Tissue-Specific Regulation of CFTR Gene Expression.

More than 2000 variations are described within the CFTR (Cystic Fibrosis Transmembrane Regulator) gene and related to large clinical issues from cystic fibrosis to mono-organ diseases. Although these CFTR-associated diseases have been well documented, a large phenotype spectrum is observed and correlations between phenotypes and genotypes are still not well established. To address this issue, we present several regulatory elements that can modulate CFTR gene expression in a tissue-specific manner. Among them, cis-regulatory elements act through chromatin loopings and take part in three-dimensional structured organization. With tissue-specific transcription factors, they form chromatin modules and can regulate gene expression. Alterations of specific regulations can impact and modulate disease expressions. Understanding all those mechanisms highlights the need to expand research outside the gene to enhance our knowledge.

3D Chromatin Organization Involving MEIS1 Factor in the cis-Regulatory Landscape of GJB2.

The human genome is covered by 8% of candidate cis-regulatory elements. The identification of distal acting regulatory elements and an understanding of their action are crucial to determining their key role in gene expression. Disruptions of such regulatory elements and/or chromatin conformation are likely to play a critical role in human genetic diseases. Non-syndromic hearing loss (i.e., DFNB1) is mostly due to GJB2 (Gap Junction Beta 2) variations and DFNB1 large deletions. Although several GJB2 cis-regulatory elements (CREs) have been described, GJB2 gene regulation remains not well understood. We investigated the endogenous effect of these CREs with CRISPR (clustered regularly interspaced short palindromic repeats) disruptions and observed GJB2 expression. To decipher the GJB2 regulatory landscape, we used the 4C-seq technique and defined new chromatin contacts inside the DFNB1 locus, which permit DNA loops and long-range regulation. Moreover, through ChIP-PCR, we determined the involvement of the MEIS1 transcription factor in GJB2 expression. Taken together, the results of our study enable us to describe the 3D DFNB1 regulatory landscape.

Whole-Genome Sequencing Improves the Diagnosis of DFNB1 Monoallelic Patients.

Hearing loss is the most common sensory defect, due in most cases to a genetic origin. Variants in the GJB2 gene are responsible for up to 30% of non-syndromic hearing loss. Today, several deafness genotypes remain incomplete, confronting us with a diagnostic deadlock. In this study, whole-genome sequencing (WGS) was performed on 10 DFNB1 patients with incomplete genotypes. New variations on GJB2 were identified for four patients. Functional assays were realized to explore the function of one of them in the GJB2 promoter and confirm its impact on GJB2 expression. Thus, in this study WGS resolved patient genotypes, thus unlocking diagnosis. WGS afforded progress and bridged some gaps in our research.

CFTR Cooperative Cis-Regulatory Elements in Intestinal Cells.

About 8% of the human genome is covered with candidate cis-regulatory elements (cCREs). Disruptions of CREs, described as "cis-ruptions" have been identified as being involved in various genetic diseases. Thanks to the development of chromatin conformation study techniques, several long-range cystic fibrosis transmembrane conductance regulator (CFTR) regulatory elements were identified, but the regulatory mechanisms of the CFTR gene have yet to be fully elucidated. The aim of this work is to improve our knowledge of the CFTR gene regulation, and to identity factors that could impact the CFTR gene expression, and potentially account for the variability of the clinical presentation of cystic fibrosis as well as CFTR-related disorders. Here, we apply the robust GWAS3D score to determine which of the CFTR introns could be involved in gene regulation. This approach highlights four particular CFTR introns of interest. Using reporter gene constructs in intestinal cells, we show that two new introns display strong cooperative effects in intestinal cells. Chromatin immunoprecipitation analyses further demonstrate fixation of transcription factors network. These results provide new insights into our understanding of the CFTR gene regulation and allow us to suggest a 3D CFTR locus structure in intestinal cells. A better understand of regulation mechanisms of the CFTR gene could elucidate cases of patients where the phenotype is not yet explained by the genotype. This would thus help in better diagnosis and therefore better management. These cis-acting regions may be a therapeutic challenge that could lead to the development of specific molecules capable of modulating gene expression in the future.

Characterization of GJB2 cis-regulatory elements in the DFNB1 locus.

