To our knowledge, this is the largest Pakistani study that comprehensively investigated the spectrum of BRCA1/2 small-range mutations and LGRs and prevalence of mutations in 539 high-risk families. Mutations were identified in 24.7% (133/539) of families. Eighteen BRCA1 and three BRCA2 mutations were recurrent and accounted for 68.2% and 34.8% of all mutations in BRCA1 and BRCA2, respectively. Nine mutations were specific to the Pakistani population, whereas other mutations had been reported elsewhere.
The most common type of identified mutations were frameshift mutations (60.6%) followed by nonsense mutations (25.4%). These data are consistent with our previous report  and a recent worldwide study . In Pakistani patients, BRCA1 mutations were about 5-times more frequent than BRCA2 mutations. A similar distribution was observed in two Asian studies from South India  and Saudi Arabia  and most studies among white populations [3,4,5, 28]. This is likely due to the predominance of recurrent BRCA1 mutations in these populations. Contradictory results were reported in other Asian studies from China, Hong Kong, Korea, and Indonesia, where BRCA2 mutations were observed at an equal or a higher frequency than BRCA1 mutations [6, 12, 15,16,17].
Among the 133 mutations identified in our study, 18 BRCA1 and three BRCA2 mutations were recurrent, accounting for 68.2% and 34.8% of all mutations in BRCA1 and BRCA2, respectively. The proportion of recurrent BRCA1 mutations to the total number of identified BRCA1 mutations is higher than our previous report , which is likely due to the larger size of the present study. Of the identified recurrent mutations, the majority was also reported as recurrent mutations in other populations [1, 4, 25], while few were exclusively identified in a specific ethnic group of Pakistan. Fourteen BRCA1 mutations (c.3770_3771del, c.5503C>T, c.4485-1G>A, c.2405_2406del, c.1793T>G, c.4508C>A, c.2603C>G, c.3339_3341del, c.3598C>T, c.5035del, c.5074+1G>A, c.5361_5362del, exon 1-2 deletion, and exon 21-24 deletion) and one BRCA2 mutation (c.92G>A) were identified only in the Punjabi ethnic group. One BRCA2 mutation (c.5682C>A) was found only in the Pathan ethnic group. Five other recurrent mutations were identified in more than one ethnic group. Our findings imply that a panel of ethnic specific recurrent mutations may be useful for initial screening of high-risk patients from these ethnic groups. Founder effects were previously suggested for six of the 18 recurrent BRCA1 mutations (c.3770_3771del, c.4065_4068del, c.4485-1G>A, c.4508C>A, c.5503C>T, exon 1-2 deletion) [9,10,11], while haplotype analyses of the remaining recurrent mutations have not been performed so far. The high percentage of recurrent BRCA1 mutations facilitates the development of a local, economical, and efficient ethnic-specific genetic testing strategy in Pakistan.
BRCA1/2 mutations were identified in 24.7% of Pakistani breast cancer families. This frequency is higher than that from our initial report (17%) , probably due to the larger study size and comprehensive mutation analyses of both genes. This frequency is also higher than those from other Asian reports from Hong Kong, Malaysia, and Korea, ranging from 9.4% to 21.7% [6,7,8, 16, 17]. These findings further support the notion that the BRCA1/2 mutation frequencies vary among different populations. Our data are similar to those reported in white populations, ranging from 17.6% to 29.8% [1, 2, 4]. We found the highest mutation frequency in breast and ovarian cancer families (55.4%), in agreement with previous studies from Pakistan , Korea , and studies in white populations [4, 28]. We observed a 2.52 fold (53.8% vs. 21.3%) increased occurrence of BRCA1 mutations in breast and ovarian cancer families compared to breast cancer only families, in line with previous reports [1, 4, 6, 28]. Our findings support the notion that the presence of ovarian cancer in Pakistani breast cancer families increases the likelihood for the occurrence of BRCA1 mutation.
In the present study on 27 families with male breast cancer, a BRCA1/2 mutation frequency of approximately 19% was observed, with BRCA2 mutations being about 4-times more common than BRCA1 mutations. Our data are in line with previous studies [4, 14]. This observed frequency is higher than that reported in our initial much smaller study, in which no mutations were identified . In agreement with the National Comprehensive Cancer Network (NCCN) guidelines, our data also warrant BRCA1/2 testing in families with male breast cancer (NCCN Guidelines Version 2.2019).
The main strength of this study is its large size of 539 high-risk families, the comprehensive screening of both genes for small-range mutations and LGRs using highly sensitive methods (allowing the identification of recurrent BRCA1/2 mutations in the Pakistani population and the more accurate estimation of BRCA1/2 mutation frequencies among high-risk families), and the confirmations of mutations in an independent patient’s sample. However, our study also has some limitations. Participants were recruited at one tertiary care cancer center in Lahore, which may have introduced selection bias. Families belonging to Punjabi and Pathan ethnic groups are over-represented and, therefore, mutations in these groups may be over-represented. Nevertheless, Punjabi (44.7%) and Pathan (15.4%) are the most common ethnic groups reported in Pakistan (The World Factbook). Further, our data are based on self-reported ethnicity of study participants, which may lead to a misclassification of the ethnic origin of some of them.