Identification of Synonymous Pathogenic Variants in Monogenic Disorders by Integrating Exome with Transcriptome Sequencing.

Exome sequencing is becoming a first-tier clinical diagnostic test for Mendelian diseases, drastically reducing the time and cost of diagnostic odyssey and improving the diagnosis rate. Despite its success, exome sequencing faces practical challenges in assessing the pathogenicity of numerous intronic and synonymous variants, leaving a significant proportion of patients undiagnosed. In this study, a whole-blood transcriptome database was constructed that showed the expression profile of 2981 Online Mendelian Inheritance in Man disease genes in blood samples. Meanwhile, a workflow integrating exome sequencing, blood transcriptome sequencing, and in silico prediction tools to identify and validate splicing-altering intronic or synonymous variants was proposed. Following this pipeline, seven synonymous variants in eight patients were discovered. Of these, the functional evidence of c.981G>A (PIGN), c.1161A>G (ALPL), c.858G>A (ATP6AP2), and c.1011G>T (MTHFR) have not been reported previously. RNA sequencing validation confirmed that these variants induced aberrant splicing, expanding the disease-causing variant spectrum of these genes. Overall, this study shows the feasibility of combining multi-omics data to identify splicing-altering variants, especially the power of RNA sequencing. It also reveals that synonymous variants, which often are overlooked in standard diagnostic approaches, comprise an important portion of unresolved genetic diseases.