Although most disease-causing variants are within coding region of genes, it is now well established that cis-acting regulatory sequences, depending on 3D-chromatin organization, are required for temporal and spatial control of gene expression. Disruptions of such regulatory elements and/or chromatin conformation are likely to play a critical role in human genetic disease. Hence, recurrent monoallelic cases, who present the most common hereditary type of nonsyndromic hearing loss (i.e., DFNB1), carry only one identified pathogenic allele. This strongly suggests the presence of uncharacterized distal cis-acting elements in the missing allele. Here within, we study the spatial organization of a large DFNB1 locus encompassing the gap junction protein beta 2 (GJB2) gene, the most frequently mutated gene in this inherited hearing loss phenotype, with the chromosome conformation capture carbon copy technology (5C). By combining this approach with functional activity reporter assays and mapping of CCCTC-binding factor (CTCF) along the DFNB1 locus, we identify a novel set of cooperating GJB2 cis-acting elements and suggest a DFNB1 three-dimensional looping regulation model.

Novel long-range regulatory mechanisms controlling PKD2 gene expression.

BACKGROUND: Cis-regulatory elements control gene expression over large distances through the formation of chromatin loops, which allow contact between enhancers and gene promoters. Alterations in cis-acting regulatory systems could be linked to human genetic diseases. Here, we analyse the spatial organization of a large region spanning the polycystic kidney disease 2 (PKD2) gene, one of the genes responsible of autosomal dominant polycystic kidney disease (ADPKD). RESULTS: By using chromosome conformation capture carbon copy (5C) technology in primary human renal cyst epithelial cells, we identify novel contacts of the PKD2 promoter with chromatin regions, which display characteristics of regulatory elements. In parallel, by using functional analysis with a reporter assay, we demonstrate that three DNAse I hypersensitive sites regions are involved in the regulation of PKD2 gene expression. CONCLUSIONS: Finally, through alignment of CCCTC-binding factor (CTCF) sites, we suggest that these novel enhancer elements are brought to the PKD2 promoter by chromatin looping via the recruitment of CTCF.

Analysis of long-range interactions in primary human cells identifies cooperative CFTR regulatory elements.

A mechanism by which control DNA elements regulate transcription over large linear genomic distances is by achieving close physical proximity with genes, and looping of the intervening chromatin paths. Alterations of such regulatory 'chromatin looping' systems are likely to play a critical role in human genetic disease at large. Here, we studied the spatial organization of a ≈790 kb locus encompassing the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Dysregulation of CFTR is responsible for cystic fibrosis, which is the most common lethal genetic disorder in Caucasian populations. CFTR is a relatively large gene of 189 kb with a rather complex tissue-specific and temporal expression profile. We used chromatin conformation at the CFTR locus to identify new DNA sequences that regulate its transcription. By comparing 5C chromatin interaction maps of the CFTR locus in expressing and non-expressing human primary cells, we identified several new contact points between the CFTR promoter and its surroundings, in addition to regions featuring previously described regulatory elements. We demonstrate that two of these novel interacting regions cooperatively increase CFTR expression, and suggest that the new enhancer elements located on either side of the gene are brought together through chromatin looping via CTCF.

The initial characteristics of marine oil emulsions and the composition of the media inflect lipid oxidation during in vitro gastrointestinal digestion.

This work aimed at evaluating the effect of simulated digestive fluids, interface and lipid droplet sizes on the oxidation of oil-in-water emulsions containing long chain n-3 fatty acyls. Emulsions stabilised by a protein or by phosphatidyl-choline/Tween 80 were submitted to gastro-intestinal in vitro conditions in presence of metmyoglobin. The gastric phase was characterised by a decrease of tocopherol amounts and moderate O2 uptake and aldehyde formation. Oxidation developed further during the intestinal phase, with tocopherols tending to zero, oxygen uptake and production of aldehydes at potentially toxic concentrations. The simulated digestive fluids reduced oxygen uptake and MDA formation only during the intestinal step of the phospholipid-stabilised emulsion. Quantitative losses of PUFA (e.g. EPA, DHA) were less than 10% even significant at the end of the digestion.

