MYH14基因突变在先天性肛门直肠畸形发生中的作用研究

Anorectal malformation (ARM) is the most common birth defect in the digestive system, and its genetic cause remains to be elucidated. In the early stage, we found that the same homozygous missense mutation (c.5393C>A /p.A1806D) exists in the MYH14 gene in two ARM children through whole exome sequencing in the ARM family of 8 person, suggesting the genetic pattern of the family is autosomal recessive. Bioinformatics prediction and molecular modeling analysis showed that the mutation was harmful. Pre-experiment showed that the non-muscle myosin heavy chain IIC (NMHC IIC) encoded by MYH14 gene was widely expressed in various stages of embryonic mouse cloaca development. Therefore, we propose the hypothesis: MYH14 gene is a novel candidate gene for ARM. In this study, we plan to investigate the effects of MYH14 gene mutation on the cytoskeleton, cell migration and adhesion of HMC cells by plasmid construction, cell transfection and immunofluorescence. At the same time, we prepare for constructing the homozygous MYH14 p.A1806D mouse to define the role of MYH14 gene in mouse cloaca development, and finally expanding the sample to identify the variation spectrum of MYH14 gene in ARM children, which is providing a new theoretical basis for the prevention and treatment of ARM.

先天性肛门直肠畸形（ARM）是最为常见的消化系统出生缺陷，其遗传学病因仍待阐明。前期我们通过一个ARM家系三代8人全外显子组测序发现两名ARM患儿在MYH14基因上存在同一纯合错义突变(c.5393C>A /p.A1806D)，提示该家系的遗传模式为常染色体隐性遗传。生物信息学预测及分子建模分析均显示该变异为有害变异，预实验显示MYH14基因编码的非肌肉肌球蛋白IIC重链蛋白（NMHC IIC）在胎鼠泄殖腔发育各个阶段均广泛表达。因此我们提出假说：MYH14基因为ARM新候选致病基因。本课题拟通过质粒构建、细胞转染和免疫荧光等观察MYH14基因突变对人直肠黏膜上皮细胞骨架、细胞迁移及黏附能力的影响；同时构建MYH14基因p.A1806D纯合型小鼠明确MYH14基因在小鼠泄殖腔发育中的作用，最后扩大样本明确MYH14基因在ARM患儿中的变异谱，为ARM的防治提供新的理论依据。

肛门直肠畸形（anorectal malformations，ARM）是最常见的先天性消化道畸形，其遗传因素多样，遗传机制不明。本项目组前期发现一个携带有MYH14基因新突变p.A1806D的ARM家系，先证患儿及其姐姐均为该新发突变纯合子。俩患儿均表现为低位肛门闭锁，患儿父母亲、祖父母、外祖父母均无相关症状。. 为明确MYH14基因新突变p.A1806D对肛门直肠发育的致病性和MYH14及相关易感基因在中国人群ARM患儿中的突变率及致病谱，进行了实验探索：1.利用CRISPR/Cas9技术模拟患者MYH14基因新突变p.A1806D构建了Myh14基因p.A1802D (c.5405C>A)突变敲入小鼠模型，未见纯合、杂合突变小鼠有明显肛门闭锁表现；2.胚胎期取出正常胎鼠与纯合突变胎鼠，正中矢状面连续切片显示纯合突变胎鼠尿道直肠隔突破肛膜的时间要晚于正常胎鼠；3.胎鼠泄殖腔区域Myh14的空间表达结果显示MYH14基因p.A1806D错义突变未改变MYH14蛋白表达；4.对67例ARM患儿的相关易感基因（MYH14、HOXA13、HOXD13、SALL1、SALL4、ZIC3）进行突变检测分析，结果显示多种基因的多个突变在ARM中均可能有致病性。. 本研究成功构建了模拟人类MYH14基因突变p.A1806D小鼠模型，明确了MYH14基因新突变p.A1806D在肛门直肠发育过程中的致病性。更是在多例ARM患儿中筛查了多个易感基因，拓宽了中国人群中ARM患儿易感基因的突变率及变异谱。