Abstract：
RNA sequencing (RNA-seq) is a complementary diagnostic tool to exome sequencing (ES), only recently clinically available to undiagnosed patients post-ES, that provides functional information on variants of unknown significance (VUS) by evaluating its effect on RNA transcription. ES became clinically available in the early 2010s and promised an agnostic platform for patients with a neurological disease, especially for those who believed to have a genetic etiology. However, the massive data generated by ES pose challenges in variant interpretation, especially for rare missense, synonymous, and deep intronic variants that may have a splicing effect. Without functional study and/or family segregation analysis, these rare variants would be likely interpreted as VUS which is difficult for clinicians to use in clinical care. Clinicians are able to assess the VUS for phenotypic overlap, but this additional information alone is usually not enough to re-classify a variant. Here, we report a case of a 14-month-old male who presented to clinic with a history of seizures, nystagmus, cerebral palsy, oral aversion, global developmental delay, and poor weight gain requiring gastric tube placement. ES revealed a previously unreported homozygous missense VUS, c.7406A > G p.(Asn2469Ser), in VPS13D. This variant has not been previously reported in genome aggregation database (gnomAD), ClinVar, or in any peer-reviewed published literature. By RNA-seq, we demonstrated that this variant mainly impacts splicing and results in a frameshift and early termination. It is expected to generate either a truncated protein, p.(Val2468fs*19), or no protein from this transcript due to nonsense-mediated mRNA decay leading to VPS13D deficiency. To our knowledge, this is the first case utilizing RNA-seq to further functionally characterize a homozygous novel missense VUS in VPS13D and confirm its impact on splicing. This confirmed pathogenicity gave the diagnosis of VPS13D movement disorder to this patient. Therefore, clinicians should consider utilizing RNA-seq to clarify VUS by evaluating its effect on RNA transcription.
摘要：
RNA测序（RNA-seq）是外显子组测序（ES）的补充诊断工具，仅在最近临床上对未确诊的ES后患者可用，通过评估其对RNA转录的影响来提供有关未知意义变体（VUS）的功能信息。ES在2010年代初成为临床可用的，并承诺为患有神经系统疾病的患者提供一个不可知的平台，尤其是那些认为有遗传病因的人。然而,ES产生的海量数据对变体解释提出了挑战，尤其是对于罕见的错觉，同义词,和可能具有剪接效应的深层内含子变体。没有功能研究和/或家庭隔离分析，这些罕见变异很可能被解释为VUS,临床医生难以在临床护理中使用.临床医生能够评估VUS的表型重叠，但是仅靠这些额外的信息通常不足以对变体进行重新分类。这里,我们报告了一例14个月大的男性，他有癫痫发作史，眼球震颤,脑瘫,口头厌恶，全球发育迟缓，和不良的体重增加需要胃管放置。ES揭示了以前未报告的纯合错义VUS，c.740A>Gp.(Asn2469Ser)，在VPS13D中。这种变异以前在基因组聚集数据库(gnomAD)中没有报道过，ClinVar,或任何同行评审的已发表文献。通过RNA-seq,我们证明了该变体主要影响剪接，并导致移码和提前终止。预计会产生截短的蛋白质，p.(Val2468fs*19)，或由于无义介导的mRNA衰变导致VPS13D缺乏，因此该转录物中没有蛋白质。据我们所知,这是利用RNA-seq进一步在功能上表征VPS13D中的纯合新型错义VUS并确认其对剪接的影响的第一个案例.这种确认的致病性为该患者诊断为VPS13D运动障碍。因此,临床医师应考虑利用RNA-seq通过评估其对RNA转录的影响来阐明VUS.
