PROKR2 (
      • Bergman J.E.
      • Janssen N.
      • Hoefsloot L.H.
      • Jongmans M.C.
      • Hofstra R.M.
      • van Ravenswaaij-Arts C.M.
      CHD7 mutations and CHARGE syndrome: the clinical implications of an expanding phenotype.
      ,
      • Bonomi M.
      • Libri D.V.
      • Guizzardi F.
      • Guarducci E.
      • Maiolo E.
      • Pignatti E.
      • et al.
      New understandings of the genetic basis of isolated idiopathic central hypogonadism.
      ,
      • Newbern K.
      • Natrajan N.
      • Kim H.G.
      • Chorich L.P.
      • Halvorson L.M.
      • Cameron R.S.
      • et al.
      Identification of HESX1 mutations in Kallmann syndrome.
      ,
      • Stark Z.
      • Storen R.
      • Bennetts B.
      • Savarirayan R.
      • Jamieson R.V.
      Isolated hypogonadotropic hypogonadism with SOX2 mutation and anophthalmia/microphthalmia in offspring.
      ,
      • Walker A.P.
      • Fowkes R.C.
      • Saleh F.
      • Kim S.H.
      • Wilkinson P.
      • Cabrera-Sharp V.
      • et al.
      Genetic analysis of NR0B1 in congenital adrenal hypoplasia patients: identification of a rare regulatory variant resulting in congenital adrenal hypoplasia and hypogonadal hypogonadism without testicular carcinoma in situ.
      ). However, only approximately 40% of IHH patients have mutations in these genes (
      • Miraoui H.
      • Dwyer A.A.
      • Sykiotis G.P.
      • Plummer L.
      • Chung W.
      • Feng B.
      • et al.
      Mutations in FGF17, IL17RD, DUSP6, SPRY4, and FLRT3 are identified in individuals with congenital hypogonadotropic hypogonadism.
      ), indicating that many genes underlying the pathogenesis of IHH remain to be discovered. In addition, the mutation profiles of IHH patients may vary among different ethnic groups, and few genetic studies have been reported in Chinese patients with IHH (
      • Fathi A.K.
      • Hu S.
      • Fu X.
      • Huang S.
      • Liang Y.
      • Ning Q.
      • et al.
      Molecular defects of the GnRH-receptor gene in Chinese patients with idiopathic hypogonadotropic hypogonadism and the severity of hypogonadism.
      ,
      • Wang F.
      • Huang G.D.
      • Tian H.
      • Zhong Y.B.
      • Shi H.J.
      • Li Z.
      • et al.
      Point mutations in KAL1 and the mitochondrial gene MT-tRNA(cys) synergize to produce Kallmann syndrome phenotype.
      ).
      The key to diagnosing IHH is excluding differential diagnoses, such as pituitary tumors or functional causes. Currently, a diagnosis of IHH is based on clinical, biochemical, and imaging investigations. It is especially challenging to differentiate IHH and constitutional delay of growth and puberty in early adolescence (
      • Boehm U.
      • Bouloux P.M.
      • Dattani M.T.
      • de Roux N.
      • Dode C.
      • Dunkel L.
      • et al.
      Expert consensus document: European Consensus Statement on congenital hypogonadotropic hypogonadism—pathogenesis, diagnosis and treatment.
      ). Genetic testing is a good method for diagnosing IHH and is necessary to determine an IHH prognosis and provide related genetic counseling (
      • Sykiotis G.P.
      • Hoang X.H.
      • Avbelj M.
      • Hayes F.J.
      • Thambundit A.
      • Dwyer A.
      • et al.
      Congenital idiopathic hypogonadotropic hypogonadism: evidence of defects in the hypothalamus, pituitary, and testes.
      ). Thus, seeking an efficient and economical tool for genetically testing for this condition is very important for clinicians and patients.
      IHH is classically categorized as a monogenic disorder, meaning that one defective gene is sufficient to cause the disease phenotype. However, the bigenic and oligogenic inheritance has been observed in many genetic diseases, including IHH (
      • Beate K.
      • Joseph N.
      • Nicolas D.R.
      • Wolfram K.
      Genetics of isolated hypogonadotropic hypogonadism: role of GnRH receptor and other genes.
      ). In addition, multiple gene defects could synergize to produce a more severe IHH phenotype. Thus, it is very important to use genetic models to explore genotype-phenotype correlations.
      In recent years, next-generation sequencing has emerged as an effective tool for sequencing large numbers of genes (
      • Rehm H.L.
      Disease-targeted sequencing: a cornerstone in the clinic.
      ). Considering its efficiency and cost, targeted sequencing is effective for detecting mutations within candidate genes for well-characterized diseases with known causal genes. In addition, targeted sequencing can yield more than a 99% coverage rate and 500 × depth data (
      • Li Z.
      • Huang J.
      • Zhao J.
      • Chen C.
      • Wang H.
      • Ding H.
      • et al.
      Rapid molecular genetic diagnosis of hypertrophic cardiomyopathy by semiconductor sequencing.
      ), ensuring ample coverage and precision when used to detect mutations in correctly diagnosed patients.
      The objective of our study was to investigate the mutation profiles of known pathogenic genes in Chinese males with IHH and to use targeted next-generation sequencing to identify new candidate causal genes for IHH. In addition, the clinical features of IHH patients carrying specific gene mutations were observed and noted. Furthermore, the data presented here suggest that targeted sequencing could act as a tool for genetic testing for IHH, which is important for the diagnosis, prognosis, and genetic counseling of IHH patients.

