Data_Sheet_1_Whole-Exome Sequencing Analysis of Human Semen Quality in Russian Multiethnic Population.XLSX

The global trend toward the reduction of human spermatogenic function observed in many countries, including Russia, raised the problem of extensive screening and monitoring of male fertility and elucidation of its genetic and ethnic mechanisms. Recently, whole-exome sequencing (WES) was developed as a powerful tool for genetic analysis of complex traits. We present here the first Russian WES study for identification of new genes associated with semen quality. The experimental 3 × 2 design of the WES study was based on the analysis of 157 samples including three ethnic groups—Slavs (59), Buryats (n = 49), and Yakuts (n = 49), and two different semen quality groups—pathozoospermia (n = 95) and normospermia (n = 62). Additionally, our WES study group was negative for complete AZF microdeletions of the Y-chromosome. The normospermia group included men with normal sperm parameters in accordance with the WHO-recommended reference limit. The pathozoospermia group included men with impaired semen quality, namely, with any combined parameters of sperm concentration <15 × 106/ml, and/or progressive motility <32%, and/or normal morphology <4%. The WES was performed for all 157 samples. Subsequent calling and filtering of variants were carried out according to the GATK Best Practices recommendations. On the genotyping stage, the samples were combined into four cohorts: three sets corresponded to three ethnic groups, and the fourth set contained all the 157 whole-exome samples. Association of the obtained polymorphisms with semen quality parameters was investigated using the χ2 test. To prioritize the obtained variants associated with pathozoospermia, their effects were determined using Ensembl Variant Effect Predictor. Moreover, polymorphisms located in genes expressed in the testis were revealed based on the genomic annotation. As a result, the nine potential SNP markers rs6971091, rs557806, rs610308, rs556052, rs1289658, rs278981, rs1129172, rs12268007, and rs17228441 were selected for subsequent verification on our previously collected population sample (about 1,500 males). The selected variants located in seven genes FAM71F1, PPP1R15A, TRIM45, PRAME, RBM47, WDFY4, and FSIP2 that are expressed in the testis and play an important role in cell proliferation, meiosis, and apoptosis.

包括俄罗斯在内的多个国家均观测到人类精子发生功能呈全球性下降趋势，这引发了男性生育力大规模筛查与监测，以及阐明其遗传与种族机制的相关研究课题。近年来，全外显子组测序（whole-exome sequencing, WES）已成为复杂性状遗传分析的强大工具。本研究报道了俄罗斯首个针对精液质量相关新基因鉴定的WES研究。该研究采用3×2实验设计，共分析157份样本，涵盖3个种族群体：斯拉夫族（59例）、布里亚特族（n=49）与雅库特族（n=49），以及2个精液质量分组：病理精子症组（n=95）与正常精子症组（n=62）。此外，本研究所有样本均未检出Y染色体完全性无精子症因子（Azoospermia Factor, AZF）微缺失。正常精子症组纳入的男性，其精子参数符合世界卫生组织（World Health Organization, WHO）推荐的参考限值标准。病理精子症组纳入的男性均存在精液质量异常，具体表现为满足以下任意一项或多项联合参数异常：精子浓度<15×10^6/ml、前向运动率<32%、正常形态率<4%。对全部157份样本均完成了WES检测。后续的变异检出与过滤流程严格遵循GATK最佳实践指南的推荐方案。在基因分型阶段，样本被划分为4个队列：3个队列分别对应上述3个种族群体，第4个队列则包含全部157份全外显子组测序样本。采用χ²检验分析所得多态性位点与精液质量参数的关联。为筛选与病理精子症相关的候选变异，采用Ensembl变异效应预测器（Ensembl Variant Effect Predictor）对其功能效应进行注释。此外，通过基因组注释筛选出在睾丸中表达的基因内的多态性位点。最终，筛选出9个潜在单核苷酸多态性（single nucleotide polymorphism, SNP）标记：rs6971091、rs557806、rs610308、rs556052、rs1289658、rs278981、rs1129172、rs12268007及rs17228441，将在我们此前收集的约1500名男性的人群样本中开展后续验证。上述候选变异位于7个基因内：FAM71F1、PPP1R15A、TRIM45、PRAME、RBM47、WDFY4及FSIP2，这些基因均在睾丸中表达，且在细胞增殖、减数分裂及细胞凋亡过程中发挥关键作用。