Kuang Xinyu, Sun Lei, Wu Ying, Huang Wenyan
Department of Nephrology and Rheumatology, Shanghai Children's Hospital, Children's Hospital of Shanghai Jiao Tong University, Shanghai, China.
Transl Pediatr. 2020 Oct;9(5):587-595. doi: 10.21037/tp-20-47.
X-linked Alport syndrome (XLAS) is the most common form of Alport syndrome (AS), involves mutations in the gene encoding the type IV collagen a5 chain. In this research, we will report the analysis of the gene in a Chinese family with XLAS, and investigate the effect of the missense mutation of this family on type IV collagen.
Targeted sequencing using next-generation sequencing (NGS) was conducted for genes (COL4A3/4/5). Normal and mutation COL4A5 plasmids were constructed and then transfected into human podocytes, none plasmid and empty plasmid transfection as control. And then real-time PCR, western blot and indirect immunofluorescence were used to detect the COL4A1/3/5 mRNA, protein, and immunofluorescence expression of each group.
In this study, we found an Alport family, and the whole exon sequencing found a new missense mutation c.1844G>C in exon 25. The results of real-time PCR, western blot and immunofluorescence showed that in the mutation group, both the mRNA and protein levels of COL4A5 were significantly reduced.
c.1844G>C is a functional variation of COL4A5, which might play a very important role in the occurrence and development of AS.
X连锁遗传性肾炎（XLAS）是遗传性肾炎（AS）最常见的类型，涉及编码IV型胶原α5链的基因突变。在本研究中，我们将报告一个中国XLAS家系的基因分析，并研究该家系错义突变对IV型胶原的影响。
采用下一代测序（NGS）对基因（COL4A3/4/5）进行靶向测序。构建正常和突变的COL4A5质粒，然后转染到人足细胞中，以无质粒和空质粒转染作为对照。然后使用实时PCR、蛋白质印迹法和间接免疫荧光法检测每组的COL4A1/3/5 mRNA、蛋白质和免疫荧光表达。
在本研究中，我们发现了一个遗传性肾炎家系，全外显子测序在外显子25中发现了一个新的错义突变c.1844G>C。实时PCR、蛋白质印迹法和免疫荧光法的结果表明，在突变组中，COL4A5的mRNA和蛋白质水平均显著降低。
c.1844G>C是COL4A5的一个功能性变异，可能在遗传性肾炎的发生和发展中起非常重要的作用。
