Liu Genyan, Tan Adrian Y, Michaeel Alber, Blumenfeld Jon, Donahue Stephanie, Bobb Warren, Parker Tom, Levine Daniel, Rennert Hanna
Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA; Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA.
Gene. 2014 Oct 15;550(1):131-5. doi: 10.1016/j.gene.2014.07.008. Epub 2014 Jul 8.
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two large genes, PKD1 and PKD2, but genetic testing is complicated by the large transcript sizes and the duplication of PKD1 exons 1-33 as six pseudogenes on chromosome 16. Long-range PCR (LR-PCR) represents the gold standard approach for PKD1 genetic analysis. However, a major issue with this approach is that it requires large quantities of genomic DNA (gDNA) material limiting its application primarily to DNA extracted from blood. In this study, we have developed a whole genome amplification (WGA)-based genotyping assay for PKD1 and PKD2, and examined whether this approach can be applied to biosamples with low DNA yield, including blood, buccal cells and urine. DNA samples were amplified by multiple displacement amplification (MDA) and a high-fidelity DNA polymerase followed by LR-PCR and exon-specific amplifications of PKD1 and PKD2 respectively, and Sanger sequencing. This method has generated large amounts of DNA with high average product length (>10 kb), which were uniformly amplified across all sequences assessed. When compared to the gDNA direct sequencing method for six ADPKD samples, a total of 89 variants were detected including all 86 variations previously reported, in addition to three new variations, including one pathogenic mutation not previously detected by the standard gDNA-based analysis. We have further applied WGA to ADPKD mutation analysis of low DNA-yield specimens, successfully detecting all 63 gene variations. Compared to the gDNA method the WGA-based assay had a sensitivity and specificity of 100%. In conclusion, WGA-based LR-PCR represents a major technical improvement for PKD genotyping from trace amounts of DNA.
常染色体显性多囊肾病（ADPKD）由两个大基因PKD1和PKD2的突变引起，但由于转录本尺寸大以及16号染色体上六个假基因导致PKD1外显子1 - 33的重复，基因检测变得复杂。长程PCR（LR-PCR）是PKD1基因分析的金标准方法。然而，该方法的一个主要问题是它需要大量的基因组DNA（gDNA）材料，这主要限制了其应用于从血液中提取的DNA。在本研究中，我们开发了一种基于全基因组扩增（WGA）的PKD1和PKD2基因分型检测方法，并研究了该方法是否可应用于DNA产量低的生物样本，包括血液、颊细胞和尿液。DNA样本通过多重置换扩增（MDA）和高保真DNA聚合酶进行扩增，随后分别进行LR-PCR以及PKD1和PKD2的外显子特异性扩增，并进行桑格测序。该方法产生了大量平均产物长度高（>10 kb）的DNA，在所有评估序列中均被均匀扩增。与六个ADPKD样本的gDNA直接测序方法相比，共检测到89个变异，包括先前报道的所有86个变异，此外还有三个新变异，包括一个基于标准gDNA分析先前未检测到的致病突变。我们进一步将WGA应用于低DNA产量标本的ADPKD突变分析，成功检测到所有63个基因变异。与gDNA方法相比，基于WGA的检测方法的灵敏度和特异性均为100%。总之，基于WGA的LR-PCR代表了从微量DNA进行PKD基因分型的一项重大技术改进。
