Table1_Novel method for the genomic analysis of PKD1 mutation in autosomal dominant polycystic kidney disease.DOCX

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Although next-generation sequencing (NGS) technology can be used to sequence tens of thousands of DNA molecules simultaneously. It has poor capture efficiency for the six PKD1 pseudogenes and GC-rich regions. Multiplex ligation-dependent probe amplification (MLPA) technology can detect consecutive deletions of exons, but it is less sensitive for single-base mutations. However, pathogenic genes might not be detected in some patients, even when using the above methods. Improving the detection rate of pathogenic genes is an important technical problem hindering clinical diagnosis of ADPKD. Four pedigrees of ADPKD patients with mutation sites not identified by NGS were examined by other methods. First, MLPA was performed. Then, pedigrees in which MLPA did not identify pathogenic genes were subjected to multiplex polymerase chain reaction (MPCR) and targeted region sequencing. Finally, the detected mutation sites were verified by Sanger sequencing. The results showed that MLPA detected the following PKD1 exonic deletion mutations in three pedigrees: PKD1-18 nt–290 nt, PKD1-up-257 nt, PKD1-up-444 nt and PKD1-3 nt–141 nt. A new mutation site was identified through targeted region sequencing in one pedigree: PKD1 NM_001009944: c.151T > C at the protein level, described as p. Cys51Arg. In summary, we established a system of genetic detection and analytical methods, from NGS to MLPA to targeted region sequencing and finally to Sanger sequencing. We combined MPCR and targeted region sequencing for the first time in ADPKD diagnosis, which further improved diagnosis accuracy. Moreover, we identified one new missense mutation and four new deletion mutations.

常染色体显性遗传性多囊肾病（Autosomal dominant polycystic kidney disease, ADPKD）是最常见的遗传性肾脏疾病。尽管下一代测序技术（next-generation sequencing, NGS）可同时对数万个DNA分子进行测序，但该技术对6种PKD1假基因及GC富集区域的捕获效率欠佳。多重连接依赖探针扩增技术（multiplex ligation-dependent probe amplification, MLPA）可检测外显子连续缺失突变，但对单碱基突变的灵敏度不足。即便采用上述两种技术，仍有部分患者无法检出致病基因，提升致病基因检出率是阻碍ADPKD临床诊断的关键技术难题。本研究对4个经NGS未检出突变位点的ADPKD患者家系开展了联合检测：首先实施MLPA检测；对于MLPA未检出致病基因的家系，进一步开展多重聚合酶链反应（multiplex polymerase chain reaction, MPCR）及靶向区域测序；最终通过桑格测序对检出的突变位点进行验证。结果显示，MLPA技术在3个家系中检出PKD1外显子缺失突变，具体为PKD1-18 nt–290 nt、PKD1-up-257 nt、PKD1-up-444 nt及PKD1-3 nt–141 nt；另有1个家系经靶向区域测序检出1个全新突变位点：PKD1 NM_001009944: c.151T>C，蛋白水平表现为p.Cys51Arg。综上，本研究建立了一套覆盖NGS、MLPA、靶向区域测序至桑格测序的遗传检测与分析体系，并首次将MPCR与靶向区域测序联合应用于ADPKD诊断，进一步提升了诊断准确率。此外，本研究还检出1个全新错义突变与4个全新缺失突变。