Abstract：
The diagnosis of Mendelian disorders has notably advanced with integration of whole exome and genome sequencing (WES and WGS) in clinical practice. However, challenges in variant interpretation and uncovered variants by WES still leave a substantial percentage of patients undiagnosed. In this context, integrating RNA sequencing (RNA-seq) improves diagnostic workflows, particularly for WES inconclusive cases. Additionally, functional studies are often necessary to elucidate the impact of prioritized variants on gene expression and protein function. Our study focused on three unrelated male patients (P1-P3) with ATP6AP1-CDG (congenital disorder of glycosylation), presenting with intellectual disability and varying degrees of hepatopathy, glycosylation defects, and an initially inconclusive diagnosis through WES. Subsequent RNA-seq was pivotal in identifying the underlying genetic causes in P1 and P2, detecting ATP6AP1 underexpression and aberrant splicing. Molecular studies in fibroblasts confirmed these findings and identified the rare intronic variants c.289-233C > T and c.289-289G > A in P1 and P2, respectively. Trio-WGS also revealed the variant c.289-289G > A in P3, which was a de novo change in both patients. Functional assays expressing the mutant alleles in HAP1 cells demonstrated the pathogenic impact of these variants by reproducing the splicing alterations observed in patients. Our study underscores the role of RNA-seq and WGS in enhancing diagnostic rates for genetic diseases such as CDG, providing new insights into ATP6AP1-CDG molecular bases by identifying the first two deep intronic variants in this X-linked gene. Additionally, our study highlights the need to integrate RNA-seq and WGS, followed by functional validation, in routine diagnostics for a comprehensive evaluation of patients with an unidentified molecular etiology.
摘要：
随着全外显子组和基因组测序（WES和WGS）在临床实践中的整合，孟德尔疾病的诊断显着进步。然而,WES在变异解释和未发现变异方面的挑战仍然使相当大比例的患者未得到诊断.在这种情况下，整合RNA测序（RNA-seq）改善了诊断工作流程，特别是对于WES不确定的情况。此外,功能研究往往是必要的，以阐明优先变异对基因表达和蛋白质功能的影响。我们的研究集中在三个无关的男性患者（P1-P3）与ATP6AP1-CDG（先天性糖基化障碍），表现为智力残疾和不同程度的肝病，糖基化缺陷，并通过WES进行初步不确定的诊断。随后的RNA-seq在确定P1和P2的潜在遗传原因，检测ATP6AP1表达不足和异常剪接方面至关重要。成纤维细胞中的分子研究证实了这些发现，并分别在P1和P2中鉴定了罕见的内含子变体c.289-233C>T和c.289-289G>A。Trio-WGS还揭示了P3中的变异c.289-289G>A，这在两名患者中是从头变化。在HAP1细胞中表达突变等位基因的功能测定通过再现在患者中观察到的剪接改变证明了这些变体的致病影响。我们的研究强调了RNA-seq和WGS在提高CDG等遗传疾病的诊断率方面的作用。通过确定该X连锁基因中的前两个深层内含子变体，为ATP6AP1-CDG分子碱基提供新的见解。此外,我们的研究强调了整合RNA-seq和WGS的必要性，其次是功能验证，在常规诊断中，对分子病因不明的患者进行全面评估。
