Zvereff Val, Yao Suxia, Ramsey Julia, Mikhail Fady M, Vijzelaar Raymon, Messiaen Ludwine
Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Genet Test Mol Biomarkers. 2010 Aug;14(4):505-10. doi: 10.1089/gtmb.2009.0188.
Mutations in the PKHD1 gene are responsible for autosomal recessive polycystic kidney disease (ARPKD). Using exon scanning by denaturing high-performance liquid chromatography (dHPLC) or bidirectional sequencing of all exons constituting the longest open reading frame, the mutation detection rate reaches approximately 82% and minor lesion mutations include truncating, splice, and missense mutations.
The main aim of this study was to screen ARPKD patients in whom only one pathogenic PKHD1 mutation was identified after bidirectional sequencing of the longest open reading frame, for gene copy number alterations by employing multiplex ligation-dependent probe amplification complemented with quantitative real-time polymerase chain reaction.
Sixteen ARPKD probands were studied in whom only one clearly pathogenic mutation was previously identified. One patient with a suspected homozygous deletion of the exons 1-37 was also included in this cohort. Three distinct PKHD1 germ-line deletions were identified. Two of these deletions encompassed multiple exons of PKHD1 extending far beyond the 5' and 3' untranslated regions of the gene, and spanning at least 170 and 470 kb, respectively. The third 3.7 kb intragenic deletion affected only exons 20-21 of the PKHD1 gene. Thus, this is the first report presenting analysis of the entire PKHD1 longest open reading frame for gene deletions/duplications in a select cohort of ARPKD patients, in whom previously only one mutation was identified after bidirectional sequencing of the entire longest open reading frame.
The data indicate that multiplex ligation-dependent probe amplification is a sensitive and rapid method to identify PKHD1 deletions. Our study demonstrates that dosage analysis will increase the PKHD1 mutation detection rate and should be performed as a complementary assay in patients suspected to have ARPKD in the absence of two clear pathogenic mutations.
PKHD1基因的突变是常染色体隐性多囊肾病（ARPKD）的病因。通过变性高效液相色谱法（dHPLC）进行外显子扫描或对构成最长开放阅读框的所有外显子进行双向测序，突变检测率约为82%，微小病变突变包括截短突变、剪接突变和错义突变。
本研究的主要目的是，对于那些在对最长开放阅读框进行双向测序后仅鉴定出一个致病性PKHD1突变的ARPKD患者，采用多重连接依赖探针扩增结合定量实时聚合酶链反应来筛查基因拷贝数改变。
对16名ARPKD先证者进行了研究，这些患者此前仅鉴定出一个明确的致病性突变。该队列中还纳入了一名疑似外显子1 - 37纯合缺失的患者。鉴定出了三种不同的PKHD1种系缺失。其中两种缺失包含PKHD1的多个外显子，远远超出该基因的5'和3'非翻译区，分别跨越至少170 kb和470 kb。第三种3.7 kb的基因内缺失仅影响PKHD1基因的外显子20 - 21。因此，这是首篇报道对一组特定ARPKD患者的整个PKHD1最长开放阅读框进行基因缺失/重复分析的文章，这些患者在对整个最长开放阅读框进行双向测序后此前仅鉴定出一个突变。
数据表明多重连接依赖探针扩增是鉴定PKHD1缺失的一种灵敏且快速的方法。我们的研究表明，剂量分析将提高PKHD1突变检测率，对于疑似患有ARPKD但未发现两个明确致病性突变的患者，应作为补充检测进行剂量分析。
