Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA, USA.
Department of Dermatology and Cutaneous Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, USA.
Clin Chem. 2021 Jun 1;67(6):876-888. doi: 10.1093/clinchem/hvab042.
Among the approximately 8000 Mendelian disorders, >1000 have cutaneous manifestations. In many of these conditions, the underlying mutated genes have been identified by DNA-based techniques which, however, can overlook certain types of mutations, such as exonic-synonymous and deep-intronic sequence variants. Whole-transcriptome sequencing by RNA sequencing (RNA-seq) can identify such mutations and provide information about their consequences.
We analyzed the whole transcriptome of 40 families with different types of Mendelian skin disorders with extensive genetic heterogeneity. The RNA-seq data were examined for variant detection and prioritization, pathogenicity confirmation, RNA expression profiling, and genome-wide homozygosity mapping in the case of consanguineous families. Among the families examined, RNA-seq was able to provide information complementary to DNA-based analyses for exonic and intronic sequence variants with aberrant splicing. In addition, we tested the possibility of using RNA-seq as the first-tier strategy for unbiased genome-wide mutation screening without information from DNA analysis.
We found pathogenic mutations in 35 families (88%) with RNA-seq in combination with other next-generation sequencing methods, and we successfully prioritized variants and found the culprit genes. In addition, as a novel concept, we propose a pipeline that increases the yield of variant calling from RNA-seq by concurrent use of genome and transcriptome references in parallel.
Our results suggest that "clinical RNA-seq" could serve as a primary approach for mutation detection in inherited diseases, particularly in consanguineous families, provided that tissues and cells expressing the relevant genes are available for analysis.
在大约 8000 种孟德尔疾病中，有>1000 种具有皮肤表现。在这些疾病中，许多疾病的潜在突变基因已经通过基于 DNA 的技术确定，但这些技术可能会忽略某些类型的突变，如外显子同义突变和深内含子序列变异。通过 RNA 测序（RNA-seq）进行全转录组测序可以识别这些突变，并提供有关其后果的信息。
我们分析了 40 个具有不同类型遗传异质性的孟德尔皮肤疾病家族的全转录组。对 RNA-seq 数据进行了变异检测和优先级排序、致病性确认、RNA 表达谱分析，以及在近亲家族中进行全基因组纯合性映射。在所检查的家族中，RNA-seq 能够提供与基于 DNA 的分析互补的信息，包括外显子和内含子序列变异与异常剪接。此外，我们还测试了在没有 DNA 分析信息的情况下，使用 RNA-seq 作为无偏基因组范围内突变筛查的一线策略的可能性。
我们发现 35 个家族（88%）的 RNA-seq 结合其他下一代测序方法可以提供致病性突变，并且我们成功地对变异进行了优先级排序，并找到了罪魁祸首基因。此外，作为一个新概念，我们提出了一种流水线，通过同时使用基因组和转录组参考，增加了从 RNA-seq 中调用变异的产量。
我们的研究结果表明，“临床 RNA-seq”可以作为遗传性疾病突变检测的主要方法，特别是在近亲家族中，只要有表达相关基因的组织和细胞可供分析。
