Virginie Moncoutier

Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2 genes as a model

To meet challenges in terms of throughput and turnaround time, many diagnostic laboratories are shifting from Sanger sequencing to higher throughput next-generation sequencing (NGS) platforms. Bearing in mind that the performance and quality criteria expected from NGS in diagnostic or research settings are strikingly different, we have developed an Ion Torrent’s PGM-based routine diagnostic procedure for BRCA1/2 sequencing. The procedure was first tested on a training set of 62 control samples, and then blindly validated on 77 samples in parallel with our routine technique. The training set was composed of difficult cases, for example, insertions and/or deletions of various sizes, large-scale rearrangements and, obviously, mutations occurring in homopolymer regions. We also compared two bioinformatic solutions in this diagnostic context, an in-house academic pipeline and the commercially available NextGene software (Softgenetics). NextGene analysis provided higher sensitivity, as four previously undetected single-nucleotide variations were found. Regarding specificity, an average of 1.5 confirmatory Sanger sequencings per patient was needed for complete BRCA1/2 screening. Large-scale rearrangements were identified by two distinct analyses, that is, bioinformatics and fragment analysis with electrophoresis profile comparison. Turnaround time was enhanced, as a series of 30 patients were sequenced by one technician, making the results available for the clinician in 10 working days following blood sampling. BRCA1/2 genes are a good model, representative of the difficulties commonly encountered in diagnostic settings, which is why we believe our findings are of interest for the whole community, and the pipeline described can be adapted by any user of PGM for diagnostic purposes.

Enhanced Mismatch Mutation Analysis: Simultaneous Detection of Point Mutations and Large Scale Rearrangements by Capillary Electrophoresis, Application to BRCA1 and BRCA2

We present the routine diagnostic application of EMMA (Enhanced Mismatch Mutation Analysis, Fluigent), a new, fast, reliable, and cost-effective method for mutation screening. This method is based on heteroduplex analysis by capillary electrophoresis and relies on the use of innovative matrices increasing the electrophoretic mobility differences between homoduplex and heteroduplex DNA, which is further enhanced by the addition of nucleosides in the separation matrix. Nucleosides interact with heteroduplex mismatched bases, hence increasing mobility difference with homoduplex. As separations are performed by multi-capillary electrophoresis, it allows for high automation, low cost, and high throughput. Moreover, EMMA, in combination with limiting PCR conditions, can be used to achieve the simultaneous detection of point mutation and large scale rearrangement in a single run.We now report on the routine diagnostic use of this method for BRCA1 and BRCA2 screening. The coding sequence and exon-intron junctions of BRCA1 and BRCA2 were amplified in 24 multiplex PCRs using a single condition. PCRs were electrophoresed with a single analytical condition on an ABI3100, and data were analyzed using dedicated software (Emmalys).The strength of this new method relies on the following assets: (1) a single condition of analysis: modeling related to melting domain is not required (2) simultaneous detection of point mutations and large scale rearrangements, (3) optimized and ready-to-use polymer that can be used on various ABI sequencers, (4) easy to use, (5) low reagent costs, and (6) throughput.

Breast and ovarian cancer predisposition due to de novo BRCA1 and BRCA2 mutations.

BRCA1 and BRCA2 are the two major genes predisposing to breast and ovarian cancer. Whereas high de novo mutation rates have been demonstrated for several genes, only 11 cases of de novo BRCA1/2 mutations have been reported to date and the BRCA1/2 de novo mutation rate remains unknown. The present study was designed to fill this gap based on a series of 12 805 consecutive unrelated patients diagnosed with breast and/or ovarian cancer who met the inclusion criteria for BRCA1/2 gene analysis according to French guidelines. BRCA1/2 mutations were detected in 1527 (12%) patients, and three BRCA1 mutations and one BRCA2 mutation were de novo. The BRCA1/2 de novo mutation rate was estimated to be 0.3% (0.1%; 0.7%). Although rare, it may be useful to take the possibility of de novo BRCA1/2 mutation into account in genetic counseling of relatives and to improve the understanding of complex family histories of breast and ovarian cancers.

