Identification of seven genes essential for male fertility through a genome-wide association study of non-obstructive azoospermia and RNA interference-mediated large-scale functional screening in Drosophila.

Non-obstructive azoospermia (NOA) is a complex and severe condition whose etiology remains largely unknown. In a genome-wide association study (GWAS) of NOA in Chinese men, few loci reached genome-wide significance, although this might be a result of genetic heterogeneity. Single nucleotide polymorphisms (SNPs) without genome-wide significance may also indicate genes that are essential for fertility, and multiple stage validation can lead to false-negative results. To perform large-scale functional screening of the genes surrounding these SNPs, we used in vivo RNA interference (RNAi) in Drosophila, which has a short maturation cycle and is suitable for high-throughput analysis. The analysis found that 7 (31.8%) of the 22 analyzed orthologous Drosophila genes were essential for male fertility. These genes corresponded to nine loci. Of these genes, leukocyte-antigen-related-like (Lar) is primarily required in germ cells to sustain spermatogenesis, whereas CG12404, doublesex-Mab-related 11E (dmrt11E), CG6769, estrogen-related receptor (ERR) and sulfateless (sfl) function in somatic cells. Interestingly, ERR and sfl are also required for testis morphogenesis. Our study thus demonstrates that SNPs without genome-wide significance in GWAS may also provide clues to disease-related genes and therefore warrant functional analysis.

Low-frequency germline variants across 6p22.2-6p21.33 are associated with non-obstructive azoospermia in Han Chinese men.

Genome-wide association studies (GWAS) have identified several common loci contributing to non-obstructive azoospermia (NOA). However, a substantial fraction of NOA heritability remains undefined, especially those low-frequency [defined here as having a minor allele frequency (MAF) between 0.5 and 5%] and rare (MAF below 0.5%) variants. Here, we performed a 3-stage exome-wide association study in Han Chinese men to evaluate the role of low-frequency or rare germline variants in NOA development. The discovery stage included 962 NOA cases and 1348 healthy male controls genotyped by exome chips and was followed by a 2-stage replication with an additional 2168 cases and 5248 controls. We identified three low-frequency variants located at 6p22.2 (rs2298090 in HIST1H1E encoding p.Lys152Arg: OR = 0.30, P = 2.40 × 10(-16)) and 6p21.33 (rs200847762 in FKBPL encoding p.Pro137Leu: OR = 0.11, P = 3.77 × 10(-16); rs11754464 in MSH5: OR = 1.78, P = 3.71 × 10(-7)) associated with NOA risk after Bonferroni correction. In summary, we report an instance of newly identified signals for NOA risk in genes previously undetected through GWAS on 6p22.2-6p21.33 in a Chinese population and highlight the role of low-frequency variants with a large effect in the process of spermatogenesis.

A Screen for Genomic Disorders of Infertility Identifies MAST2 Duplications Associated with Nonobstructive Azoospermia in Humans.

Since the cytogenetic identification of azoospermia factor regions 40 years ago, the Y chromosome has dominated research on the genetics of male infertility. We hypothesized that hotspots of structural rearrangement, which are dispersed across the genome, may mediate rare, recurrent copy number variations (CNVs), leading to severe infertility. We tested this hypothesis by contrasting patterns of rare CNVs in 970 Han Chinese men with idiopathic nonobstructive azoospermia and 1661 ethnicity-matched 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 (odds ratio [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), combined data). 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.

The rs3807989 G/A polymorphism in CAV1 is associated with the risk of atrial fibrillation in Chinese Han populations.

BACKGROUND: A recent meta-analysis of several genome-wide association studies identified six new susceptibility single nucleotide polymorphisms (SNPs) for atrial fibrillation (AF) in individuals of the European ancestry. We aimed to replicate the associations between these SNPs and the risk of AF in a Chinese Han population. METHODS: We genotyped six SNPs (rs3903239 in PRRX1, rs3807989 in CAV1, rs10821415 in C9orf3, rs10824026 in SYNPO2L, rs1152591 in SYNE2, and rs7164883 in HCN4) using the middle-throughput iPLEX Sequenom MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated in logistic regression models. RESULTS: We enrolled a total of 1,593 Chinese Han origin individuals in the study, including 597 AF patients and 996 non-AF controls. Among the six SNPs analyzed in the study, the SNP rs3807989 in CAV1 on chromosome 7q31 was found to be significantly associated with a decreased risk of AF (crude OR = 0.76, 95% CI: 0.64-0.89, P = 0.001; adjusted OR = 0.75, 95% CI: 0.63-0.89, P = 0.001). There were no significant associations between the other five loci and AF risk. CONCLUSION: Our results confirmed that CAV1 rs3807989 may contribute to a decreased AF risk in Chinese Han populations. However, further validation studies with different ethnic backgrounds and biological function analyses are warranted to confirm our finding.

