Search

• Exonic variants may have various molecular mechanisms of pathogenicity. • The impact on splicing of exonic sequence variations should be systemically assessed. • Splicing default of exonic variants may hamper the efficiency of targeted pharmacotherapy. • Functional in vitro experiments are key tools to classify CFTR rare variants in order to offer personalized therapies. The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR -France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments. [Display omitted] [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), 2 incurable neurodegenerative disorders, share the same pathological hallmark named TDP43 (TAR DNA binding protein 43) proteinopathy. This event is characterized by a consistent cytoplasmic mislocalization and aggregation of the protein TDP43, which loses its physiological properties, leading neurons to death. Antibody-based approaches are now emerging interventions in the field of neurodegenerative disorders. Here, we tested the target specificity, in vivo distribution, and therapeutic efficacy of a monoclonal full-length antibody, named E6, in TDP43-related conditions. We observed that the antibody recognizes specifically the cytoplasmic fraction of TDP43. We demonstrated its ability in targeting large neurons in the spinal cord of mice and in reducing TDP43 mislocalization and NF-κB activation. We also recognized the proteasome as well as the lysosome machineries as possible mechanisms used by the antibody to reduce TDP43 proteinopathy. To our knowledge, this is the first report showing the therapeutic efficacy and feasibility of a full-length antibody against TDP43 in reducing TDP43 proteinopathy in spinal neurons of an ALS/FTLD mouse model.

To investigate the role of neuronal NF-κB activity in pathogenesis of amyotrophic lateral sclerosis (ALS), we generated transgenic mice with neuron-specific expression of a super-repressor form of the NF-κB inhibitor (IκBα-SR), which were then crossed with mice of both sexes, expressing ALS-linked gene mutants for TAR DNA-binding protein (TDP-43) and superoxide dismutase 1 (SOD1). Remarkably, neuronal expression of IκBα-SR transgene in mice expressing TDP-43 A315T or TDP-43 G348C mice led to a decrease in cytoplasmic to nuclear ratio of human TDP-43. The mitigation of TDP-43 neuropathology by IκBα-SR, which is likely due to an induction of autophagy, was associated with amelioration of cognitive and motor deficits as well as reduction of motor neuron loss and gliosis. Neuronal suppression of NF-κB activity in SOD1 G93A mice also resulted in neuroprotection with reduction of misfolded SOD1 levels and significant extension of life span. The results suggest that neuronal NF-κB signaling constitutes a novel therapeutic target for ALS disease and related disorders with TDP-43 proteinopathy. SIGNIFICANCE STATEMENT This study reports that neuron-specific expression of IκB super-repressor mitigated behavioral and pathologic changes in transgenic mouse models of amyotrophic lateral sclerosis expressing mutant forms of either Tar DNA-binding protein 43 or superoxide dismutase. The results suggest that neuronal NF-κB signaling constitutes a novel therapeutic target for amyotrophic lateral sclerosis and related disorders with Tar DNA-binding protein 43 proteinopathy., (Copyright © 2020 Dutta et al.)

To test the hypothesis that the cerebrospinal fluid (CSF) could provide a spreading route for pathogenesis of amyotrophic lateral sclerosis (ALS), we have examined the effects of intraventricular infusion during 2 weeks of pooled CSF samples from sporadic ALS patients or control CSF samples into transgenic mice expressing human TDP43 WT which do not develop pathological phenotypes. Infusion of ALS-CSF, but not of control CSF, triggered motor and cognitive dysfunction, as well as ALS-like pathological changes including TDP43 proteinopathy, neurofilament disorganization and neuroinflammation. In addition, the neuron-specific translational profiles from peptide analyses of immunoprecipitated ribosomes revealed dysregulation of multiple protein networks in response to ALS-CSF altering cytoskeletal organization, vesicle trafficking, mitochondrial function, and cell metabolism. With normal mice, similar ALS-CSF infusion induced mild motor dysfunction but without significant TDP43 pathology in spinal neurons. We conclude that the CSF from sporadic ALS contains factors that can transmit and disseminate disease including TDP43 proteinopathy into appropriate recipient animal model expressing human TDP43. These findings open new research avenues for the discovery of etiogenic factors for sporadic ALS and for the testing of drugs aiming to neutralize the ALS-CSF toxicity.

