Identification of KISS1R gene mutations in disorders of non‐obstructive azoospermia in the northeast population of China

Abstract Background Non‐obstructive azoospermia (NOA), a serious phenotype of male spermatogenesis failure, is a multifactorial disease which is regulated by genetic, epigenetic, and environmental factors. Some gene structural variants have been demonstrated to be related to NOA. Loss‐of‐function mutations of KISS1R cause normosmic idiopathic hypogonadotropic hypogonadism (IHH) which result in azoospermia at the pre‐testicular level. The objective of this research was to investigate genetic variants of KISS1R in NOA patients. Methods The entire coding region of 52 spermatogenesis‐associated genes (KISS1R included) was sequenced from 200 NOA patients. Mutation screening was performed to identify genetic variations of these genes by targeted exome sequencing. Sequencing data analysis was carried out by a series of bioinformatics tools. Candidate variants confirmation was performed by Sanger sequencing. Functional analysis of candidate variants was evaluated using SIFT and PolyPhen‐2. Results Three heterozygous missense variants in KISS1R were identified in three patients, respectively. No deleterious variations in other candidate genes were found in the three patients. Two of these three variants, p.A211T and p.G186E, had been reported in the ExAC and dbSNP database, respectively, while the other variant p.A301D was novel. These variants were all predicted to be likely pathogenic by in silico analysis. Conclusion Our study revealed three heterozygous missense variants in KISS1R which expanded the mutation spectrum of KISS1R in infertile men with NOA in the northeast of China.


| INTRODUC TI ON
Azoospermia, which affects up to 1% of men in the general population, 1 is a type of male infertility with a lack of spermatozoa in the ejaculate. 2 Types of azoospermia include obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). 2 Non-obstructive azoospermia, a serious phenotype of male spermatogenesis failure, is a multifactorial disease which is regulated by genetic, epigenetic, and environmental factors. The genetic causes of NOA including Y-chromosome microdeletions, karyotype abnormality and autosomal single-gene or polygenic mutations or polymorphisms in multiple biological pathways are involved in the development of NOA. 3 The prevalence of structural and numerical chromosomal abnormalities in the NOA population can up to 20%. 4 Many other genetic causes of NOA remain unknown, although several genes have been reported based on an association between genetic variants of genes and NOA etiologyincluding PRM1, HSF2, KLHL10, SPATA16, AURKC, and ATM. 5 The original purpose of our study was to investigate the contribution of genetic variations in some known causative genes associated with spermatogenesis to the development of NOA. Then in this study, we discovered and clarified genetic mutations of KISS1R gene in a population of infertile men with NOA.
The human KISS1R gene, located at 19p13.3, was initially identified as an orphan G protein-coupled receptor (GPCR), and kisspeptin, a product of the KISS1 gene, was its endogenous ligand. 6 Loss-of-function mutations of KISS1R cause normosmic idiopathic hypogonadotropic hypogonadism (IHH) which result in azoospermia at the pre-testicular level. 7,8 Kisspeptin binds to KISS1R stimulating GnRH release by hypothalamic neurons, leading to secretion of sexual steroids and pituitary gonadotropins, which in turn will play roles in producing the gametes. 9 In the immature animals, precocious activation of the gonadotropic axis and pubertal development was able to induce by intermittent kisspeptin administration. 7 In addition, animal studies suggested the targeted deletion of KISS1R led to hypogonadotropic hypogonadism, abnormal sexual maturation, and infertility. 6,9 Nevertheless, researches on kisspeptin and infertility are scarce. 9 The understanding of genetic variations in KISS1R with NOA may lead to the use of KISS1R as a biomarker for diagnosis and treatments of male infertility.

| MATERIAL S AND ME THODS
Our study consisted of 200 Chinese patients with idiopathic NOA.
Mean age was 30.25 ± 5.24 years. All patients were diagnosed with NOA based on the following examination, including a detailed Mutation screening of genes associated with spermatogenesis was carried out by targeted exome sequencing as described previously. 10 Genomic DNA was isolated from peripheral blood samples of all patients and subjected to exome capture using the in-house targeted genes panel (Peking Medriv Academy of Genetics and Reproduction, Peking). Capture procedure was performed in solution that enriched the exonic sequences of 52 spermatogenesis-associated genes ( Genomes Project or in the dbSNP databases were excluded.

