| Summary: | 碩士 === 國立臺灣大學 === 分子醫學研究所 === 104 === ADPKD stands for autosomal dominant polycystic kidney disease, it affects 1/400~1/1000 individuals worldwide. It is a late on-set disease and patients often require dialysis or renal transplant when progressed into ESRD (end-stage renal disease). PKD1 and PKD2 are the causative genes for ADPKD. The large number of exons and homologous pseudogenes of PKD1 makes genetic testing challenging. In this study we perform both LRPCR (long-range polymerase chain reaction), the gold standard testing for PKD1, and probe capture as target enrichment methods, combined with region enhancement to improve coverage on high GC-content exons for NGS (next-generation sequencing) testing. All possible pathogenic variants were classified according to PKDB (PKD database) or ACMG guideline followed by Sanger sequencing for validation. We also perform bioinformatics analysis to estimate the interference of the pseudogenes.
In the 61-family cohorts, we gave 70% diagnostic rate (41/59) for ADPKD patients and 50% (1/2) for ARPKD patients. With 27% (16/59) of the ADPKD patients in our study carried the same variant, we also developed a screening method of RFLP for ADPKD hotspot (PKD2 c.2407C>T, p.R803X), that allowed us to perform fast diagnostic in less than 2.5 hours. The bioinformatics analysis also showed that with longer read length with paired-end reads, we can minimize the interference of pseudogenes to as low as 4% of total reads. In conclusion, we are able to provide confident genetic testing result utilizing NGS with diagnostic rate of 70% that requires no intensive labor work prior or additional sequencing after NGS, great coverage for high GC-content with double capture for region enhancement. Finally, we develop a speedy screening test that can be performed within 2.5 hours targeting PKD2 hotspot in our population that greatly reduces costs and may be carried in most testing laboratories.
|
|---|
