RNA-Seq of Cultured Peripheral Blood Lymphocytes Improves Identification of Cryptic Splicing Defects in Rare Disease...

Area of Science:

• Genomics and Transcriptomics
• Rare Disease Diagnostics
• Reproductive Genetics

Background:

• Whole-exome sequencing (WES) and whole-genome sequencing (WGS) have limitations in diagnosing rare diseases.
• A significant portion of families remain undiagnosed after WES/WGS, impacting genetic counseling.
• Clinical implementation of RNA sequencing (RNA-seq) for preconception counseling is underexplored.

Purpose of the Study:

• To optimize an RNA-seq workflow for enhanced rare disease diagnosis in preconception settings.
• To evaluate the diagnostic yield of the optimized RNA-seq pipeline in families with previously negative WES/WGS results.
• To assess the utility of phytohemagglutinin-activated peripheral blood cells (PHACs) as a robust RNA source.

Main Methods:

• Developed an optimized RNA-seq pipeline integrating transcript isoform distribution (TID) analysis, MANE reference transcriptome realignment, and pharmacological induction-based cryptic splicing detection.
• Utilized PHACs as the RNA source, demonstrating reduced interindividual variability and higher RNA integrity compared to peripheral blood mononuclear cells (PBMCs).
• Applied the workflow to 55 rare disease families undergoing preconception genetic counseling with prior negative WES/WGS results.

Main Results:

• The optimized RNA-seq workflow achieved a 2.2-fold increase in diagnostic yield (20% vs. 9% baseline).
• Positive diagnostic rates varied by family grouping: 71% for VUS, 40% for suspected-gene/variant-negative, and 5.2% for unsolved/no-candidate.
• Ten out of eleven positive cases received definitive diagnoses, informing diverse reproductive decisions.

Conclusions:

• The enhanced RNA-seq workflow is clinically applicable and scalable for improving molecular diagnostics in reproductive and preconception care.
• This approach offers a valuable model for future clinical transcriptomics applications in rare disease diagnosis.
• Improved diagnostic capabilities through RNA-seq empower families with crucial information for reproductive decision-making.