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Variants of uncertain significance (VUS) are a significant issue for the molecular diagnosis of rare diseases. The publication of episignatures as effective biomarkers of certain Mendelian neurodevelopmental disorders has raised hopes to help classify VUS. However, prediction abilities of most published episignatures have not been independently investigated yet, which is a prerequisite for an informed and rigorous use in a diagnostic setting. We generated DNA methylation data from 102 carriers of (likely) pathogenic variants in ten different genes, 58 VUS carriers, and 25 healthy controls. Combining published episignature information and new validation data with a k-nearest-neighbour classifier within a leave-one-out scheme, we provide unbiased specificity and sensitivity estimates for each of the signatures. Our procedure reached 100% specificity, but the sensitivities unexpectedly spanned a very large spectrum. While ATRX, DNMT3A, KMT2D, and NSD1 signatures displayed a 100% sensitivity, CREBBP-RSTS and one of the CHD8 signatures reached less than 40% sensitivity on our dataset. Remaining Cornelia de Lange syndrome, KMT2A, KDM5C and CHD7 signatures reached 70%-100% sensibility at best with unstable performances, suffering from heterogeneous methylation profiles among cases and rare discordant samples. Our results call for cautiousness and demonstrate that episignatures do not perform equally well. Some signatures are ready for confident use in a diagnostic setting. Yet, it is imperative to characterise the actual validity perimeter and interpretation of each episignature with the help of larger validation sample sizes and in a broader set of episignatures.

Over the last 20 years since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, more than 1,600 different putatively pathological CFTR mutations have been identified. Until now, however, copy number mutations (CNMs) involving the CFTR gene have not been methodically analyzed, resulting almost certainly in the under-ascertainment of CFTR gene duplications compared with deletions. Here, high-resolution array comparative genomic hybridization (averaging one interrogating probe every 95 bp) was used to analyze the entire length of the CFTR gene (189 kb) in 233 cystic fibrosis chromosomes lacking conventional mutations. We succeeded in identifying five duplication CNMs that would otherwise have been refractory to analysis. Based upon findings from this and other studies, we propose that deletion and duplication CNMs in the human autosomal genome are likely to be generated in the proportion of approximately 2-3:1. We further postulate that intragenic gene duplication CNMs in other disease loci may have been routinely underascertained. Finally, our analysis of +/-20 bp flanking each of the 40 CFTR breakpoints characterized at the DNA sequence level provide support for the emerging concept that non-B DNA conformations in combination with specific sequence motifs predispose to both recurring and nonrecurring genomic rearrangements. Hum Mutat 31:421-428, 2010. (C) 2010 Wiley-Liss, Inc.

One of the causes of chronic pancreatitis is the duplication and triplication of a approximately 605 kb segment containing the trypsinogen locus. Employing array-comparative genomic hybridization, we fully characterized the triplication copy number mutation (CNM) and found it to be part of a complex rearrangement that also contains a triplicated approximately 137 kb segment and 21 bp sequence tract. This triplication allele therefore constitutes a gain of two tandemly arranged composite duplication blocks, each comprising a copy of the approximately 605 kb segment, a copy of the inverted approximately 137 kb segment and a copy of the inverted 21 bp sequence tract. As such, it represents the first characterization of a human complex triplication CNM at the DNA sequence level. All triplications and duplications identified were found to arise from a common founder chromosome. A two-step process is proposed for the generation of this highly unusual triplication CNM. Thus, the first composite duplication block is envisaged to have been generated by break-induced serial replication slippage during mitosis. This duplication would have provided the sequence homology required to promote non-allelic homologous recombination (NAHR) during meiosis which would then, in a second step, have generated the complex triplication allele. Our data provide support for the view that many human germline copy number variants arise through replication-based mechanisms during the premeiotic mitotic divisions of germ cells. The low copy repeats thereby generated could then serve to promote NAHR during meiosis, giving rise to amplified DNA sequences which would themselves predispose to further recombinational events during both mitosis and meiosis.