      Materials and methods

       Study Subjects

      This study included 138 sporadic male IHH patients and 10 IHH families. All patients were admitted to the outpatient department of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from 2005 to 2013. All of the included patients were diagnosed with IHH and classified as KS or nIHH according to standard procedures and previously described diagnostic criteria (
      • Laitinen E.M.
      • Vaaralahti K.
      • Tommiska J.
      • Eklund E.
      • Tervaniemi M.
      • Valanne L.
      • et al.
      Incidence, phenotypic features and molecular genetics of Kallmann syndrome in Finland.
      ,
      • Sykiotis G.P.
      • Plummer L.
      • Hughes V.A.
      • Au M.
      • Durrani S.
      • Nayak-Young S.
      • et al.
      Oligogenic basis of isolated gonadotropin-releasing hormone deficiency.
      ).
      The patients recruited in the cohort or their adult parents signed an informed consent form. The experiments performed on the tissue obtained from the patients and 100 controls were approved by the ethics committee of Tongji Hospital.

       Clinical Measurements

      Body weight, height, Tanner stage, testicular volume, and penis length were evaluated at each visit. Body height was measured as the distance between the sole and the highest point of the head. Body weight was measured after the patient removed articles and clothing under comfortable conditions. Testicular volume (including the scrotum) was measured using a Prada orchidometer. Tanner stages were evaluated by a senior physician. Penis length was measured in the standing position after emptying the bladder.

       Laboratory Test

      Serum FSH, LH, T, and E2 levels were measured by chemiluminescence immunoassays (UniCel DXI 800, Beckman Coulter). The normal ranges for serum FSH, LH, T, and E2 levels were 1.27–19.26 mIU/mL, 1.24–8.62 mIU/mL, 1.75–7.81 ng/mL, and 20–75 pg/mL, respectively.

       Designing Gene Panel and Sequencing

      A search of well-known databases (OMIM, GTR-NCBI, and HGMD) resulted in the identification of 83 genes that were used as targets in the next step of the study. These genes included 31 known disease-causing genes and 52 candidate genes. All the known disease-causing genes were reported to be discovered in KS or nIHH patients and were validated by functional experiments. The candidate genes were classified into two modules: [1] genes that were confirmed in mouse models to affect GnRH neuron migration and function and [2] family members or paralogs of known genes that were predicted to result in GnRH neuron abnormality. All the genes and their corresponding references are shown in Supplemental Table 1. Next-generation sequencing was conducted on the IonTorrent PGM platform (
      • Tan L.
      • Li Z.
      • Zhou C.
      • Cao Y.
      • Zhang L.
      • Li X.
      • et al.
      FBN1 mutations largely contribute to sporadic non-syndromic aortic dissection.
      ). Detailed quality control information is summarized in Supplemental Table 2.

       Bioinformatics Analysis

      All variants were compared with entries in known databases (dbSNP, UCSC, ESP, and ExAC) to remove common single nucleotide polymorphisms (minor allele frequency ≥ 1%). Then all the intron variants were discarded. The retained variants were searched in the control samples, resulting in a collection of potential pathogenic variants. The putative disease-causing single nucleotide variants were then imported into online scoring databases (SIFT, Proven, Polyphen2, and MutationTaster) to evaluate the possibility of pathogenicity (in SIFT, score < 0.05; in Proven, score < –2.5; in Polyphen2, score > 0.5; and in MutationTaster, disease causing or disease causing automatically; shown in Supplemental Table 3). Pathogenic variants met the standard that the SNV was assessed as a disease-causing variant in more than two of the databases. Finally, the yielded variants were classified into two groups (known and novel) by referring to the HGMD (2014.04, professional version) and ClinVar databases. The bioinformatics pipeline is shown in Supplemental Figure 1. The protein sequences were imported into the online PHYRE2server (
      • Kelley L.A.
      • Mezulis S.
      • Yates C.M.
      • Wass M.N.
      • Sternberg M.J.
      The Phyre2 web portal for protein modeling, prediction and analysis.
      ) for protein homology modeling. The resulting PDB files were then opened using SPDBV_4.10_PC software (
      • Guex N.
      • Peitsch M.C.
      SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.
      ) to view the three-dimensional protein model.

       Mutation Validation

      All the putative pathogenic variants were validated by Sanger sequencing performed on an ABI 3130XL automated genetic analyzer (Applied Biosystems). The primers designed for polymerase chain reaction and sequencing are listed in Supplemental Table 4. Additionally, the coverage of <20 mutations and the missed region in the final panel of the target genes was also provided by supplemental Sanger sequencing (primers provided in Supplemental Table 4). False-positive results were deleted.

       Burden Test

      To compare the gene burden between 148 probands and 100 controls in our cohort, all the candidate genes were tested with the sequence kernel association test using R packages (
      • Ionita-Laza I.
      • Lee S.
      • Makarov V.
      • Buxbaum J.D.
      • Lin X.
      Sequence kernel association tests for the combined effect of rare and common variants.
      ). In addition, within each candidate gene we identified the number of rare exonic variants (ExAC minor allele frequency ≤ 0.01) in the probands and ExAC data sets that were predicted to be disruptive (nonsense, frameshift, or splice site) or missense variants with bioinformatics prediction of pathogenic (two out of four methods [SIFT, Proven, MutationTaster, and Polyphen2] classified as damaging). We analyzed these data for significant differences in the proportion of exonic variants in individual genes between this cohort and ExAC controls using Fisher's exact test (
      • Clarke C.M.
      • Fok V.T.
      • Gustafson J.A.
      • Smyth M.D.
      • Timms A.E.
      • Frazar C.D.
      • et al.
      Single suture craniosynostosis: identification of rare variants in genes associated with syndromic forms.
      ).

