A complete mutation screen of the ADPKD genes by DHPLC
A complete mutation screen of the ADPKD genes by DHPLC
复制标题
DOI：
10.1046/j.1523-1755.2002.00326.x
复制
发表时间：
2002-05-01
影响因子：
19.6
通讯作者：
Harris, PC
中科院分区：
文献类型：
--
作者：
Rossetti, S;Chauveau, D;Harris, PC
背景遗传分析是常染色体显性遗传性多囊肾病（ADPKD）的一种有用的诊断工具，特别是当影像学结果不明确时。然而，由于主要基因座PKD 1的遗传和等位基因异质性和复杂性，通过直接突变筛选的分子诊断已被证明在这种疾病中是困难的。开发了一种方案，以从基因组DNA中特异性扩增PKD 1和PKD 2的外显子作为150至450 bp的扩增子。使用Wave Fragment Analysis System（Transgenomics）通过变性高效液相色谱（DHPLC）技术分析这些片段，以检测贯穿两个基因的碱基对变化，DHPI-C检测到的变化通过测序表征，结果。优化了DHPLC对PKD 1和PKD 2整个编码区的成本有效且敏感的突变筛选。我们先前表征的这些基因的所有碱基对突变被检测为改变的DHPLC谱。为了评估该方法的常规诊断用途，分析了来自45名遗传学未表征的ADPKD患者的样本。检测到29个明确的突变，26个PKD 1，3个PKD 2和另外5个可能的错义突变，其特征在于导致最大检出率为76%。PKD 1的多态性水平也很高，定义了71种不同的变化。DHPLC图谱的可重复性使许多常见多态性的识别成为可能，而无需重新测序。DHPLC已被证明是一种高效、有效的ADPKD基因分子诊断方法。区分错义突变和多态性仍然是一个挑战，但基于家族的分离分析很有帮助。
Background. Genetic analysis is a useful diagnostic tool in autosomal dominant polycystic kidney disease (ADPKD), especially when imaging results are equivocal. However, molecular diagnostics by direct mutation screening has proved difficult in this disorder due to genetic and allelic heterogeneity and complexity of the major locus, PKD1.Methods. A protocol was developed to specifically amplify the exons of PKD1 and PKD2 from genomic DNA as 150 to 450 bp amplicons. These fragments were analyzed by the technique of denaturing high-performance liquid chromatography (DHPLC) using a Wave Fragment Analysis System (Transgenomics) to detect base-pair changes throughout both genes, DHPI-C-detected changes were characterized by sequencing,Results. Cost effective and sensitive mutation screening of the entire coding regions of PKD1 and PKD2 by DHPLC was optimized. All base-pair mutations to these genes that we previously characterized were detected as an altered DHPLC profile. To assess this method for routine diagnostic use, samples from a cohort of 45 genetically uncharacterized ADPKD patients were analyzed. Twenty-nine definite mutations were detected, 26 PKD1, 3 PKD2 and a further five possible missense mutations were characterized leading to a maximal detection rate of 76%. A high level of polymorphism of PKD1 also was detected, with 71 different changes defined. The reproducibility of the DHPLC profile enabled the recognition of many common polymorphisms without the necessity for re-sequencing.Conclusions. DHPLC has been demonstrated to be an efficient and effective means for gene-based molecular diagnosis of ADPKD. Differentiating missense mutations and polymorphisms remains a challenge, but family-based segregation analysis is helpful.