Germline pathogenic variants in E-cadherin (CDH1) confer high risk of developing lobular breast cancer and diffuse gastric cancer (DGC). The cumulative risk of DGC in CDH1 carriers has been recently reassessed (from 40–83% by age 80 to 25–42%) and varies according to the presence and number of gastric cancers in the family. As there is no accurate estimate of the risk of gastric cancer in families without DGC, the International Gastric Cancer Linkage Consortium recommendation is not straightforward: prophylactic gastrectomy or endoscopic surveillance should be proposed for these families. The inclusion of CDH1 in constitutional gene panels for hereditary breast and ovarian cancer and for gastrointestinal cancers, recommended by the French Genetic and Cancer Consortium in 2018 and 2020, leads to the identification of families with lobular cancer without DGC but also to incidental findings of pathogenic variants. Management of CDH1 carriers in case of incidental findings is complex and causes dilemmas for both patients and providers. We report eleven families (47 CDH1 carriers) from our oncogenetic department specialized in breast and ovarian cancer, including four incidental findings. We confirmed that six families did not have diffuse gastric cancer in their medical records. We discuss the management of the risk of diffuse gastric cancer in Hereditary Lobular Breast Cancer (HLBC) through a family of 11 CDH1 carriers where foci were identified in endoscopic surveillance. We also report a new colon signet ring cancer case in a CDH1 carrier, a rare aggressive cancer included in CDH1-related malignancies.
DNA methylation is thought to induce transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators unable to bind their target sites when methylated, and the recruitment of transcriptional repressors with specific affinity for methylated DNA. The Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. Here, we present MBD2 ChIPseq data obtained from the endogenous MBD2 in an isogenic cellular model of oncogenic transformation of human mammary cells. In immortalized (HMEC-hTERT) or transformed (HMLER) cells, MBD2 was found in a large proportion of methylated regions and associated with transcriptional silencing. A redistribution of MBD2 on methylated DNA occurred during oncogenic transformation, frequently independently of local DNA methylation changes. Genes downregulated during HMEC-hTERT transformation preferentially gained MBD2 on their promoter. Furthermore, depletion of MBD2 induced an upregulation of MBD2-bound genes methylated at their promoter regions, in HMLER cells. Among the 3,160 genes downregulated in transformed cells, 380 genes were methylated at their promoter regions in both cell lines, specifically associated by MBD2 in HMLER cells, and upregulated upon MBD2 depletion in HMLER. The transcriptional MBD2-dependent downregulation occurring during oncogenic transformation was also observed in two additional models of mammary cell transformation. Thus, the dynamics of MBD2 deposition across methylated DNA regions was associated with the oncogenic transformation of human mammary cells.
A, Closed symbols indicate patients affected with cancer. Open symbols indicate healthy individuals. The type of cancer and age at presentation are given in brackets. Blue circle represents c.4471_4474del variant and red circle represents the c.9648 + 1G > A. B, RNA was extracted from blood of patient III-3 and his sisters III-1 and III-4. RT-PCR analysis was performed with primers mapping to exons 25 and 27, and PCR products were separated by Bioanalyzer electrophoresis. The sizes of the DNA marker (M) are indicated to the left. LM, lower marker; UM, upper marker. C, Each RT-PCR product from patient III-3 was gel-purified and analyzed by Sanger sequencing. The 297-bp band corresponds to the reference BRCA2 transcript and the 150-bp band corresponds to a BRCA2 transcript lacking exon 26. Less than 10% of hereditary colorectal cancers (CRC) can be explained by well-defined syndromes, including Lynch syndrome, familial adenomatous polyposis, MUTYH-associated polyposis, and hamartomatous polyposis.1 BRCA2 codes for a DNA repair protein involved in homologous recombination. Heterozygous germline pathogenic variants in BRCA2 are involved in hereditary breast and ovarian cancer (HBOC) syndrome and biallelic pathogenic variants are responsible for Fanconi anemia (FA), characterized by congenital abnormalities, chromosome instability, bone marrow failure and predisposition to cancer.2 Some conflicting studies suggest the implication of BRCA2 in CRC risk.3 Here we report a family presenting early-onset CRC associated with biallelic BRCA2 variants but without signs of FA. The proband, patient III-3 (Figure 1A), presented a colorectal adenocarcinoma