Cost of exome analysis in patients with intellectual disability: a micro-costing study in a French setting.

BACKGROUND: With the development of next generation sequencing technologies in France, exome sequencing (ES) has recently emerged as an opportunity to improve the diagnosis rate of patients presenting an intellectual disability (ID). To help French policy makers determine an adequate tariff for ES, we aimed to assess the unit cost per ES diagnostic test for ID from the preparation of the pre-analytical step until the report writing step and to identify its main cost drivers. METHODS: A micro-costing bottom-up approach was conducted for the year 2018 in a French setting as part of the DISSEQ study, a cost-effectiveness study funded by the Ministry of Health and performed in collaboration with the GAD (Génétique des Anomalies du Développement), a genetic team from the Dijon University Hospital, and a public sequencing platform, the Centre National de Recherche en Génomique Humaine (CNRGH). The analysis was conducted from the point of view of these two ES stakeholders. All of the resources (labor, equipment, disposables and reagents, reusable material) required to analyze blood samples were identified, collected and valued. Several sensitivity analyses were performed. RESULTS: The unit nominal cost per ES diagnostic test for ID was estimated to be €2,019.39. Labor represented 50.7% of the total cost. The analytical step (from the preparation of libraries to the analysis of sequences) represented 88% of the total cost. Sensitivity analyses suggested that a simultaneous price decrease of 20% for the capture kit and 50% for the sequencing support kit led to an estimation of €1,769 per ES diagnostic test for ID. CONCLUSION: This is the first estimation of ES cost to be done in the French setting of ID diagnosis. The estimation is especially influenced by the price of equipment kits, but more generally by the organization of the centers involved in the different steps of the analysis and the time period in which the study was conducted. This information can now be used to define an adequate tariff and assess the efficiency of ES. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT03287206 on September 19, 2017.

Genetic polymorphisms of Th2 interleukins, history of asthma or eczema and childhood acute lymphoid leukaemia: Findings from the ESCALE study (SFCE).

BACKGROUND: Previous studies on the putative role of allergy in the aetiology of childhood leukaemia have reported contradictory results. The present study aimed to analyse the relation between a medical history of asthma or eczema and childhood acute lymphoid leukaemia (ALL) in light of potential candidate gene-environment interactions. METHODS: Analyses were based on a subset of 434 cases of ALL and 442 controls successfully genotyped and of European ancestry children enrolled in a French population-based case-control study conducted in 2003-2004. Information about medical history was obtained during a standardized interview with the mothers. Candidate polymorphisms in genes of the Th2 cytokines IL4, IL10, IL13 and IL4-receptor, were genotyped or imputed. RESULTS: None of the variant alleles were directly associated with childhood acute lymphoid leukaemia. A medical history of asthma or eczema was reported more often in the control group (OR = 0.7 [0.5-1.0]). This association was mostly seen in the group of children not carrying the IL13-rs20541 variant allele (Interaction Odds Ratio IOR 1.9, p-interaction = 0.07) and in those carrying the IL10 triple variant haplotype (IOR 0.5, p-interaction = 0.04). No interaction was observed with the candidate polymorphisms in IL4 and IL4R. CONCLUSION: This study provides a new insight into the relationship between allergic symptoms and childhood acute lymphoid leukaemia, by suggesting this inverse association could be limited to children carrying certain genetic polymorphisms. If confirmed, these results could help better understand the biological mechanisms involved in the development of childhood acute lymphoid leukaemia.

Single-nucleotide polymorphism discovery and validation in high-density SNP array for genetic analysis in European white oaks.

An Illumina Infinium SNP genotyping array was constructed for European white oaks. Six individuals of Quercus petraea and Q. robur were considered for SNP discovery using both previously obtained Sanger sequences across 676 gene regions (1371 in vitro SNPs) and Roche 454 technology sequences from 5112 contigs (6542 putative in silico SNPs). The 7913 SNPs were genotyped across the six parental individuals, full-sib progenies (one within each species and two interspecific crosses between Q. petraea and Q. robur) and three natural populations from south-western France that included two additional interfertile white oak species (Q. pubescens and Q. pyrenaica). The genotyping success rate in mapping populations was 80.4% overall and 72.4% for polymorphic SNPs. In natural populations, these figures were lower (54.8% and 51.9%, respectively). Illumina genotype clusters with compression (shift of clusters on the normalized x-axis) were detected in ~25% of the successfully genotyped SNPs and may be due to the presence of paralogues. Compressed clusters were significantly more frequent for SNPs showing a priori incorrect Illumina genotypes, suggesting that they should be considered with caution or discarded. Altogether, these results show a high experimental error rate for the Infinium array (between 15% and 20% of SNPs potentially unreliable and 10% when excluding all compressed clusters), and recommendations are proposed when applying this type of high-throughput technique. Finally, results on diversity levels and shared polymorphisms across targeted white oaks and more distant species of the Quercus genus are discussed, and perspectives for future comparative studies are proposed.

