Next-generation sequencing and recombinant expression characterized aberrant splicing mechanisms and provided correction strategies in factor VII deficiency

Despite the exhaustive screening of F7 gene exons and exon-intron boundaries and promoter region, a significant proportion of mutated alleles remains unidentified in patients with coagulation factor VII deficiency. Here, we applied next-generation sequencing to thirteen FVII-deficient patients displaying genotype-phenotype discrepancies upon conventional sequencing, and identified six rare intronic variants. Computational analysis predicted splicing effects for three of them, which would strengthen (c.571+78G>A; c.806-329G>A) or create (c.572-392C>G) intronic 5' splice sites (5'ss). In F7 minigenes assays the c.806-329G>A was ineffective while the c.571+78G>A change led to usage of the +79 cryptic 5'ss with only trace levels of correct transcripts (3% of wild-type), in accordance with factor VII activity levels in homozygotes (1-3% of normal). The c.572-392C>G change led to pseudo-exonization and frame-shift, but also substantial levels of correct transcripts (~70%). However, this variant was associated with the common F7 polymorphic haplotype predicted to further decrease factor VII levels, thus roughly explaining the factor VII levels of 10% in the homozygous patient. Intriguingly, the effect of the c.571+78G>A and c.572-392C>G changes, and particularly of the former, the severest and well-represented in our cohort, was counteracted by antisense U7snRNA variants targeting the intronic 5'ss, thus demonstrating their pathogenic role. In conclusion, the combination of next-generation sequencing of the entire F7 gene with the minigene expression studies elucidated the molecular bases of factor VII deficiency in ten out of thirteen patients, thus improving diagnosis and genetic counselling, and provided a potential therapeutic approach based on antisense molecules, successfully exploited in other disorders

CT-pro-AVP as a tool for assessment of intravascular volume depletion in severe hyponatremia

International audienceBackground: Assessment of volume status is essential to best manage hyponatremic patients but is not always accurate in clinical practice. The aim of this study was to evaluate the reliability of C-terminal portion of pro-arginine-vasopressin (CT-pro-AVP), a surrogate biomarker of vasopressin release, in assessing intravascular volume (IVV) depletion in hypoosmolar hyponatremic patients.Methods: Plasma CT-pro-AVP and urea-to-creatinine ratio (Ur/Cr) were performed in 131 hospitalized patients presenting chronic severe hypoosmolar hyponatremia. At hospital discharge, their IVV was evaluated regardless of CT-pro-AVP concentrations. All patients were then classified as decreased or as normal/expanded IVV group.Results: Plasma CT-pro-AVP levels were higher in patients with decreased IVV (34.6 vs. 11.3 pmol/L, p < 0.001) and exhibited a reliable performance for assessment of decreased IVV (ROC AUC at 0.717 [95% CI 0.629–0.805]). The combination of CT-pro-AVP and Ur/Cr resulted in an improved ROC AUC up to 0.787 (95% CI 0.709–0.866).Conclusions: Our findings support the hypothesis that CT-pro-AVP plasma level may reflect IVV and would be a tool for its assessment. This performance has been magnified by its combination with Ur/Cr. A dual-marker strategy may help clinicians to optimize the management of severe hyponatremia especially in case of confusing clinical presentations

Evaluation of QMS Everolimus Assay Using Indiko Analyzer

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Genotyping of five Pakistani patients with severe inherited factor X deficiency

International audience: Congenital factor X deficiency is a rare coagulation defect characterized by variable bleeding tendency. The aim of the study was to give a first insight of F10 gene mutations in Pakistani probands. Direct sequencing and/or next-generation sequencing was performed on the coding regions, boundaries and 5' and 3' untranslated regions of the F10 gene in five severe factor X-deficient patients from Pakistan. All patients were born from consanguineous marriages and displayed FX:C levels below 2%. Sequencing revealed five different substitutions, including three previously reported p.Ala15Asp, p.Gly406Ser, and p.Gly420Arg missense variants, and also two novel variants: p.Cys57Arg and p.Gln175*. Though one genotype could not be characterized, we were able to confirm the inherited nature of the defect using familial studies. As the copy number variations were ruled out, we hypothesized the presence of deep intronic mutants that might have escaped detection from sequencing or abnormalities in epigenetic regulation. Three patients presented with severe clinical symptoms, in the early days of life, whereas two presented only with trauma-provoked bleeds and bruises later in life. Those patients with milder forms bore the p.Gly406Ser at the homozygous state and F10 unknown alleles, respectively. F10 mutation spectrum in Pakistan is heterogeneous as seen in other populations. Identification of the F10 mutations is important for genetic counseling and prenatal diagnosis in subsequent pregnancies

Variants du facteur VII de la coagulation : quelle thromboplastine utiliser pour doser son activité ?

