The French National Registry of patients with Facioscapulohumeral muscular dystrophy

Background

Facioscapulohumeral muscular dystrophy is a rare inherited neuromuscular disease with an estimated prevalence of 1/20,000 and France therefore harbors about 3000 FSHD patients. With research progress and the development of targeted therapies, patients’ identification through registries can facilitate and improve recruitment in clinical trials and studies.

Results

The French National Registry of FSHD patients was designed as a mixed model registry involving both patients and physicians, through self-report and clinical evaluation questionnaires respectively, to collect molecular and clinical data. Because of the limited number of patients, data quality is a major goal of the registry and various automatic data control features have been implemented in the bioinformatics system. In parallel, data are manually validated by molecular and clinical curators. Since its creation in 2013, data from 638 FSHD patients have been collected, representing about 21% of the French FSHD population. The mixed model strategy allowed to collect 59.1% of data from both patients and clinicians; 26 and 14.9% from respectively patients and clinicians only. With the identification of the FSHD1 and FSHD2 forms, specific questionnaires have been designed. Though FSHD2 patients are progressively included, FSHD1 patients still account for the majority (94.9%). The registry is compatible with the FAIR principles as data are Findable, Accessible and Interoperable. We thus used molecular standards and standardized clinical terms used by the FILNEMUS French network of reference centers for the diagnosis and follow-up of patients suffering from a rare neuromuscular disease. The implemented clinical terms mostly map to dictionaries and terminology systems such as SNOMED-CT (75% of terms), CTV3 (61.7%) and NCIt (53.3%). Because of the sensitive nature of data, they are not directly reusable and can only be accessed as aggregated data after evaluation and approval by the registry oversight committee.

Conclusions

The French National Registry of FSHD patients belongs to a national effort to develop databases, which should now interact with other initiatives to build a European and/or an international FSHD virtual registry for the benefits of patients. It is accessible at www.fshd.fr and various useful information, links, and documents, including a video, are available for patients and professionals.

     

A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules

With the aim of quickly and easily characterizing new estrogen or anti-estrogen molecules, we developed a cellular model in which estrogenic action can be detected by bioluminescence. This model is based on MCF-7 cells stably transfected with a receptor gene which allows expression of the firefly luciferase enzyme under control of the estrogen regulatory element of the Xenopus vitellogenin A2 gene. A stably transfected cell line (cultured for more than eight months without loss of the chimeric estrogenic response) was established by cotransfection of a neomycin resistance gene and cloning under selective pressure. Subcloning luminescent clones was accomplished by using a single-photon detecting camera. This cellular model allowed the study of an estrogenic activity either in whole-cell or in cell-free experiments by detection of the induced luciferase. Estradiol induced the luciferase activity in a dose-dependent manner at subnanomolar concentrations. The induced luciferase activity reached a maximum level as early as 24 hours after the cells were incubated with estradiol. The antiestrogen 4-hydroxy-tamoxifen inhibited the luciferase activity induced by estradiol. The cross-reactivity of ligands, such as dexamethasone, progesterone, testosterone, aldosterone, calcitriol, oxysterol and retinoic acid, were also studied, showing an estradiol specificity for a 24-hour incubation time.     

Low-serum medium development for human diploid fibroblast microcarrier cultures

A new medium supplement mixture, PPRF92, has been developed to enable the serial subculture of human diploid fibroblasts (MRC-5 cells) on microcarriers. Furthermore, the PPRF92 supplements enable cell growth at serum levels as low as 1%. Through an optimization programme, the PPRF92 supplements have evolved into a simple mixture with the concentrations of key components at a level that makes the overall cost very competitive with medium containing 10% foetal bovine serum (FBS). Furthermore, the PPRF92 supplement mixture is most efficacious when FBS is replaced with the cheaper, and more widely available, adult bovine serum (ABS). Although medium exchange with serum is necessary in order to achieve confluence on microcarriers, the PPRF92 mixture is only necessary at the initiation of each passage. Using the medium replinishment protocol that has been developed in our laboratory, MRC-5 cells were successfully serially passaged through 13 bead-to-bead transfers on microcarriers in DMEM/F12 medium enriched with the PPRF92 supplement mixture reported here, and 1% ABS.Correspondence to: L. A. Behie     

