A Study About Establishing The Genetic Test Platform For Citrin Deficiency Based On Targeted Sequence Capture And High-throughput Sequencing And Its Clinical Applications

Citrin deficiency, also known as Type II citrullinaemia is an autosomal recessive metabolic disorder, which is caused b y pathogenic mutations in the SLC25A13 gene on chromosome 7q21.3. In recent years, the study found that the genetic disease is cosmopolitan, but in East Asia especially frequent, including china. The identification of biallelic pathogenic variants in SLC25A13 confirms the diagnosis of citrin deficiency. Sanger sequence anal ysis is the most common method for citrin deficiency genetic testing but its shortcomings have complex operation, low-throughput and so on. PCR-RFLP also is used for citrin deficiency genetic testing but it can not detect the new mutation. In this study, we created a new test platform for SLC25A13 gene, combined with targeted sequence capture and next-generation sequencing.In this study, a new test platform combined with high-throughput sequencing technology and target sequence capture technique is tested using the Chinese YH g DNA for straightening out the whole test process and determin ing reads data filtering method of the platform. Subsequently, the platform detections 100 g DNA samples of normal human and g DNA samples of 2 patients of citrin deficiency. In order to screen ing pathogenic new candid ate mutations, a series of prediction software（such as SIFT, Polyphen, GERP, LRT etc.） are used for harmful and conservative prediction of new gene variations detected to eventually find new mutations associated with the disease. The results include 100 normal SLC25A13 and ASS1 Chinese gene variations frequency table and that four heterozygous mutations are found in the SLC25A13 gene of 2 patients of citrin deficiency: c.851₈54del GTAT/c.1177+1G>A and c.754G>A/c.1177+1G>A. The c.851₈54del GTAT and c.1177+1G>A mutations have been reported. The c.754G>A（p.Glu252Lys） has not been previously reported at home and abroad, forecasting anal ysis that the position is highly conserved and th is new mutation is harmful or pathogenicity. The classical Sanger sequencing is used for patients and their parents to validate the mutations detected b y high-throughput sequencing combinated with target sequence capture, and then the results of Sanger sequencing are consistent with high-throughput sequencing. At the same time, the results of Sanger sequencing also show that the patients are given a pathogenic mutation from both of the parents and have compound heterozygous mutations in SLC25A13 gene, and it consistents with autosomal recessive manner of citrin deficiency.Targeted next-generation sequencing platform has shown considerable potential and value in both clinical and research applications in this study. In our research, targeted sequence capture and Hi Seq2000 high-throughput sequencing were emplo yed. The combination of targeted sequence capture and next-generation sequencing with bioinformatics anal yses can be an effective method of genetic testing for some diseases with a clear nosogenesis, including Citrin deficiency examined in this study.