Cédrick Lefol

Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants.

Assessing the impact of variants of unknown significance (VUS) on splicing is a key issue in molecular diagnosis. This impact can be predicted by in silico tools, but proper evaluation and user guidelines are lacking. To fill this gap, we embarked upon the largest BRCA1 and BRCA2 splice study to date by testing 272 VUSs (327 analyses) within the BRCA splice network of Unicancer. All these VUSs were analyzed by using six tools (splice site prediction by neural network, splice site finder (SSF), MaxEntScan (MES), ESE finder, relative enhancer and silencer classification by unanimous enrichment, and human splicing finder) and the predictions obtained were compared with transcript analysis results. Combining MES and SSF gave 96% sensitivity and 83% specificity for VUSs occurring in the vicinity of consensus splice sites, that is, the surrounding 11 and 14 bases for the 5′ and 3′ sites, respectively. This study was also an opportunity to define guidelines for transcript analysis along with a tentative classification of splice variants. The guidelines drawn from this large series should be useful for the whole community, particularly in the context of growing sequencing capacities that require robust pipelines for variant interpretation. Hum Mutat 33:1228–1238, 2012.

Dismantling papillary renal cell carcinoma classification: The heterogeneity of genetic profiles suggests several independent diseases.

Papillary renal cell carcinoma (pRCC) is the second most frequent renal cell carcinoma (RCC) after clear cell RCC. In contrast to clear cell RCC, there is no consensual protocol using targeted therapy for metastatic pRCC. Moreover, diagnosis of some pRCC, especially pRCC of type 2 (pRCC2) may be challenging. Our aim was to identify molecular biomarkers that could be helpful for the diagnosis and treatment of pRCC. We studied the clinical, histological, immunohistological, and comprehensive genetic features of a series of 31 pRCC including 15 pRCC1 and 16 pRCC2. We aimed to determine whether pRCC represents a unique entity or several diseases. In addition, we compared the genetic features of pRCC2 to those of eight RCC showing various degrees of tubulo‐papillary architecture, including three TFE‐translocation RCC and five unclassified RCC. We demonstrate that pRCC is a heterogeneous group of tumors with distinct evolution. While most pRCC2 had genetic profiles similar to pRCC1, some shared genomic features, such as loss of 3p and loss of chromosome 14, with clear cell RCC, TFE‐translocation RCC, and unclassified RCC. We identified variants of the MET gene in three pRCC1. A mutation in the BRAF gene was also identified in one pRCC1. In addition, using next‐generation sequencing (NGS), we identified several variant genes. Genomic profiling completed by NGS allowed us to classify pRCC2 in several groups and to identify novel mutations. Our findings provide novel information on the pathogenesis of pRCC that allow insights for personalized treatment.

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.

High-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.

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.

A diagnostic genetic test for the physical mapping of germline rearrangements in the susceptibility breast cancer genes BRCA1 and BRCA2

The BRCA1 and BRCA2 genes are involved in breast and ovarian cancer susceptibility. About 2 to 4% of breast cancer patients with positive family history, negative for point mutations, can be expected to carry large rearrangements in one of these two genes. We developed a novel diagnostic genetic test for the physical mapping of large rearrangements, based on molecular combing (MC), a FISH‐based technique for direct visualization of single DNA molecules at high resolution. We designed specific Genomic Morse Codes (GMCs), covering the exons, the noncoding regions, and large genomic portions flanking both genes. We validated our approach by testing 10 index cases with positive family history of breast cancer and 50 negative controls. Large rearrangements, corresponding to deletions and duplications with sizes ranging from 3 to 40 kb, were detected and characterized on both genes, including four novel mutations. The nature of all the identified mutations was confirmed by high‐resolution array comparative genomic hybridization (aCGH) and breakpoints characterized by sequencing. The developed GMCs allowed to localize several tandem repeat duplications on both genes. We propose the developed genetic test as a valuable tool to screen large rearrangements in BRCA1 and BRCA2 to be combined in clinical settings with an assay capable of detecting small mutations. Hum Mutat 33:998–1009, 2012.

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.