SYCE1 R16*突变是导致人类无精子症的致病原因

Azoospermia is termed as the absence of sperm in more than two different ejaculate samples.Approximately 1% of all men in the general population suffer from azoospermia, of which about 87% are caused by testicular azoospermia. However, the molecular mechanisms of testicular azoospermia are largely unknown. Our previous results found SYCE1 nonsense mutation((SYCE1 R16*/ R16*)), a synaptonemal complex component gene, in azoospermia with spermatocyte development arrest. The pathogenesis of SYCE1 mutation in azoospermia is still unknown. Accordingly, we will firstly construct the plasmid with SYCE1 normal or mutant coding sequencing(CDS), and then transfect into cell line to check their mRNA and protein expression respectively. Then, the knock-in mice expressing human SYCE1 normal or mutant CDS will be generated from the Syce1+/- mice and analysis the phenotype related with synapsis and recombination. Finally, to confirm the function of mutant SYCE1, we will check SYCE1 and its associated molecules expression in the patient. Based on the results, we will confirm the SYCE1 mutation function and how it cause azoospermia. It will be used as diagnostic and genetic screening biomarkers for azoospermia patients and embryos derived from assistant reproductive technology by testicular sperm from azoospermia patients.

无精子症是两次以上精液检查均未发现精子，约占所有男性的1%，其中约87%由睾丸自身因素所致。但对人类无精子症的致病机理，了解甚少。我们发现精母细胞发育停滞的无精子症患者中出现联会复合体关键基因SYCE1纯合无义突变（SYCE1 R16*/ R16*）。但对SYCE1该突变的致病原因仍不清楚。因此，拟开展以下工作：1.体外表达检测-构建SYCE1突变质粒，转染细胞系或培养睾丸组织，检测突变对SYCE1表达的影响； 2.小鼠体内功能鉴定-在基因型为Syce1+/-小鼠背景下，构建分别含表达人SYCE1正常及突变CDS的基因敲入小鼠，分析精子发生和联会重组情况及相关蛋白质表达和定位； 3. 患者病理确认-利用携带突变患者睾丸组织，对SYCE1及相关分子进行表达定位分析；以期确认SYCE1突变的致病原因，揭示其致病机理，为无精子症患者基因诊断和辅助生殖中胚胎遗传筛查，提供分子标志。

本项目利用全外显子、体外模型等方法，从散发减数分裂异常患者的潜在致病突变筛选中发现SYCE1突变，进一步的患者组织特征分析，发现患者的睾丸内没有任何减数分裂分裂后细胞，最晚只能看到精母细胞；对从患者中测序发现的突变，我们同时还对外显子测序结果进行了验证，确认了该突变位点的正确性。针对SYCE1所携带的nonsense突变，我们构建了包含突变位点的cDNA表达载体，转染细胞系后检测蛋白的表达情况。通过对GFP报告基因的检测，发现SYCE1基因携带突变后将导致蛋白无法正常表达等。并通过项目实施，发表论文1篇，培养硕士生1人。