       Statistics

      All statistical calculation in the article was conducted using SPSS version 18. P<.05 was considered statistically significant. The gene burden test was performed using Fisher's exact test. Other statistics calculations were carried out using t-test as a default.

      Results

       Clinical Characteristics

      A total of 153 males with IHH, including 77 KS patients, 74 nIHH patients, and two AHH patients, were involved in the primary study. In addition, one female with KS was included. The mean age at diagnosis was 20.86 years old (SD ± 4.58). Additionally, 17% (26/153) of the patients exhibited other clinical features of KS, including cryptorchidism, renal agenesis, and cleft lip/palate ichthyosis. Cryptorchidism was the most common accompanying symptom (17/153, 11.11%). No difference in accompanying symptoms was detected between the two subgroups. Detailed information regarding the clinical characteristics of the subjects is shown in Table 1.
      Table 1The baseline clinical characteristics of the subjects.
      CharacteristicSummary (n = 151)KS (n = 77)nIHH (n = 74)P value
      Age, y20.86 ± 4.5620.79 ± 3.7420.46 ± 4.46.62
      BMI, kg/m221.57 ± 3.6021.35 ± 3.3821.74 ± 3.78.554
      Testis, ml3.92 ± 3.453.45 ± 2.864.35 ± 3.87.235
      Penis length, cm3.86 ± 1.803.72 ± 2.083.98 ± 1.52.121
      Pubic hair stage (median)111.074
      Genitals stage (median)111.561
      FSH, IU/L1.32 ± 1.031.13 ± 0.841.48 ± 1.15.045
      LH, IU/L0.69 ± 0.730.57 ± 0.580.80 ± 0.82.067
      T, ng/mL0.46 ± 0.510.35 ± 0.310.55 ± 0.61.024
      E2, ng/mL21.28 ± 16.0820.15 ± 12.1622.31 ± 19.04.458
      Note: Data presented as mean ± standard deviation, unless stated otherwise. BMI = body mass index; FHS = follicle-stimulating hormone; KS = Kallmann syndrome; LH = luteinizing hormone; nIHH = normosmic isolated hypogonadotropic hypogonadism.

       Mutation Profiles

      By deep sequencing of 83 genes in 153 IHH patients and 58 relatives, as well as 100 paired healthy males, 145 loci, including 130 missense variant loci and 9 loss-of-function variant loci (including nonsense, frameshift, and splice site variants), could be defined as putative pathogenic variants. These variants were distributed uniformly throughout 52 genes (52/83, 62.65%), including 16 (16/31, 51.61%) causal genes and 36 (36/52, 69.23%) candidate genes. In the 148 probands, the most frequently mutated candidate gene in this panel was RELN (21/148, 14.19%), which contained 10 loci shared by 21 samples. RELN, p.Lys1481Gln, which was found in 10 samples, was not detected in the 1,000 genomes and ExAC database nor in the 100 healthy controls. Other variants in RELN were found in 12 samples. Detailed variant information is provided in Supplemental Table 5, and the variant distribution within genes is shown in Supplemental Figure 2.