QTL analysis of bread-making quality in wheat using a doubled haploid population

A set of 187 doubled haploid lines derived from the cross between cvs. Courtot and Chinese Spring was explored for QTLs for three bread-making quality tests: hardness, protein content and strength of the dough (W of alveograph). The scores of the parental lines were quite different except for protein content, and the population showed a wide range of variation. About 350 molecular and biochemical markers were used to establish the genetic map, and technological criteria were evaluated in 1 to 3 years. QTL detection was performed by the ”marker regression” method. The most significant unlinked markers were used in the model as covariates, and the results were tested by bootstrap resampling. For hardness, we confirmed a previously tagged major QTL on chromosome 5DS, and two additional minor QTLs were found on chromosome 1A and 6D, respectively. For protein content two main QTLs were identified on chromosomes 1B and 6A, respectively. For W, three consistent QTLs were detected: two at the same location as those for hardness, on chromosomes 1A and 5D; the third one on chromosome 3B. Therefore, it appeared that except for the Glu-1A locus, storage protein loci were not clearly involved in the genetic control of the criteria studied in the present work. Despite the reasonable size of the population no QTL with interactive effects could be substantially established as measured. All computations were carried out using home-made programmes in Splus language, and these are available upon request. Received: 16 May 1999 / Accepted: 15 October 1999     

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.     

Differential proliferation rates generate patterns of mechanical tension that orient tissue growth

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal–distal (P–D) axis by the Dachsous‐Fat‐Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P–D axis despite strong Dachs polarization. Here, we show that these circumferential divisions are oriented by circumferential mechanical forces that influence cell shapes and thus orient the mitotic spindle. We propose that this circumferential pattern of force is not generated locally by polarized constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localized overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape.     

In vivo calpain/caspase cross-talk during 3-nitropropionic acid-induced striatal degeneration: implication of a calpain-mediated cleavage of active caspase-3

The role of caspases and calpains in neurodegeneration remains unclear. In this study, we focused on these proteases in a rat model of Huntington's disease using the mitochondrial toxin 3-nitropropionic acid (3NP). Results showed that 3NP-induced death of striatal neurons was preceded by cytochrome c redistribution, transient caspase-9 processing, and activation of calpain, whereas levels of the active/processed form of caspase-3 remained low and were even reduced as compared with control animals. We evidenced here that this decrease in active caspase-3 levels could be attributed to calpain activation. Several observations supported this conclusion. 1) Pharmacological blockade of calpain in 3NP-treated rats increased the levels of endogenous processed caspase-9 and caspase-3. 2) Cell-free extracts prepared from the striatum of 3NP-treated rats degraded in vitro the p34 and p20 subunits of active recombinant caspase-9 and caspase-3, respectively. 3) This degradation of p34 and p20 could be mimicked by purified mu-calpain and was prevented by calpain inhibitors. 4) mu-Calpain produced a loss of the DEVDase (Asp-Glu-Val-Asp) activity of active caspase-3. 5) Western blot analysis and experiments with 35S-radiolabeled caspase-3 showed that mu-calpain cleaved the p20 subunit of active caspase-3 near its catalytic site. 6) mu-Calpain activity was selectively inhibited (IC50 of 100 mum) by a 12 amino acid peptide corresponding to the C terminus of p20. Our results showed that calpain can down-regulate the caspase-9/caspase-3 cell death pathway during neurodegeneration due to chronic mitochondrial defects in vivo and that this effect may involve, at least in part, direct cleavage of the caspase-3 p20 subunit.     

Influence of sparkle and saccades on tongue electro-stimulation-based vision substitution of 2D vectors

Vision substitution by electro-stimulation has been studied since the 60s beginning with P. Bach-y-Rita. Camera pictures or movies encoded in gray levels are displayed using an electro-stimulation display device on the surface of a body part, such as the skin or the tongue. Medical-technical devices have been developed on this principle to compensate for sensory-motor disabilities such as blindness or loss of balance, or to guide specific actions, such as surgery. However, the electrical signals of stationary or moving slowly moving objects, displayed on a Tongue display unit (TDU), are quickly lost due to saturation of receptors undergoing electrostimulation. We propose to add random saccades or sparkle to the displayed visual scene to increase the quality of pattern recognition by the subjects. In the present experimental trimodal study (normal vision, TDU vision substitution, or both), we show that the presence of a moderate sparkle level enhances the perception of the direction of lines drawn on a TDU and reduces the response time.     

Backseat drivers: the hidden influence of microbial viruses on disease

Because viral replication depends on the vigour of its host, many viruses have evolved incentives of fitness to pay their keep. When the viral host is a human pathogen, these fitness factors can surface as virulence: creating a Russian doll of pathogenesis where pathogens within pathogens complicate the disease process. Microbial viruses can even be independently immunogenic, as we recently reported for leishmania-virus. Thus, the incidence of this 'hyperpathogenism' is becoming an important clinical consideration and by appreciating the microbial-virus as a backseat driver of human disease, we could exploit its presence as a diagnostic biomarker and molecular target for therapeutic intervention. Here we discuss the prevalence of clinically relevant hyperpathogenism as well as the environmental sanctuaries that breed it.