Identification of BRAF Variants and Their Frequency in Colorectal Cancer Patients from Iraq

Abstract

Introduction: Colorectal cancer (CRC) is a biologically heterogeneous disease characterized by diverse molecular alterations that influence tumor behavior and clinical outcomes. This study aimed to investigate BRAF gene alterations in Iraqi CRC patients.

Materials and Methods: A total of 110 tissue biopsy samples were collected from CRC patients attending Ghazi Al-Hariri Specialized Surgery Hospital in Baghdad between November 2023 and August 2024, along with 36 tissue samples from individuals without colorectal cancer serving as controls. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissues. A 209 bp fragment of the BRAF gene was amplified by PCR and analyzed using bidirectional Sanger sequencing. Identified variants were annotated using public databases (dbSNP and ClinVar).

Results: Demographic analysis revealed no significant difference in gender distribution between cases and controls (p > 0.05), whereas age differed significantly (p = 0.001), with CRC patients being older. Tumors were most frequently located in the colon and rectum, and moderately differentiated adenocarcinoma represented the predominant histological subtype. Four BRAF variants were identified among CRC cases: two missense substitutions, AGT→AGG (p.Ser→Arg) and GAC→GTC (p.Asp→Val), and two intronic variants located outside the coding region. In addition, two single nucleotide polymorphisms (SNPs), rs2128998532 and rs2128998070, were examined in a subset of cases. The rs2128998532 T>C variant was observed predominantly in the heterozygous CT genotype (93.75%), while rs2128998070 (A>G) showed a heterozygous AG genotype frequency of 75% among analyzed samples. Two were missense coding variants resulting in amino acid substitutions, and two were intronic variants. All identified variants were previously reported in public databases. The affected cases included tumors from different anatomical sites and disease stages, with no evident clustering by stage or histological grade.

Conclusion: Two common BRAF SNPs showed high prevalence in the limited subset analyzed; however, no association analysis with disease stage or risk was performed due to sample size limitations.