Exome sequencing used for molecular diagnosis of Mendelian disorders considerably increases the number of missense variants of unclear significance, whose pathogenicity can be assessed by a variety of prediction tools. As the performance of algorithms may vary according to the datasets, complementary specific resources are needed to improve variant interpretation. As a model, we were interested in the cystic fibrosis transmembrane conductance regulator gene (CFTR) causing cystic fibrosis, in which at least 40% of missense variants are reported. Cystic fibrosis missense analysis (CYSMA) is a new web server designed for online estimation of the pathological relevance of CFTR missense variants. CYSMA generates a set of computationally derived data, ranging from evolutionary conservation to functional observations from three-dimensional structures, provides all available allelic frequencies, clinical observations, and references for functional studies. Compared to software classically used in analysis pipelines on a dataset of 141 well-characterized missense variants, CYSMA was the most efficient tool to discriminate benign missense variants, with a specificity of 85%, and very good sensitivity of 89%. These results suggest that such integrative tools could be adapted to numbers of genes involved in Mendelian disorders to improve the interpretation of missense variants identified in the context of diagnosis., (© 2019 Wiley Periodicals, Inc.)

Background: Despite the increasing use of medical records to measure quality of care, studies have shown that their validity is suboptimal. The objective of this study is to assess the concordance of cardiovascular care processes evaluated through medical record review and patient self-administered questionnaires (SAQs) using ten quality indicators (TRANSIT indicators). These indicators were developed as part of a participatory research program (TRANSIT study) dedicated to TRANSforming InTerprofessional clinical practices to improve cardiovascular disease (CVD) prevention in primary care., Methods: For every patient participating in the TRANSIT study, the compliance to each indicator (individual scores) as well as the mean compliance to all indicators of a category (subscale scores) and to the complete set of ten indicators (overall scale score) were established. Concordance between results obtained using medical records and patient SAQs was assessed by prevalence-adjusted bias-adjusted kappa (PABAK) coefficients as well as intraclass correlation coefficients (ICCs) and 95% confidence intervals (95% CI). Generalized linear mixed models (GLMM) were used to identify patients' sociodemographic and clinical characteristics associated with agreement between the two data sources., Results: The TRANSIT study was conducted in a primary care setting among patients (n = 759) with multimorbidity, at moderate (16%) and high risk (83%) of cardiovascular diseases. Quality of care, as measured by the TRANSIT indicators, varied substantially between medical records and patient SAQ. Concordance between the two data sources, as measured by ICCs (95% CI), was poor for the subscale (0.18 [0.08-0.27] to 0.46 [0.40-0.52]) and overall (0.46 [0.40-0.53]) compliance scale scores. GLMM showed that agreement was not affected by patients' characteristics., Conclusions: In quality improvement strategies, researchers must acknowledge that care processes may not be consistently recorded in medical records. They must also be aware that the evaluation of the quality of care may vary depending on the source of information, the clinician responsible of documenting the interventions, and the domain of care.

Mutations in the gene encoding ubiquilin-2 (UBQLN2) are linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). UBQLN2 plays a central role in ubiquitin proteasome system (UPS) and UBQLN2 up-regulation exacerbates TDP-43 cytoplasmic aggregates. To analyze interaction between UBQLN2 and TDP-43 and to produce a relevant ALS animal model, we have generated a new transgenic mouse expressing UBQLN2 P497H under the neurofilament heavy (NFH) gene promoter. The UBQLN2 P497H mice were then bred with our previously described TDP-43 G348C mice to generate double-transgenic UBQLN2 P497H ; TDP-43 G348C mice. With low-expression levels of UBQLN2, the double-transgenic mice developed TDP-43 cytosolic accumulations in motor neurons starting at 5 months of age. These double-transgenic mice exhibited motor neuron loss, muscle atrophy, as well as motor and cognitive deficits during aging. The microglia from double-transgenic mice were hyperresponsive to intraperitoneal injection of lipopolysaccharide (LPS). In vivo and in vitro analyses suggested that extra UBQLN2 proteins can exacerbate cytoplasmic TDP-43 accumulations by competing with the UPS for binding to ubiquitin. Thus, increasing the pool of ubiquitin promoted the UPS function with ensuing reduction of TDP-43 cytosolic accumulations. In conclusion, the double-transgenic UBQLN2 P497H ; TDP-43 G348C mice provides a unique mouse model of ALS/FTD with enhanced TDP-43 pathology that can be exploited for drug testing.

