Completing genetic analysis of the ABCA4 locus

描述（由申请人提供）：ABCA 4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-视杆营养不良（CRD）和色素性视网膜炎（RP）。“轻度”、“中度”和“重度”ABCA 4突变等位基因的不同组合导致疾病表现连续体中的不同表型。ABCA 4基因座的变异已成为孟德尔视网膜疾病最常见的原因，估计所有人群中有1/20的人携带该基因中潜在的疾病相关变异。临床和等位基因异质性使ABCA 4相关视网膜疾病的遗传分析变得非常复杂。整个ABCA 4开放阅读框（ORF）的直接测序目前可检测到高达80%的等位基因，导致在约50%的患者中鉴定出2种预期突变，在30%的患者中鉴定出1种突变，而在20%的患者中在ABCA 4编码区中未发现疾病相关等位基因。 由于ABCA 4是唯一已知的负责隐性STGD的基因，因此很明显，许多疾病相关等位基因必须位于编码区之外，特别是在ORF中发现1个突变的患者中。该提案的主要目标是检验以下假设：ABCA 4基因组基因座的非编码序列中存在显著数量的突变，这些突变导致ABCA 4相关疾病负荷，以及其他已知和新基因中的突变导致其余具有ABCA 4样表型的疾病。 为了研究这些假设，我们将使用我们的大型散发性和家族性ABCA 4相关疾病患者的临床和遗传数据库，通过利用下一代测序技术和拷贝数变异（CNV）分析的逐步方法来确定1 p21上整个ABCA 4基因座中的所有疾病相关变异。首先，我们将对所有具有1个突变的患者的整个ABCA 4 ORF进行测序，然后对这些患者的整个ABCA 4基因组位点进行深度测序和CNV分析，其中不会发现第二个突变。在用尽ABCA 4位点分析并通过统计和功能测试确认变体后，我们将通过已知基因座的靶向重测序来确定在ABCA 4基因座中未发现突变的患者中哪些其他基因是ABCA 4样表型的基础，然后在选定的家族中进行全基因组外显子组测序。这些研究的结果将大大有助于疾病的诊断和预后，并将作为一个平台，为新兴的临床试验选择患者，以延迟ABCA 4相关疾病的发作或阻止其进展。 公共卫生相关性：由ABCA 4基因突变引起的疾病代表了多种常染色体隐性视网膜变性表型，包括Stargardt病（STGD）、视锥-视杆细胞营养不良（CRD）和色素性视网膜炎（RP）; ABCA 4基因座的变异已成为孟德尔视网膜疾病的最常见原因。目前的突变检测方法能够鉴定ABCA 4基因座中70-80%的所有致病等位基因。正如本提案所建议的，通过尖端技术鉴定ABCA 4基因座中的所有遗传变异将大大有助于疾病诊断和预后，并将作为选择患者进行新兴临床试验的平台，这些临床试验旨在延迟ABCA 4相关疾病的发作或阻止其进展。

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. PUBLIC HEALTH RELEVANCE: Diseases caused by mutations in the ABCA4 gene represent a variety of autosomal recessive retinal degeneration phenotypes, including Stargardt disease (STGD), cone-rod dystrophy (CRD) and retinitis pigmentosa (RP); variation in the ABCA4 locus has emerged as the most prevalent cause of Mendelian retinal disease. Current mutation detection methods are able to identify 70-80% of all disease-causing alleles in the ABCA4 locus. Identification of all genetic variation in the ABCA4 locus by cutting-edge technologies, as suggested in this proposal, 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.