Detection of a pathogenic Alu element insertion in PALB2 gene from targeted NGS diagnostic data.

Despite routine analysis of a large panel of genes, pathogenic variants are only detected in approximately 20% of families with hereditary breast and/or ovarian cancer. Mobile element insertions (MEI) are known to cause genetic diseases in humans, but remain challenging to detect. Retrospective analysis of targeted next-generation sequencing (NGS) data from 359 patients was performed using a dedicated MEI detection pipeline. We detected one MEI in exon 9 of the PALB2 gene in a woman with a family history of breast cancer. The pathogenic variant, c.2872_2888delins114AluL2, disrupts the PALB2 coding sequence and leads to the production of a truncated protein, p.(Gln958Valfs*38). This is the first report of a pathogenic MEI in PALB2. This study illustrates that MEI analysis may help to improve molecular diagnostic yield and can be performed from targeted NGS data used for routine diagnosis.

TUMOSPEC: A Nation-Wide Study of Hereditary Breast and Ovarian Cancer Families with a Predicted Pathogenic Variant Identified through Multigene Panel Testing.

Assessment of age-dependent cancer risk for carriers of a predicted pathogenic variant (PPV) is often hampered by biases in data collection, with a frequent under-representation of cancer-free PPV carriers. TUMOSPEC was designed to estimate the cumulative risk of cancer for carriers of a PPV in a gene that is usually tested in a hereditary breast and ovarian cancer context. Index cases are enrolled consecutively among patients who undergo genetic testing as part of their care plan in France. First- and second-degree relatives and cousins of PPV carriers are invited to participate whether they are affected by cancer or not, and genotyped for the familial PPV. Clinical, family and epidemiological data are collected, and all data including sequencing data are centralized at the coordinating centre. The three-year feasibility study included 4431 prospective index cases, with 19.1% of them carrying a PPV. When invited by the coordinating centre, 65.3% of the relatives of index cases (5.7 relatives per family, on average) accepted the invitation to participate. The study logistics were well adapted to clinical and laboratory constraints, and collaboration between partners (clinicians, biologists, coordinating centre and participants) was smooth. Hence, TUMOSPEC is being pursued, with the aim of optimizing clinical management guidelines specific to each gene.

National recommendations of the French Genetics and Cancer Group - Unicancer on the modalities of multi-genes panel analyses in hereditary predispositions to tumors of the digestive tract.

In case of suspected hereditary predisposition to digestive cancers, next-generation sequencing can analyze simultaneously several genes associated with an increased risk of developing these tumors. Thus, "Gastro Intestinal" (GI) gene panels are commonly used in French molecular genetic laboratories. Lack of international recommendations led to disparities in the composition of these panels and in the management of patients. To harmonize practices, the Genetics and Cancer Group (GGC)-Unicancer set up a working group who carried out a review of the literature for 31 genes of interest in this context and established a list of genes for which the estimated risks associated with pathogenic variant seemed sufficiently reliable and high for clinical use. Pancreatic cancer susceptibility genes have been excluded. This expertise defined a panel of 14 genes of confirmed clinical interest and relevant for genetic counseling: APC, BMPR1A, CDH1, EPCAM, MLH1, MSH2, MSH6, MUTYH, PMS2, POLD1, POLE, PTEN, SMAD4 and STK11. The reasons for the exclusion of the others 23 genes have been discussed. The paucity of estimates of the associated tumor risks led to the exclusion of genes, in particular CTNNA1, MSH3 and NTHL1, despite their implication in the molecular pathways involved in the pathophysiology of GI cancers. A regular update of the literature is planned to up-grade this panel of genes in case of new data on candidate genes. Genetic and epidemiological studies and international collaborations are needed to better estimate the risks associated with the pathogenic variants of these genes either selected or not in the current panel.

[Inherited predisposition to breast cancer (2) : risks and surveillance]. / Prédisposition héréditaire au cancer du sein (2) - Risques et prise en charge.

Recent progres in massive deep sequencing have paved the way to routine exploration of the current panorama of inherited predisposition to breast cancer, through multigene panel analysis. Cancer risks and spectrum vary according to the gene at stake. Surveillance modalities are thus adapted to the risk-level : (1) very high, which follow specific recommendations from french NCI (INCa) ; (2) high and (3) moderate ; in the latter case, patients'follow-up compares to women of the general population. In the absence of causal mutation, other risk factors may be considered and professional scores be calculated. Of note and according to french HAS recommendations issued in 2014, the family history prevails ; on this basis, INCa has set up a national coverage of follow-up networks which specifically address high risk individuals, with or without mutation. Finally, an innovative path of specific targeted-therapies is currently opening for mutation carriers.