The cytoplasmic aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of degenerating neurons in amyotrophic lateral sclerosis (ALS) and subsets of frontotemporal dementia (FTD). In order to reduce TDP-43 pathology, we generated single-chain (scFv) antibodies against the RNA recognition motif 1 (RRM1) of TDP-43, which is involved in abnormal protein self-aggregation and interaction with p65 NF-κB. Virus-mediated delivery into the nervous system of a scFv antibody, named VH7Vk9, reduced microgliosis in a mouse model of acute neuroinflammation and mitigated cognitive impairment, motor defects, TDP-43 proteinopathy, and neuroinflammation in transgenic mice expressing ALS-linked TDP-43 mutations. These results suggest that antibodies targeting the RRM1 domain of TDP-43 might provide new therapeutic avenues for the treatment of ALS and FTD.

Background: Cardiovascular disease morbidity and mortality are largely influenced by poor control of hypertension, dyslipidemia, and diabetes. Process indicators are essential to monitor the effectiveness of quality improvement strategies. However, process indicators should be validated by demonstrating their ability to predict desirable outcomes. The objective of this study is to identify an effective method for building prediction models and to assess the predictive validity of the TRANSIT indicators., Methods: On the basis of blood pressure readings and laboratory test results at baseline, the TRANSIT study population was divided into 3 overlapping subpopulations: uncontrolled hypertension, uncontrolled dyslipidemia, and uncontrolled diabetes. A classic statistical method, a sparse machine learning technique, and a hybrid method combining both were used to build prediction models for whether a patient reached therapeutic targets for hypertension, dyslipidemia, and diabetes. The final models' performance for predicting these intermediate outcomes was established using cross-validated area under the curves (cvAUC)., Results: At baseline, 320, 247, and 303 patients were uncontrolled for hypertension, dyslipidemia, and diabetes, respectively. Among the 3 techniques used to predict reaching therapeutic targets, the hybrid method had a better discriminative capacity (cvAUCs=0.73 for hypertension, 0.64 for dyslipidemia, and 0.79 for diabetes) and succeeded in identifying indicators with a better capacity for predicting intermediate outcomes related to cardiovascular disease prevention., Conclusions: Even though this study was conducted in a complex population of patients, a set of 5 process indicators were found to have good predictive validity based on the hybrid method.

Objective: To assess a selection of psychometric properties of the TRANSIT indicators., Design: Using medical records, indicators were documented retrospectively during the 14 months preceding the end of the TRANSIT study., Setting: Primary care in Quebec, Canada., Participants: Indicators were documented in a random subsample (n = 123 patients) of the TRANSIT study population (n = 759)., Interventions: For every patient, the mean compliance to all indicators of a category (subscale score) and to the complete set of indicators (overall scale score) were established. To evaluate test-retest and inter-rater reliabilities, indicators were applied twice, two months apart, by the same evaluator and independently by different evaluators, respectively. To evaluate convergent validity, correlations between TRANSIT indicators, Burge et al. indicators and Institut national d'excellence en santé et en services sociaux (INESSS) indicators were examined., Main Outcome Measures: Test-retest reliability, inter-rater reliability, and convergent validity., Results: Test-retest reliability, as measured by intraclass correlation coefficients (ICCs) was equal to 0.99 (0.99-0.99) for the overall scale score while inter-rater reliability was equal to 0.95 (0.93-0.97) for the overall scale score. Convergent validity, as measured by Pearson's correlation coefficients, was equal to 0.77 (P < 0.001) for the overall scale score when the TRANSIT indicators were compared to Burge et al. indicators and to 0.82 (P < 0.001) for the overall scale score when the TRANSIT indicators were compared to INESSS indicators., Conclusions: Reliability was excellent except for eleven indicators while convergent validity was strong except for domains related to the management of CVD risk factors., (© The Author 2017. Published by Oxford University Press in association with the International Society for Quality in Health Care. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Most of the 2,000 variants identified in the CFTR (cystic fibrosis transmembrane regulator) gene are rare or private. Their interpretation is hampered by the lack of available data and resources, making patient care and genetic counseling challenging. We developed a patient-based database dedicated to the annotations of rare CFTR variants in the context of their cis- and trans-allelic combinations. Based on almost 30 years of experience of CFTR testing, CFTR-France (https://cftr.iurc.montp.inserm.fr/cftr) currently compiles 16,819 variant records from 4,615 individuals with cystic fibrosis (CF) or CFTR-RD (related disorders), fetuses with ultrasound bowel anomalies, newborns awaiting clinical diagnosis, and asymptomatic compound heterozygotes. For each of the 736 different variants reported in the database, patient characteristics and genetic information (other variations in cis or in trans) have been thoroughly checked by a dedicated curator. Combining updated clinical, epidemiological, in silico, or in vitro functional data helps to the interpretation of unclassified and the reassessment of misclassified variants. This comprehensive CFTR database is now an invaluable tool for diagnostic laboratories gathering information on rare variants, especially in the context of genetic counseling, prenatal and preimplantation genetic diagnosis. CFTR-France is thus highly complementary to the international database CFTR2 focused so far on the most common CF-causing alleles., (© 2017 Wiley Periodicals, Inc.)

