Characterization of five associations of F8 missense mutations containing FVIII B domain mutations.

INTRODUCTION: Haemophilia A (HA) is a bleeding disorder due to an absence or a reduced activity of coagulation factor VIII (FVIII) caused by mutations in F8 gene. Missense mutations represent approximately 45% of the reported molecular defects in HA. However, only few missense mutations in FVIII B domain have been described. AIM: The aim of this study was to characterize five genetic variations (three novels and two previously reported) localized in the FVIII B domain. In all cases, an additional missense variation located outside the FVIII B domain was found. We investigated each of these variations separately and in combination too for their contribution to HA phenotype. METHODS: F8 variants were transiently expressed in COS-1 cells. Media and cell lysates were collected after 72 h. Then, FVIII activity, secretion and thermostability were analysed and compared to FVIII wild-type. RESULTS: The 5 FVIII B domain variants showed normal FVIII: C (98.5-128.5%) and FVIII: Ag (97.7-154%). No synergistic effect was observed between the B domain variant and their associated mutations. In contrast, the variants located outside the B domain, p.V682L, p.S714L, p.V592D and p.C573F revealed significantly decrease of FVIII: C with values in the range 3.5-44.5% (p < 0.05). However, the p.G224R variant showed FVIII: C and FVIII: Ag values no significantly different from FVIII-WT. CONCLUSION: The FVIII B domain variants, p.D963N, p.S806T, p.G873D, p.H998Q and p.Q1225R may be considered as polymorphism or non-pathologic mutations. In five patients, clinical phenotype could be explained by the additional causative missense mutation. For the p.G224T variant further splicing studies are necessary to determine its pathogenicity.

Usefulness of an in vitro cellular expression model for haemophilia A carrier diagnosis: illustration with five novel mutations in the F8 gene in women with isolated factor VIII:C deficiency.

This study aims to determine the way to predict the haemophilia A (HA) carrier status and the potential severity in six females with low FVIII: C levels (<0.50 IU mL(-1) ), F8 gene variations and without family history of HA. Except p.Ser577Tyr, F8 gene variations that we reported have never been described (p.Leu107His, p.Pro521Leu, p.Val682Leu, p.Leu2032Pro, p.Ala315dup). Prediction of their potential causal impact was studied by two strategies: bioinformatics approaches and site-directed mutagenesis followed by FVIII cellular expression into COS-1 cell. FVIII clotting assay ( FVIII: C) and antigen ( FVIII: Ag) were assayed in vitro. In silico analysis showed the probably damaging effect of all substitutions and the full conservation of the residues across mammalian species, except for p.Leu2032Pro. The in vitro variant expression model showed abnormal intra and/or extracellular FVIII: C and FVIII: Ag levels for five mutations, which suggest their causality in HA and provide informations about the involved mechanism. We suspect a defect in synthesis and secretion for p.Leu107His, p.Ala315dup and p.Pro521Leu. The mutation p.Val682Leu only affects the FVIII function while p.Ser577Tyr alters function and synthesis. The variant p.Leu2032Pro is probably a polymorphism because no alteration of the FVIII protein expression was observed in vitro. In vitro results suggest that mutations p.Ser577Tyr and p.Ala315dup could led to a severe HA in men. This study demonstrates the ability of this in vitro cellular expression model to contribute to the diagnosis strategy for female suspected of being HA carrier, without HA family history and with a novel F8 gene variation and to provide new criteria for the genetic counselling.

Characterization of four novel molecular changes in the promoter region of the factor VIII gene.

