Department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan.
Department of Immunology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan.
Asian Pac J Cancer Prev. 2021 Mar 1;22(3):719-724. doi: 10.31557/APJCP.2021.22.3.719.
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.
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.
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.
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.
BRCA1/2 种系致病性突变构成了全球范围内大多数遗传性乳腺癌和/或卵巢癌。巴基斯坦女性的乳腺癌和卵巢癌发病率和死亡率都很高。因此，为了在巴基斯坦建立针对特定疗法的诊断，我们进行了基于下一代测序的种系测试，以检测与乳腺癌和卵巢癌亚型相关的 BRCA1/2 致癌变体。
在征得进行种系基因检测的同意后，从确诊患有乳腺癌和上皮性卵巢癌的 24 名女性患者中采集外周血。从外周血中提取 DNA，并进行索引 BRCA 面板文库。使用 AmpliSeq for Illumina BRCA 面板和 Illumina MiSeq 测序仪（放置在 AFIP）对 BRCA1 和 BRCA2 基因的所有编码区和剪接位点进行靶向 NGS。使用 Illumina 生物信息学工具对测序结果进行分析。
在所有研究病例中，我们在所有病例中均检测到了 421 个质量得分为 100 的变体。在过滤掉质量得分低于 q30 的变体和基于 gnomAD 浏览器的次要等位基因频率（MAF）>0.05 的变体后，缩小了在 BRCA1 和 BRCA2 基因中鉴定的变体列表。为了对这些变体进行分类，使用外部精心策划的数据库预测了临床意义。结果，我们在 BRCA1 中解释了（n=4）16.7%的致病性变体，在两个基因中解释了（n=6）25%的不确定意义（VUS）变体。描述性统计数据表明，BRCA 阳性病例的年龄和 BMI 均低于 BRCA 阴性病例。
我们的发现展示了在巴基斯坦进行基于 NGS 的癌症遗传测试的初步报告。这将使我们能够利用巴基斯坦本地最新的最先进技术进行遗传性 BRCA 突变的筛查和诊断，最终实现针对癌症的靶向治疗。
