Completing genetic analysis of the ABCA4 locus

描述（由申请人提供）：ABCA4基因突变可导致多种视网膜变性表型，如常染色体隐性Stargardt病（STGD）、锥杆营养不良（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在连续的疾病表现中导致不同的表型。ABCA4位点的变异已成为孟德尔视网膜疾病最普遍的原因，估计在所有人群中有1/20的人携带该基因的潜在疾病相关变异。临床和等位基因异质性使abca4相关视网膜疾病的遗传分析变得非常复杂。对整个ABCA4开放阅读框（ORF）的直接测序目前检测到高达80%的等位基因，结果在约50%的患者中鉴定出2个预期突变，在30%的患者中鉴定出1个突变，而在20%的患者中未在ABCA4编码区发现与疾病相关的等位基因。由于ABCA4是唯一已知的与隐性STGD有关的基因，很明显，许多疾病相关的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。本提案的主要目标是验证以下假设：ABCA4基因组位点的非编码序列中存在大量突变，这些突变有助于ABCA4相关的疾病负荷，以及其他已知和新基因的突变是导致其余ABCA4样表型疾病的原因。为了研究这些假设，我们将利用散发性和家族性ABCA4相关疾病患者的大型临床和遗传数据库，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位点的所有疾病相关变异。首先，我们将对所有1个突变的患者进行整个ABCA4 ORF测序，然后对这些没有发现第二个突变的患者进行整个ABCA4基因组位点的深度测序和CNV分析。在耗尽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的靶向重测序，然后在选定的家庭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，还有哪些其他基因是ABCA4样表型的基础。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，旨在延缓abca4相关疾病的发病或阻止其进展。

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.