Gene copy number alterations in the azoospermia-associated AZFc region and their effect on spermatogenic impairment.

The azoospermia factor c (AZFc) region in the long arm of human Y chromosome is characterized by massive palindromes. It harbors eight multi-copy gene families that are expressed exclusively or predominantly in testis. To assess systematically the role of the AZFc region and these eight gene families in spermatogenesis, we conducted a comprehensive molecular analysis (including Y chromosome haplogrouping, AZFc deletion typing and gene copy quantification) in 654 idiopathic infertile men and 781 healthy controls in a Han Chinese population. The b2/b3 partial deletion (including both deletion-only and deletion-duplication) was consistently associated with spermatogenic impairment. In the subjects without partial AZFc deletions, a notable finding was that the frequency of DAZ and/or BPY2 copy number alterations in the infertile group was significantly higher than in the controls. Combined patterns of DAZ and/or BPY2 copy number abnormality were associated with spermatogenic impairment when compared with the pattern of all AZFc genes with common level copies. In addition, in Y chromosome haplogroup O1 (Y-hg O1), the frequency of copy number alterations of all eight gene families was significantly higher in the case group than that in the control group. Our findings indicate that the DAZ, BPY2 genes may be prominent players in spermatogenesis, and genomic rearrangements may be enriched in individuals belonging to Y-hg O1. Our findings emphasize the necessity of routine molecular analysis of AZFc structural variation during the workup of azoospermia and/or oligozoospermia, which may diminish the genetic risk of assisted reproduction.

A smartphone-based crowd-sourced real-time surveillance platform (apple snail inspector) for the invasive snails: a design and development study.

BACKGROUND: The large amphibious freshwater apple snail is an important invasive species in China, but there is currently no method available for their surveillance. The development and popularization of smartphones provide a new platform for research on surveillance technologies for the early detection and effective control of invasive species. METHODS: The ASI surveillance system was developed based on the infrastructure of the WeChat platform and Amap. The user can directly enter the game interface through the WeChat port on their mobile phone, and the system automatically obtains their location. The user can then report the location of apple snails. The administrator can audit the reported information, and all information can be exported to Microsoft Excel version 2016 for analysis. The map was generated by ArcGIS 10.2 and was used to characterize the spatial and temporal distribution of apple snails in Jiangsu Province. RESULTS: The architecture of ASI consists of three parts: a mobile terminal, a server terminal and a desktop terminal. We published more than 10 tweets on the official WeChat account of the system to announce it to the public, and a total of 207 users in 2020 and 2021 correctly reported sightings of apple snails. We identified 550 apple snails breeding sites in 2020 and 2021, featuring ponds (81%), parks (17%) and farmland (2%). In addition, most of the locations contained snail eggs, and the reporting times mainly occurred between May and September. CONCLUSIONS: The ASI is an effective surveillance system that can be used to identify the breeding locations of apple snails and provides the basis of prevention and control for its dispersal. Its successful development and operation provide new potential avenues for surveillance of other public health issues.

Multi-intervention integrated deworming strategy for sustained control of soil-transmitted helminths infections: a case study in Jiangsu Province, China.

BACKGROUND: Soil-transmitted helminths (STH) infections still present a global health problem. Mass drug administration (MDA) is a widely applied strategy to reduce morbidity and mortality caused by STH. Yet, this approach has some shortcomings. In this study, we analyzed the impact of a multi-intervention integrated deworming approach including MDA, health education (HE), and environmental sanitation improvements (ESI) for sustained STH control in Jiangsu Province of China that was applied from 1989 to 2019. METHODS: Data, including infection rate of STH, medications used, coverage of the medication, non-hazardous lavatory rate, and household piped-water access rate in rural areas, and actions related to HE and ESI were collected (from archives) and analyzed in this retrospective descriptive study. Pearson's correlation analysis was applied to test correlations. RESULTS: There was a dramatic decline in the infection rate of STH from 1989 (59.32%) to 2019 (0.12%). From 1995 to 1999, MDA and HE were recommended in rural areas. A negative correlation was observed between infection rate and medication from 1994 to 1998 (r = - 0.882, P = 0.048). From 2000 to 2005, targeted MDA was given to high-risk populations with HE continuously promoting good sanitation behaviors. From 2006 to 2014, targeted MDA + HE and ESI were used to consolidate the control effect. ESI was strengthened from 2006, and a negative correlation was observed between the coverage rate of the non-hazardous lavatory and the infection rate from 2006 to 2019 (r = - 0.95, P < 0.001). The targeted MDA was interrupted in 2015, while continuous efforts like HE and ESI contributed in sustaining STH control. CONCLUSIONS: Multi-intervention integrated deworming strategy contributes to the reduction of STH infections. This approach is a valuable example of how different interventions can be integrated to promote durable STH control.

