Akademik Veri Yönetim Sistemi
2nd International Hereditary Cancer Congress, Antalya, Türkiye, 5 - 08 Şubat 2026, ss.25, (Özet Bildiri)
Re-evaluation of a Single-Exon Deletion Detected by MLPA in the MLH1 Gene Using Next-Generation Sequencing: A Case Report
Introduction: Copy number variants (CNVs) constitute a component of the diagnostic process in hereditary cancer predisposition syndromes. The MLH1 gene is one of the main mismatch repair genes associated with Lynch syndrome, and exon-level deletions and duplications detected in this gene are of clinical significance. Although Multiplex Ligation-dependent Probe Amplification (MLPA) is a sensitive method for the detection of copy number changes, it may yield false-positive results, particularly in cases involving single-exon deletions. One reason for this limitation is the presence of sequence variants within MLPA probe-binding regions, which may interfere with probe hybridization and ligation to the target sequence. In this study, we present a case of a patient with colon cancer in whom an MLPA-detected deletion of exon 15 in the MLH1 gene was shown to be associated with a heterozygous variant localized to the probe-binding region identified by Next-Generation Sequencing (NGS), highlighting the complementary roles of MLPA and NGS. Case: A patient diagnosed with colon cancer and evaluated for hereditary cancer predisposition initially underwent an NGS-based hereditary cancer gene panel. No significant pathogenic or likely pathogenic variants were detected in the NGS analysis. Due to persistent suspicion, MLPA analysis targeting the MLH1/MSH2 genes was subsequently performed, revealing a copy number decrease consistent with a deletion of exon 15 in the MLH1 gene. Given that this finding involved a single exon, the possibility of a technical artifact was considered, and the patient’s NGS data were re-evaluated. Reanalysis identified a heterozygous nucleotide variant, c.1693A>T, localized to the probe-binding region corresponding to the exon in which the deletion had been detected by MLPA. The MLH1 c.1693A>T heterozygous variant identified by NGS was reported with a population frequency of 0.1294% in the Turkish Variome database. The variant was found to be positioned in a manner likely to disrupt binding of the MLPA probe to its target sequence, leading to the conclusion that the deletion signal observed by MLPA represented a false-positive result caused by a sequence variant within the probe-binding region rather than a true genomic deletion. Discussion: Single-exon deletions require cautious interpretation in MLPA analyses. While MLPA is a method for detecting exon-level copy number changes, heterozygous nucleotide variants located within probe-binding regions may interfere with probe ligation and generate false deletion signals. As demonstrated in this case, identification of c.1693A>T was crucial in reclassifying the MLPA result from a pathogenic deletion to a technical artifact. This scenario underscores the importance of utilizing NGS data not just for SNV detection, but as a mandatory quality control step for validating isolated MLPA findings. Such a combined approach is vital for preventing the clinical mismanagement of patients suspected of having Lynch syndrome. Conclusion: Sequence variants within MLPA probe-binding regions represent an analytical pitfall that can mimic true genomic deletions. This report highlights that “single-exon” decreases in MLPA signals require mandatory confirmation to avoid misdiagnosis. The integration of NGS data into the MLPA interpretation process is essential for distinguishing technical artifacts from true copy number variants, ensuring accurate genetic counseling and clinical decision-making.