A MALDI-TOF mass spectrometry-based method for detection of copy number variations in BRCA1 and BRCA2 genes

Identifying germline mutations in and genes ( ) would benefit the carriers in multiple aspects. In addition to single-nucleotide variations and small indels, copy number variations (CNVs) is also an indispensable component of identifiable mutations in . A sensitive, rapid and throughput-flexible method to detect CNVs would be preferred to meet the rising clinical requirements for testing. We developed a MALDI-TOF-MS-based method (MS assay) which included three steps: first, multiplex end-point PCR followed by a single base extension reaction; second, automated analyte transfer and data acquisition; third, data analysis. We applied MS assay to detect CNVs in in 293 Chinese patients with ovarian or pancreatic cancer. All the samples were examined by targeted next-generation sequencing (TS) simultaneously. Samples were further cross-validated by multiplex ligation-dependent probe amplification (MLPA) if the results from MS assay and TS were inconsistent. Long range PCR was then applied to identify the exact breakpoints in . MS assay introduced highly multiplexed panels to detect CNVs of semi-quantitatively. Simplified on-board data analysis was available for MS assay and no complex bioinformatics was needed. The turnaround time of MS assay was less than 8 hours with a hands-on time of only 40 min. Compared to TS, MS assay exhibited higher sensitivity (100% vs. 75%) and was more flexible in throughput, with the reagent cost per sample remaining constant no matter how many samples were examined per assay. A total of eight CNVs in were detected from the 293 samples, and the molecular breakpoints were successfully identified in five samples through long-range PCR followed by Sanger sequencing. Our results suggested that MS assay might be an effective method in primary screening for CNVs in genes such as , especially when short turnaround time and/or high sensitivity is a top priority.