The effect of introducing IGRA to screen French healthcare workers for tuberculosis and potential conclusions for the work organisation.

INTRODUCTION: In France, pre-employment screening for tuberculosis (TB) is performed for healthcare workers (HCW). Screening is repeated when exposure to TB patients or infectious material occurs. The results of these TB screenings were analysed in a retrospective analysis. METHOD: Tuberculin skin tests (TST) and interferon-gamma release assays (QuantiFERON® Gold In-Tube - QFT) were used to perform the TB screenings. The screening results of 637 HCWs on whom QFT was performed were taken from the records of the University Hospital of Nantes. RESULTS: In three (0.5%) HCW, the QFT was indeterminate. In 22.2%, the QFT was positive. A second QFT was performed in 118 HCWs. The reversion rate was 42% (5 out of 17). The conversion rate was 6% (6 out of 98). A TST was performed on 466 (73.5%) of the HCWs. Results for TST > 10 mm were 77.4%. In those with a TST < 10 mm, QFT was positive in 14% and in those with a TST ≥ 10 mm, QFT was positive in 26.7%. Depending on the definition for conversion in the QFT, the annual attack rate was 4.1% or 7.3%. X-ray and pneumology consultation was based on positive QFT rather than TST alone (52 out of 56). No active TB was detected. CONCLUSION: The TST overestimated the prevalence of LTBI in this cohort. The decision about X-ray and consultation regarding preventive treatment should be based on the QFT rather than the TST results. The high reversion rate should be taken into consideration when consulting with HCWs regarding preventive treatment. The high conversion rate seems to indicate that preventive measures such as wearing masks should be improved.

Silica-associated limited systemic sclerosis after occupational exposure to calcined diatomaceous earth.

Silica-associated systemic sclerosis can occur in persons using calcined diatomaceous earth for filtration purpose. A limited systemic sclerosis was diagnosed in a 52-year-old male winegrower who had a combination of Raynaud's phenomenon, oesophageal dysfunction, sclerodactyly and telangectasia. The anti-centromere antibodies titre was 1/5000. The patient was frequently exposed to high atmospheric concentrations of calcined diatomaceous earth when performing the filtration of wines. Calcined diatomaceous earth is almost pure crystalline silica under the cristobalite form. The diagnosis of silica-associated limited systemic sclerosis after exposure to calcined diatomaceous earth was made. The patient's disease met the medical, administrative and occupational criteria given in the occupational diseases list 22 bis of the agriculture Social Security scheme and thence was presumed to be occupational in origin, without need to be proved. The diagnosis of occupational disease had been recognized by the compensation system of the agricultural health insurance.

Surveillance program of neck and upper limb musculoskeletal disorders: assessment over a 4 year period in a large company.

OBJECTIVE: A company-wide surveillance program for musculoskeletal disorders (MSDs) based on the assessment of health and risk factors was implemented between 1996 and 2000 in a large shoe factory. The study aimed to compare the results of the surveillance program in 1996 and 1997 with the occurrence of MSDs in 2000. METHODS: A health and ergonomic assessment of workstations was performed for 253 workers in 1996. Of these, 166 were examined again in both 1997 and 2000. A set of criteria was used to predict whether or not a job category could be predicted to have a potentially high risk of MSDs in 1996 and 1997 and the results were compared with the incidence rate of MSDs in the job category in 2000. RESULTS: The criteria based on prevalence data in 1996 were unable to detect the job categories characterized by the occurrence of MSDs in 2000. The criterion based on an incidence rate >1% in 1997 was sensitive and specific. The agreement between the ranking of the job categories according to incidence rate of MSDs in 1997 and 2000 was good (rho=0.57, P=0.11). Agreement of the prediction based on ergonomic exposure was lower than that based on incidence data. CONCLUSION: The efficacy of the surveillance program to predict on a collective basis the job categories in which numerous cases of MSDs occurred in 2000 depended on the decision criteria used. The criteria based on the incidence of MSDs were more reliable to predict the risk of MSDs than those based on prevalence data or on exposure assessment. Because exposure assessment plays a greater role in determining the priorities for ergonomic intervention, surveillance of health and exposure must nevertheless be combined to predict the risk of MSDs in the plant in the short and middle terms.