Chromosome 9p-linked families with frontotemporal dementia associated with motor neuron disease.

BACKGROUND: Frontotemporal dementia associated with motor neuron disease (FTD-MND) is a rare neurodegenerative disorder that may be inherited by autosomal dominant trait. No major gene has been identified but a locus was mapped on chromosome 9 (9p21.3-p13.3). METHODS: Ten French families with FTD-MND were tested for linkage to the 9p21.3-p13.3 region. We report extensive mutation screening in 9p-linked families and their clinical characteristics. RESULTS: We identified six new families with evidence for linkage to the chromosome 9p. Cumulative multipoint LOD score values were positive between markers D9S1121 and D9S301, reaching a peak of 8.0 at marker D9S248. Haplotype reconstruction defined the telomeric boundary at marker AFM218xg11, slightly narrowing the candidate interval. We found no disease-causing mutations by sequencing 29 candidate genes including IFT74 and no copy number variations in the 9p region. The mean age at onset was 57.9 +/- 10.3 years (range, 41-84), with wide heterogeneity within and among families suggesting age-dependant penetrance. The patients presented isolated FTD (32%), isolated MND (29%), or both disorders (39%). The general characteristics of the disease did not differ, except for an older age at onset and shorter disease duration in the 9p-linked compared to nonlinked families. TDP-43-positive neuronal cytoplasmic inclusions were found in cortex and spinal cord in 3 patients. CONCLUSIONS: This study increases the number of 9p-linked families now reported and shows that this locus may have a major effect on frontotemporal dementia (FTD) and motor neuron disease (MND). Considering our results, the causative gene might be implicated in at least 60% of the families with FTD-MND disorder.

Tuberculosis in Siberia: 2. Diagnosis, chemoprophylaxis and treatment.

OBJECTIVE: To assess tuberculosis diagnosis, chemoprophylaxis and therapy in Siberia as a paradigm for the Russian Federation. DESIGN: Data was obtained from official sources and through visits to dispensaries and hospitals in 1994. RESULTS: Tuberculosis disease and cure is classified according to a Dispensary Group Register based principally on clinical and radiological criteria. Isoniazid is widely used for chemoprophylaxis and post-therapy and may be linked to high levels of isoniazid resistance. Combination drug therapy is individualized, frequently changed, and given orally, parenterally or intra-bronchially. Galvanization, autotransfusion of ultra-violet irradiated blood, antioxidants and steroids are used as adjunct treatment. Ambulatory treatment is uncommon. Surgical treatments including lobectomy and pneumonectomy are used in 5-10% of patients. CONCLUSION: Tuberculosis is increasing in Siberia. An improved drug supply using short course standardized regimens is required supported by high quality co-ordinated bacteriological services. Surgery retains a useful role, but many adjunct therapies should be abandoned.

Tuberculosis in Siberia: 1. An epidemiological and microbiological assessment.

SETTING: Siberia, Russian Federation. OBJECTIVE: To assess the situation regarding tuberculosis as a paradigm for the Russian Federation. DESIGN: Data was obtained from official sources and through visits to dispensaries and hospitals in 1994. RESULTS: The downward trend in notifications of tuberculosis throughout Russia reversed in 1990/91, the rate increasing from 34/100,000 to 42.9/100,000 in 1993. Incidence rates are higher in Siberia, varying from approximately 43 to 108/100,000; prevalence is 250-300/100,000. The tuberculosis service is centralized and based on specialized polyclinics and dispensaries. An extensive surveillance system employs regular fluorography and tuberculin testing: half of the cases diagnosed are detected by fluorography, against 1% through contact tracing. Patients are classified principally on clinical and radiological grounds. Bacille Calmette-Guérin immunisation is performed at birth and at age 7, and again at 13, 21, and 28 years if Mantoux test is negative. Microscopy and culture services are organisationally separate, and direct comparison of smear and culture data is not possible. Drug resistance to isoniazid and streptomycin is probably high and resistance to rifampicin low, but data on susceptibility of isolates from new cases are not available. CONCLUSION: Tuberculosis is increasing in Siberia. Homelessness, unemployment and alcoholism are important factors, but concurrent human immunodeficiency virus (HIV) infection appears to be uncommon. Prisons probably form a significant reservoir of infectious cases.

