[CFTR gene variant screening in gamete donation candidates in France: Which indications? How to screen? Why?].

Objectives: According to French recommendations, only the caryotype is carried out as a first line in candidates for gamete donation. The prescription of additional genetic tests for variants responsible for serious monogenic diseases is only recommended in the case of call points. However, cystic fibrosis remains the most common genetic disease with serious consequences in childhood.

The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy.

Background: 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.

Performance and impact of a multiplex PCR in ICU patients with ventilator-associated pneumonia or ventilated hospital-acquired pneumonia.

Background: Early appropriate antibiotic therapy reduces morbidity and mortality of severe pneumonia. However, the emergence of bacterial resistance requires the earliest use of antibiotics with the narrowest possible spectrum. The Unyvero Hospitalized Pneumonia (HPN, Curetis) test is a multiplex PCR (M-PCR) system detecting 21 bacteria and 19 resistance genes on respiratory samples within 5 h.

OPLS3e: Extending Force Field Coverage for Drug-Like Small Molecules.

Building upon the OPLS3 force field we report on an enhanced model, OPLS3e, that further extends its coverage of medicinally relevant chemical space by addressing limitations in chemotype transferability. OPLS3e accomplishes this by incorporating new parameter types that recognize moieties with greater chemical specificity and integrating an on-the-fly parametrization approach to the assignment of partial charges. As a consequence, OPLS3e leads to greater accuracy against performance benchmarks that assess small molecule conformational propensities, solvation, and protein-ligand binding.

The importance of functional tests to assess the effect of a new variant when genotype-phenotype correlation is not possible.

In vitro functional tests aimed to investigate CFTR dysfunction appear critical to help elucidate the functional impact of new variants of uncertain clinical significance and solve inconclusive cases, especially in early deceased newborns.

OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins.

The parametrization and validation of the OPLS3 force field for small molecules and proteins are reported. Enhancements with respect to the previous version (OPLS2.1) include the addition of off-atom charge sites to represent halogen bonding and aryl nitrogen lone pairs as well as a complete refit of peptide dihedral parameters to better model the native structure of proteins.

Tracking the extramedullary PML-RARα-positive cell reservoirs in a preclinical model: biomarker of long-term drug efficacy.

Using an acute promyelocytic leukemia (APL) preclinical model, we show that oncogene-specific PCR (Polymerase Chain Reaction)-based assays allow to evaluate the efficacy of immunotherapy combining all-trans retinoic acid (ATRA) and a DNA-based vaccine targeting the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) oncogene. Kaplan-Meier survival analysis according to the peripheral blood PML-RARα normalized copy number (NCN) clearly shows that ATRA + DNA-treated mice with an NCN lower than 10 (43%) formed the group with a highly significant (p < 0.0001) survival advantage.

Human connexin channel specificity of classical and new gap junction inhibitors.

Connexins are transmembrane proteins involved in gap junction intercellular communication. They present cell- and tissue-specific expression, with own electric and metabolic coupling specificities. These proteins are involved in numerous physiological processes in the brain and among them neuronal synchronization and trafficking of glucose.

DNA vaccination with all-trans retinoic acid treatment induces long-term survival and elicits specific immune responses requiring CD4+ and CD8+ T-cell activation in an acute promyelocytic leukemia mouse model.

DNA vaccination and all-trans retinoic acid (ATRA) result in a survival advantage in a mouse model of acute promyelocytic leukemia (APL). Depletion of CD4(+) or CD8(+) cells abolished this effect. CD4(+) depletions of long-term survivors resulted in relapse and death within 3 months, thus demonstrating the need of both CD4(+) and CD8(+) subsets for the generation of DNA-driven antileukemic immune responses and underscoring a crucial role of CD4(+) cells in the maintenance of durable remissions.

Influence of the duplication of CFTR exon 9 and its flanking sequences on diagnosis of cystic fibrosis mutations.

The DNA sequences of seven regions in the human genome were examined for sequence identity with exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is mutated in cystic fibrosis, and its intronic boundaries. These sequences were 95% to 96% homologous. Based on this nucleotide sequence similarity, PCR primers for CFTR exon 9 can potentially anneal with other homologous sequences in the human genome.

New inhibitors of prion replication that target the amyloid precursor.

At present, there is no effective therapy for any of the neurodegenerative amyloidoses, despite renewed efforts to identify compounds active against the various implicated pathogenetic molecules. We have screened a library of 2960 natural and synthetic compounds in two cell lines chronically infected with mouse prions, and have identified eight new inhibitors of prion replication in vitro. They belong to two distinct chemical families that have not previously been recognised as effective in the field of transmissible spongiform encephalopathies: seven are 3-aminosteroids and one is a derivative of erythromycin A with an oxime functionality.