Several protocols have illustrated the possibility of deriving cells, such as fibroblasts, from different organs. These techniques generally concern organs sampled from living persons, but have already been described for cadavers, especially concerning the skin and tendons. We present, for the first time, an easy way to derive pulmonary fibroblasts from a lung tissue sampled from a cadaver and directly culture plated. The fibroblast output was checked daily. We obtained lung fibroblasts from 3 (60%) cadavers and 2 (100%) living persons. The fibroblast output took about 3 days for cells from living persons and took up to 39 days for those from cadavers. We did not clearly identify any parameters that could explain these differences. Nevertheless, these derived cells had the same features as the source cells, especially in terms of morphology and proliferation, and could potentially be used in different research domains such as forensic or regeneration medicine., (© 2017 American Academy of Forensic Sciences.)

Cytoplasmic TDP-43 aggregation is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Here we investigated the role of exosomes in the secretion and propagation of TDP-43 aggregates. TDP-43 was detected in secreted exosomes from Neuro2a cells and primary neurons but not from astrocytes or microglia. Evidence is presented that protein aggregation and autophagy inhibition are factors that promote exosomal secretion of TDP-43. We also report that levels of exosomal TDP-43 full length and C-terminal fragment species are upregulated in human amyotrophic lateral sclerosis brains. Exposure of Neuro2a cells to exosomes from amyotrophic lateral sclerosis brain, but not from control brain, caused cytoplasmic redistribution of TDP-43, suggesting that secreted exosomes might contribute to propagation of TDP-43 proteinopathy. Yet, inhibition of exosome secretion by inactivation of neutral sphingomyelinase 2 with GW4869 or by silencing RAB27A provoked formation of TDP-43 aggregates in Neuro2a cells. Moreover, administration of GW4869 exacerbated the disease phenotypes of transgenic mice expressing human TDP-43 A315T mutant. Thus, even though results suggest that exosomes containing pathological TDP-43 may play a key role in the propagation of TDP-43 proteinopathy, a therapeutic strategy for amyotrophic lateral sclerosis based on inhibition of exosome production would seem inappropriate, as in vivo data suggest that exosome secretion plays an overall beneficial role in neuronal clearance of pathological TDP-43., (© The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Facilitation is a powerful approach to support practice change. The purpose of this study is to better understand the facilitation roles exercised by both external facilitators and interprofessional facilitation teams to foster the implementation of change. Building on Dogherty et al.'s taxonomy of facilitation activities, this study uses an organizational development lens to identify and analyze facilitation roles. It includes a concise definition of what interprofessional facilitation teams actually do, thus expanding our limited knowledge of teams that act as change agents. We also investigate the facilitation dynamics between change actors., Methods: We carried out a qualitative analysis of a 1-year process of practice change implementation. We studied four family medicine groups, in which we constituted interprofessional facilitation teams. Each team was supported by one external facilitator and included at least one family physician, one case manager nurse, and health professionals located on or off the family medicine group's site (one pharmacist, plus at least one nutritionist, kinesiologist, or psychologist). We collected our data through focus group interviews with the four teams, individual interviews with the two external facilitators, and case audit documentation. We analyzed both predetermined (as per Dogherty et al., 2012) and emerging facilitation roles, as well as facilitation dynamics., Results: A non-linear framework of facilitation roles emerged from our data, based on four fields of expertise: change management, project management, meeting management, and group/interpersonal dynamics. We identified 72 facilitation roles, grouped into two categories: "implementation-oriented" and "support-oriented." Each category was subdivided into themes (n = 6; n = 5) for clearer understanding (e.g., legitimation of change/project, management of effective meetings). Finally, an examination of facilitation dynamics revealed eight relational ties occurring within and/or between groups of actors., Conclusions: Facilitation is an approach used by appointed individuals, which teams can also foster, to build capacity and support practice change. Increased understanding of facilitation roles constitutes an asset in training practitioners such as organizational development experts, consultants, facilitators, and facilitation teams. It also helps decision makers become aware of the multiple roles and dynamics involved and the key competencies needed to recruit facilitators and members of interprofessional facilitation teams.