[Hereditary predisposition to breast cancer (1): genetics]. / Prédisposition héréditaire au cancer du sein (1) - Génétique.

The main objective of oncogenetics is to characterize a subpopulation of patients at high risk of cancer development at an early age in order to provide specific recommendations for an optimized follow-up and care path. Oncogenetic counselling helps to assess individual risk from a family history. By a family approach of formal genetics, the key issue is to identify families with a strong aggregation of cancers, and, in particular, suggesting a specific syndrome of inherited predisposition to cancer. This approach can lead to the proposal of germline genetic testing in search of causal mutations. As up to know, the search for a constitutional mutation in the BRCA genes has led to the identification of a causal deleterious mutation in less than 10% of index-cases analyzed. It is therefore important to evaluate the impact of new genes in the current panorama of inherited predisposition to breast and ovarian cancer.

Familial breast cancer and DNA repair genes: Insights into known and novel susceptibility genes from the GENESIS study, and implications for multigene panel testing.

Pathogenic variants in BRCA1 and BRCA2 only explain the underlying genetic cause of about 10% of hereditary breast and ovarian cancer families. Because of cost-effectiveness, multigene panel testing is often performed even if the clinical utility of testing most of the genes remains questionable. The purpose of our study was to assess the contribution of rare, deleterious-predicted variants in DNA repair genes in familial breast cancer (BC) in a well-characterized and homogeneous population. We analyzed 113 DNA repair genes selected from either an exome sequencing or a candidate gene approach in the GENESIS study, which includes familial BC cases with no BRCA1 or BRCA2 mutation and having a sister with BC (N = 1,207), and general population controls (N = 1,199). Sequencing data were filtered for rare loss-of-function variants (LoF) and likely deleterious missense variants (MV). We confirmed associations between LoF and MV in PALB2, ATM and CHEK2 and BC occurrence. We also identified for the first time associations between FANCI, MAST1, POLH and RTEL1 and BC susceptibility. Unlike other associated genes, carriers of an ATM LoF had a significantly higher risk of developing BC than carriers of an ATM MV (ORLoF = 17.4 vs. ORMV = 1.6; p Het = 0.002). Hence, our approach allowed us to specify BC relative risks associated with deleterious-predicted variants in PALB2, ATM and CHEK2 and to add MAST1, POLH, RTEL1 and FANCI to the list of DNA repair genes possibly involved in BC susceptibility. We also highlight that different types of variants within the same gene can lead to different risk estimates.

[The French Genetic and Cancer Consortium guidelines for multigene panel analysis in hereditary breast and ovarian cancer predisposition]. / Recommandations françaises du Groupe Génétique et Cancer pour l'analyse en panel de gènes dans les prédispositions héréditaires au cancer du sein ou de l'ovaire.

INTRODUCTION: Next generation sequencing allows the simultaneous analysis of large panel of genes for families or individuals with a strong suspicion of hereditary breast and/or ovarian cancer (HBOC). Because of lack of guidelines, several panels of genes potentially involved in HBOC were designed, with large disparities not only in their composition but also in medical care offered to mutation carriers. Then, homogenization in practices is needed. METHODS: The French Genetic and Cancer Group (GGC) - Unicancer conducted an exhaustive bibliographic work on 18 genes of interest. Only publications with unbiased risk estimates were retained. RESULTS: The expertise of each 18 genes was based on clinical utility criteria, i.e. a relative risk of cancer of 4 and more, available medical tools for screening and prevention of mutation carriers, and pre-symptomatic genetic tests for relatives. Finally, 13 genes were selected to be included in a HBOC diagnosis gene panel: BRCA1, BRCA2, PALB2, TP53, CDH1, PTEN, RAD51C, RAD51D, MLH1, MSH2, MSH6, PMS2, EPCAM. The reasons for excluding NBN, RAD51B, CHEK2, STK11, ATM, BARD1, BRIP1 from the HBOC diagnosis panel are presented. Screening, prevention and genetic counselling guidelines were detailed for each of the 18 genes. DISCUSSION: Due to the rapid increase in knowledge, the GGC has planned a yearly update of the bibliography to take into account new findings. Furthermore, genetic-epidemiological studies are being initiated to better estimate the cancer risk associated with genes which are not yet included in the HBOC diagnosis panel.

Diagnosis of Constitutional Mismatch Repair-Deficiency Syndrome Based on Microsatellite Instability and Lymphocyte Tolerance to Methylating Agents.