       Known Pathogenic Variants

      The gene panel included 31 known causal genes. The distribution of the variants within the genes is shown in Supplemental Figure 3. Pathogenic variants reported in articles or databases (Clinvar or HGMD), and LOF variants in known causal genes were defined as pathogenic variants. The rest were categorized as variants of unknown significance. Eighty-six variants, including 34 pathogenic variants and 52 variants of unknown significance, were discovered in the group of known causal genes. The 34 pathogenic variants accounted for 31 patients. If all the causal gene variants were taken into account, the total number of patients with genetic causes could reach 70. In all 153 patients, the detection rate (70/153, 45.75%) was consistent with previously reported data. Approximately 19% (13/70) of the patients carried more than one variant. Oligogenic inheritance was more common than in a previous report (
      • Sykiotis G.P.
      • Plummer L.
      • Hughes V.A.
      • Au M.
      • Durrani S.
      • Nayak-Young S.
      • et al.
      Oligogenic basis of isolated gonadotropin-releasing hormone deficiency.
      ). The frequency of known causal genes in Chinese HAN males with IHH is shown in Table 2.
      Table 2The frequency of known causal genes in the 148 probands.
      GeneOMIMPhenotypeInheritanceMutated rate, n/N, %
      AXL109135KS/nIHHAD/AR3/148, 2.03
      CHD7608892KS/nIHHAD13/148, 8.78
      FGFR1136350KS/nIHHAD8/148, 5.41
      GNRH1152760nIHHAR1/148, 0.68
      GNRHR138850nIHHAR4/148, 2.70
      IL17RD606807KS/nIHHDD, AR, AD5/148, 3.38
      ANOS1300836KSXLR5/72, 6.94
      KISS1R604161nIHHAR4/74, 5.41
      LEPR601007nIHH1/74, 1.35
      LHB152780KS/nIHHAR8/148, 5.41
      NSMF608137KS/nIHHAD1/148, 0.68
      PROK2607002KS/nIHHAD1/148, 0.68
      PROK2/PROKR2607123KS/nIHHAD23/148, 15.54
      SEMA3A603961KS/nIHHAD2/148, 1.35
      TACR3162332KS/nIHHAR1/148, 0.68
      WDR11606417KS/nIHHAD1/148, 0.68
      Note: AD = autosomal dominant; AR = autosomal recessive; DD = digenic dominant; XLR = X-linked recessive.
      Among the known causal genes, PROKR2 was the most frequently mutated. A total of 10 variant loci were detected in 23 patients. These included a reported causal mutation, PROKR2, p.Trp178Ser (
      • Dode C.
      • Teixeira L.
      • Levilliers J.
      • Fouveaut C.
      • Bouchard P.
      • Kottler M.L.
      • et al.
      Kallmann syndrome: mutations in the genes encoding prokineticin-2 and prokineticin receptor-2.
      ), a heterozygous form of which was shared by 13 patients. The other three variants, p.Arg80His, p.Arg80fs, and p.Pro302Leu, can also be classified as pathogenic or likely pathogenic according to the The American College of Medical Genetics and Genomics guidelines for the interpretation of sequence variants (
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      ), based on functional experiments that were performed in the study of Cox et al. (
      • Cox K.H.
      • Oliveira L.
      • Plummer L.
      • Corbin B.
      • Gardella T.
      • Balasubramanian R.
      • et al.
      Modeling mutant/wild-type interactions to ascertain pathogenicity of PROKR2 missense variants in patients with isolated GnRH deficiency.
      ) (Supplemental Table 13). Other variants dispersed throughout the PROKR2 gene were predicted to be deleterious and were shared by only one or two patients (Supplemental Fig. 4A). In the 148 probands, 22 patients carried PROKR2 mutations (22/148, 14.86%), resulting in a higher incidence than in a previous report indicating that PROKR2 accounts for approximately 10% of IHH mutations. Another important gene identified in the cohort was CHD7 (Supplemental Fig. 4B), an extensively studied gene known to cause CHARGE syndrome (coloboma, heart defect, choanal atresia, retardation of growth and/or development, genital hypoplasia, and ear anomalies) (
      • Bergman J.E.
      • de Ronde W.
      • Jongmans M.C.
      • Wolffenbuttel B.H.
      • Drop S.L.
      • Hermus A.
      • et al.
      The results of CHD7 analysis in clinically well-characterized patients with Kallmann syndrome.
      ). Hypogonadotropic hypogonadism is a minor feature of CHARGE syndrome; however, in recent years, the CHD7 mutation has been reported to be involved in the etiology of IHH by many scientists (
      • Balasubramanian R.
      • Choi J.H.
      • Francescatto L.
      • Willer J.
      • Horton E.R.
      • Asimacopoulos E.P.
      • et al.
      Functionally compromised CHD7 alleles in patients with isolated GnRH deficiency.
      ,
      • Laitinen E.M.
      • Tommiska J.
      • Sane T.
      • Vaaralahti K.
      • Toppari J.
      • Raivio T.
      Reversible congenital hypogonadotropic hypogonadism in patients with CHD7, FGFR1 or GNRHR mutations.
      ,
      • Kim H.G.
      • Kurth I.
      • Lan F.
      • Meliciani I.
      • Wenzel W.
      • Eom S.H.
      • et al.
      Mutations in CHD7, encoding a chromatin-remodeling protein, cause idiopathic hypogonadotropic hypogonadism and Kallmann syndrome.
      ), or it has been reported to contribute to IHH in an oligogenic manner with other known IHH-causing genes (
      • Cariboni A.
      • Andre V.
      • Chauvet S.
      • Cassatella D.
      • Davidson K.
      • Caramello A.
      • et al.
      Dysfunctional SEMA3E signaling underlies gonadotropin-releasing hormone neuron deficiency in Kallmann syndrome.
      ). Thirteen variants were detected in the gene. Two indel mutations, CHD7, c.26_26delT and c.1223_1225delCTC, were identified in the two samples. The former deletion mutation resulted in a 32 amino acid–truncated CHD7 protein, which means that most of the functional motifs were lost. However, with the exception of hypogonadotropic hypogonadism, no other signs of system abnormalities due to CHARGE syndrome were found in this patient in a comprehensive examination. However, a previous study had reported that missense mutations were more frequent than truncating mutations in KS (
      • Marcos S.
      • Sarfati J.
      • Leroy C.
      • Fouveaut C.
      • Parent P.
      • Metz C.
      • et al.
      The prevalence of CHD7 missense versus truncating mutations is higher in patients with Kallmann syndrome than in typical CHARGE patients.
      ).
      ANOS1 was the first reported gene associated with IHH. It follows an X-linked inheritance model such that mutations were more often detected in male than in female patients. Four variant loci were found in ANOS1, and of these, one previously reported locus was shared by seven samples. The p.Val560Ile variant was shared by 3 KS patients and four of their relatives, one of whom was male. Furthermore, three male controls were reported to carry the point mutation. Thus, it is reasonable to conclude that this mutation is not a causal variant or that it needs other chaperon variants to cause this disease. ANOS1 only causes KS other than nIHH, and the mutation rate is about 10% in KS patients, in concordance with previously reported data.

       Candidate Gene Variants

      Ninety-three predicted pathogenic mutated loci were detected in 36 candidate genes. In all, 69.23% of candidate genes had positive results. A total of 136 variants of candidate genes were discovered in 87 patients, and 56.86% patients had positive results for candidate genes. The oligogenic phenomenon was found in more than one third (31/87, 35.63%) of mutation-carrying patients. Except for RELN, the genes carrying the most variants were PLXNB1, ERBB4, and EGFR. The number of variants detected in PLXNB1, ERBB4, and EGFR was 9, 8, and 8, respectively. Thus, RELN, PLXNB1, ERBB4, and EGFR warrant further research because of the high prevalence of variants in the genes in the IHH cohort.

