Muhammad Usman Rashid

Genetic landscape of Pakistani familial breast cancer patients using multigene panel testing

Abstract Pathogenic/likely pathogenic (P/LP) variants in high‐, moderate‐, and low‐penetrance genes account for approximately half of all familial breast cancer (BC) cases. In Pakistan, data on P/LP variants beyond BRCA1/2 remain limited. This study investigated the frequency and distribution of P/LP variants in Pakistani familial BC patients using a 14‐gene hereditary breast and ovarian cancer (HBOC) core panel. A total of 160 familial BC patients previously tested negative for protein‐truncating variants in BRCA1 , BRCA2 , CHEK2 , PALB2 , RAD51C , RAD51D , and TP53 using conventional methods were included. Next‐generation sequencing (NGS) was performed using the Illumina MiSeq platform, and all identified P/LP variants were validated by Sanger sequencing. Twenty‐four unique P/LP variants were identified across seven genes: BRCA1 ( n  = 10), BRCA2 ( n  = 6), TP53 ( n  = 3), CHEK2 ( n  = 2), PALB2 , ATM , and RAD51C ( n  = 1 each). Two recurrent BRCA1 variants, p.Gln169Ter and p.Val757Phefs*8, were identified in three patients each. NGS‐detected P/LP variants were identified in 18.1% (29/160) of patients. When combined with previous germline testing in the same cohort, the overall detection rate increased to 50.2% (132/263): BRCA1 (101/263; 38.4%), BRCA2 (22/263; 8.4%), TP53 (3/263; 1.1%), CHEK2 (2/263; 0.8%), PALB2 (2/263; 0.8%), ATM (1/263; 0.4%) and RAD51C (1/263; 0.4%). Among these, BRCA1/2 variants accounted for 93.2% (123/132) of all P/LP variants. Our findings demonstrate that P/LP variants are concentrated in a limited number of genes, with BRCA1/2 as the predominant contributors. We propose a cost‐effective, first‐tier genetic testing panel comprising seven genes ( ATM , BRCA1 , BRCA2 , CHEK2 , RAD51C , PALB2 , and TP53 ) for familial BC risk assessment in Pakistan.

Prevalence of FANCM germline variants in BRCA1/2 negative breast and/or ovarian cancer patients from Pakistan

The Fanconi anemia complementation group M (FANCM) gene is a potential candidate for breast/ovarian cancer susceptibility in European populations. Here, we examined the contribution of FANCM germline variants to hereditary breast and/or ovarian cancer in Pakistan. Comprehensive FANCM variant screening was performed in 201 BRCA1 and BRCA2 (BRCA1/2) negative Pakistani patients with and without triple-negative breast cancer (TNBC) and/or ovarian cancer, using denaturing high-performance liquid chromatography analysis (DHPLC) followed by DNA sequencing. Novel variants were tested for their potential effect on protein function using in silico tools. Reverse transcription (RT)-PCR analysis of RNA extracted from one deletion/insertion (delins) variant (p.K1780delinsNGIT) carrier and three non-carriers was performed to evaluate the impact of this variant on splicing. Furthermore, potentially functional variants were evaluated in 200 healthy female controls. A missense variant (p.V1857M) was identified in a 50-year-old TNBC patient with a family history of breast cancer. It was also identified in the index patient´s daughter, who was diagnosed with osteosarcoma at 15 years of age. Further, one delins variant (p.K1780delinsNGIT) was identified in a 45-year-old non-TNBC patient, but not detected in her brother, who was diagnosed with Hodgkin's lymphoma at 38 years of age. Based on in silico and RNA analyses, p.V1857M and p.K1780delinsNGIT were predicted as variants of uncertain significance (VUS), respectively. Both variants were absent in 200 healthy controls. Our findings suggest a marginal contribution of FANCM variants to hereditary breast/ovarian cancer in Pakistan, which need to be confirmed in larger studies.

Chasing the origin of 23 recurrent BRCA1 mutations in Pakistani breast and ovarian cancer patients

AbstractKnowledge of population specific BRCA1/2 founder mutations provides a valuable and cost‐effective genetic testing strategy. Twenty‐three recurrent BRCA1 mutations have been identified previously in 100 Pakistani breast and/or ovarian cancer families. These accounted for 72.5% of all BRCA1 mutations identified. In our study, we investigated whether these mutations (identified in ≥2 unrelated patients) have a common ancestral origin and estimated the ages of these mutations. Haplotype analyses were performed in 188 individuals (100 index patients, 88 relatives) from Pakistani breast/ovarian cancer families, all harboring one of the 23 recurrent BRCA1 mutations, and 90 healthy controls. Six microsatellite markers (D17S800, D17S1801, D17S855, D17S1322, D17S1323, and D17S951) were analyzed. Mutation ages were estimated using DMLE+2.3 software. An identical haplotype of different length was found in families harboring the same BRCA1 mutation and suggested founder effects for all 23 mutations. Sixteen founder mutations were ethnicity‐specific: 15 occurred in families of Punjabi background and one in a family of Pathan background. The remaining seven mutations occurred in families with two ethnic backgrounds. All BRCA1 founder mutations were estimated to have arisen approximately 147 to 159 generations ago. Our findings suggest founder effects for all 23 recurrent BRCA1 mutations. This knowledge allows the design and development of a cost effective local genetic testing strategy in Pakistan.