CRISPR Detection and Research on Screening Mutant Gene TTN of Moyamoya Disease Family Based on Whole Exome Sequencing.

Yilei Xiao,Zhaogang Dong,Liyong Zhang,Jiyue Wang,Xingbang Wang,Weidong Liu,Jiheng Hao,Donghu Yu,Qunlong Jiang

doi:10.3389/fmolb.2022.846579

Abstract

Moyamoya disease (MMD) has a high incidence in Asian populations and demonstrates some degree of familial clustering. Whole-exome sequencing (WES) is useful in establishing key related genes in familial genetic diseases but is time-consuming and costly. Therefore, exploring a new method will be more effective for the diagnosis of MMD. We identified familial cohorts showing MMD susceptibility and performed WES on 5 affected individuals to identify susceptibility loci, which identified point mutation sites in the titin (TTN) gene (rs771533925, rs559712998 and rs72677250). Moreover, TTN mutations were not found in a cohort of 50 sporadic MMD cases. We also analyzed mutation frequencies and used bioinformatic predictions to reveal mutation harmfulness, functions and probabilities of disease correlation, the results showed that rs771533925 and rs72677250 were likely harmful mutations with GO analyses indicating the involvement of TTN in a variety of biological processes related to MMD etiology. CRISPR-Cas12a assays designed to detect TTN mutations provided results consistent with WES analysis, which was further confirmed by Sanger sequencing. This study recognized TTN as a new familial gene marker for moyamoya disease and moreover, demonstrated that CRISPR-Cas12a has the advantages of rapid detection, low cost and simple operation, and has broad prospects in the practical application of rapid detection of MMD mutation sites.

Highlights

Moyamoya disease (MMD) is a chronic progressive, cerebrovascular, and occlusive disease of unknown etiology first reported by Suzuki in 1969 (Kuroda and Houkin, 2008)
According to age analysis of these three loci within the global population, we found that the rs72677250 mutation site was predominant in subjects aged 50–55 years, the rs559712998 mutation site was predominant in subjects aged 30–80 years, and the rs771533925 mutation site was predominant in subjects aged 65–70 years (Figures 5A–C)
The results showed that rs112735431 and rs148731719 mutations of the ring finger protein 213 (RNF213) gene were successfully detected by the CRISPR-Cas12a system with 100% agreement with the results of Sanger sequencing

Summary

Introduction

Moyamoya disease (MMD) is a chronic progressive, cerebrovascular, and occlusive disease of unknown etiology first reported by Suzuki in 1969 (Kuroda and Houkin, 2008). The incidence of MMD is higher in China, Korea and Japan, among which MMD is the main cause of stroke in children and adolescents (Kim, 2016; Zhao et al, 2018; Deng et al, 2021). Previous studies have shown a higher incidence of moyamoya disease in East Asia, among which, in China, the incidence of moyamoya disease in the north is significantly higher than in the south (Hu et al, 2017). 10–15% of MMD patients have a family history, and the prevalence of these people with a family history is 30–40% higher than that of ordinary people (Kim, 2016). It is easier to obtain potential genetic related genes through the research on family patient

Methods

Discussion

Conclusion