近亲家系中FANCM突变导致无精子症的研究

Azoospermia, which is defined as no sperm in semen for at least couple of examinations, is reported in up to 10-20% of infertile men. Its molecular basis and mechanism are needed to be elucidated. Previously, we found homologous mutation of FANCM gene in azoospermia patients from a Pakistani consanguineous family. Thus, we hypothesize that mutation in FANCM may induce azoospermia. To prove this, we will first conduct the histological examination of testicular tissue from patients and analyse spermatogenesis; then we will try to locate the disease causing gene through exon sequencing and bioinformatic analysis; this will be followed up by generation of FANCM knock-out and knock-in mouse model to confirm the specific role of FANCM in azoospermia patients and finally we will try to determine the expression and localization of FANCM in FANCM mutated testicular tissue. Our work will confirm that mutation in FANCM can cause azoospermia in human and will reveal its pathogenic mechanism, providing molecular markers for genetic diagnosis of azoospermia patients and in early embryos derived from assisted reproductive technology.

无精子症是至少两次精液检查均未发现精子，约占男性不育的10-20%。但对人无精子症的分子基础和机制，了解甚少。我们在巴基斯坦近亲结婚家系的无精子症患者中发现了FANCM纯合突变，并假设：FANCM突变导致了人无精子症。为证明FANCM是人无精子症致病突变，拟开展以下工作：1.家系鉴定、收集-通过睾丸组织病理和精子发生进程分析，发现无精子症患者；2.遗传筛选-用全外显子测序和生物信息学分析，发现候选无精子症致病突变；3.功能鉴定-分析FANCM敲除和突变敲入小鼠的精子发生，确定突变是否致无精子症；4.病理确认-用FANCM突变携带者睾丸组织，对FANCM及关联分子进行表达定位分析。以期发现并证实FANCM突变是人类无精子症致病原因、揭示致病机制，为无精子症患者基因诊断和人工辅助生殖所获胚胎遗传诊断，提供分子标志。

本项目中，我们对一个巴基斯坦近亲结婚后代发生男性不育的家系进行了研究，以期揭示其不育的原因。该家系父母为表兄妹结婚，生育四个儿子，其中三个不育，两位为少弱精子症患者，另一位是无精子症患者。通过全外显子组和Sanger测序，筛选到范可尼贫血（Fanconi Anemia, FA）核心复合物成分FANCM的一个移码突变（c.1946_1958del，p.P648Lfs*16）与不育症共分离，认为是该家系不育患者的潜在致病突变。我们检测患者外周血淋巴细胞对MMC诱发染色体断裂的敏感性，发现相比于其父亲，患者的淋巴细胞呈现染色体断裂增加，且断裂频率随MMC浓度的增高而增多，提示该FANCM突变破坏其功能，并导致ICL损伤修复缺陷。进一步的HEK293T细胞实验证实，该FANCM移码突变可导致截短蛋白形成，使FANCM 不能定位于ICL 位点，进而引起FANCD2不能被单泛素化、FA通路不能激活，最终导致ICL无法修复和细胞死亡。此外，我们制备了携带与患者几乎一致的Fancm突变（Fancm∆C/∆C）小鼠，发现纯合突变小鼠与患者表型类似，即：精子发生障碍、生育力大幅降低。具体表现为精子数目显著减少、运动能力降低，各级生精细胞大量丢失和圆形精细胞停滞。因此，本项目第一次发现FANCM纯合突变导致生精障碍，引起男性不育。