Bioscientia Center for Human Genetics, Ingelheim, Germany.
PLoS One. 2013 Nov 12;8(11):e78496. doi: 10.1371/journal.pone.0078496. eCollection 2013.
Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover "hidden mutations" such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5' exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.
色素性视网膜炎（RP）和莱伯先天性黑蒙（LCA）是导致失明的主要原因。它们是由许多基因突变引起的，这长期以来一直阻碍了全面的遗传分析。最近，靶向下一代测序（NGS）已被证明有助于克服这一限制。为了发现“隐藏的突变”，如拷贝数变异（CNVs）和非编码区域的突变，我们扩展了 NGS 数据的用途，对 126 名患者的 55 个 RP 和 LCA 基因的外显子进行了定量读出，并包括了非编码 5'外显子。我们检测到了几个致病的 CNVs，这些 CNVs 是解决迄今未解的基因组合中关键的诊断因素，例如在近亲家庭中发现的半合子点突变，以及 CNVs 互补的明显单等位基因隐性等位基因。EYS 非编码外显子 1 的突变揭示了其对疾病的贡献。鉴于视网膜疾病基因突变在普通人群中的高携带频率，我们考虑了每个患者的总体变异负荷，以评估突变是否是致病的，还是反映了多个基因或单个隐性等位基因突变患者的偶然携带。例如，RP1 基因中的截断突变，该基因与隐性和显性 RP 都有关，在双等位基因的情况下是致病的，与另一个基因的双等位基因突变背景下的杂合子无关，甚至在靠近 C 末端时是非致病性的。多个基因位点突变的患者很常见，但没有证据表明存在双基因或寡基因遗传。虽然与以前的研究相比，靶向基因的数量较少，但突变检测率最高（70%），这可能是由于完整和深度的覆盖范围，以及定量数据分析。CNV 分析应常规应用于靶向 NGS，并且非编码外显子中的突变也有理由系统地包含在疾病基因或外显子组中。考虑所有变体是必不可少的，因为即使是截断突变也可能具有误导性。
