RNF115在左右不对称建立及内脏异位综合征发生中作用机制的研究

Heterotaxy syndrome results from failure of the developing embryo to establish normal left-right asymmetry, seriously endangers the health of children. Its manifestations often include complex congenital heart defects. And its molecular mechanism still needs further elucidation. Previously we recruited a cohort of children suffered from heterotaxy syndrome, and analyzed the known causal genes mutations and copy number variants profiles of them. A rare heterozygous deletion of 302kb in 1q21.1 chromosome was validated in one of patients. Preliminary study using the zebrafish morpholino model showed that the E3 ubiquitin ligase RNF115 in the deletion fragment may be a new candidate gene of heterotaxy syndrome. To further illustrate the funcion of RNF115 on the establishment of left-right asymmetry and the heterotaxy syndrome pathogenesis, the following experiments will be performed: (1) RNF115 mutation identification in heterotaxy syndrome and functional analysis of mutant proteins; (2) the establishment of rnf115 knock-out zebrafish model to identify the molecular mechanism of RNF115 in the establishment of left-right asymmetry and the heterotaxy syndrome pathogenesis. This study is expected to not only intensity our understanding of molecular machenism underlying the establishment of left-right asymmetry and heterotaxy syndrome, but also provide evidences for early diagnosis, genetic counseling and treatment for heterotaxy syndrome and complex congenital heart defects.

内脏异位是常合并复杂心脏畸形的出生缺陷，严重危害新生儿健康，其分子机制尚远未阐明。本课题基于前期工作中在内脏异位患儿发现的不含已知致病基因的1q21.1区段 302kb片段缺失，通过初步的斑马鱼morpholino干扰基因表达实验分析，发现片段内RNF115可能为新的内脏异位致病基因。为进一步阐明RNF115在左-右不对称建立及内脏异位发生中的作用，本课题首先筛查内脏异位患儿中RNF115突变，分析突变对蛋白功能的影响；并建立rnf115基因敲除斑马鱼模型，检测左-右不对称标志性基因表达模式，Kuffer’s囊泡内纤毛数量、长度及运动能力，左右不对称信号通路及纤毛相关基因有无泛素化修饰等明确rnf115的作用。本项目研究将拓宽对左-右不对称建立及内脏异位发生机制的认识，为今后开发内脏异位临床早期诊断、遗传咨询及治疗提供一定的依据，同时为理解与内脏异位紧密相关的复杂先心病发生机制提供线索。

内脏异位（HTX）是常合并复杂心脏畸形的出生缺陷，严重危害新生儿健康，其分子机制尚远未阐明。本研究通过外显子测序分析中国汉族内脏异位综合征患儿相关基因变异，结果显示：1、通过病例-对照分析，Sanger测序验证，及生物信息学软件分析，确认中国汉族内脏异位综合征患儿DNAI1和DNAH5基因突变发生率分别为4.94%和2.5%，DNAI1及DNAH5基因编码区变异与内脏异位的发生相关。2、通过家系样本Sanger测序证实HTX患儿MMP21-G244E，MMP21-L277F，MMP21-K487E突变为新发突变。通过真核及原核细胞过表达野生型及突变型MMP21质粒构建，MMP21野生型及突变型表达分析显示三个变异位点不影响MMP21蛋白的表达。通过斑马鱼反义寡核苷酸（morpholino, MO）实验及MMP21野生型及突变型mRNA回补实验结果显示在HTX患儿中发现的mRNA(MMP21-G244E)和mRNA(MMP21-K487E)变异不能有效回补斑马鱼心管环化异常，且杂合子mRNA(WT&p.G244E)、mRNA(WT&p.K487E)也不能有效回补心管环化异常。提示杂合子的MMP21变异与中国汉族儿童HTX发生相关。3、通过建立home-made外显子分析pipeline，分析HTX及房室间隔缺损（AVSD）患儿外显子测序数据，发现DUSP27是潜在中国HTX及AVSD新致病基因，Sanger测序及生物信息学软件证实突变位点在患儿中存在且具有致病性。利用Crispr-Cas9技术构建Dusp27敲除小鼠，证实Dusp27在小鼠胚胎发育中发挥重要作用，且纯合敲除小鼠出现房室间隔缺损及内脏异位相关的心脏畸形。本课题进一步完善了中国汉族内脏异位患儿突变谱，为进一步理解内脏异位发病机制，研究用于疾病早期诊断的分子标志提供基础。