       Pedigree Investigation

      This cohort included 10 pedigrees. Two pedigrees (F9 and F8, shown in Fig. 1) were previously reported by our group (the proband carried ANOS1 and STS gene deletions and ANOS1 p.V560I) (
      • Xu H.
      • Li Z.
      • Wang T.
      • Wang S.
      • Liu J.
      • Wang D.W.
      Novel homozygous deletion of segmental KAL1 and entire STS cause Kallmann syndrome and X-linked ichthyosis in a Chinese family.
      ,
      • Zhang S.
      • Wang T.
      • Yang J.
      • Liu Z.
      • Wang S.
      • Liu J.
      A fertile male patient with Kallmann syndrome and two missense mutations in the KAL1 gene.
      ). To study genotype and phenotype cosegregation, all accessible DNA of the family members was collected for next-generation sequencing. In the two reported families (F8 and F9), in addition to previous mutations, new variants of causal genes (PROKR2, TACR3) were also detected. Four pedigrees (F2, F4, F5, and F6) had pathogenic or predicted pathogenic variants in causal genes. In these families, predicted deleterious variants were also found in three cases. SEMA4D p.Ala515Val was found in one family (F5), in which it exhibited autosomal recessive inheritance. Additionally, in two families, only variants of candidate genes (RELN, EFNA5, and DCC) were detected in two families, and there were no valuable findings in three pedigrees.
      Figure thumbnail gr1
      Figure 1Pedigrees of IHH patients carrying variants. Eight pedigrees with KS were collected to analyze the cosegregation of phenotype and genotype. All available subjects were genotyped. The proband is indicated by the arrow.

       Homozygosity/Compound Heterozygosity

      Except for hemizygotes, homozygosity/compound heterozygosity was very rare in this cohort. Only six (6/153, 3.92%) samples showed homozygosity/compound heterozygosity, and among these, two patients carried two heterozygous variants in one gene. Only one person carried a homozygous mutation in SEMA4D, which was inherited from his heterozygous parents. Another 19-year-old male with anosmia was the only KS patient with compound heterozygous variants. He inherited the mutated chromosomes from his parents, resulting in a compound heterozygous mutation in WDR11. Other compound heterozygous mutations were carried by four nIHH patients, and these were located in the ERBB4, ROBO3, RELN, and FGFR1 genes.

       Oligogenic Inheritance

      Oligogenic inheritance was a common phenomenon in this cohort. In total, 153 patients with the previously defined phenotype were included in an oligogenic inheritance model. When the 83 identified genes were analyzed, 69 patients carried at least two mutations in different genes, and these accounted for approximately half of the variants detected in individuals (69/153, 45.10%). In this cohort, 28 individuals carried more than three mutations, and among these, seven patients (four KS, three nIHH) carried four potential pathogenic mutations. Interestingly, more oligogenic variants were detected in patients with KS than in patients with nIHH. Detailed information for KS and nIHH patients with oligogenic variants is provided in Table 3.
      Table 3The oligogenic inheritance of allele with rare putative pathogenic variants.
      No. alleles with rare protein-altering variantsIHH patients (n = 153)Controls (n = 100)
      Known causal genes
       0v3350
       1v4639
       2, same gene50
       ≥2, different genes6911
      All genes in the panel
       0v8377
       1v5519
       2, same gene20
       ≥2, different genes134
      Note: IHH = isolated hypogonadotropic hypogonadism.
      When the known gene subset was evaluated, two or more variants of different disease-causing genes were carried by 8.50% (13/153) of the patients but only by four of the 100 controls (Table 3). When the gene panel was considered, more patients carried at least two variants compared with controls (69/153 vs. 11/100).

       Genotype-Phenotype Correlation

      Seventeen patients, including seven (7/77, 9.09%) patients with KS and 10 (10/74, 13.51%) with nIHH, demonstrated clinical features related to cryptorchidism. Within this group, no variants were detected in four subjects, five patients had pathogenic variants of known causal genes, and the genetic cause of the disease was identified in 29.41% of the patients in the group. In addition, another four patients carried predicted deleterious variants in causal genes. The genotypes and phenotypes of the patients with cryptorchidism are shown in Supplemental Table 6. The other four patients had variants in candidate genes, including RELN, NOS1, CNTN2, B3GNT1, and DCC. Two cases with cleft lip carried FGFR1, p.Gly379Glu, and FGFR1, p.Arg240Cys, respectively (Supplemental Table 7). The results indicate the relationship between FGFR1 and cleft lip.

       Gene Burden Test

      In addition to assessing variant number, we used gene-based burden testing to prioritize candidate genes for sequential functional experiments. The standard was set in that burden test were significant and the mutation rate was higher in patients than in ExAC controls. EGFR, ERBB4, and PAX6 were identified as the prioritized genes for future research (Supplemental Table 8). When only the East Asian population was taken into consideration, IGF1 was also the possible candidate gene. Sequence kernel association test results indicated that NR5A1, SEMA4D, NTN1, EBF2, EMX2, KLF7, CNTN2, GAS6, RELN, and SEMA7A could be promising candidate genes for IHH, although there was bias because of the limited number of controls (Supplemental Table 9). SEMA7A also passed the burden test compared with the ExAC East Asian population controls, although the mutation rate was lower in patients than in controls. Therefore, from the results of the burden test, SEMA7A, EGFR, ERBB4, PAX6, and IGF1 could be considered candidate genes for IHH.

