Mutations detection and classification in ADPKD

描述（由申请人提供）：常染色体显性多囊肾病（ADPKD）是一种常见的遗传性肾病，其特征是囊性肾脏逐渐增大并导致终末期肾脏疾病。ADPKD疾病谱系与心血管异常和其他器官（特别是肝脏）的囊肿有关。ADPKD是由两个基因PKD1和PKD2突变引起的。在我们之前对ADPKD的研究中，我们开发了用于PKD1和PKD2基因突变分析的分子工具，描述了ADPKD队列中基因型-表型相关性（突变位置与肾脏疾病严重程度和血管表型发展的关联），并开发了一个评分矩阵来分类这两个基因的错义突变。Sanger法对PKD1和PKD2基因的测序使用了大量的扩增子，并且主要集中在编码外显子和近内含子区域周围。这种方法没有探索深层内含子，并且由于使用的引物数量过多，可能容易导致等位基因脱落。我们现在建议利用Illumina Genome Analyzer II下一代测序平台作为PKD基因全面突变分析的工具（Specific Aim 1）。我们建议开发远程PCR扩增子，覆盖PKD1和PKD2基因的整个基因组结构。我们将利用这些远程PCR扩增子和下一代测序来探索30例经Sanger法测序为突变阴性的ADPKD患者的PKD1和PKD2基因的所有深层内含子区域。这将允许确定这组突变阴性的ADPKD患者（可能富含非典型突变）是否携带影响剪接的深内含子突变，或者在Sanger分析中这些患者是否发生等位基因脱落。我们建议通过计算机和体外方法评估特异性Aim 1中发现的所有内含子变化（特异性Aim 2）。在该队列中发现的深层内含子变异将使用群体数据（NCBI dbSNP，正常内含子变异模式和分离分析）来评估致病性，以从私有变异中过滤出共同多态性；在计算机预测工具（4个预测工具）中，预测剪接是否可能受到这些变异的影响；以及体外功能分析（cDNA和minigene分析），以验证剪接是否实际上受到通过前两个步骤选择的内含子变异的影响。对这一突变阴性队列的进一步表征将有助于研究深内含子突变在ADPKD中的作用，研究PKD基因中剪接调节的新机制，并分析基因型-表型相关性。

DESCRIPTION (provided by applicant): Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common inherited nephropathy, characterized by progressively enlarged cystic kidneys and leading to end stage renal disease. The ADPKD disease spectrum is associated with cardiovascular abnormalities and cysts in other organs, particularly the liver. ADPKD is caused by mutations in two genes, PKD1 and PKD2. In our previous studies in ADPKD, we have developed molecular tools for the mutation analysis of the PKD1 and PKD2 genes, described genotype- phenotype correlations in ADPKD cohorts (association of mutation position with severity of renal disease and with development of a vascular phenotype), and developed a scoring matrix to classify missense mutations in these two genes. Sequencing by Sanger method of the PKD1 and PKD2 genes uses a large number of amplicons, and it is focused around the coding exons and close intronic regions. Such approach does not explore deep introns and may be prone to allele drop-out due to the high number of primers used. We now propose to utilize the Illumina Genome Analyzer II next-generation sequencing platform as a tool for comprehensive mutation analysis of the PKD genes (Specific Aim 1). We propose to develop long-range PCR amplicons that cover the whole genomic structure of both the PKD1 and PKD2 genes. We will utilize these long-range PCR amplicons and next-generation sequencing for exploring all the deep intronic regions of the PKD1 and PKD2 genes in a group of 30 ADPKD patients, who are mutation-negative after sequencing by Sanger method. This will allow determining whether this group of mutation-negative ADPKD patients, likely enriched for atypical mutations, does carry deep intronic mutations affecting splicing, or whether allele drop-out occurred in these patients during Sanger analysis. We propose to evaluate all the intronic changes found in Specific Aim 1 by in silico and in vitro approaches (Specific Aim 2). Deep intronic variants found in this cohort will be evaluated for pathogencity using population data (the NCBI dbSNP, the pattern of normal intronic variation, and segregation analysis), to filter out common polymorphisms from private variants; in silico predictive tools (4 predictive tools), to predict whether splicing is likely affected by these variants; and in vitro functional assays (cDNA and minigene analysis), to verify if splicing is actually affected by the intronic variants selected through the two previous steps. Further characterization of this mutation-negative cohort will allow to investigate the role of deep intronic mutations in ADPKD, investigate new mechanisms by which splicing is regulated in the PKD genes, and analyze genotype-phenotype correlations. PUBLIC HEALTH RELEVANCE: Autosomal Dominant Polycystic Kidney Disease is a frequent inherited nephropathy that leads to end stage renal disease by mid-life. The disease is caused by mutations at two genes, PKD1 and PKD2. ADPKD type1 patients reach ESRD typically 20 years earlier than ADPKD type2 patients (54 years of age versus 74 years of age). Several hundreds different mutations account for ADPKD in both genes, and they are mostly specific to a single family. The PKD1 and PKD2 genes are large and complex genes. Complete analysis of these genes requires the amplification by PCR and sequencing by Sanger methods of a large number of fragments. However, such analysis is focused around coding exons and flanking introns, leaving deep introns unexplored. Allele drop-out may also occur when using a large number of PCR primers, due to the presence of SNPs in the target sequence. Sequencing by Sanger methods of large cohorts reveals that ~10% of ADPKD patients remain mutation-negative. These patients are likely to be enriched for atypical mutations, like deep intronic mutations that lead to abnormal splicing. Since deep introns are not currently explored, such mutation type has not yet been descried in ADPKD. Here we propose to utilize the Illumina Genome Analyzer II next-generation platform for deep sequencing a group of 30 ADPKD patients who are mutation-negative after extensive molecular characterization. We will utilize long-range amplicons to cover the whole genomic structure of both the PKD1 and PKD2 genes, and next-generation sequencing to obtain a complete molecular signature of both genes in these mutation-negative ADPKD patients. The intronic variants found in these patients will be evaluated using population data (the NCBI dbSNP and the pattern of normal intronic variation), in silico tools (to predict the activation of cryptic splicing sites) and in vitro assays (to functionally validate sequence variants strongly predicted to affect normal splicing). In conclusion, we propose to utilize deep genomic sequencing and multi-step validation of intronic variants in order to determine whether these mutation-negative ADPKD patients do carry a deep intronic variant. Such finding will validate this assay as a comprehensive genotyping assay for ADPKD, particularly in mutation- negative patients, and allow novel interesting genotype-phenotype correlations. The finding of deep intronic mutations affecting splicing may lead to new understanding on how proper gene splicing is regulated.