Seroprevalence and Risk Factors of Toxoplasma gondii Infection Among High-Risk Populations in Jiangsu Province, Eastern China.

Toxoplasma gondii, an opportunistic protozoan, infects one-third of people worldwide and could lead to serious outcomes in immunodeficient or immunocompromised populations. The present study aimed to investigate the prevalence and risk factors for T. gondii infection among high-risk populations in Jiangsu Province, eastern China. We conducted a cross-sectional survey among 4 categories of populations in 13 prefectures including HIV/AIDS patients, livestock breeding/processing (B/P) staff, pregnant women, and cancer patients. We detected specific immunoglobulin G and M (IgG and IgM) levels for each participant using enzyme-linked immunosorbent assay (ELISA) and asked to complete a questionnaire for each participant that covered sociodemographic information as well as the basic knowledge of attitudes toward and the practices for the prevention of toxoplasmosis. A total of 5231 participants distributed across 13 prefecture-level cities was surveyed, including 2455 males and 2776 females. Total seropositivity rate in each population category was as follows: 9.08% (HIV/AIDS patients), 11.65% (livestock B/P staff), 5.50% (pregnant women), and 12.89% (cancer patients). We detected IgM positivity in HIV/AIDS patients (0.47%, 6/1289), livestock B/P staff (0.08%, 1/1330), and cancer patients (0.46%, 6/1303) but not in pregnant women. Further, we detected IgM+IgG positivity only in cancer patients (0.31%, 4/1303). The seropositivity rate for pregnant women was significantly lower, while cancer patients were significantly higher. Higher educational levels were associated with lower seropositivity rates for T. gondii infection. High seropositivity rates were associated with long period of HIV infection among HIV/AIDS patients, frequent contact with livestock among livestock breeding/processing staff and male older patients among cancer patients, respectively. Analysis of practices across all participants showed that frequent contact with pets in everyday life or using the same cutting board for both raw and cooked foods leads to higher seropositivity rates. Therefore, we obtained the seroprevalence and risk factors of toxoplasmosis among high-risk populations in Jiangsu Province which could provide evidence for the implementation of control measures in the near future.

Evaluation of regulatory genetic variants in POU5F1 and risk of congenital heart disease in Han Chinese.

OCT4 is a transcription factor of the POU family, which plays a key role in embryonic development and stem cell pluripotency. Previous studies have shown that Oct4 is required for cardiomyocyte differentiation in mice and its depletion could result in cardiac morphogenesis in embryo. However, whether the genetic variations in OCT4 coding gene, POU5F1, confer the predisposition to congenital heart disease (CHD) is unclear. This study sought to investigate the associations between low-frequency (defined here as having minor allele frequency (MAF) between 0.1%-5%) and rare (MAF below 0.1%) variants with potential function in POU5F1 and risk of CHD. We conducted association analysis in a two-stage case-control study with a total of 2,720 CHD cases and 3,331 controls in Chinese. The low-frequency variant rs3130933 was observed to be associated with a significantly increased risk of CHD [additive model: adjusted odds ratio (OR) = 2.15, adjusted P = 3.37 × 10(-6)]. Furthermore, luciferase activity assay showed that the variant A allele led to significantly lower expression levels as compared to the G allele. These findings indicate for the first time that low-frequency functional variant in POU5F1 may contribute to the risk of congenital heart malformations.

Association analysis identifies new risk loci for congenital heart disease in Chinese populations.