      Discussion

      In this study, 211 individuals of Chinese Han ancestry were recruited for an investigation of the genetic mechanisms underlying IHH. A total of 83 genes were fully sequenced with semiconductor next-generation sequencing technology, and the results were further confirmed by Sanger sequencing. Thirty-four pathogenic variants accounted for the phenotypes observed in 31 patients. In addition, 52 predicted pathogenic variants were detected in causal genes, and several candidate genes were identified for further study. We concluded that SEMA7A, PAX6, IGF1, ERBB4, EGFR, and SEMA4D should be prioritized as candidate genes for further research. Our results indicate that approximately half of the patients carried at least two variants, in accordance with the results from the previously reported oligogenic inheritance model in IHH (
      • Sykiotis G.P.
      • Pitteloud N.
      • Seminara S.B.
      • Kaiser U.B.
      • Crowley W.J.
      Deciphering genetic disease in the genomic era: the model of GnRH deficiency.
      ).
      The genes included in an IHH genetic testing panel should be screened as accurately and concisely as possible. Of the genes reported to cause IHH, a large number of were ligands and corresponding receptors and were associated with GnRH neuron migration and development (
      • Laitinen E.M.
      • Vaaralahti K.
      • Tommiska J.
      • Eklund E.
      • Tervaniemi M.
      • Valanne L.
      • et al.
      Incidence, phenotypic features and molecular genetics of Kallmann syndrome in Finland.
      ). The genes, which participate in GnRH neuronal ontogenesis and migration, could play an important role in IHH. In addition, mutations affecting GnRH neurons could also disturb other neurons, resulting in syndromes that manifest as the clinical features of IHH. These include, CHD7 in CHARGE syndrome and SOX10 in Waardenburg syndrome (
      • Sykiotis G.P.
      • Plummer L.
      • Hughes V.A.
      • Au M.
      • Durrani S.
      • Nayak-Young S.
      • et al.
      Oligogenic basis of isolated gonadotropin-releasing hormone deficiency.
      ). Thus, we infer that many candidate genes of IHH participate in axon guidance pathways and the associated pathways of other developmental syndromes. Of the 31 known genes, 45.75% (70/153) of the patients carried at least one variant. The 5.75% difference may be explained by the fact that the panel included more causal genes that have been discovered in recent years. However, when the total gene panel is taken into consideration, at least one mutation was detected in approximately 78.43% (120/153) of the IHH patients. These results imply that the candidate genes have a role in IHH pathogenesis and that our sequencing panel may perform better than currently available tests when used for clinical diagnosis.
      ANOS1 was the first gene identified to cause KS, and more genes were later found to be associated with IHH. However, these genes only account for no more than 40% patients. In most of the previously reported cohorts, such as those produced by the Harvard IHH Center and the Europa IHH Institute, the majority of the patient populations were white, and there may be some differences between different ethnic backgrounds. Our study focused on the mutation spectrum in the Chinese Han population, and the results showed that the mutation rates in known genes in this population were different from those found in white populations (ANOS1, 10.38% vs. 10%–14% (
      • Sato N.
      • Katsumata N.
      • Kagami M.
      • Hasegawa T.
      • Hori N.
      • Kawakita S.
      • et al.
      Clinical assessment and mutation analysis of Kallmann syndrome 1 (KAL1) and fibroblast growth factor receptor 1 (FGFR1, or KAL2) in five families and 18 sporadic patients.
      ); FGFR1, 5.41% vs. 10% (
      • Villanueva C.
      • de Roux N.
      FGFR1 mutations in Kallmann syndrome.
      ); and PROK2/PROKR2, 15.54% vs. 9% (
      • Sarfati J.
      • Dode C.
      • Young J.
      Kallmann syndrome caused by mutations in the PROK2 and PROKR2 genes: pathophysiology and genotype-phenotype correlations.
      )).
      At least six candidate genes were identified as being likely causal genes for IHH. RELN, PLXNB1, ERBB4, and EGFR were the genes with the highest mutation rate in our cohort compared with the control groups. RELN has been reported to be transiently expressed in human neural cells during olfactory placode development (
      • Antal M.C.
      • Samama B.
      • Ghandour M.S.
      • Boehm N.
      Human neural cells transiently express reelin during olfactory placode development.
      ). The olfactory placode is the origin of the GnRH neuron. Mutations in RELN are associated with schizophrenia, epilepsy, and myoclonus-dystonia. The 22 predicted deleterious variants in the cohort had not been reported before to be associated with any known diseases. However, RELN did not pass the gene burden test, and the correlation between RELN and IHH remains unclear. Cell-cell signaling of semaphoring ligands through interaction with plexin receptors is an important mechanism in neural connectivity, cell migration, axon guidance, and repulsion (
      • Janssen B.J.
      • Robinson R.A.
      • Perez-Branguli F.
      • Bell C.H.
      • Mitchell K.J.
      • Siebold C.
      • et al.
      Structural basis of semaphorin-plexin signalling.
      ). In this mechanistic pathway, SEMA3A was reported to be an autosomal dominant gene associated with IHH (
      • Hanchate N.K.
      • Giacobini P.
      • Lhuillier P.
      • Parkash J.
      • Espy C.
      • Fouveaut C.
      • et al.
      SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome.
      ). SEMA7A has been suspected of causing the disease, and it was recently reported that rare variants of SEMA7A were detected in patients with congenital hypogonadotropic hypogonadism via oligogenic inheritance (
      • Kansakoski J.
