The Association Between RNF213 , MMP3 , and Moyamoya Disease: Screening the Full Exons of Susceptibility Genes in Families with Moyamoya Disease

Abstract

Background: In this study, we aimed to investigate the association between polymorphisms in the RNF213 and MMP3 genes and Moyamoya disease (MMD) and to screen susceptibility genes for families with MMD.Methods: The mutation of RNF213 gene (rs112735431,rs148731719) and MMP3 gene rs3025058 were detected by first generation sequencing and CRISPR-Cas12a. The genetic susceptibility genes of MMD families samples were studied by whole exome sequencing (WES). Results: The application of CRISPR-Cas12a and Sanger sequencing identified rs112735431 and rs148731719 mutations in the RNF213 gene, and an insertion mutation in the MMP3 gene (rs3025058) with significant regional differences (P<0.05). Additionally, we identified 100% agreement between CRISPR-Cas12a and Sanger sequencing with regards to the detection of these mutations in samples, thus indicating that CRISPR-Cas12a can be used for the detection of MMD mutations. Next, we carried out whole exon screening for family members with MMD and identified multiple recessive genes associated with genetic disease. Notably, the Titin (TTN) gene (rs771533925, rs559712998 and rs72677250) was significantly associated with point mutations. Screening of the Exome Aggregation Consortium (EXAC) database identified significant differences between these populations with respect to frequency. PROVEAN，nvariant Feature Transform (SIFT) and PolyPhen algorithms further demonstrated that rs771533925 and rs72677250 were potentially damaging in three databases, rs559712998 was considered to be tolerated in above mentioned databases. Scale Invariant Feature Transform (SIFT) and PolyPhen algorithms further demonstrated that rs771533925 and rs72677250 were potentially damaging by all three databases and that rs559712998 was considered to be tolerated in above mentioned databases. Gene Ontology (GO) analysis demonstrated that the target of TTN is involved in many important biological processes. The screening of 50 sporadic MMD samples failed to identify any mutations, thus indicating that single nucleotide polymorphism (SNP) mutation sites (rs771533925, rs559712998, and rs72677250) within the TTN gene may play an important role in the inheritance of MMD. Conclusions: In summary, our study investigated susceptibility genes for MMD from multiple perspectives and found that CRISPR-Cas12a technology can efficiently detect MMD mutation sites. Our findings provide a theoretical basis for future investigations of the molecular mechanisms that underlie MMD.