Haemophilia A (HA) is an X-linked recessive bleeding disorder, caused by a wide variety of mutations in the factor VIII (F8) gene, leading to deficiency in the activity of coagulation FVIII. These mutations can affect all the F8 exons from the initiation codon to the termination codon, however, only few molecular changes in the promoter region of the F8 gene were reported so far. Here, we describe six nucleotide variations (c.-51G>A, c.-218T>C, c.-219C>T, c.-219delC, c.-221T>A and c.-664G>A) detected in the F8 promoter and their correlation with clinical phenotype of the patients. Potential role of these mutations in HA was also assessed. Causality was demonstrated with transient transfection experiments using luciferase reporter gene plasmids and computational analysis. Two molecular changes (c.-51G>A and c.-664G>A) did not seem to affect the promoter function of the F8 gene whereas c.-218T>C, c.-219C>T, c.-219delC, c.-221T>A mutations had an impact on the F8 promoter function and were responsible for HA. Furthermore, these mutations were associated with resistance to 1-deamino-8-D-argininevasopressin (desmopressin) therapy when they were causative. When molecular variation was detected in F8 promoter, we propose to use prediction software and to verify predictions by reporter gene analysis. If the mutation is causative, it will be probably associated with a lack of therapeutic response to desmopressin and this clinical implication should be considered by clinicians.

Significance of the serum CTX-II level in an osteoarthritis animal model: a 5-month longitudinal study.

OBJECTIVE: The aim of this study was to investigate the clinical value of serum measurement of C-telopeptide of type II collagen (CTX-II). In correlation with late stages of osteoarthritis (OA) evaluated with histological assessment, the evolution of serum CTX-II concentration was followed during a 20-week longitudinal study in rabbit anterior cruciate ligament transection (ACLT) OA model in adult and growing animals. METHODS: OA was induced in five adult and nine growing rabbits. Four adult and four young rabbits were unoperated. Serum sampling was made at week 0, 1, 2, 3, 4, 6, 8, 10, 12, 14, 16 and 20 after the surgery in all rabbits. Animals were euthanized 20 weeks after the surgery. Serum CTX-II levels were analyzed with a recently available enzyme-linked immunosorbent assay (ELISA) kit, the protocol of which has been modified to increase the sensitivity of the test. RESULTS: Significant differences for the CTX-II levels at W3, W6, W8, W10, W12, W14, W16 and W20 were observed between the adult ACLT and the control groups. A negative correlation between CTX-II levels and cartilage thickness of the medial compartment of the knee at W8, W10, W12 and a positive correlation between the CTX-II levels and the histomorphological score of the medial compartment of the knee at W3, W6, W8, W10, W12 were noted in adult animals. In young animals, operated or not, we observed high CTX-II levels at the beginning of the study, which decreased until the end. CONCLUSION: Our results suggest the interest of the serum CTX-II monitoring for the OA progression and the relevance of the multiple time point analysis of this biomarker. Moreover, they address the question of the importance of correctly choosing the age of the animals used in the pre-clinical studies of OA.

Knee joint ultrasonography of the ACLT rabbit experimental model of osteoarthritis: relevance and effectiveness in detecting meniscal lesions.

OBJECTIVES: To develop a protocol for rabbit knee joint ultrasonography (US); to grade ultrasonographically the meniscal injuries of the anterior cruciate ligament transection (ACLT) rabbit model of osteoarthritis (OA); to assess with US the effectiveness of the ACLT; to compare final US with macroscopy for the evaluation of medial and lateral meniscal injuries depending on the age and weight when ACLT is performed. METHODS: Twenty-two skeletally mature and adolescent New Zealand white rabbits were housed during the same period at the Institut Claude-Bourgelat, Lyon, France. Surgical ACLT was performed in the left knee of nine adolescent and five adult rabbits. Final US and macroscopic semi-quantitative grading of the meniscal injuries were compared 5 months after ACLT. RESULTS: A standardised protocol was developed to evaluate the rabbit knee joint. US was performed in both control and ACLT knees. Normal and abnormal meniscal US appearances were described. A semi-quantitative scale to grade US meniscal injuries was created. Macroscopic and US total meniscal scores were significantly positively correlated (P<0.001, r=0.70). US detection of meniscal injuries was 92% sensitive and 87.5% specific compared to macroscopy. Positive and negative predictive values of US were, respectively, 92% and 87.5%. US detection of the ACLT effectiveness was 100% specific and 78.5% sensitive. CONCLUSION: A significant relationship was found between ultrasonographic and macroscopic grading of meniscal injuries. US was both specific and sensitive in detecting meniscal lesions. We propose US as a non-invasive, non-expensive, in vivo imaging technique for preclinical studies in the ACLT rabbit OA model.