at 37 years old. Two years later, colonoscopic investigation revealed a second colorectal adenocarcinoma and one sessile serrated adenoma. Immunohistochemistry analyses showed normal expression of the MMR proteins MLH1, MSH2, MSH6 and PMS2. His brother, patient III-2, presented at 38 years of age a sigmoid adenocarcinoma with 17 adenomatous colorectal polyps. Retrospective clinical examination did not reveal any pathogenic features of FA. Chromosome-breakage analysis revealed no chromosomal breaks and exchanges typical of FA, particularly radial configurations from DEB-treated cultures. Informed consent was obtained for each patient and principles outlined in the Helsinki Declaration were followed. Study ethics approval was obtained on July 11, 2019 (CECIC Rhône-Alpes-Auvergne, Grenoble, IRB 5921). Multigene panel analysis was performed on DNA extracted from peripheral blood of patients III-3 and III-2 and did not reveal any variant in the major genes known to be involved in CRC risk (MMR, APC/MUTYH, POLD1/POLE, BMPR1A/SMAD4) and in three additional genes recently described in CRC risk (NTHL1, GREM1 and RNF43). Two BRCA2 (LRG_293) variants were observed: one causal c.4471_4474del; p.(Leu1491Lysfs*12) in exon 11 and one of unknown significance, c.9648 + 1G > A, a splice site variant in intron 26. RT-PCR analysis of patient RNA revealed that the c.9648 + 1G > A variant resulted in skipping of the second to last exon (exon 26), leading to an in-frame deletion of 46 amino acids (r.9502_9648del; p.Asn3168_Leu3216del) (Figure 1B). Minigene analysis showed that exon 26 skipping is total for this allele (Figure 1C). Three other variants causing skipping of exon 26 were previously reported as pathogenic or likely pathogenic for HBOC in the ClinVar database and this isoform was not listed in the naturally occurring alternate splicing events.4 However, loss of exon 26 does not seem to impact the tertiary structure and BRCA2 exon 26 is not a highly conserved region. BRCA2 protein without exon 26 could thus represent a partially functional isoform. The fact that both brothers declared early-onset CRC may indicate a role for the splice variant. Degrolard-Courcet et al (2014)5 already described a family of CRC predisposition with a BRCA2 pathogenic variant associated in trans with a variant leading to a partial splicing defect. This study and ours suggest that these splice variants could be hypomorphic, representing low-penetrance or disease-modifier alleles. The constitutional absence of any fully functional BRCA2 allele may also result in unexpected pleiotropic effects. In conclusion, multigene panel testing can reveal genetic predisposition, irrespective of the familial clinical phenotype. Our observations suggest that biallelic BRCA2 variants could be implicated in familial CRC inheritance but this has to be confirmed in large scale studies. The authors declare no conflict of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
Breast cancer is the most common cancer and leading cause of cancer death among women worldwide, encompassing a wide heterogeneity of subtypes with different clinical features. During the last two decades, the use of targeted therapies has emerged in clinical research in order to increase treatment efficiency, improve prognosis and reduce recurrence. However, the triple negative breast cancer (TNBC) subtype remains a clinical challenge, with poor prognosis since no therapeutic targets have been identified. This aggressive breast cancer entity lacks expression of oestrogen receptor (ER) and progesterone receptor (PR), and it does not overexpress human epidermal growth factor receptor 2 (HER2). The major reason for TNBC poor prognosis is early therapeutic escape from conventional treatments, leading to aggressive metastatic relapse. Metastases occur after an epithelial-mesenchymal transition EMT of epithelial cells, allowing them to break free from the primary tumour site and to colonize distant organs. Cancer-associated EMT consists not only of acquired migration and invasion ability, but involves complex and comprehensive reprogramming, including changes in metabolism, expression levels and epigenetic. Recently, many studies have considered epigenetic alterations as the primary initiator of cancer development and metastasis. This review builds a picture of the epigenetic modifications implicated in the EMT of breast cancer. It focuses on TNBC and allows comparisons with other subtypes. It emphasizes the role of the main epigenetic modifications lncRNAs, miRNAs, histone and DNA- modifications in tumour invasion and appearance of metastases. These epigenetic alterations can be considered biomarkers representing potential diagnostic and prognostic factors in order to define a global metastatic signature for TNBC.
