Short-Term Lymphocyte Culture Improves the Diagnostic Yield of Targeted RNA NGS in Cancer Predisposition Testing

Area of Science:

• Genomics
• Molecular Biology
• Cancer Genetics

Background:

• Next-generation RNA sequencing (RNA-seq) is valuable for germline cancer predisposition testing.
• Low gene expression in whole blood limits RNA-seq diagnostic utility for key cancer genes.
• Optimizing RNA yield and transcript representation is crucial for improving diagnostic accuracy.

Purpose of the Study:

• To develop and validate a simple protocol for enhanced RNA yield and gene expression from whole blood for targeted RNA-seq.
• To assess the impact of short-term lymphocyte culture on the expression of clinically relevant cancer predisposition genes.
• To evaluate sample handling and storage variability for routine diagnostic applications.

Main Methods:

• Optimized a short-term lymphocyte culture protocol from whole blood collected in Li-heparin tubes.
• Systematically evaluated biological reproducibility and pre-analytical sample handling variability.
• Compared RNA yield and gene expression from whole blood versus cultured lymphocytes.
• Investigated the effect of culture duration (3-5 days) on gene expression.
• Assessed the protocol's ability to inhibit nonsense-mediated decay for variant analysis.

Main Results:

• Short-term lymphocyte culture substantially increased expression of key cancer predisposition genes (e.g., BRCA1, BRCA2, RAD51C/D, PALB2, CHEK2, Fanconi anaemia genes).
• Whole blood can be stored at 4°C for up to 5 days without compromising RNA quality or gene expression.
• Gene expression levels were comparable between K2EDTA and Tempus tubes.
• Culturing lymphocytes for 3-5 days provided flexibility without significantly affecting gene expression.
• The protocol facilitated the detection of a splicing variant in the FANCA gene, crucial for variant interpretation.

Conclusions:

• Short-term lymphocyte culture is a simple, flexible, and effective method to enhance RNA yield and gene expression for clinical RNA-seq.
• This approach improves the diagnostic utility of RNA-seq in cancer predisposition testing by increasing the detectability of low-expressed genes.
• The optimized protocol offers practical advantages for routine diagnostics, including sample storage flexibility and improved variant analysis capabilities.