Digital multiplex ligation-dependent probe amplification identifies exon-level copy number variants in patients with suspected hereditary cancer and negative next-generation sequencing results.

BACKGROUND: Next-generation sequencing (NGS) has improved hereditary cancer diagnostics, but exon-level copy number variants (CNVs) may remain undetected because of uneven read depth or limitations of CNV-calling algorithms. Digital multiplex ligation-dependent probe amplification (digital MLPA) offers exon-level CNV assessment across multiple genes in a single experiment. This study evaluated whether digital MLPA can resolve cases with strong hereditary cancer features but negative multigene NGS results. METHODS AND RESULTS: Forty-eight patients with clinical suspicion of hereditary cancer and previously negative NGS panel testing were analyzed using the Hereditary Cancer Panel 1 (D001–D1, MRC Holland). Internal positive and negative controls were used to confirm assay performance. Digital MLPA identified clinically relevant CNVs in four unrelated families, corresponding to an additional diagnostic yield of 8.5% (4/47 informative families). The detected CNVs involved MLH1, and CDKN2A, each showing clear molecular or clinical correlation through tumor immunohistochemistry, segregation patterns, or personal/family history. All CNVs would have remained undetected if testing relied solely on sequencing-based pipelines. Additionally, a single-exon PMS2 deletion initially detected by both digital and conventional MLPA was subsequently re-evaluated and reclassified as a technical artifact following identification of a ligation-site SNV by NGS read inspection. CONCLUSIONS: Digital MLPA revealed clinically relevant CNVs in a subset of NGS-negative patients, demonstrating that structural variants continue to represent an important diagnostic blind spot. While the method proved practical for routine workflow integration, our findings highlight a critical pitfall regarding single-exon deletions. Since concordance between MLPA platforms does not rule out artifacts caused by ligation-site variants, visual inspection of NGS data is an essential validation step. Incorporating digital MLPA as a second-tier assay, with rigorous confirmation of single-probe findings, offers a robust strategy to improve genetic counseling and risk management.