Cultured rat skeletal muscle cells treated with cytochalasin exhibit normal dystrophin expression and intracellular free calcium control.

Many studies performed to elucidate the molecular and cellular processes involved in muscular dystrophies have led to the working hypothesis of a key role for the cytoskeleton elements linking the extracellular matrix to myofibrils. It was recently suggested that cytochalasin B treatment of mouse soleus muscle promoted cell damage mediated by a cytosolic increase in free calcium concentration. Since intracellular calcium overload may be a primary event resulting from the alteration of cytoskeletal structure, this study was intended to evaluate whether or not the integrity of the F-actin microfilament network is necessary for calcium homeostasis. The developmental establishment of the normal cytoarchitecture was altered by treatment of myoblasts with the actin-disrupting agents cytochalasin B and D, and the effects were compared with those in myoblasts treated with colchicine. These drugs modified the morphogenesis in that they prevented the formation of elongated myotubes by myoblast fusion, but did not prevent the maturation of contractile myogenic cells. The subcellular organisation of actin filaments visualised by confocal fluorescence microscopy was modified by colchicine and cytochalasins, but appearance of contractile apparatus and mechanical activity were not precluded. Sarcolemmal addressing of dystrophin, the subsarcolemmal protein lacking in Duchenne muscular dystrophy, was not prevented by cytochalasin. The evaluation of the basal activity of cytosolic calcium measured with indo-1 suggested that the disruption of actin or microtubules did not prevent developing muscle cells to maintain a low basal calcium activity. We propose that the global integrity of the cytoskeleton network is not crucial for the maintenance of calcium homeostasis in muscle cells developing in vitro. These results are discussed with regard to current theories attempting to understand the functional consequences of an abnormal expression of the dystrophin-glycoprotein complex interacting with the extracellular matrix and the cytoskeleton.

Genetic mapping of a susceptibility locus for insulin-dependent diabetes mellitus on chromosome 11q.

Loci in the major histocompatibility complex (MHC) on chromosome 6 and the insulin (INS) region on chromosome 11 have been implicated in susceptibility to insulin-dependent diabetes mellitus (IDDM) through candidate gene investigations, but they may account for less than 50% of genetic risk for the disease. Genome-wide linkage studies have led to localization of more than 10 susceptibility loci for insulin-dependent diabetes in the non-obese diabetic (NOD) mouse and the BB rat. Similar studies are now possible in humans through the development of dense genetic maps of highly informative microsatellite loci obtained using polymerase chain reaction analysis. We have applied microsatellite markers from recent Généthon maps, and other highly informative markers, in a genome-wide linkage study in IDDM. Here we report evidence for the localization of a previously undetected susceptibility locus for IDDM in the region of the FGF3 gene on chromosome 11q. Our results shows the potential of genome-wide linkage studies to detect susceptibility loci in IDDM and other multifactorial disorders.

A study of structure and degradation of nonpolymeric biomaterials implanted in bone using reflected and transmitted light microscopy.

Orthopedic biomaterials currently are made of metal alloy coated with one or more thin layers of dense or porous ceramic or metal. Sections of these materials implanted in human bone were made without altering the implant or bone-implant interfaces. Bone containing an implant was fixed and then embedded in polymethylmethacrylate. Thick sections were made using a cooled, low speed diamond saw, then ground and polished. Some were stained by fuchsin-toluidine staining solution, others were acid etched to reveal the structure of the metal contained in the prosthesis. Observation by reflected and transmitted light microscopy revealed microstructure of the implant material as well as features of the surrounding tissues.

The anticonvulsant hypersensitivity syndrome.

