Improving Mutation Screening in Patients with Colorectal Cancer Predisposition Using Next-Generation Sequencing

Area of Science:

• Genetics
• Oncology
• Molecular Biology

Background:

• Accurate identification of genetic alterations is crucial for colorectal cancer (CRC) family risk assessment.
• Next-generation sequencing (NGS) offers a powerful approach for simultaneous genetic analysis of multiple genes and samples.

Purpose of the Study:

• To validate a multiplex PCR-based NGS assay for simultaneous detection of mutations in mismatch repair (MMR) and familial adenomatous polyposis (FAP) genes.
• To assess the utility of this NGS assay for clinical diagnosis and individual cancer risk assessment.

Main Methods:

• Validation of Multiplicom MASTR designs for MMR and FAP genes using NGS on a MiSeq platform.
• Simultaneous analysis of eight DNA samples per run.
• Testing of 224 patient blood samples.

Main Results:

• The assay correctly identified 97 known mutations in a validation cohort.
• NGS detected single-nucleotide variations in PMS2, with copy number variation (CNV) analysis confirmed for clinical use.
• In routine diagnostics, 27 variants of unknown significance, 21 disease-causing variants, and two CNVs were identified.
• MUTYH mutations were found in two patients undergoing MMR testing, and one patient had both MUTYH and MSH6 mutations.

Conclusions:

• The validated NGS assay enables accurate and rapid individual risk assessment for colorectal cancer.
• The method effectively identifies disease-causing mutations and copy number variations in MMR and FAP genes.
• This approach aids in identifying potential cancer risk modifiers and optimizing clinical management strategies.