Our previous genome-wide association study (GWAS) identified two susceptibility loci for congenital heart disease (CHD) in Han Chinese. Here we identify additional loci by testing promising associations in an extended 3-stage validation consisting of 6,053 CHD cases and 7,410 controls. We find GW significant (P<5.0 × 10(-8)) evidence of 4 additional CHD susceptibility loci at 4q31.22 (rs1400558, upstream of EDNRA, Pall=1.63 × 10(-9)), 9p24.2 (rs7863990, close to SMARCA2, Pall=3.71 × 10(-14)), 12q24.13 (rs2433752, upstream of TBX3 and TBX5, Pall=1.04 × 10(-10)) and 20q12 (rs490514, in PTPRT, Pall=1.20 × 10(-13)). Moreover, the data from previous European GWAS supports that rs490514 is associated with the risk of CHD (P=3.40 × 10(-3)). These results enhance our understanding of CHD susceptibility.

Genetic variants in Ser-Arg protein-coding genes are associated with the risk of nonobstructive azoospermia in Chinese men.

OBJECTIVE: To evaluate the association between genetic variants in Ser-Arg (SR) protein-coding genes and the susceptibility of nonobstructive azoospermia (NOA) in Chinese men. DESIGN: Case-control study. SETTING: State Key Laboratory of Reproductive Medicine in Nanjing Medical University conducted the genotyping and examined the expression levels of genes. PATIENT(S): The study included 962 NOA patients and 1,931 control subjects. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genotyping of 16 single-nucleotide polymorphisms (SNPs) of eight "canonic" SR protein-coding genes were performed with the use of the Illumina Infinium Beadchip platform. Odds ratios were calculated by logistic regression analysis in the additive model. Expression levels were measured by quantitative reverse-transcription polymerase chain reaction. RESULT(S): Rs17431717 near SFRS9 and rs12046213 near SFRS4 were significantly associated with a decreased risk of NOA, whereas rs10849753 near SFRS9 and rs6103330 in SFRS6 were associated with an increased risk of NOA. Of the two SNPs in SFRS9, only rs17431717 remained significant after conditioning on another. Combined analysis of three promising SNPs (rs17431717, rs12046213, and rs6103330) showed that compared with individuals with "0-2" risk alleles, those carrying "3," "4," and "≥ 5" risk alleles had 1.22-, 1.38-, and 1.90-fold increased risk of NOA, respectively. CONCLUSION(S): Polymorphisms in SR protein-coding genes may contribute to the risk of NOA in Chinese men. The findings of this study can help us to further understand the etiology of spermatogenic impairment, and they provide more evidence for the role of splicing activity in human spermatogenesis.

Maternal lifestyle factors in pregnancy and congenital heart defects in offspring: review of the current evidence.

The prognosis of children with congenital heart defects(CHDs) continues to improve with advancing surgical techniques; however, lack of information about modifiable risk factors for malformations in cardiovascular development impeded the prevention of CHDs. We investigated an association between maternal lifestyle factors and the risk of CHDs, because epidemiological studies have reported conflicting results regarding maternal lifestyle factors and the risk of CHDs recently. A review published on 2007 provided a summary of maternal exposures associated with an increased risk of CHDs. As part of noninherited risk factors, we conducted a brief overview of studies on the evidence linking common maternal lifestyle factors, specifically smoking, alcohol, illicit drugs, caffeine, body mass index and psychological factors to the development of CHDs in offspring. Women who smoke and have an excessive body mass index(BMI) during pregnancy are suspected to be associated with CHDs in offspring. Our findings could cause public health policy makers to pay more attention to women at risk and could be used in the development of population-based prevention strategies to reduce the incidence and burden of CHDs. However, more prospective studies are needed to investigate the association between maternal lifestyle factors and CHDs.

Association of aminoacyl-tRNA synthetases gene polymorphisms with the risk of congenital heart disease in the Chinese Han population.