International audienceLe déficit constitutionnel en facteur VII de la coagulation (FVII), caractérisé par une grande hétérogénéité clinique et biologique, a pour particularité de présenter des variants FVII dont le dosage de l’activité (FVII:C) varie selon la thromboplastine utilisée. Il s’agit en particulier des variants p.Arg364Gln et p.Arg139Gln, très fréquents sur le continent africain et dans le sud de l’Europe, mais la liste de ces variants s’allonge avec la caractérisation moléculaire des déficits en FVII. Les hypothèses physiopathologiques reposent sur la différence de la séquence protéique des facteurs tissulaires humains ou de lapin composant les thromboplastines. L’analyse des phénotypes cliniques associés conforte l’option consistant à retenir la valeur obtenue avec une thromboplastine humaine, c’est-à-dire composée du facteur tissulaire physiologique. Il est ainsi recommandé de contrôler toute valeur de FVII:C dans le cadre d’un déficit constitutionnel en FVII afin de ne pas surtraiter ces patients

LAIR1, an ITIM-Containing Receptor Involved in Immune Disorders and in Hematological Neoplasms

Leukocyte-associated immunoglobulin (Ig)-like receptor 1 (LAIR1, CD305) belongs to the family of immune-inhibitory receptors and is widely expressed on hematopoietic mature cells, particularly on immune cells. Four different types of ligands of LAIR1 have been described, including collagens, suggesting a potential immune-regulatory function on the extracellular matrix. By modulating cytokine secretion and cellular functions, LAIR1 displays distinct patterns of expression among NK cell and T/B lymphocyte subsets during their differentiation and cellular activation and plays a major negative immunoregulatory role. Beyond its implications in physiology, the activity of LAIR1 can be inappropriately involved in various autoimmune or inflammatory disorders and has been implicated in cancer physiopathology, including hematological neoplasms. Its action as an inhibitory receptor can result in the dysregulation of immune cellular responses and in immune escape within the tumor microenvironment. Furthermore, when expressed by tumor cells, LAIR1 can modulate their proliferation or invasion properties, with contradictory pro- or anti-tumoral effects depending on tumor type. In this review, we will focus on its role in normal physiological conditions, as well as during pathological situations, including hematological malignancies. We will also discuss potential therapeutic strategies targeting LAIR1 for the treatment of various autoimmune diseases and cancer settings

Estimation of residual renal function using Beta‐Trace Protein: Impact of dialysis procedures

International audienceAbstractBeta‐trace protein (BTP), a low molecular weight protein of 23‐29 kDa, has been proposed as a promising biomarker to estimate residual renal function (RRF) in patients on maintenance hemodialysis (HD). Indeed, BTP is cleared by native kidney but not during conventional HD session. By contrast, the removal rate of BTP using convective processes (mainly hemodiafiltration [HDF]) and peritoneal dialysis (PD) has been little or not investigated. Therefore, an aim of this study was to evaluate the impact of dialysis procedures (high‐flux HD, on‐line post‐dilution HDF and PD) on BTP removal in comparison with beta‐2 microglobulin (B2M) and cystatin C (CYSC) removals after a single session. In addition, the ability of BTP to predict RRF in PD was assessed. This observational cross‐sectional study included a total of 82 stable chronic kidney disease patients, 53 patients were on maintenance dialysis (with n = 26 in HD and n = 27 in HDF) and 29 were on PD. Serum concentrations of BTP, B2M, and CYSC were measured (a) before and after a single dialysis session in HD and HDF anuric patients to calculate reduction percentages, (b) in serum, 24‐hour‐dialysate and 24‐hour‐urine in PD patients to compute total, peritoneal, and urinary clearance. RRF was estimated using four equations developed for dialysis patients without urine collection and compared to the mean of the urea and creatinine clearances in PD. The concentrations of the three studied molecules were significantly reduced (P < .001) after dialysis session with significantly higher reduction ratio using HDF compared to HD modality (P < .001): BTP 49.3% vs 17.5%; B2M 82.3% vs 69.7%; CYSC 77.4% vs 66% in HDF and HD, respectively. In non‐anuric PD patients, B2M and CYSC were partly removed by peritoneal clearance (72.3% and 57.6% for B2M and CYSC, respectively). By contrast, BTP removal by the peritoneum was negligible and a low bias for the BTP‐based equation to estimate RRF (−1.4 mL/min/1.73 m2) was calculated. BTP is significantly removed by high‐flux HD or HDF, thereby compromising its use to estimate RRF. By contrast, BTP appears as a promising biomarker to estimate RRF in PD patients since it is not affected by peritoneal clearance, unlike B2M and CYSC, and it is well correlated to RRF