A Screen For Genomic Disorders Of Infertility And Genome-wide Association Study On Chinese Non-obstructive Azoospermia

Infertility is serious affectting the human reproductive health.10%-15% of the couples worldwide have reproductive difficulties and about 30-55% of them were caused by male factors. The causes of male infertility were various, among them, spermatogenic failure is one of the important reasons. Non-obstructive azoospermia (NOA) is known as the most common and serious cause of male infertility. The incidence rate of NOA is about 1% of the total number of male and nearly a quarter of male infertility patients in our country are caused by NOA. NOA is defined as no sperm in semen as a result of congenital dysfunction in spermatogenesis after excluding obstructive azoospermia and retrograde ejaculation. Genetic factors accounted for about 15-30% in male infertility.In recent years, with the rapid development of genetics research methods, Copy number variations (CNV) is concerned gradually. CNV is a common type of structural variation, with variations in the size of 1 kilobase (Kb) to several megabases (Mb). Due to the large area covered by the CNV and contained a large number of genes and some functional elements. Since CNV covers a large area and usually contains a large number of genes, the study of CNV is important valuable. Since there is currently no large CNV study in Chinese population, we performed two stages of the case-control study of genome-wide copy number variation. Different from the previous candidate gene study, genome-wide association study does not require hypothesis before research. The GWAS study using high-density chip to achieve large-scale systematic screening to identify potential pathogenic sites. GWAS has become a powerful tool for genetic scientists in the past 10 years. The strategy of GWAS is to uncover the SNPs which occur differently in people with or without a particular disease like cancer, Alzheimer disease, obesity, etc. Using this solution, we have identified several potential risk genomic regions of NOA in our previous studies.The Y chromosome has high genetic variability with low rates of parallel and back mutations, which make up the most informative haplotyping system. Due to the limit of statistical methods employed in genome-wide association study (GWAS), genotypes analyzed in such studies are mainly on autosomes, and those located on sex chromosomes are often excluded, especially the Y chromosome, which plays important roles in sex determination and spermato genes is. GWAS focus on common SNPs and neglect rare mutations; and SNPs on sex chromosomes are commonly excluded from most GWAS due to the law of linkage disequilibrium. However, sex chromosomes, especially the Y chromosome, play central roles in sex determination, and it is improper to overlook effects of sex chromosomes when understanding the genetic etiology of a disease. The neglected sex chromosomes in GWAS are believed to contribute to the "missing" heritability in the etiology of complex diseases. Y chromosome, in contrast to the rest of the genome, is confined to males and contains the smallest number of genes, most of which locate in the male specific region (MSY). Y chromosome has the most informative haplotyping system with applications in evolutionary studies, medical genetics and genealogical reconstruction. Considering the function of the Y chromosome in sex determination, it has been reported that some Y chromosome haplogroups (Y-hgs) may increase the danger of spermatogenic impairment across different populations. Our efforts using this strategy have proved that some Y-hgs, such as Y-hg K, Q1, are potential risk factors for male infertility.Therefore, we conducted a GWAS of NOA to test the hypothesis that some of the ’missing’ CNVs at these hotspots may cause severe infertility, and thus are purged rapidly from the population. And in the second part of this study we recruited 1,000 NOA cases and 1,703 fertile controls in Han-Chinese, compared the distributions of Y-hgs in both groups and analyzed the interactions between Y-hgs and autosomal variants in GWAS.Part Ⅰ:A Screen for Genomic Disorders of Infertility in Nonobstructive Azoospermia in HumansWe have previously reported that deletions of DMRT1 are associated with NOA, and it is well known that DMRT1 loss-of-function can lead to a variety of derangements in gonad development. Since the sample size is small and can not reveal the copy number variation in Chinese male, so we have assembled a cohort of men that received a diagnosis of nonobstructive azoospermia (NOA), more than 2500 men in total, and have performed genome-wide screens for novel, recurrent CNVs causing NOA.We tested this hypothesis by contrasting patterns of rare CNVs in 1,000 Han Chinese men with NOA and 1,703 controls. Our results strongly support our previous claim that sperm production is modulated by genetic variation across the entire genome. The X chromosome in particular was enriched for loci modulating spermatogenesis-rare X-linked deletions larger than 100 kb were twice as common in patients compared with controls (OR=2.05, P=0.01).At rearrangement hotspots across the genome, we observed a 2.4-fold enrichment of singleton CNVs in patients (P< 0.02), and we identified 117 testis genes, such as SYCE1, contained within 47 hotspots that may plausibly mediate genomic disorders of fertility. In our discovery sample we observed 3 case-specific duplications of the autosomal gene MAST2, and in a replication phase we found another 11 duplications in 1457 patients and 1 duplication in 1590 controls (P<5×10-5). With a large, polygenic genetic basis, new ways of establishing the pathogenicity of rare, large-effect mutations will be needed to fully reap the benefit of genome data in the management of azoospermia.Our findings clearly show that there was an increased load of rare deleterious CNVs in cases compared with controls, affecting genes other than those on the Y chromosome.Part Ⅱ:Y chromosome haplogroups based genome-wide association study pinpoints revelation for interactions on Chinese non-obstructive azoospermiaThe Y chromosome has high genetic variability with low rates of parallel and back mutations, which make up the most informative haplotyping system. Due to the limit of statistical methods employed in genome-wide association study (GWAS), genotypes analyzed in such studies are mainly on autosomes, and those located on sex chromosomes are often excluded, especially the Y chromosome, which plays important roles in sex determination and spermatogenesis. Thus, to examine whether Ychromosome haplogroups (Y-hg) could modify the effects of autosomal variants on non-obstructive azoospermia (NOA), we conducted a genetic interaction analysis in 1,000 individuals with NOA and 1,703 fertile male controls, who have been reported in our previous GWAS study. Using a logistic regression model, we found that Y-hg O3e* showed a protective effect on spermatogenic impairment [odds ratio (OR)= 0.68; 95% confidence interval (CI)=0.52-0.89; P=5.55 x 10"3].Then, we explored the potential interaction between Y-hg O3e* and autosomal variants, our results demonstrated that there was a synergistic interaction between Y-hg O3e* and rs11135484 (OR=2.07; P=9.89 × 10"5) on NOA. In addition, bioinformatic analysis revealed that genes annotated by significant single nucleotide polymorphisms (SNPs) were mainly enriched in immuno logical pathways. This is the first study of interactions between Y-hgs and autosomal variants on a genome-wide scale. It addresses part of the missing heritability in azoospermia risk and sheds new light on the pathogenesis of male infertility.In summary, we combined Y-hgs with GWAS to investigate potential interactions between them on NOA, and observed that Y-hg O3e* may modify the risk of some SNPs. These results suggest that both the Ychromosome and autosomes may jointly contribute to the reproductive outcomes. We cannot always divide them into two distinct aspects, and the combination may shed new light on the pathogenesis of male infertility.