Background: Seven chronic disease prevention and management programs were implemented across Quebec with funding support from a provincial-private industry funding initiative. Given the complexity of implementing integrated primary care chronic disease management programs, a knowledge transfer meeting was held to share experiences across programs and synthesize common challenges and success factors for implementation., Methods: The knowledge translation meeting was held in February 2014 in Montreal, Canada. Seventy-five participants consisting of 15 clinicians, 14 researchers, 31 knowledge users, and 15 representatives from the funding agencies were broken up into groups of 10 or 11 and conducted a strengths, weaknesses, opportunities, and threats analysis on either the implementation or the evaluation of these chronic disease management programs. Results were reported back to the larger group during a plenary and recorded. Audiotapes were transcribed and summarized using pragmatic thematic analysis., Results and Discussion: Strengths to leverage for the implementation of the seven programs include: (1) synergy between clinical and research teams; (2) stakeholders working together; (3) motivation of clinicians; and (4) the fact that the programs are evidence-based. Weaknesses to address include: (1) insufficient resources; (2) organizational change within the clinical sites; (3) lack of referrals from primary care physicians; and (4) lack of access to programs. Strengths to leverage for the evaluation of these programs include: (1) engagement of stakeholders and (2) sharing of knowledge between clinical sites. Weaknesses to address include: (1) lack of referrals; (2) difficulties with data collection; and (3) difficulties in identifying indicators and control groups. Opportunities for both themes include: (1) fostering new and existing partnerships and stakeholder relations; (2) seizing funding opportunities; (3) knowledge transfer; (4) supporting the transformation of professional roles; (5) expand the use of health information technology; and (6) conduct cost evaluations. Fifteen recommendations related to mobilisation of primary care physicians, support for the transformation of professional roles, and strategies aimed at facilitating the implementation and evaluation of chronic disease management programs were formulated based on the discussions at this knowledge translation event., Conclusion: The results from this knowledge translation day will help inform the sustainability of these seven chronic disease management programs in Quebec and the implementation and evaluation of similar programs elsewhere.

Implementing interprofessional collaborative practices in primary care is challenging, and research about its facilitating factors remains scarce. The goal of this participatory action research study was to better understand the driving forces during the early stage of the implementation process of a community-driven and patient-focused program in primary care titled "TRANSforming InTerprofessional cardiovascular disease prevention in primary care" (TRANSIT). Eight primary care clinics in Quebec, Canada, agreed to participate by creating and implementing an interprofessional facilitation team (IFT). Sixty-three participants volunteered to be part of an IFT, and 759 patients agreed to participate. We randomized six clinics into a supported facilitation ("supported") group, with an external facilitator (EF) and financial incentives for participants. We assigned two clinics to an unsupported facilitation ("unsupported") group, with no EF or financial incentives. After 3 months, we held one interview for the two EFs. After 6 months, we held eight focus groups with IFT members and another interview with each EF. The analyses revealed three key forces: (1) opportunity for dialogue through the IFT, (2) active role of the EF, and (3) change implementation budgets. Decision-makers designing implementation plans for interprofessional programs should ensure that these driving forces are activated. Further research should examine how these forces affect interprofessional practices and patient outcomes.