Aminoacyl-tRNA synthetases (ARSs) are in charge of cellular protein synthesis and have additional domains that function in a versatile manner beyond translation. Eight core ARSs (EPRS, MRS, QRS, RRS, IRS, LRS, KRS, DRS) combined with three nonenzymatic components form a complex known as multisynthetase complex (MSC).We hypothesize that the single-nucleotide polymorphisms (SNPs) of the eight core ARS coding genes might influence the susceptibility of sporadic congenital heart disease (CHD). Thus, we conducted a case-control study of 984 CHD cases and 2953 non-CHD controls in the Chinese Han population to evaluate the associations of 16 potentially functional SNPs within the eight ARS coding genes with the risk of CHD. We observed significant associations with the risk of CHD for rs1061248 [G/A; odds ratio (OR) = 0.90, 95% confidence interval (CI) = 0.81-0.99; P = 3.81×10(-2)], rs2230301 [A/C; OR = 0.73, 95%CI = 0.60-0.90, P = 3.81×10(-2)], rs1061160 [G/A; OR = 1.18, 95%CI = 1.06-1.31; P = 3.53×10(-3)] and rs5030754 [G/A; OR = 1.39, 95%CI = 1.11-1.75; P = 4.47×10(-3)] of EPRS gene. After multiple comparisons, rs1061248 conferred no predisposition to CHD. Additionally, a combined analysis showed a significant dosage-response effect of CHD risk among individuals carrying the different number of risk alleles (Ptrend = 5.00×10(-4)). Compared with individuals with "0-2" risk allele, those carrying "3", "4" or "5 or more" risk alleles had a 0.97-, 1.25- or 1.38-fold increased risk of CHD, respectively. These findings indicate that genetic variants of the EPRS gene may influence the individual susceptibility to CHD in the Chinese Han population.

Genetic polymorphisms in ZFHX3 are associated with atrial fibrillation in a Chinese Han population.

BACKGROUND: The gene zinc finger homeobox 3 (ZFHX3) encodes a transcription factor with cardiac expression and its genetic variants are associated with atrial fibrillation (AF). We aimed to explore the associations between single nucleotide polymorphisms (SNPs) of ZFHX3 and the risk of AF in a Chinese Han population. METHODS: We genotyped eight SNPs, including seven potentially functional SNPs and one previously reported SNP by using the middle-throughput iPLEX Sequenom MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated in logistic regression models. RESULTS: We enrolled a total of 1,593 Chinese Han origin individuals in the study, including 597 AF patients and 996 non-AF controls. Logistic regression analyses revealed that potentially functional SNPs rs6499600 and rs16971436 were associated with a decreased risk of AF (adjusted OR  = 0.73, 95% CI: 0.63-0.86, P = 1.07×10-4; adjusted OR  = 0.74, 95% CI: 0.56-0.98, P = 0.039, respectively). In addition, rs2106261 showed a robust association with an increased risk of AF (adjusted OR  = 1.71, 95% CI: 1.46-2.00, P = 1.85×10-11). After multiple comparisons, rs16971436 conferred a borderline significant association with the risk of AF. Stratification analysis indicated that the risks of AF were statistically different among subgroups of age for rs2106261, and the effect for rs16971436 was more evident in subgroups of patients with coronary artery disease. CONCLUSION: In summary, our study investigated the role of genetic variants of ZFHX3 in AF and two SNPs (rs2106261, rs6499600) showed significant associations while rs16971436 conferred a borderline significant association with AF risk in Chinese Han populations. However, further large and functional studies are warranted to confirm our findings.

Replication of the 4p16 susceptibility locus in congenital heart disease in Han Chinese populations.

Congenital heart disease (CHD) is the most common form of congenital human birth anomalies and a leading cause of perinatal and infant mortality. Some studies including our published genome-wide association study (GWAS) of CHD have indicated that genetic variants may contribute to the risk of CHD. Recently, Cordell et al. published a GWAS of multiple CHD phenotypes in European Caucasians and identified 3 susceptibility loci (rs870142, rs16835979 and rs6824295) for ostium secundum atrial septal defect (ASD) at chromosome 4p16. However, whether these loci at 4p16 confer the predisposition to CHD in Chinese population is unclear. In the current study, we first analyzed the associations between these 3 single nucleotide polymorphisms (SNPs) at 4p16 and CHD risk by using our existing genome-wide scan data and found all of the 3 SNPs showed significant associations with ASD in the same direction as that observed in Cordell's study, but not with other subtypes- ventricular septal defect (VSD) and ASD combined VSD. As these 3 SNPs were in high linkage disequilibrium (LD) in Chinese population, we selected one SNP with the lowest P value in our GWAS scan (rs16835979) to perform a replication study with additional 1,709 CHD cases with multiple phenotypes and 1,962 controls. The significant association was also observed only within the ASD subgroup, which was heterogeneous from other disease groups. In combined GWAS and replication samples, the minor allele of rs16835979 remained significant association with the risk of ASD (OR = 1.22, 95% CI = 1.08-1.38, P = 0.001). Our findings suggest that susceptibility loci of ASD identified from Cordell's European GWAS are generalizable to Chinese population, and such investigation may provide new insights into the roles of genetic variants in the etiology of different CHD phenotypes.