Completing genetic analysis of the ABCA4 locus

描述(申请人提供)：ABCA4基因突变导致多种视网膜变性表型，如常染色体隐性遗传性Stargardt病(STGD)、视锥-视杆细胞营养不良(CRD)和视网膜色素变性(RP)。“轻度”、“中度”和“重度”ABCA4突变等位基因的不同组合导致一系列疾病表现的不同表型。ABCA4基因的变异已成为孟德尔人视网膜疾病最常见的原因，估计在所有人群中，有1/20的人携带该基因潜在的疾病相关变异。临床和等位基因的异质性使ABCA4相关视网膜疾病的遗传分析变得非常复杂。目前，对整个ABCA4开放阅读框架(ORF)的直接测序可以检测到高达80%的等位基因，导致在~50%的患者中识别出2个预期的突变，在30%的患者中发现1个突变，而在20%的患者的ABCA4编码区中没有发现与疾病相关的等位基因。由于ABCA4是唯一已知的导致隐性STGD的基因，很明显，许多疾病相关等位基因必须位于编码区之外，特别是在ORF发现1个突变的患者中。这项建议的主要目标是检验以下假设，即ABCA4基因组座位的非编码序列中有大量突变导致ABCA4相关疾病负载，以及其他已知和新基因的突变是导致ABCA4样表型疾病的剩余原因的假设。为了研究这些假说，我们将使用我们的大型临床和遗传数据库，利用我们的散发性和家族性ABCA4相关疾病患者的数据库，通过利用下一代测序技术和拷贝数变异(CNV)分析，逐步确定1p21上整个ABCA4基因座的所有疾病相关变异。首先，我们将对所有有1个突变的患者的整个ABCA4 ORF进行测序，然后对这些患者的整个ABCA4基因组进行深度测序和CNV分析，在这些患者中不会发现第二个突变。在完成ABCA4基因座分析并通过统计和功能测试确认变异后，我们将通过对已知基因座进行有针对性的重新测序，然后在选定的家系中进行全基因组外显子组测序，确定在ABCA4基因座未发现突变的患者中，还有哪些基因是ABCA4样表型的潜在基因。这些研究的结果将大大有助于疾病的诊断和预后，并将成为为新的临床试验挑选患者的平台，这些临床试验旨在推迟或阻止ABCA4相关疾病的发病或进展。

DESCRIPTION (provided by applicant): Mutations in the ABCA4 gene are responsible for a wide variety of retinal degeneration phenotypes, such as autosomal recessive Stargardt disease (STGD), cone-rod dystrophy (CRD) and retinitis pigmentosa (RP). Different combinations of "mild", "moderate", and "severe" ABCA4 mutant alleles result in distinct phenotypes in a continuum of disease manifestations. Variation in the ABCA4 locus has emerged as the most prevalent cause of Mendelian retinal disease, with an estimated 1/20 people across all populations carrying a potential disease-associated variant in this gene. Clinical and allelic heterogeneity has substantially complicated genetic analyses of ABCA4-associated retinal disease. Direct sequencing of the entire ABCA4 open reading frame (ORF) currently detects up to 80% of the alleles, resulting in the identification of the 2 expected mutations in ~50% of patients, 1 mutation in 30%, while no disease-associated allele is found in the ABCA4 coding region in 20% patients. Since ABCA4 is the only known gene responsible for recessive STGD, it is clear that many disease- associated alleles must be located outside of the coding regions, especially in patients where 1 mutation is found in the ORF. The major goal of this proposal is to test the hypotheses that there are significant numbers of mutations in non-coding sequences of the ABCA4 genomic locus that contribute to ABCA4-associated disease load and that mutations in additional known and novel genes are responsible for the remainder of diseases with ABCA4-like phenotypes. To investigate these hypotheses, we will use our large clinical and genetic databases of sporadic and familial patients with ABCA4-associated diseases to determine all disease-associated variants in the entire ABCA4 locus on 1p21 by a step-by-step approach utilizing the next generation sequencing technologies and copy number variation (CNV) analyses. First, we will sequence the entire ABCA4 ORF in all patients with 1 mutation, followed by deep sequencing and CNV analyses of the entire ABCA4 genomic locus in these patients where no 2nd mutation will be found. After exhausting the ABCA4 locus analysis and confirming the variants by statistical and functional tests, we will determine what other genes are underlying ABCA4-like phenotypes in patients where no mutation is found in the ABCA4 locus by targeted resequencing of known loci, followed by full genome exome sequencing in selected families. The outcome of these studies will substantially aid in disease diagnosis, prognosis and will serve as a platform for selecting patients for emerging clinical trials geared to delay the onset, or arrest the progression, of ABCA4-associated diseases.