From variant of unknown significance to likely pathogenic: Characterization and pathogenicity determination of a large genomic deletion in the MLH1 gene

Abstract Background The MLH1 gene is one of the DNA mismatch repair genes (MMR), implicated in Lynch syndrome (LS), an autosomal dominant hereditary tumor susceptibility disease. The advent of next‐generation sequencing (NGS) technologies has accelerated the diagnosis of inherited diseases and increased the percentage of diagnosis of inherited cancers. However, some complex genomic alterations require the combination of several analytical strategies to allow correct biological interpretations. Here, we describe a novel MLH1 deletion and its pathogenicity determination in a patient suspected of LS. Methods The index case was a French 73‐year‐old man diagnosed with colorectal cancer displaying microsatellite instability and the loss of MLH1 and PMS2 expression. NGS analysis was used as the primary method for MMR genes screening. Long‐range PCR and reverse transcriptase polymerase chain reaction (RT‐PCR) were used for breakpoints and pathogenicity determinations. Results A large genomic deletion was detected which removed the last six nucleotides of MLH1 exon 11 together with a large part of intron 11. It was initially considered as a variant of unknown significance (VUS). Genomic breakpoints were subsequently characterized defining the deletion as c.1033_1039‐248del. Further RNA analysis demonstrated that this variant activated a cryptic donor splice site at the 5′ of the breakpoint, leading to a premature truncated protein: p.Thr345Alafs*13. Conclusion Our finding suggested that although NGS technologies have increased variant detection yield, combined approaches were still needed for complex variant characterization and pathogenicity assessment

A One-Step Prescreening for Point Mutations and Large Rearrangement in BRCA1 and BRCA2 Genes Using Quantitative Polymerase Chain Reaction and High-Resolution Melting Curve Analysis

International audienceHigh-resolution melting (HRM) of DNA is a versatile method for mutation scanning that monitors the fluorescence of double-strand DNA with saturating dye. Performing HRM on a real-time thermocycler enables semiquantitative analysis (quantitative polymerase chain reaction, qPCR) to be associated to HRM analysis for detection of both large gene rearrangements and point mutations (qPCR-HRM). We evaluated this method of mutation screening for the two major breast and ovarian cancer susceptibility genes BRCA1 and BRCA2. Screening of these two genes is time-consuming and must include exploration of large rearrangements that represent 5% to 15% of the alterations observed in these genes. To assess the reliability of the HRM technology, 201 known nucleotide variations scattered over all amplicons were tested. The sensitivity of qPCR was evaluated by analyzing seven large rearrangements. All previously identified variants tested were detected by qPCR-HRM. A retrospective study was done with 45 patients: qPCR-HRM allowed all the variants previously tested by denaturing high-performance liquid chromatography to be identified. qPCR analysis showed three cases of allele dropout (due to a 104-bp deletion, SNP primer mismatch, and an Alu insertion). A prospective study was done with 165 patients allowing 22 deleterious mutations, 16 unclassified variants, and 2 rearrangements to be detected. qPCR-HRM is a simple, sensitive, and fast method that does not require modified PCR primers. Thus, this method allows in one step the detection of point mutation, gene rearrangements, and prevention of missing a mutation due to primer mismatch

A One-Step Prescreening for Point Mutations and Large Rearrangement in BRCA1

International audienceHigh-resolution melting (HRM) of DNA is a versatile method for mutation scanning that monitors the fluorescence of double-strand DNA with saturating dye. Performing HRM on a real-time thermocycler enables semiquantitative analysis (quantitative polymerase chain reaction, qPCR) to be associated to HRM analysis for detection of both large gene rearrangements and point mutations (qPCR-HRM). We evaluated this method of mutation screening for the two major breast and ovarian cancer susceptibility genes BRCA1 and BRCA2. Screening of these two genes is time-consuming and must include exploration of large rearrangements that represent 5% to 15% of the alterations observed in these genes. To assess the reliability of the HRM technology, 201 known nucleotide variations scattered over all amplicons were tested. The sensitivity of qPCR was evaluated by analyzing seven large rearrangements. All previously identified variants tested were detected by qPCR-HRM. A retrospective study was done with 45 patients: qPCR-HRM allowed all the variants previously tested by denaturing high-performance liquid chromatography to be identified. qPCR analysis showed three cases of allele dropout (due to a 104-bp deletion, SNP primer mismatch, and an Alu insertion). A prospective study was done with 165 patients allowing 22 deleterious mutations, 16 unclassified variants, and 2 rearrangements to be detected. qPCR-HRM is a simple, sensitive, and fast method that does not require modified PCR primers. Thus, this method allows in one step the detection of point mutation, gene rearrangements, and prevention of missing a mutation due to primer mismatch

Combining Homologous Recombination and Phosphopeptide-binding Data to Predict the Impact of BRCA1 BRCT Variants on Cancer Risk

WOS:000454834200006International audienceBRCA1 mutations have been identified that increase the risk of developing hereditary breast and ovarian cancers. Genetic screening is now offered to patients with a family history of cancer, to adapt their treatment and the management of their relatives. However, a large number of BRCA1 variants of uncertain significance (VUS) are detected. To better understand the significance of these variants, a high-throughput structural and functional analysis was performed on a large set of BRCA1 VUS. Information on both cellular localization and homology-directed DNA repair (HR) capacity was obtained for 78 BRCT missense variants in the UMD-BRCA1 database and measurement of the structural stability and phosphopeptide-binding capacities was performed for 42 mutated BRCT domains. This extensive and systematic analysis revealed that most characterized causal variants affect BRCT-domain solubility in bacteria and all impair BRCA1 HR activity in cells. Furthermore, binding to a set of 5 different phosphopeptides was tested: all causal variants showed phosphopeptide-binding defects and no neutral variant showed such defects. A classification is presented on the basis of mutated BRCT domain solubility, phosphopeptide-binding properties, and VUS HR capacity. These data suggest that HR-defective variants, which present, in addition, BRCT domains either insoluble in bacteria or defective for phosphopeptide binding, lead to an increased cancer risk. Furthermore, the data suggest that variants with a WT HR activity and whose BRCT domains bind with a WT affinity to the 5 phosphopeptides are neutral. The case of variants with WT HR activity and defective phosphopeptide binding should be further characterized, as this last functional defect might be sufficient per se to lead to tumorigenesis. Implications: The analysis of the current study on BRCA1 structural and functional defects on cancer risk and classification presented may improve clinical interpretation and therapeutic selection