      • Fagerholm R.
      • Laitinen E.M.
      • Vaaralahti K.
      • Hackman P.
      • Pitteloud N.
      • et al.
      Mutation screening of SEMA3A and SEMA7A in patients with congenital hypogonadotropic hypogonadism.
      ). SEMA4D, a paralog of SEMA3A, was reported to cause abnormal GnRH neuron migration through interactions with PLXNB1 and MET based on cell culture and animal experiments (
      • Giacobini P.
      • Messina A.
      • Morello F.
      • Ferraris N.
      • Corso S.
      • Penachioni J.
      • et al.
      Semaphorin 4D regulates gonadotropin hormone-releasing hormone-1 neuronal migration through PlexinB1-Met complex.
      ). In our study, one patient carried a homozygous SEMA4D variant inherited from his healthy parents. This finding implies that SEMA4D may function in an autosomal recessive manner. From the results of the gene burden test, EGFR, ERBB4, PAX6, and IGF1 were prioritized as the most promising candidate genes. The pubertal increase in luteinizing hormone-releasing hormone secretion in female animals also requires neuron-glia signaling mediated by growth factors of the epidermal growth factor (EGF) family and their astrocytic erbB receptors (
      • Prevot V.
      • Rio C.
      • Cho G.J.
      • Lomniczi A.
      • Heger S.
      • Neville C.M.
      • et al.
      Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes.
      ). Dellovade et al. reported that PAX6, which was a transcription factor critical for pituitary development and function, was very important for the migration and differentiation of specific neuronal progenitor cells (
      • Dellovade T.L.
      • Pfaff D.W.
      • Schwanzel-Fukuda M.
      The gonadotropin-releasing hormone system does not develop in small-eye (Sey) mouse phenotype.
      ). IGF1-IGF1R signaling played an important role in GnRH neuronal morphology and regulated pubertal timing (
      • Divall S.A.
      • Williams T.R.
      • Carver S.E.
      • Koch L.
      • Bruning J.C.
      • Kahn C.R.
      • et al.
      Divergent roles of growth factors in the GnRH regulation of puberty in mice.
      ). Nevertheless, according to the guideline for implicating variants and genes as causative for a disease reported by MacArthur et al. (
      • MacArthur D.G.
      • Manolio T.A.
      • Dimmock D.P.
      • Rehm H.L.
      • Shendure J.
      • Abecasis G.R.
      • et al.
      Guidelines for investigating causality of sequence variants in human disease.
      ), the association of SEMA4D, SEMA7A, EGFR, ERBB4, PAX6, and IGF1 with IHH needs to be verified in vivo and in vitro.
      IHH is reported to be a complex genetic disease with diversity in its inheritance model, including X-linked, autosomal dominant, and autosomal recessive inheritance. On the other hand, digenic and oligogenic inheritance are common phenomena reported in IHH cohorts. Oligogenic inheritance was first reported in Bardet-Biedl syndrome (
      • Beales P.L.
      • Badano J.L.
      • Ross A.J.
      • Ansley S.J.
      • Hoskins B.E.
      • Kirsten B.
      • et al.
      Genetic interaction of BBS1 mutations with alleles at other BBS loci can result in non-Mendelian Bardet-Biedl syndrome.
      ) to explain its complex phenotypes, and it was soon introduced into other genetic diseases to interpret the causes of rare diseases. In 2010, Sykiotis and his colleagues first used an oligogenic model to study the genetic mechanism of IHH (
      • Sykiotis G.P.
      • Plummer L.
      • Hughes V.A.
      • Au M.
      • Durrani S.
      • Nayak-Young S.
      • et al.
      Oligogenic basis of isolated gonadotropin-releasing hormone deficiency.
      ). The discovery of an SEMA3E mutation in KS patients carrying a CHD7 mutation provided a good example of oligogenic variants in IHH (
      • Cariboni A.
      • Andre V.
      • Chauvet S.
      • Cassatella D.
      • Davidson K.
      • Caramello A.
      • et al.
      Dysfunctional SEMA3E signaling underlies gonadotropin-releasing hormone neuron deficiency in Kallmann syndrome.
      ). In our study, oligogenic inheritance was more often implicated under conditions in which a known pathogenic mutation is accompanied by a candidate gene variant also involved in the KS pathogenesis pathway. Through pathway analysis, one possible conclusion is that variants located in genes that participate in related pathways interact with each other to cause disease. The resulting condition may occur at the gene or protein level. In the gene ontology enrichment of the mutated genes performed in this study, the axon guidance pathway was highlighted. Axon guidance is a complex biological procedure consisting of different molecular processes, including axon attraction, axon repulsion, and axon outgrowth (Supplemental Fig. 5). The Netrin, Ephrin, Slit, Semaphorin, Plexin, and Robo families were the most common molecular families participating in the pathway.
      In our study, 10 pedigrees were included to clarify the genetic basis of IHH pathogenesis. More than half of the pedigrees (6/10) had pathogenic variants in known causal genes. However, no variants were found in three families in the genes on the 83-gene panel. This result may be explained in part by the limited number of genes included in the study. Recently, more genes, such as CCDC141 and FEZF1, have been reported to cause IHH (
      • Hutchins B.I.
      • Kotan L.D.
      • Taylor-Burds C.
      • Ozkan Y.
      • Cheng P.J.
      • Gurbuz F.
      • et al.
      CCDC141 mutation identified in anosmic hypogonadotropic hypogonadism (Kallmann syndrome) alters GnRH neuronal migration.
      ,
      • Kotan L.D.
      • Hutchins B.I.
      • Ozkan Y.
      • Demirel F.
      • Stoner H.
      • Cheng P.J.
      • et al.
      Mutations in FEZF1 cause Kallmann syndrome.
      ). These genes may explain some of the unknown genetic causes of IHH.