About 95% of the CML patients with chronic myeloid leukemia (CML) have a Philadelphia chromosome resulting from a reciprocal translocation between bands 9q34 and 22q11.2 that juxtaposes the 3' region of the ABL gene to the 5' region of BCR. Over the past few years, submicroscopic deletions due to the loss of sequences proximal to chromosome 9 breakpoint or distal to chromosome 22 breakpoint have been found using fluorescence in situ hybridization (FISH). Among 150 CML bone marrow samples analyzed by molecular cytogenetics in our laboratory, 11 had a der(9) deletion detectable by FISH (deletion of the 5'ABL region and 3'BCR region in 10 samples and deletion of the 5'ABL region solely in 1 sample). To delineate the size of the deletions, FISH mapping was performed using 22 bacterial artificial chromosomes (BACs), 11 on either side of the breakpoints, the mean distance between BACs being 0.5 Mb. The deletion size of the 5'ABL region on the der(9) extended from 2 to 5 Mb, the minimal deletion size being localized between BACs RP11-101E3 and RP11-83J21. In two patients, the deletion size of the 3'BCR region was about 500 kb (between RP11-80O7 and RP11-681C06). The poor prognosis associated with these deletions was postulated by several workers to be explained by haploinsufficiency of a tumor suppressor gene. However, in our cases, the hypothetical deletion of one or more tumor suppressor genes is not sufficient to explain the poor response to interferon therapy, but the good response to imatinib treatment. We think that there could be one or more genes coding for interferon receptors or for proteins acting directly or indirectly with these receptors in the deleted regions.

Objectives/Hypothesis: Wood dust is a well-established risk factor for intestinal type sinonasal adenocarcinoma. The 5-year overall survival has varied from 20% to 80% according T1-T4 stages; 5-year survival according to histologic subtype has varied from 20% to 50%. To date, no study has evaluated whether environmental, occupational, and personal risk factors have any impact on both overall and cancer-specific survival. We aimed to determine whether exposure to carcinogenic risk factors besides wood exposure can influence the survival of patients with sinonasal ethmoid carcinoma. Study Design: Retrospective cohort study of the association of survival data and occupational and personal carcinogenic risk factors. Methods: All patients hospitalized for ethmoid adenocarcinoma at the Nantes University Hospital between 1988 and 2004 were included . Data concerning TNM classification, histology, type and quality of tumor resection at the macro- and microscopic level, and occupational and personal exposure to carcinogens were collected. Statistical analysis was conducted using univariate and multivariate linear regression. Results: A total of 98 patients were included with a response rate of 98%. Data showed 86% of patients had been exposed to wood dust. The 5-year survival was 62%. We first identified four factors that independently influenced overall survival: diplopia (P = .0159), spread to the orbit (P = .0113), bilateral involvement (P = .0134), TNM stage (P < .001). When the analysis included all occupational environmental factors (wood dust, solvent, and metals exposure) as well as personal risk factors, the length of exposure to metals (P = .0307) and tobacco exposure (P = .0031) also were found to influence 5-year overall survival. We identified high prevalence of colon cancer (4%) and double cancer (18%). Conclusions: We showed exposure to both environmental (tobacco) and occupational (metal dust) factors could influence survival in the diagnosis of a cancer. Our study suggests that screening for colon cancer should be offered to wood dust workers. A prospective multicentric study should be necessary to confirm our results.

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Background Sinonasal adenocarcinomas are uncommon tumors which develop in the ethmoid sinus after exposure to wood dust. Although the etiology of these tumors is well defined, very little is known about their molecular basis and no diagnostic tool exists for their early detection in high-risk workers. Methods To identify genes involved in this disease, we performed gene expression profiling using cancer-dedicated microarrays, on nine matched samples of sinonasal adenocarcinomas and non-tumor sinusal tissue. Microarray results were validated by quantitative RT-PCR and immunohistochemistry on two additional sets of tumors. Results Among the genes with significant differential expression we selected LGALS4, ACS5, CLU, SRI and CCT5 for further exploration. The overexpression of LGALS4, ACS5, SRI, CCT5 and the downregulation of CLU were confirmed by quantitative RT-PCR. Immunohistochemistry was performed for LGALS4 (Galectin 4), ACS5 (Acyl-CoA synthetase) and CLU (Clusterin) proteins: LGALS4 was highly up-regulated, particularly in the most differentiated tumors, while CLU was lost in all tumors. The expression of ACS5, was more heterogeneous and no correlation was observed with the tumor type. Conclusion Within our microarray study in sinonasal adenocarcinoma we identified two proteins, LGALS4 and CLU, that were significantly differentially expressed in tumors compared to normal tissue. A further evaluation on a new set of tissues, including precancerous stages and low grade tumors, is necessary to evaluate the possibility of using them as diagnostic markers.

Hemochromatosis is predominantly associated with the HFE p.C282Y homozygous genotype, which is carried by approximately 1 person in 200 in Northern European populations. However, p.C282Y homozygosity is often characterized by incomplete penetrance. Here, we describe the case of a woman who had a major structural alteration in the HFE gene. Molecular characterization revealed an Alu-mediated recombination leading to the loss of the entire HFE gene sequence. Although homozygous for the HFE deleted allele, the woman had a phenotype similar to that seen in most women homozygous for the common p.C282Y mutation. Contrasting with previously reported results in Hfe knockout and Hfe knockin mice, our report gives further evidence that progression of the disease depends on modifying factors.