Synonymous variants and variants in genes with unknown clinical
phenotypes were filtered out. The remaining variants were evaluated for correlation with patient's phenotype. As our research objective, pathogenicity of the candidate variants was evaluated using SIFT (https ://sift.bii.a-star.edu.sg/) and PolyPhen-2 (https :// genet ics.bwh.harva rd.edu/pph2/).  patients were found to have KISS1R variations, and we identified three KISS1R heterozygous missense variants in three cases, respectively (Table 2). No deleterious variations in other 51 candidate genes were found in the three patients. The p.A211T and p.G186E missense mutations had been reported in the ExAC and dbSNP database, respectively. The other variant p.A301D had never previously been reported, which was not found in the public databases including 1000 Genome Project, dbSNP, and ExAC database. According to SIFT software, the three variants were all predicted to be deleterious to the protein's function. The p.A211T and p.A301D variants were both possibly damaging, and the p.G186E variant was probably damaging according to PolyPhen-2 (Table 3). We performed PCR and Sanger sequencing on the three patients and confirmed the three heterozygous missense mutations in these patients, respectively (Figure 1 and Figure 2). The relevant clinical and hormone data of these patients were summarized in Table 4. Color Doppler ultrasonography for scrotal of these three patients with missense variants revealed that Pat2 and Pat3 showed normal testicular volume, while Pat1 carrying p.A211T showed small testes in the scrotal sac. Hormone levels were normal or slightly lower than normal in the two patients with KISS1R mutations (Pat2 and Pat3).

| RE SULTS
The hormonal level of the other one patient carrying p.A211T (Pat1) was obviously abnormal which exhibited high FSH level and a low T level.

| D ISCUSS I ON
In the present study, we have described three heterozygous mis- Males can be clinically manifested as absent or incomplete puberty, small penis, cryptorchidism, and infertility. 12 In the past few years, IHH has classically been categorized as a single-gene disease, 13 but the phenotypic presentation of this disease and its genetic background are highly heterogeneous. 14 A few genes that are involved in the pathogenesis of IHH have been identified at various sites, including TAC3, TACR3, GnRHR, FGFR1, GNRH1, FGF8, KISS1, and KISS1R. 11 However, these genetic defects account for less than 30% of patients with IHH. 15 AR  AURKC  CATSPER1  CCDC39  CFTR  CHD7  DNAAF1  DNAAF2   DNAAF3  DNAH1  DNAH11  DNAH5  DNAI1  DNAI2  DPY19L2  DYX1C1   ETV5  FGF8  FGFR1  GNRHR  HEATR2  HSF2  HYDIN  KAL1   KISS1R  LEP  LEPR  NANOS1  NELF  NR5A1  PLCZ1  PROK2   PROKR2  RHOXF1  RHOXF2  RSPH1  RSPH4A  RSPH9  SEPT12  SLC26A8   SOHLH2  SPATA16  SUN5  SYCE1  SYCP3  TAC3  TACR3  TEX11   USP26   In previous studies, KISS1R was one of the major genes that implicated in IHH in the autosomal-recessive form. 16  mutation. We hope that our report will contribute to the ongoing genetic characterization of NOA.

ACK N OWLED G EM ENTS
We are sincerely grateful to the staff at the Peking Medriv Academy of Genetics and Reproduction, Beijing, China, for their hard work and technical support.

CO N S E NT TO PA RTI CI PATE
Performed after obtaining written informed consent from the participants.