Evaluation of pathogenic variants detected in high homology regions of the PMS2 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 PMS2 gene are associated with <15% of all cases. The PMS2CL pseudogene presents high homology with PMS2, challenging molecular diagnosis by next-generation sequencing (NGS). Due to the high methodological complexity required to distinguish variants between PMS2 and PMS2CL, 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 PMS2 gene in a Brazilian sample. Methods: An orthogonal and gold standard long-range PCR (LR-PCR) methodology to separate variants detected in the PMS2 gene from those detected in the pseudogene. Results: A total of 74 samples with a PMS2 GPV detected by NGS in exons with high homology with PMS2CL 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 PMS2. Conclusion: In conclusion, the use of LR-PCR was demonstrated to be a reliable approach for accurate molecular analysis of PMS2 variants in segments with high homology with PMS2CL. We highlight that our laboratory is a pioneer in routine diagnostic complementation of the PMS2 gene in Brazil, directly contributing to a more assertive molecular diagnosis and adequate genetic counseling for these patients and their families.

Parental segregation study reveals rare benign and likely benign variants in a Brazilian cohort of rare diseases.

Genomic studies may generate massive amounts of data, bringing interpretation challenges. Efforts for the differentiation of benign and pathogenic variants gain importance. In this article, we used segregation analysis and other molecular data to reclassify to benign or likely benign several rare clinically curated variants of autosomal dominant inheritance from a cohort of 500 Brazilian patients with rare diseases. This study included only symptomatic patients who had undergone molecular investigation with exome sequencing for suspected diseases of genetic etiology. Variants clinically suspected as the causative etiology and harbored by genes associated with highly-penetrant conditions of autosomal dominant inheritance underwent Sanger confirmation in the proband and inheritance pattern determination because a "de novo" event was expected. Among all 327 variants studied, 321 variants were inherited from asymptomatic parents. Considering segregation analysis, we have reclassified 51 rare variants as benign and 211 as likely benign. In our study, the inheritance of a highly penetrant variant expected to be de novo for pathogenicity assumption was considered as a non-segregation and, therefore, a key step for benign or likely benign classification. Studies like ours may help to identify rare benign variants and improve the correct interpretation of genetic findings.

Frequency of carriers for rare recessive Mendelian diseases in a Brazilian cohort of 320 patients.

Several Mendelian disorders follow an autosomal recessive inheritance pattern. Epidemiological information on many inherited disorders may be useful to guide health policies for rare diseases, but it is often inadequate, particularly in developing countries. We aimed to calculate the carrier frequencies of rare autosomal recessive Mendelian diseases in a cohort of Brazilian patients using whole exome sequencing (WES). We reviewed the molecular findings of WES from 320 symptomatic patients who had carrier status for recessive diseases. Using the Hardy-Weinberg equation, we estimated recessive disease frequencies (q2 ) considering the respective carrier frequencies (2pq) observed in our study. We calculated the sensitivity of carrier screening tests based on lists of genes from five different clinical laboratories that offer them in Brazil. A total of 425 occurrences of 351 rare variants were reported in 278 different genes from 230 patients (71.9%). Almost half (48.8%) were carriers of at least one heterozygous pathogenic/likely pathogenic variant for rare metabolic disorders, while 25.9% of epilepsy, 18.1% of intellectual disabilities, 15.6% of skeletal disorders, 10.9% immune disorders, and 9.1% of hearing loss. We estimated that an average of 67% of the variants would not have been detected by carrier screening panels. The combined frequencies of autosomal recessive diseases were estimated to be 26.39/10,000 (or ~0.26%). This study shows the potential research utility of WES to determine carrier status, which may be a possible strategy to evaluate the clinical and social burden of recessive diseases at the population level and guide the optimization of carrier screening panels.