Analysis of the ABCA4 Gene by Next-Generation Sequencing
Analysis of the ABCA4 Gene by Next-Generation Sequencing
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DOI：
10.1167/iovs.11-8182
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发表时间：
2011-10-01
影响因子：
4.4
通讯作者：
Allikmets, Rando
中科院分区：
文献类型：
--
作者：
Zernant, Jana;Schubert, Carl;Allikmets, Rando
目的。在一大群被诊断患有 ABCA4 相关疾病的患者中找到 ABCA4 基因编码序列中所有可能的疾病相关变异。方法。使用 ABCA4 微阵列对 168 名临床诊断患有 Stargardt 病、视杆细胞营养不良和其他 ABCA4 相关表型的患者进行了 ABCA4 突变的预筛查，结果在 111 名患者中发现了 2 种预期突变中的 1 种，在 57 名患者中发现了 2 种突变中的 0 种。对这些患者应用下一代测序（NGS）策略，对ABCA4基因的整个编码区和剪接位点进行测序。通过桑格测序确认或拒绝识别出的新变体，并通过计算机程序分析可能的致病性，并在可能的情况下通过分离分析。结果。对 168 名患者中的 159 名进行了成功测序，并在 103 名患者中的 49 名（相当于 48%）具有先前已发现的突变的患者中鉴定出了第二个与疾病相关的等位基因。在没有突变的患者中，56 名患者中有 4 名检测到两种与疾病相关的等位基因，56 名患者中有 10 名检测到一种突变。作者总共检测到 57 个以前未知的、可能致病的变体：29 个错义变体、4 个无义变体、9 个小缺失变体和 15 个剪接位点改变变体。通过预测方法和分离分析的结合，其中 55 个变异被认为是致病的。结论。 ABCA4 基因编码序列中的许多突变仍然未知，并且许多可能位于 ABCA4 基因座的非编码区域。尽管 ABCA4 阵列仍然是一个很好的首次筛选选择，但 NGS 平台是筛选大型群体的一种省时且经济高效的工具。 （投资眼科可见科学。2011；52：8479-8487）DOI：10.1167/iovs.11-8182
PURPOSE. To find all possible disease-associated variants in coding sequences of the ABCA4 gene in a large cohort of patients diagnosed with ABCA4-associated diseases.METHODS. One hundred sixty-eight patients who had been clinically diagnosed with Stargardt disease, cone-rod dystrophy, and other ABCA4-associated phenotypes were pre-screened for mutations in ABCA4 with the ABCA4 microarray, resulting in finding 1 of 2 expected mutations in 111 patients and 0 of 2 mutations in 57 patients. The next-generation sequencing (NGS) strategy was applied to these patients to sequence the entire coding region and the splice sites of the ABCA4 gene. Identified new variants were confirmed or rejected by Sanger sequencing and analyzed for possible pathogenicity by in silico programs and, where possible, by segregation analyses.RESULTS. Sequencing was successful in 159 of 168 patients and identified the second disease-associated allele in 49 of 103 (similar to 48%) of patients with one previously identified mutation. Among those with no mutations, both disease-associated alleles were detected in 4 of 56 patients, and one mutation was detected in 10 of 56 patients. The authors detected a total of 57 previously unknown, possibly pathogenic, variants: 29 missense, 4 nonsense, 9 small deletions and 15 splice-site-altering variants. Of these, 55 variants were deemed pathogenic by a combination of predictive methods and segregation analyses.CONCLUSIONS. Many mutations in the coding sequences of the ABCA4 gene are still unknown, and many possibly reside in noncoding regions of the ABCA4 locus. Although the ABCA4 array remains a good first-pass screening option, the NGS platform is a time-and cost-efficient tool for screening large cohorts. (Invest Ophthalmol Vis Sci. 2011;52:8479-8487) DOI: 10.1167/iovs.11-8182