Background: Analysis of circulating cell-free fetal DNA (cffDNA) in maternal plasma is very promising for early diagnosis of monogenic diseases. However, this approach is not yet available for routine use and remains technically challenging because of the low concentration of cffDNA, which is swamped by the overwhelming maternal DNA., Methods: To make clinical applications more readily accessible, we propose a new approach based on mutant enrichment with 3'-modified oligonucleotides (MEMO) PCR along with real-time PCR to selectively amplify from the maternal blood the paternally inherited fetal allele that is not present in the maternal genome., Results: The first proof of concept of this strategy was displayed for cystic fibrosis by the accuracy of our detection of the p.Gly542* mutation used as the initial developmental model. Subsequently, a retrospective study of plasmas originating from two pregnant women carrying a fetus with private mutation confirmed the effectiveness of our method. We confirmed the presence of cffDNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit., Conclusions: This new non-invasive prenatal diagnosis test offers numerous advantages over current methods: it is simple, cost effective, time efficient and does not require complex equipment or bioinformatics settings. Moreover, our assays for different private mutations demonstrate the viability of this approach in clinical settings for monogenic disorders.

There is emerging evidence that the misfolding of superoxide dismutase 1 (SOD1) may represent a common pathogenic event in both familial and sporadic amyotrophic lateral sclerosis (ALS). To reduce the burden of misfolded SOD1 species in the nervous system, we have tested a novel therapeutic approach based on adeno-associated virus (AAV)-mediated tonic expression of a DNA construct encoding a secretable single-chain fragment variable (scFv) antibody composed of the variable heavy and light chain regions of a monoclonal antibody (D3H5) binding specifically to misfolded SOD1. A single intrathecal injection of the AAV encoding the single-chain antibody in SOD1(G93A) mice at 45 days of age resulted in sustained expression of single-chain antibodies in the spinal cord, and it delayed disease onset and extension of life span by up to 28%, in direct correlation with scFv titers in the spinal cord. The treatment caused attenuation of neuronal stress signals and reduction in levels of misfolded SOD1 in the spinal cord of SOD1(G93A) mice. From these results, we propose that an immunotherapy based on intrathecal inoculation of AAV encoding a secretable scFv against misfolded SOD1 should be considered as potential treatment for ALS, especially for individuals carrying SOD1 mutations.

Background: The chronic care model provides a framework for improving the management of chronic diseases. Participatory research could be useful in developing a chronic care model-based program of interventions, but no one has as yet offered a description of precisely how to apply the approach., Objectives: An innovative, structured, multi-step participatory process was applied to select and develop (1) chronic care model-based interventions program to improve cardiovascular disease prevention that can be adapted to a particular regional context and (2) a set of indicators to monitor its implementation., Methods: Primary care clinicians (n = 16), administrative staff (n = 2), patients and family members (n = 4), decision makers (n = 5), researchers, and a research coordinator (n = 7) took part in the process. Additional primary care actors (n = 26) validated the program., Results: The program targets multimorbid patients at high or moderate risk of cardiovascular disease with uncontrolled hypertension, dyslipidemia or diabetes. It comprises interprofessional follow-up coordinated by case-management nurses, in which motivated patients are referred in a timely fashion to appropriate clinical and community resources. The program is supported by clinical tools and includes training in motivational interviewing. A set of 89 process and clinical indicators were defined., Conclusion: Through a participatory process, a contextualized interventions program to optimize cardiovascular disease prevention and a set of quality indicators to monitor its implementation were developed. Similar approach might be used to develop other health programs in primary care if program developers are open to building on community strengths and priorities.

Molecular diagnosis of cystic fibrosis and cystic fibrosis transmembrane regulator (CFTR)-related disorders led to the worldwide identification of nearly 1,900 sequence variations in the CFTR gene that consist mainly of private point mutations and small insertions/deletions. Establishing their effect on the function of the encoded protein and therefore their involvement in the disease is still challenging and directly impacts genetic counseling. In this context, we built a decision tree following the international guidelines for the classification of variants of unknown clinical significance (VUCS) in the CFTR gene specifically focused on their consequences on splicing. We applied general and specific criteria, including comprehensive review of literature and databases, familial genetics data, and thorough in silico studies. This model was tested on 15 intronic and exonic VUCS identified in our cohort. Six variants were classified as probably nonpathogenic considering their impact on splicing and eight as probably pathogenic, which include two apparent missense mutations. We assessed the validity of our method by performing minigenes studies and confirmed that 93% (14/15) were correctly classified. We provide in this study a high-performance method that can play a full role in interpreting the results of molecular diagnosis in emergency context, when functional studies are not achievable., (© 2013 Wiley Periodicals, Inc.)

