Abstract
BACKGROUND: Pathogenic germline mutations in BRCA1/2 constitute the majority of hereditary breast and/or ovarian cancers worldwide. Incidence and mortality rate of breast and ovarian cancers in Pakistani women is high. Thus, to establish the diagnosis for targeted therapy in Pakistan, we conducted Next-generation sequencing-based germline testing for the detection of BRCA1/2 oncogenic variants associated with breast and ovarian cancer subtype. METHODS: Peripheral blood of 24 women, diagnosed with breast and epithelial ovarian cancers, was taken from the recruited cases with the consent of performing germline genetic testing. DNA was isolated from the peripheral blood and subjected to indexed BRCA Panel libraries. Targeted NGS was performed for all coding regions and splicing sites of BRCA1 and BRCA2 genes using AmpliSeq for Illumina BRCA Panel and Illumina MiSeq sequencer (placed at AFIP). Analysis of the sequencing results has been done by using Illumina bioinformatics tools. RESULTS: We detected 421 variants having a quality score of 100 in all cases under study. The list of identified variants in BRCA1 and BRCA2 genes was narrowed down after filtering out those which did not pass q30 and those with a minor allele frequency (MAF) > 0.05 based on gnomAD browser. To classify these variants, clinical significance was predicted using external curated databases. As a result, we interpreted (n = 4) 16.7% pathogenic variants in BRCA1 and (n = 6) 25% variants of uncertain significance (VUS) in both genes. Descriptive statistics depicted that the age and BMI of BRCA positive cases are less than BRCA negative cases. CONCLUSION: Our findings exhibit an initial report for the NGS based cancer genetic testing in Pakistan. This will enable us to pursue screening and diagnosis of hereditary BRCA mutation utilizing the latest state-of-the-art technique locally available in Pakistan ultimately resulting in targeted cancer therapy.