Sporadic endometrial adenocarcinoma with MMR deficiency due to biallelic MSH2 somatic mutations

The invalidation of the Mismatch Repair (MMR) system is responsible for a so-called “deficient MMR” phenotype (dMMR) characterized by microsatellite instability and abnormal pattern of expression of MMR proteins in tumor tissue. This phenotype occurs in at least 20% of sporadic endometrial adenocarcinomas by epigenetic silencing of MLH1 gene. It is also observed in virtually all tumors occurring in patients with Lynch syndrome by monoallelic germline mutation in one of the MMR genes. The determination of this phenotype (dMMR vs. proficient MMR—pMMR) has therefore a pivotal place in the diagnosis algorithm for Lynch syndrome by monoallelic germline mutation in one of the MMR genes. The determination of this phenotype (dMMR vs. proficient MMR—pMMR) has therefore a pivotal place in the diagnosis algorithm for Lynch syndrome. We report the case of a woman with an early-onset endometrial adenocarcinoma who was suspected to be affected with Lynch syndrome based on tumor dMMR phenotype (MSI associated with loss of expression of MSH2 and MSH6 proteins). After complete germline and somatic evaluations, this phenotype was eventually explained by two MSH2 somatic mutations and the diagnosis of Lynch-like syndrome due to an unidentified MSH2 germline mutation was ruled out. Somatic mosaicism at low mutation rate was unlikely as no mutation was detected by DNA analysis from various tissue samples. Nevertheless, the three patient’s children were tested for the two mutations and these tests were negative. Biallelic somatic mutations of one MMR gene is a mechanism of invalidation of the MMR system in sporadic cases. Clinicians have to be aware of this mechanism because of the great clinical implication for the patients and their relatives.

Contribution of germline deleterious variants in the RAD51 paralogs to breast and ovarian cancers

RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have recently been involved in breast and ovarian cancer predisposition: RAD51B, RAD51C, and RAD51D in ovarian cancer, RAD51B and XRCC2 in breast cancer. The aim of this study was to estimate the contribution of deleterious variants in the five RAD51 paralogs to breast and ovarian cancers. The five RAD51 paralog genes were analyzed by next-generation sequencing technologies in germline DNA from 2649 consecutive patients diagnosed with breast and/or ovarian cancer. Twenty-one different deleterious variants were identified in the RAD51 paralogs in 30 patients: RAD51B (n = 4), RAD51C (n = 12), RAD51D (n = 7), XRCC2 (n = 2), and XRCC3 (n = 5). The overall deleterious variant rate was 1.13% (95% confidence interval (CI): 0.72–1.55%) (30/2649), including 15 variants in breast cancer only cases (15/2063; 0.73% (95% CI: 0.34–1.11%)) and 15 variants in cases with at least one ovarian cancer (15/570; 2.63% (95% CI: 1.24–4.02%)). This study is the first evaluation of the five RAD51 paralogs in breast and ovarian cancer predisposition and it demonstrates that deleterious variants can be present in breast cancer only cases. Moreover, this is the first time that XRCC3 deleterious variants have been identified in breast and ovarian cancer cases.

Full in-frame exon 3 skipping of BRCA2 confers high risk of breast and/or ovarian cancer

Germline pathogenic variants in the BRCA2 gene are associated with a cumulative high risk of breast/ovarian cancer. Several BRCA2 variants result in complete loss of the exon-3 at the transcript level. The pathogenicity of these variants and the functional impact of loss of exon 3 have yet to be established. As a collaboration of the COVAR clinical trial group (France), and the ENIGMA consortium for investigating breast cancer gene variants, this study evaluated 8 BRCA2 variants resulting in complete deletion of exon 3. Clinical information for 39 families was gathered from Portugal, France, Denmark and Sweden. Multifactorial likelihood analyses were conducted using information from 293 patients, for 7 out of the 8 variants (including 6 intronic). For all variants combined the likelihood ratio in favor of causality was 4.39*1025. These results provide convincing evidence for the pathogenicity of all examined variants that lead to a total exon 3 skipping, and suggest that other variants that result in complete loss of exon 3 at the molecular level could be associated with a high risk of cancer comparable to that associated with classical pathogenic variants in BRCA1 or BRCA2 gene. In addition, our functional study shows, for the first time, that deletion of exon 3 impairs the ability of cells to survive upon Mitomycin-C treatment, supporting lack of function for the altered BRCA2 protein in these cells. Finally, this study demonstrates that any variant leading to expression of only BRCA2 delta-exon 3 will be associated with an increased risk of breast and ovarian cancer.