Transactive response DNA-binding protein 43 ubiquitinated inclusions are a hallmark of amyotrophic lateral sclerosis and of frontotemporal lobar degeneration with ubiquitin-positive inclusions. Yet, mutations in TARDBP, the gene encoding these inclusions are associated with only 3% of sporadic and familial amyotrophic lateral sclerosis. Recent transgenic mouse studies have revealed a high degree of toxicity due to transactive response DNA-binding protein 43 proteins when overexpressed under the control of strong neuronal gene promoters, resulting in early paralysis and death, but without the presence of amyotrophic lateral sclerosis-like ubiquitinated transactive response DNA-binding protein 43-positive inclusions. To better mimic human amyotrophic lateral sclerosis, we generated transgenic mice that exhibit moderate and ubiquitous expression of transactive response DNA-binding protein 43 species using genomic fragments that encode wild-type human transactive response DNA-binding protein 43 or familial amyotrophic lateral sclerosis-linked mutant transactive response DNA-binding protein 43 (G348C) and (A315T). These novel transgenic mice develop many age-related pathological and biochemical changes reminiscent of human amyotrophic lateral sclerosis including ubiquitinated transactive response DNA-binding protein 43-positive inclusions, transactive response DNA-binding protein 43 cleavage fragments, intermediate filament abnormalities, axonopathy and neuroinflammation. All three transgenic mouse models (wild-type, G348C and A315T) exhibited impaired learning and memory capabilities during ageing, as well as motor dysfunction. Real-time imaging with the use of biophotonic transactive response DNA-binding protein 43 transgenic mice carrying a glial fibrillary acidic protein-luciferase reporter revealed that the behavioural defects were preceded by induction of astrogliosis, a finding consistent with a role for reactive astrocytes in amyotrophic lateral sclerosis pathogenesis. These novel transactive response DNA-binding protein 43 transgenic mice mimic several characteristics of human amyotrophic lateral sclerosis-frontotemporal lobar degeneration and they should provide valuable animal models for testing therapeutic approaches.

New technologies, which constantly become available for mutation detection and gene analysis, have contributed to an exponential rate of discovery of disease genes and variation in the human genome. The task of collecting and documenting this enormous amount of data in genetic databases represents a major challenge for the future of biological and medical science. The Locus Specific Databases (LSDBs) are so far the most efficient mutation databases. This review presents the main types of databases available for the analysis of mutations responsible for genetic disorders, as well as open perspectives for new therapeutic research or challenges for future medicine. Accurate and exhaustive collection of variations in human genomes will be crucial for research and personalized delivery of healthcare., (Copyright © 2009 Elsevier Masson SAS. All rights reserved.)

With the increasing knowledge of cystic fibrosis (CF) and CFTR-related diseases (CFTR-RD), the number of sequence variations in the CFTR gene is constantly raising. CF and particularly CFTR-RD provide a particular challenge because of many unclassified variants and identical genotypes associated with different phenotypes. Using the Universal Mutation Database (UMD) software we have constructed UMD-CFTR (freely available at the URL: http://www.umd.be/CFTR/), the first comprehensive relational CFTR database that allows an in-depth analysis and annotation of all variations identified in individuals whose CFTR genes have been analyzed extensively. The system has been tested on the molecular data from 757 patients (540 CF and 217 CBAVD) including disease-causing, unclassified, and nonpathogenic alterations (301 different sequence variations) representing 3,973 entries. Tools are provided to assess the pathogenicity of mutations. UMD-CFTR also offers a number of query tools and graphical views providing instant access to the list of mutations, their frequencies, positions and predicted consequences, or correlations between genotypes, haplotypes, and phenotypes. UMD-CFTR offers a way to compile not only disease-causing genotypes but also haplotypes. It will help the CFTR scientific and medical communities to improve sequence variation interpretation, evaluate the putative influence of haplotypes on mutations, and correlate molecular data with phenotypes., (Copyright 2010 Wiley-Liss, Inc.)

