Evaluation of pathogenic variants detected in high homology regions of the gene. How effective is long-range PCR?

Introduction: Lynch syndrome (LS) is an inherited cancer predisposition syndrome characterized by a high risk of colorectal and extracolonic tumors. Germline pathogenic variants (GPV) in the gene are associated with <15% of all cases. The pseudogene presents high homology with , challenging molecular diagnosis by next-generation sequencing (NGS). Due to the high methodological complexity required to distinguish variants between and , most laboratories do not clearly report the origin of this molecular finding.

Objective: The aim of this study was to confirm the GPVs detected by NGS in regions of high homology segments of the gene in a Brazilian sample.

Methods: An orthogonal and gold standard long-range PCR (LR-PCR) methodology to separate variants detected in the gene from those detected in the pseudogene.

Results: A total of 74 samples with a GPV detected by NGS in exons with high homology with pseudogene were evaluated. The most common was NM_000535.6:c.2182_2184delinsG, which was previously described as deleterious mutation in a study of African-American patients with LS and has been widely reported by laboratories as a pathogenic variant associated with the LS phenotype. Of all GPVs identified, only 6.8% were confirmed by LR-PCR. Conversely, more than 90% of GPV were not confirmed after LR-PCR, and the diagnosis of LS was ruled out by molecular mechanisms associated with

Conclusion: In conclusion, the use of LR-PCR was demonstrated to be a reliable approach for accurate molecular analysis of variants in segments with high homology with . We highlight that our laboratory is a pioneer in routine diagnostic complementation of the gene in Brazil, directly contributing to a more assertive molecular diagnosis and adequate genetic counseling for these patients and their families.