Ura Hiroki, Togi Sumihito, Niida Yo
Center for Clinical Genomics, Kanazawa Medical University Hospital, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 920-0923, Japan.
Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 920-0923, Japan.
Biology (Basel). 2021 Mar 24;10(4):256. doi: 10.3390/biology10040256.
There are two combinations of heterozygous mutation, i.e., which carries mutations on different alleles, and which carries mutations on the same allele. Because only compound heterozygous mutations have been implicated in autosomal recessive diseases, it is important to distinguish them for clinical diagnosis. However, conventional phase analysis is limited because of the large target size of genomic DNA. Here, we performed a genetic analysis on a patient with Wilson disease, and we detected two heterozygous mutations chr13:51958362;G>GG (NM_000053.4:c.2304dup r.2304dup p.Met769HisfsTer26) and chr13:51964900;C>T (NM_000053.4:c.1841G>A r.1841g>a p.Gly614Asp) in the causative gene . The distance between the two mutations was 6.5 kb in genomic DNA but 464 bp in mRNA. Targeted double-stranded cDNA sequencing-based phase analysis was performed using direct adapter ligation library preparation and paired-end sequencing, and we elucidated they are compound heterozygous mutations. Trio analysis showed that the mutation (chr13:51964900;C>T) derived from the father and the other mutation from the mother, validating that the mutations are composition. Furthermore, targeted double-stranded cDNA sequencing-based phase analysis detected the differential allelic expression, suggesting that the mutation (chr13:51958362;G>GG) caused downregulation of expression by nonsense-mediated mRNA decay. Our results indicate that targeted double-stranded cDNA sequencing-based phase analysis is useful for determining compound heterozygous mutations and confers information on allelic expression.
存在两种杂合突变组合，即携带不同等位基因上突变的组合，以及携带相同等位基因上突变的组合。由于只有复合杂合突变与常染色体隐性疾病有关，因此在临床诊断中区分它们很重要。然而，由于基因组DNA的靶标尺寸较大，传统的相位分析受到限制。在这里，我们对一名威尔逊病患者进行了基因分析，在致病基因中检测到两个杂合突变，分别是chr13:51958362;G>GG（NM_000053.4:c.2304dup r.2304dup p.Met769HisfsTer26）和chr13:51964900;C>T（NM_000053.4:c.1841G>A r.1841g>a p.Gly614Asp）。这两个突变在基因组DNA中的距离为6.5 kb，但在mRNA中为464 bp。使用直接衔接子连接文库制备和双末端测序进行基于靶向双链cDNA测序的相位分析，我们阐明它们是复合杂合突变。三联体分析表明，突变（chr13:51964900;C>T）来自父亲，另一个突变来自母亲，证实这些突变是复合组成。此外，基于靶向双链cDNA测序的相位分析检测到差异等位基因表达，表明突变（chr13:51958362;G>GG）通过无义介导的mRNA降解导致表达下调。我们的结果表明，基于靶向双链cDNA测序的相位分析可用于确定复合杂合突变，并提供等位基因表达信息。
