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

Area of Science:

• Genomics and Transcriptomics
• Clinical Diagnostics
• Human Genetics

Background:

• Exome sequencing is a primary clinical tool for Mendelian diseases, but challenges remain in diagnosing variants within introns or synonymous codons.
• A significant number of patients remain undiagnosed due to limitations in current genetic diagnostic approaches for specific variant types.

Purpose of the Study:

• To develop and validate a workflow integrating exome sequencing, whole-blood transcriptome sequencing, and in silico tools.
• To identify and functionally validate splicing-altering intronic and synonymous variants in patients with unresolved genetic diseases.

Main Methods:

• Construction of a whole-blood transcriptome database for 2981 Online Mendelian Inheritance in Man (OMIM) disease genes.
• Integration of exome sequencing, blood transcriptome sequencing, and in silico prediction for variant identification.
• RNA sequencing for functional validation of identified splicing-altering variants.

Main Results:

• Seven synonymous variants were identified in eight patients using the integrated multi-omics pipeline.
• New functional evidence for splicing alterations was established for four variants: c.981G>A (PIGN), c.1161A>G (ALPL), c.858G>A (ATP6AP2), and c.1011G>T (MTHFR).
• RNA sequencing confirmed aberrant splicing induced by these variants, expanding the known variant spectrum for these genes.

Conclusions:

• Combining multi-omics data, particularly RNA sequencing, is feasible and powerful for identifying splicing-altering variants.
• Synonymous variants are a crucial, often overlooked, contributor to unresolved genetic diseases.
• The proposed workflow enhances diagnostic yield for Mendelian disorders by addressing limitations of standard exome sequencing.