Anticonvulsants can cause a characteristic hypersensitivity reaction. This multisystem reaction typically presents as fever, mucocutaneous eruptions, lymphadenopathy and hepatitis. There is cross-reactivity between different anticonvulsants, which complicates subsequent therapy. We report three cases to illustrate both the typical features, and less common complications, of this under-recognized and life-threatening syndrome.

Appearance and evolution of calcium currents and contraction during the early post-fusional stages of rat skeletal muscle cells developing in primary culture.

Primary cultures from enzymatically dissociated satellite cells of newborn rat skeletal muscles enabled developmental in vitro studies of mechanical and electrical properties during the first steps of myogenesis. The present work focused on the appearance, evolution and roles of two types of calcium currents (ICa,T and ICa,L) and of depolarization-induced contractile activity during the early stages of muscle cell development in primary culture. Prefusional mononucleated cells (myoblasts), young myotubes of 1 day (with less than 10 nuclei) or 2-3 days (more than 9 nuclei) and myoballs from 4-6, 7-9, 10-12 and 13-16 days cultures were patch-clamped (whole-cell configuration), and calcium currents and contraction simultaneously recorded. Sodium but not calcium currents could be recorded at the myoblast stage. In young myotubes (1 day), ICa,L was present with high incidence as compared to ICa,T, which was poorly expressed. Contractile responses appeared at the next stage (2-3 days) while the occurrence of ICa,T progressively increased. This developmental evolution of the calcium currents and contraction expression was accompanied by some changes in their characteristics: the ICa,T/ICa,L amplitudes ratio progressively increased and the time-to-peak of contraction progressively decreased with the age of myoballs. Physiological functions for calcium currents in developing muscle are suggested and discussed: ICa,T, which is transiently expressed, could be involved in the pacemaker-like activity while ICa,L could serve as an early contraction triggering mechanism and/or initially to fill and then to maintain the intracellular calcium stores.

Pineal photoreceptor cells in culture: fine structure, and light/ dark control of cyclic nucleotide levels and melatonin secretion.

Trout pineal photoreceptor cells were dissociated by trypsin-DNase digestion and further purified by a Percoll gradient centrifugation. Total cells or purified photoreceptor cells were then embedded in a collagen gel, or layered on culture-treated polycarbonate membranes, or maintained in suspension, with RPMI 1640 medium or BGjb medium. It has been shown that cells maintain a rhythmic production of melatonin for at least seven 24 h light/dark cycles under these conditions. In this complementary study, the morphofunctional state of the photoreceptor cells was examined 1) by electron (transmission, scanning) microscopy, and 2) by pharmacological tests under different lighting conditions. Using polycarbonate membranes together with RPMI 1640 medium appeared the most suitable. The segmented organization of photoreceptor cells was well preserved when using the culture-treated membranes. It tended to disappear in cells embedded in the collagen gel and was lost after passage through the Percoll gradient. However, this one allowed obtention of an homogeneous population of photoreceptors, as recognized by their intracellular components. Intracellular organelles were rather well preserved in the embedded photoreceptors. The study also provides novel information on the nature of second messengers involved in the photoperiodic control of melatonin production in photoreceptor cells. From the effects of an adenylyl cyclase activator and a phosphodiesterase inhibitor it appeared that 1) total cells and Percoll-selected cells behaved similarly, 2) the nocturnal rise in melatonin secretion was associated with an increase in cAMP content, and 3) a fall in cAMP may be a mechanism through which light reduces melatonin secretion by photoreceptor cells. Cyclic GMP, the metabolism of which also appeared to be controlled by light, did not seem involved in the photoperiodic control of melatonin production. The method proposed herein offers interesting perspectives for the study of the photoneuroendocrine properties of isolated photoreceptor cells.

Cytochemical evidence of an organized microtubular cytoskeleton in Xenopus laevis oocytes: involvement in the segregation of mitochondrial populations.

