Abstract
Breast cancer (BC) is the most frequently diagnosed and the leading cause of cancer-related deaths among women worldwide. It is crucial to develop a cost-effective BC genetic panel for detection and diagnosis. In this study, tissue samples from 52 BC patients and peripheral blood samples from 18 healthy volunteers were collected in western China, followed by gDNA extraction. H&E and IHC analysis were employed to detect the expression of invasive BC tissues. We analyzed data using public databases such as COSMIC/ClinVar/HGMD along with our own previously published data and queried commercial BC panels to select high-risk genes. Using Illumina DesignStudio, gene panel primers consisting of 13 genes were designed with 696 primer pairs. The specificity of all primers was validated through common PCR assays. Once the gene panel was completed, multiple polymerase chain reactions (MPCR) were performed using the designed panel primers. The resulting MPCR products were purified to enrich them as library templates. Subsequently, after passing quality tests for library integrity assessment, Next-generation sequencing (NGS) was conducted. Through bioinformatics analysis of the NGS data, 4,571 variants were identified in the annotation files from 52 samples, classified into different types. Among these variants, 358 (approximately 7.8%) were newly discovered and distributed across 11 genes in 52 patients without in the ExAC database. The KMT2C gene exhibited the highest frequency of variants, presenting in 83.0% of 52 patient samples. Variants in BRCA2 (71%), BRCA1 (48%), PALB2 (40%), PIK3CA (23%), and RNF40 (21%) genes were found in over 20% of patients. Additionally, variants were observed in the AKT1 (12%), ERBB2 (10%), ESR1 (8%), TWIST1 (8%), and PIK3R1 (4%) genes. Further analysis using PolyPhen-2, SIFT, CADD, and Mutation Taster tools analysis showed that out of these new variants, 49 (49/358) had potential pathogenic effects on protein functions and structure across 52 patients. Consequently, a high-risk gene panel has been preliminarily established for early detection/diagnosis that will contribute to earlier prevention and treatment strategies for individuals with BC, particularly those residing in developing or underdeveloped countries. The identification of novel pathogenic variants within our cohort not only expands knowledge regarding genetic diagnosis applications for BC patients but also facilitates genetic counseling services for affected individuals and their families.