Screening for BRCA mutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of the BRCA genes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.
Triple negative breast cancer (TNBC) accounts for 10-20% of breast cancers but has no specific therapy. While TNBC may be more sensitive to chemotherapy than other types of breast cancer, it has a poor prognosis. Most TNBC relapses occur during the five years following treatment, however predictive biomarkers of metastatic relapse are still lacking. High tumour-infiltrating lymphocytes (TILs) levels before and after neo-adjuvant chemotherapy (NAC) are associated with lower relapse risk and longer survival but TILs assessment is highly error-prone and still not introduced into the clinic. Therefore, having reliable biomarker of relapse, but easier to assess, remains essential for TNBC management. Searching for such biomarkers among serum/plasma proteins, circulating tumoral DNA (ctDNA) and blood cells appear relevant.This single-centre and prospective study aims to discover predictive biomarkers of TNBC relapse and particularly focuses on plasma proteins. Blood samples will be taken at diagnosis, on the day of first-line or post-NAC surgery, on the day of radiotherapy start, then 6 months and one year after radiotherapy. A blood sample will be taken at the time of metastatic relapse diagnosis. Blood samples will be used for circulating protein quantification, blood cell counts and circulating tumour DNA quantification. A tumour RNA signature, based on the analysis of the RNA expression of 6 genes, will also be tested from the initial biopsy taken routinely. In NAC patients, TILs quantity will be assessed on TNBC pre-treatment biopsy and surgical specimen.INSTIGO belongs to category 2 interventional research on humans. This study has been approved by the SUD-EST IV ethics committee and is conducted in accordance with the Declaration of Helsinki and General Data Protection Regulation (GDPR). Study findings will be published in peer-reviewed medical journals.ClinicalTrials.gov, identifier NCT04438681.
Sequence-specific endonucleases have been key to the study of the mechanisms and control of DNA double-strand break (DSB) repair and recombination, and the availability of CRISPR-Cas nucleases over the last decade has driven rapid progress in the understanding and application of targeted recombination in many organisms, including plants. We present here an analysis of recombination at targeted chromosomal 5' overhang DSB generated by the FnCas12a endonuclease in the plant,
Myoblast fusion follows a defined sequence of events that is strikingly similar in vertebrates and invertebrates. Genetic analysis in Drosophila has identified many of the molecules that mediate the different steps in the fusion process; by contrast, the molecular basis of myoblast fusion during vertebrate embryogenesis remains poorly characterised. A key component of the intracellular fusion pathway in Drosophila is the protein encoded by the myoblast city (mbc) gene, a close homologue of the vertebrate protein dedicator of cytokinesis 1 (DOCK1,formerly DOCK180). Using morpholino antisense-oligonucleotide-mediated knockdown of gene activity in the zebrafish embryo, we show that the fusion of embryonic fast-twitch myoblasts requires the activities of Dock1 and the closely related Dock5 protein. In addition, we show that the adaptor proteins Crk and Crk-like (Crkl), with which Dock proteins are known to interact physically, are also required for myoblast fusion.
