A Systematic Method for Detecting Abnormal mRNA Splicing and Assessing Its Clinical Impact in Individuals Undergoing...

Area of Science:

• Genetics
• Molecular Biology
• Bioinformatics

Background:

• Splicing disruption accounts for nearly 14% of disease-causing germline variants.
• A majority (67%) of in silico predicted splicing-disrupting DNA variants are classified as variants of uncertain significance, complicating genetic testing interpretation.

Purpose of the Study:

• To develop and validate an analytical workflow, splice effect event resolver (SPEER), for assessing mRNA splicing alterations.
• To determine the impact of SPEER-identified splicing alterations on variant pathogenicity classification.
• To investigate the potential of mRNA splicing analysis to reduce ethnic disparities in genetic testing.

Main Methods:

• Developed and validated the SPEER workflow utilizing mRNA sequencing.
• Applied SPEER to analyze leukocyte RNA from 20,317 patients, focusing on 63 hereditary cancer syndrome-related genes.
• Integrated SPEER results into variant interpretation to reclassify variants of uncertain significance.

Main Results:

• SPEER identified altered splicing with deleterious effects in 4.8% of patients with predicted splicing-affecting DNA variants.
• Integration of SPEER results led to reclassification of variants of uncertain significance to pathogenic/likely pathogenic in 0.4% and benign/likely benign in 5.9% of patients.
• SPEER-based evidence showed a greater impact on variant classification in nonwhite versus non-Hispanic white patients, suggesting a role in reducing ethnic disparities.

Conclusions:

• The SPEER workflow effectively identifies pathogenic variants through mRNA splicing analysis.
• SPEER improves the classification of variants of uncertain significance, enhancing diagnostic accuracy.
• mRNA splicing analysis holds promise for mitigating ethnic and ancestral disparities in genetic testing outcomes.