BCL-2 and mutant NRAS interact physically and functionally in a mouse model of progressive myelodysplasia.

Myelodysplastic syndromes (MDS) are clonal stem cell hematologic disorders that evolve to acute myeloid leukemia (AML) and thus model multistep leukemogenesis. Activating RAS mutations and overexpression of BCL-2 are prognostic features of MDS/AML transformation. Using NRASD12 and BCL-2, we created two distinct models of MDS and AML, where human (h)BCL-2 is conditionally or constitutively expressed.

Reactive oxygen species, DNA damage, and error-prone repair: a model for genomic instability with progression in myeloid leukemia?

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop acute myelogenous leukemia (AML). The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is loss of chromosomal material (genomic instability). Using our two-step mouse model for myeloid leukemic disease progression involving overexpression of human mutant NRAS and BCL2 genes, we show that there is a stepwise increase in the frequency of DNA damage leading to an increased frequency of error-prone repair of double-strand breaks (DSB) by nonhomologous end-joining.

Mosaic maternal uniparental isodisomy for chromosome 7q21-qter.

Uniparental disomy (UPD) for several human chromosomes is associated with clinical abnormalities. We report the case of a 2-year-old boy with severe intrauterine and post-natal growth retardation (IUGR/PNGR) and highly variable sweat chloride concentrations. The patient was identified as heterozygous for the F508del mutation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene.

Disruption of T cell regulatory pathways is necessary for immunotherapeutic cure of T cell acute lymphoblastic leukemia in mice.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. In recent years, the outcome has been globally improved by current therapies, but it remains poor in patients with high, persistent residual disease following the first course of chemotherapy, prompting evaluation of the possible beneficial effects of immunotherapy protocols. In this study, we hypothesized that the disruption of two immunoregulatory pathways controlling the auto-reactive T cell response might synergize with dendritic cell-based immunotherapy of the disease, which is considered to be poorly immunogenic.

Rapid and accurate detection of the CFTR gene mutation 1811+1.6 kbA>G by real-time fluorescence resonance energy transfer PCR.

The CFTR gene mutation 1811+1.6 kbA>G has been reported as associated with a severe phenotype of cystic fibrosis with pancreatic insufficiency. This mutation has been identified as a rather common one in the South West of France and in the Iberian Peninsula.

Diagnosis accuracy of a new challenger for thrombosis exclusion, the Stratus CS DDMR.

Background: The diagnostic accuracy of a new Point of Care, rapid and quantitative D-dimer assay (Stratus CS DDMR from Dade Behring) was evaluated.

Methods: Vidas test from bioMerieux was used as reference method in 279 patients recruited from a management study in progress in our institution.

Results: Both assays show comparable reproducibility (2.

Isolated idiopathic chronic pancreatitis associated with a compound heterozygosity for two mutations of the CFTR gene.

We report the case of a patient suffering from idiopathic chronic pancreatitis (ICP) and compound heterozygous for mutations G542X and S1235R of the cystic fibrosis transmembrane regulator (CFTR) gene. The patient had normal sweat test and no other clinical sign usually linked with a typical or moderate pathology (bronchiectasis, nasal polyposis, congenital absence of the vas deferens) of the CFTR gene. G542X is a severe mutation, which is usually found in classical cystic fibrosis when associated with other severe mutations.

Protein C and protein S assessment in hospital laboratories: which strategy and what role for DNA sequencing?

This paper presents a critical assessment of protein C (PC) and protein S (PS) functional and immunological approaches with regard to DNA sequencing in a large hospital recruitment for thrombosis exploration in more than 1700 consecutive patients. After examination of clinical status and PC and PS phenotype, a genotypic study was implemented for 17 PC-deficient and 28 PS-deficient patients (activity < 70%). Sixty-five percent of the genotyped PC-deficient patients were found to have heterozygous mutations.

Characterization of monoclonal antibodies recognizing HLA-G or HLA-E: new tools to analyze the expression of nonclassical HLA class I molecules.

Nonclassical major histocompatibility complex (MHC) class I human leukocyte antigen E (HLA-E) and HLA-G molecules differ from classical ones by specific patterns of transcription, protein expression, and immunotolerant functions. The HLA-G molecule can be expressed as four membrane-bound (HLA-G1 to -G4) and three soluble (HLA-G5 to -G7) proteins upon alternative splicing of its primary transcript. In this study, we describe a new set of monoclonal antibodies (mAbs) called MEM-G/01, -G/04, -G/09, -G/13, MEM-E/02, and -E/06 recognizing HLA-G or HLA-E.