PhD Scientific Days 2023

Molecular Sciences III.

RNA-based analyses might optimize the diagnosis of Lynch syndrome

Text of the abstract

Introduction
Lynch syndrome (LS) is the most common cause of hereditary colorectal cancer. Histopathological examination of LS-associated cancers reveal the decreased expression of proteins involved in the mismatch repair machinery necessitating genetic counseling and diagnosis to detect if germline pathogenic variants in the genes encoding MLH1, MSH2, PMS2, MSH6 are responsible for mismatch repair deficiency. Although DNA-based multigene panel testing detects the vast majority of LS cases, RNA-based investigation might reveal additional pathogenic (e.g.intronic and structural) variants.
Aims
In the present study our aim was to prospectively analyze if RNA-based analyses reveal novel germline pathogenic variants in LS.
Methods
32 patients (mean age: 57.97±13.63 years) with mismatch repair deficient (dMMR) cancers (21 colorectal cancer (CRC), 9 endometrial cancer, 2 gastric cancer) verified by immunohistochemistry (decreased MLH1 and PMS2 expression: 21 cases, decreased MSH2 and MSH6 expression: 2 cases) who were negative for germline pathogenic single nucleotide and copy number variants for the LS genes were involved in this study.
RNA was isolated from peripheral blood mononuclear cells using the Macherey-Nagel Nucleospin RNA plus kit. Reverse transcription was performed by the application of Protoscript II (New England Biolabs). Overlapping amplicons of 700-850 base pairs were amplified from cDNA and differences in amplicon lengths were screened with agarose gel electrophoresis and Agilent BioAnalyzer DNA1000 Chip. PCR products demonstrating altered amplicon length profile were sequenced on an Applied biosystems 3500 Genetic Analyzer instrument.
Result
A cDNA fragment in one patient revealed a deletion of 95 nucleotides in MLH1. This patient had CRC at age 22, while other LS-associated cancers (mother CRC, maternal grandfather gastrointestinal cancer) were present in the proband’s family. Further analysis performed on the genomic DNA revealed the same breakpoints, thus confirming the variant’s germline origin.
Conclusion
RNA-based analysis in conjunction with structural variation calling can detect additional germline pathogenic variations leading to LS. The inclusion of individuals living with these hereditary cancer predispositions into adequate clinical screening protocols allow early cancer detection and optimal care.
Funding
NLP-17, 2020-1.1.6-JÖVŐ-2021-00008, NKFIH-FK-138377, BO/00141/21, ÚNKP-22-5-SE-25.