      IHH is often accompanied by many different nongenital and genital phenotypes, such as cleft lip/palate, uterus deficiency, ichthyosis, renal agenesis, teeth dysplasia, trichomadesis, scrotal dysplasia, and cryptorchidism. Some of the accompanying phenotypes are caused by a unique gene mutation, such as the FGFR1 mutations associated with cleft lip/palate and the ANOS1 mutation associated with renal agenesis (
      • Xu H.
      • Niu Y.
      • Wang T.
      • Liu S.
      • Xu H.
      • Wang S.
      • et al.
      Novel FGFR1 and KISS1R mutations in Chinese Kallmann syndrome males with cleft lip/palate.
      ,
      • Georgopoulos N.A.
      • Koika V.
      • Galli-Tsinopoulou A.
      • Spiliotis B.E.
      • Adonakis G.
      • Keramida M.K.
      • et al.
      Renal dysgenesis and KAL1 gene defects in patients with sporadic Kallmann syndrome.
      ). Most of the genes mentioned above are involved in early embryo development. However, it is interesting that not all of the mutations detected in these genes could cause the accompanying phenotypes. Thus, it is possible that the phenotypes are variant specific or are associated with another gene. However, we cannot ignore the epigenetic effect of gene imprinting. In this study, cryptorchidism (17/151, 11.26%) was the most common accompanying phenotype, which is consistent with a previous report (
      • Bhagavath B.
      • Podolsky R.H.
      • Ozata M.
      • Bolu E.
      • Bick D.P.
      • Kulharya A.
      • et al.
      Clinical and molecular characterization of a large sample of patients with hypogonadotropic hypogonadism.
      ). In our cohort, cryptorchidism was more common in the nIHH subgroup than in the KS subgroup (13.51% vs. 9.09%). The results of our genetic analysis showed that no single gene in the panel was associated with this abnormality. INSL3 and HOXA10 have been reported to be associated with isolated cryptorchidism (
      • Canto P.
      • Escudero I.
      • Soderlund D.
      • Nishimura E.
      • Carranza-Lira S.
      • Gutierrez J.
      • et al.
      A novel mutation of the insulin-like 3 gene in patients with cryptorchidism.
      ,
      • Kolon T.F.
      • Wiener J.S.
      • Lewitton M.
      • Roth D.R.
      • Gonzales E.J.
      • Lamb D.J.
      Analysis of homeobox gene HOXA10 mutations in cryptorchidism.
      ). Cryptorchidism observed in our cohort may be explained by the fact that mutations in IHH genes may affect testis descent via a disturbance in steroid hormone metabolism through a perturbed hypothalamic-pituitary-gonadal axis. In addition, the cleft lip may be associated with mutations in FGFR1, which is consistent with a previous study (
      • Jarzabek K.
      • Wolczynski S.
      • Lesniewicz R.
      • Plessis G.
      • Kottler M.L.
      Evidence that FGFR1 loss-of-function mutations may cause variable skeletal malformations in patients with Kallmann syndrome.
      ).
      However, little cosegregation was observed in pedigrees in our cohort, implying that other genes may underlie the pathogenesis of IHH, and whole-exome sequencing or whole-genome sequencing may be a good choice for further investigation of the pedigrees. Moreover, the function of the newly discovered variants was not confirmed by biochemical experiments to result in a deleterious outcome. Therefore, a larger cosegregation study combined with functional experiments is needed to better understand the genetic causes of IHH. Another limitation of our study is that it was a single-center study with a limited number of patients, although we sequenced as many genes as possible. A multicenter cohort study with a different population is needed to further validate the genetic basis of IHH. We will collaborate with other researchers and seek for help in the public consortium, such as GeneMatcher, in the future (
      • Dellovade T.L.
      • Pfaff D.W.
      • Schwanzel-Fukuda M.
      The gonadotropin-releasing hormone system does not develop in small-eye (Sey) mouse phenotype.
      ).
      In conclusion, our cohort included 10 pedigrees and 138 sporadic nIHH/KS patients, and this is the largest cohort of Han Chinese subjects to be analyzed with next-generation sequencing. Six new pathogenic variants (PROKR2, p.Arg80fs; FGFR1, c.1430+1G>T; CHD7, p.Phe10fs; ANOS1, p.Trp589Ter; ANOS1 p.Gln45Ter; ANOS1, p.Glu451fs) and 52 likely pathogenic variants were identified in 16 genes known to cause nIHH/KS. In addition, six new candidate genes including EGFR, ERBB4, PAX6, IGF1, SEMA4D, and SEMA7A, were prioritized for future study by pedigree study and variant analysis. In the 148 probands, PROKR2 (22/148, 14.86%) and RELN (21/148, 14.19%) are the genes in the causal gene and candidate gene subgroup, respectively. Moreover, cryptorchidism is very common in nIHH/KS, although no single gene is associated with the phenotype. The results of this study expand the mutation profile of IHH and provide a useful tool for the genetic diagnosis and study of IHH.

      Acknowledgments

      The authors thank the patients and their families for their cooperation.

      Appendix

      Figure thumbnail fx1
      Supplemental Figure 1The flow diagram of bioinformatics analysis.
      Figure thumbnail fx2
      Supplemental Figure 2The distribution of variants in all genes in the panel.
      Figure thumbnail fx3
      Supplemental Figure 3The distribution of variants in known causal genes.
      Figure thumbnail fx4
      Supplemental Figure 4The distribution of variants in PROKR2 and CHD7. (A) Schematic diagram of prokr2 in the cell membrane and the corresponding three-dimensional model and the distribution of variants in PROKR2; (B) schematic diagram of chd7 in the cell membrane and the distribution of variants in CHD7.
      Figure thumbnail fx5
      Supplemental Figure 5Molecular processes, including axon attraction, axon repulsion, and axon outgrowth in axon guidance.

      Supplementary data

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