Molecular pathophysiology of facioscapulohumeral muscular dystrophy (FSHD) involves the heterozygous contraction of the number of tandemly repeated D4Z4 units at chromosome 4q35.2. FSHD is associated with a range of 1-10 D4Z4 units instead of 11-150 in normal controls. Several factors complicate FSHD molecular diagnosis, especially the cis-segregation of D4Z4 contraction with a 4qA allele, whereas D4Z4 shortening is silent both on alleles 4qB and 10q. Discrimination of pathogenic 4q-D4Z4 alleles from highly homologous 10q-D4Z4 arrays requires the use of the conventional Southern blot, which is not suitable at the single-cell level. Preimplantation genetic diagnosis (PGD) is a frequent request from FSHD families with several affected relatives. We aimed to develop a rapid and sensitive PCR-based multiplex approach on single cells to perform an indirect familial segregation study of pathogenic alleles. Among several available polymorphic markers at 4q35.2, the four most proximal (D4S2390, D4S1652, D4S2930 and D4S1523, <1.23 Mb) showing the highest heterozygote frequencies (67-91%) were selected. Five recombination events in the D4S2390-D4S1523 interval were observed among 144 meioses. In the D4S2390-D4Z4 interval, no recombination event occurred among 28 FSHD meioses. Instead, a particular haplotype segregated with both clinical and molecular status, allowing the characterization of an at-risk allele in each tested FSHD family (maximal LOD score 2.98 for theta=0.0). This indirect protocol can easily complement conventional techniques in prenatal diagnosis. Although our multiplex PCR-based approach technically fulfils guidelines for single-cell analysis, the relatively high recombination risk hampers its application to PGD.

Available commercial kits only screen for the most common cystic fibrosis transmembrane conductance regulator (CFTR) mutations causing classic cystic fibrosis and for the Tn variant in IVS8. However, full scanning of CFTR is needed for the diagnosis of patients with cystic fibrosis or CFTR-related disorders (including congenital bilateral absence of the vas deferens) bearing rare mutations. Standard strategies for detecting point mutations rely on extensive scanning of the gene by denaturing gradient gel electrophoresis or denaturing high performance liquid chromatography, which are time-consuming. Moreover, the haplotyping of IVS8-(TG)m and Tn tracts is still challenging despite several recent improvements. We have optimized both the detection of mutations and the haplotyping of IVS8 polyvariants in developing two methods: i) a rapid and robust direct sequence analysis of all exons/flanking introns of the CFTR gene based on single condition touchdown amplification/sequencing in 96-well plates, and ii) a fluorescent assay that allows haplotyping of IVS8-(TG)mTn even without family linkage study. Combined with search for rare large rearrangements, this strategy detected 87.9% of CFTR defects in congenital bilateral absence of the vas deferens patients, a proportion considerably higher than those usually reported. These highly efficient tests, scanning each sample in a few days, greatly improve the genotyping of patients with CFTR-related symptoms and may be particularly important in emergency situations such as fetus with hyperechogenic bowel suggestive of cystic fibrosis.

Purpose: In patients with advanced colorectal cancer, leucovorin, fluorouracil, and irinotecan (FOLFIRI) is considered as one of the reference first-line treatments. However, only about half of treated patients respond to this regimen, and there is no clinically useful marker that predicts response. A major clinical challenge is to identify the subset of patients who could benefit from this chemotherapy. We aimed to identify a gene expression profile in primary colon cancer tissue that could predict chemotherapy response., Patients and Methods: Tumor colon samples from 21 patients with advanced colorectal cancer were analyzed for gene expression profiling using Human Genome GeneChip arrays U133. At the end of the first-line treatment, the best observed response, according to WHO criteria, was used to define the responders and nonresponders. Discriminatory genes were first selected by the significance analysis of microarrays algorithm and the area under the receiver operating characteristic curve. A predictor classifier was then constructed using support vector machines. Finally, leave-one-out cross validation was used to estimate the performance and the accuracy of the output class prediction rule., Results: We determined a set of 14 predictor genes of response to FOLFIRI. Nine of nine responders (100% specificity) and 11 of 12 nonresponders (92% sensitivity) were classified correctly, for an overall accuracy of 95%., Conclusion: After validation in an independent cohort of patients, our gene signature could be used as a decision tool to assist oncologists in selecting colorectal cancer patients who could benefit from FOLFIRI chemotherapy, both in the adjuvant and the first-line metastatic setting.