An organized microtubular cytoskeleton was discovered in the cytoplasm of Xenopus laevis oocytes. The microtubules were observed in 10- to 30-micron cryostat sections by indirect immunoperoxidase labeling using an antibody to tubulin. A gradual extraction of cells with a nonionic detergent was essential for good penetration of the antibody into the cells. In the cytoplasm of all previtellogenic oocytes, a dense network of criss-crossed long microtubules was associated in a basket-like structure surrounding the mitochondrial mass. At the beginning of vitellogenesis, the network meshes enlarged, while clusters of mitochondria migrated, in close association with microtubule bundles. At the beginning of vitellogenesis, the reorganization of the microtubular network, mostly in the vegetal hemisphere, occurred during the segregation of the mitochondrial populations. Reorganization is characterized by (1) a temporary enlargement of the network and close association of mitochondrial clusters with microtubular bundles, and (2) a progressive organization of a ring-shaped microtubular structure in the crown elaboration area. It is hypothesized that these modifications of the microtubular cytoskeleton contribute to the maintenance of cell shape and the polarized organization of the cell.

Acetylated low density lipoprotein endocytosis by human syncytiotrophoblast in culture.

Low density lipoproteins (LDL) were chemically modified (acetyl LDL) and then conjugated to colloïdal gold (gold acetyl LDL), firstly, to visualize the acetyl LDL binding sites, and secondly, to demonstrate a possible internalization by human syncytiotrophoblast in culture. Cells were obtained by a trypsin DNase method followed by a Percoll gradient centrifugation. After 3 days of culture the syncytiotrophoblast characterization was performed by using ultramicroscopy, immunohistochemistry, and by studying the secretion of gestational hormones during culture. Binding experiments showed gold acetyl LDL attached to the membrane with random distribution. After incubation at 37 degrees C, gold acetyl LDL was internalized by the syncytiotrophoblast following the classical receptor mediated endocytosis process and a non-specific internalization process. These results suggest the existence in the placenta of a 'scavenger pathway' concomittant of the classical LDL internalization. This phenomenon may be related to the high amount of cholesterol required by the human placenta for its cellular growth and intensive progesterone synthesis.

An efficient isolation procedure of Ca-tolerant ventricular myocytes from ferret heart for applications in electrophysiological studies.

A simple procedure which provides a large yield of isolated ferret ventricular myocytes is described. The enzymatic dissociation was performed by perfusion of the whole heart with the "Langendorff method" at 37 degrees C, without an incubation period. Special attention was given to the period of perfusion with Ca-free or low-calcium containing solutions and to the proportion of both collagenase and elastase used. The viability and calcium tolerance of the isolated cells were tested by ultrastructural and electrophysiological studies. Photo-microscopy showed that 60 to 80% of the isolated cells had an elongated shape (18 microns in diameter, 150 microns in length) and did not beat spontaneously in normal Tyrode solution. The morphological and ultrastructural integrity of these cells was shown in SEM by their smooth surface with regularly spaced T-tubule openings and in TEM by the regular distribution of the transverse tubular system, mitochondrium and sarcomeres. Using the whole-cell patch-clamp technique, they had a resting membrane potential of -72 mV, two types ("Purkinje like" and "ventricular like") of action potentials could be elicited and they were correctly affected by well-known modulators of calcium channels. This technique was successfully applied to the rat heart and could be used for heart dissociation of small mammals. It can simultaneously provide isolated cells of different regions of the heart and can be easily and routinely used by any investigator.

5'-Nucleotidase activity in the pineal organ of the pike. An electron-microscopic study.

To date, it is still unknown whether the metabolism of purine nucleotides and nucleosides plays an important role in the pineal organ of lower vertebrates. We have therefore investigated the sites of 5'-nucleotidase activity in the pineal organ of the pike (Esox lucius L.). Various ultracytochemical procedures were used. An intense ecto-5'-nucleotidase activity was characteristic of the entire plasma membrane of the phototransducers (cone-like and modified photoreceptor elements) and the interstitial cells, with exception of the portions facing the basal lamina of the pericapillary spaces. Additionally, intracellular sites of activity were also visualized in the inner segment and the pedicle of the phototransducers. Most of the intracellular deposits were apparently cytosolic and only few seemed to be associated with the membrane of the clear "synaptic" vesicles of the pedicle. Phagocytotic cells in the pineal lumen also showed a strong enzymatic activity on the outer surface of their plasmalemma (in ectoposition). This was apparently not the case for the cell types of the tissues surrounding the pineal vesicle. The present study emphasizes the importance of the occurrence and metabolism of purine nucleotides and nucleosides in a photoreceptive pineal organ.