BACKGROUND & AIMS: Patients with bi-allelic germline mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2) develop a rare but severe variant of Lynch syndrome called constitutional MMR deficiency (CMMRD). This syndrome is characterized by early-onset colorectal cancers, lymphomas or leukemias, and brain tumors. There is no satisfactory method for diagnosis of CMMRD because screens for mutations in MMR genes are noninformative for 30% of patients. MMR-deficient cancer cells are resistant to genotoxic agents and have microsatellite instability (MSI), due to accumulation of errors in repetitive DNA sequences. We investigated whether these features could be used to identify patients with CMMRD. METHODS: We examined MSI by PCR analysis and tolerance to methylating or thiopurine agents (functional characteristics of MMR-deficient tumor cells) in lymphoblastoid cells (LCs) from 3 patients with CMMRD and 5 individuals with MMR-proficient LCs (controls). Using these assays, we defined experimental parameters that allowed discrimination of a series of 14 patients with CMMRD from 52 controls (training set). We then used the same parameters to assess 23 patients with clinical but not genetic features of CMMRD. RESULTS: In the training set, we identified parameters, based on MSI and LC tolerance to methylation, that detected patients with CMMRD vs controls with 100% sensitivity and 100% specificity. Among 23 patients suspected of having CMMRD, 6 had MSI and LC tolerance to methylation (CMMRD highly probable), 15 had neither MSI nor LC tolerance to methylation (unlikely to have CMMRD), and 2 were considered doubtful for CMMRD based on having only 1 of the 2 features. CONCLUSION: The presence of MSI and tolerance to methylation in LCs identified patients with CMMRD with 100% sensitivity and specificity. These features could be used in diagnosis of patients.

Uncovering and dissecting the genotoxicity of self-inactivating lentiviral vectors in vivo.

Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.

Technological overview of iPS induction from human adult somatic cells.

The unlimited proliferation capacity of embryonic stem cells (ESCs) combined with their pluripotent differentiation potential in various lineages raised great interest in both the scientific community and the public at large with hope for future prospects of regenerative medicine. However, since ESCs are derived from human embryos, their use is associated with significant ethical issues preventing broad studies and therapeutic applications. To get around this bottleneck, Takahashi and Yamanaka have recently achieved the conversion of adult somatic cells into ES-like cells via the forced expression of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. This first demonstration attracted public attention and opened a new field of stem cells research with both cognitive - such as disease modeling - and therapeutic prospects. This pioneer work just received the 2012 Nobel Prize in Physiology or Medicine. Many methods have been reported since 2006, for the generation of induced pluripotent stem (iPS) cells. Most strategies currently under use are based on gene delivery via gamma-retroviral or lentiviral vectors; some experiments have also been successful using plasmids or transposons- based systems and few with adenovirus. However, most experiments involve integration in the host cell genome with an identified risk for insertional mutagenesis and oncogenic transformation. To circumvent such risks which are deemed incompatible with therapeutic prospects, significant progress has been made with transgene-free reprogramming methods based on e.g.: sendai virus or direct mRNA or protein delivery to achieve conversion of adult cells into iPS. In this review we aim to cover current knowledge relating to both delivery systems and combinations of inducing factors including chemicals which are used to generate human iPS cells. Finally, genetic instability resulting from the reprogramming process is also being considered as a safety bottleneck for future clinical translation and stem cell-therapy prospects based on iPS.

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.

Comprehensive genomic access to vector integration in clinical gene therapy.

Retroviral vectors have induced subtle clonal skewing in many gene therapy patients and severe clonal proliferation and leukemia in some of them, emphasizing the need for comprehensive integration site analyses to assess the biosafety and genomic pharmacokinetics of vectors and clonal fate of gene-modified cells in vivo. Integration site analyses such as linear amplification-mediated PCR (LAM-PCR) require a restriction digest generating unevenly small fragments of the genome. Here we show that each restriction motif allows for identification of only a fraction of all genomic integrants, hampering the understanding and prediction of biological consequences after vector insertion. We developed a model to define genomic access to the viral integration site that provides optimal restriction motif combinations and minimizes the percentage of nonaccessible insertion loci. We introduce a new nonrestrictive LAM-PCR approach that has superior capabilities for comprehensive unbiased integration site retrieval in preclinical and clinical samples independent of restriction motifs and amplification inefficiency.

In vivo repopulation ability of genetically corrected bone marrow cells from Fanconi anemia patients.

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo, knockout animal models are not sufficient to guide preclinical steps, and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA, we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for >120 days, and after cultured cell transplantation into NOD/SCID mice, clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison, untransduced cells died in culture by 15 days. Of necessity for ethical reasons, experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements, a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition, we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA.