Molecular basis of quantitative fibrinogen disorders in 27 patients from India.

Congenital fibrinogen deficiency is an extremely rare (1:1 000 000) hereditary bleeding disorder caused by defects in genes coding for fibrinogen Aα-, Bß- and γ-chains, respectively. We report here the molecular basis of fibrinogen deficiency in a large series of patients from India. Twenty-seven patients with clinical features suggestive of fibrinogen deficiency and with prolonged plasma clotting times and low fibrinogen levels were studied. Genomic DNA was screened for mutations in the fibrinogen alpha (FGA), beta (FGB), gamma (FGG) genes by PCR and conformation sensitive gel electrophoresis. Fourteen different disease-causing mutations including frameshifts (51.9%), splice site (22.2%), missense (18.5%) and nonsense mutation (7.4%) were identified in 27 patients. Thirteen of them were novel, including seven frameshifts (fibrinogen Aα: p.Asp296 fs*59, p.Thr466 fs*17 and p.Lys575 fs*74; fibrinogen Bß: p.Gly414 fs*2 and fibrinogen γ: p.Ser81 fs*5, p.Lys185 fs*13 and p.Asp278_279 fs*17), three splice site mutations (FGA gene c.364+1G>A; c.510+2 T>G; FGB gene c.851+1G>A), two missense substitutions (fibrinogen Bß: p.Gly288Ser; p.Arg445Thr) and a nonsense mutation in fibrinogen Aα (p.Tyr127*). Two common mutations (FGA: c.364+1G>A, n = 6, FGG: p.Lys185 fs*13, n = 7) affecting 13 patients were identified in this series, suggesting that these mutations could be screened first in Indian patients with fibrinogen deficiency. The molecular data presented here is the largest series of patients with fibrinogen deficiency reported so far, adding significantly to the mutation database of this condition. It also helps create an algorithm for its genetic diagnosis in India.

Nuclear factor (NF)-κB and its associated pathways are major molecular regulators of blood-induced joint damage in a murine model of hemophilia.

BACKGROUND: The present study was designed to investigate the molecular signaling events from onset of bleeding through the development of arthropathy in a murine model of hemophilia A. METHODS AND RESULTS: A sharp-injury model of hemarthrosis was used. A global gene expression array on joint-specific RNA isolated 3 h post-injury revealed nuclear factor-kappa B (NF-κB) as the major transcription factor triggering inflammation. As a number of genes encoding the cytokines, growth factors and hypoxia regulating factors are known to be activated by NF-κB and many of these are part of the pathogenesis of various joint diseases, we reasoned that NF-κB-associated pathways may play a crucial role in blood-induced joint damage. To further understand its role, we screened NF-κB-associated pathways between 1 h to 90 days after injury. After a single articular bleed, distinct members of the NF-κB family (NF-κB1/NF-κB2/RelA/RelB) and their responsive pro-inflammatory cytokines (IL-1ß/IL-6/IFNγ/TNFα) were significantly up-regulated (> 2 fold, P < 0.05) in injured vs. control joints at the various time-points analyzed (1 h/3 h/7 h/24 h). After multiple bleeds (days 30/60/75/90), there was increased expression of NF-κB-associated factors that contribute to hypoxia (HIF-1α, 3.3-6.5 fold), angiogenesis (VEGF-α, 2.5-4.4 fold) and chondrocyte damage (matrix metalloproteinase-13, 2.8-3.8 fold) in the injured joints. Micro RNAs (miR) that are known to regulate NF-κB activation (miRs-9 and 155), inflammation (miRs-16, 155 and 182) and apoptosis (miRs-19a, 155 and 186) were also differentially expressed (-4 to +13-fold) after joint bleeding, indicating that the small RNAs could modulate the arthropathy phenotype. CONCLUSIONS: These data suggest that NF-κB-associated signaling pathways are involved in the development of hemophilic arthropathy.

Genetics of haemostasis.

Congenital defects of platelets or plasma proteins involved in blood coagulation generally lead to bleeding disorders. In some of these disorders, patients with a severe phenotype are prone to spontaneous bleeds with critical consequences. This situation occurs more commonly in haemophilia A and haemophilia B and to a certain extent in severe forms (type 3) of von Willebrand disease. Defects in other plasma coagulation proteins and platelet factors are relatively rare, with an incidence of ≤ 1: 1-2 million. Molecular genetic studies of the human coagulation factors, especially factors VIII and IX, have contributed to a better understanding of the biology of these genetic disorders, the accurate detection of carriers and genetic counselling, and have also fostered new therapeutic strategies. This article reviews the evolution of genetics over the last five decades as a tool for bleeding disorder investigations, the recent advances in molecular techniques that have contributed to improved genetic diagnosis of this condition, and the development and utility of proficiency testing programmes and reference materials for genetic diagnosis of bleeding disorders.

Molecular basis of Bernard-Soulier syndrome in 27 patients from India.

BACKGROUND: Bernard-Soulier syndrome (BSS) is an extremely rare (1:1 million) bleeding disorder of platelet adhesion, caused by defects in the glycoprotein (GP)Ib/IX/V complex. PATIENTS AND METHODS: The diagnosis in 27 patients was based on low platelet count, presence of giant platelets and aggregometry studies. Flow cytometry to assess the surface GPIb/IX/V complex showed reduced (7.7-57%) expression. gDNA was screened for mutations in the GPIBA, GPIBB, GP9 genes using PCR-conformation sensitive gel electrophoresis (CSGE). RESULTS: Thirteen different disease-causing mutations, including missense (54%), frameshifts (38%) and nonsense (8%) mutations, were identified in 27 patients. Eleven of them were novel including five novel frameshifts (GPIbα: p.Gln97_98fsX113, p.Pro402_403fsX52; GPIbß: p.Arg17fsX14; GPIX: p.Gly24fsX43, p. Pro130fs, a nonsense mutation (GPIX, p.94, Gln>X) and five novel missense mutations (GPIbα: p.492, Tyr>His; GPIbß: p.65, Pro>Arg, p.129, Gln>His, p.132, Leu>Pro; GPIX: p.55, Phe>Cys). Interestingly, four common mutations, Cys8Arg (n = 6) and Phe55Ser (n = 2), Phe55Cys (n = 2) in GPIX and a novel 22-bp deletion in the GPIBB gene predicting p.Arg17fsX 14 (n = 10) were seen in 20 patients. CONCLUSION: The molecular data presented here is the largest series of BSS patients to be reported so far, adding significantly to the mutation database of this condition and also useful for its genetic diagnosis in India.

Polymorphism in factor VII gene modifies phenotype of severe haemophilia.

The basis for 10-15% of patients with severe haemophilia having clinically mild disease is not fully understood. We hypothesized that polymorphisms in various coagulant factors may affect frequency of bleeding while functionally significant polymorphisms in inflammatory and immunoregulatory genes may also contribute to variations in the extent of joint damage. These variables were studied in patients with severe haemophilia, who were categorized as 'mild' (<5 bleeds in the preceding year, <10 World Federation of Haemophilia clinical and <10 Pettersson scores, n = 14) or 'severe' (all others, n = 100). A total of 53 parameters were studied in each individual for their association with the clinical severity. Age, F8:c activity and the incidence of thrombotic markers were comparable between the groups while the median number of bleeds, number of affected joints, clinical, radiological and functional joint scores (P < or = 0.001) and life-time clotting factor use (P < or = 0.007) were different. Patients with severe molecular defects had a 4.1-fold increased risk for a severe phenotype (95% CI: 1.18-14.42, P = 0.026) compared with other mutations. Of the polymorphisms studied, the FVII353Q (RR = 3.5, 95% CI: 1.04-12.05, P = 0.044) allele was associated with a severe phenotype. This data shows that apart from the F8/F9 genotype, functional polymorphisms in FVII gene affect the phenotype of patients with severe haemophilia.

Strategies for improving the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo.

Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to approximately 5% of murine hepatocytes. Viral second-strand DNA synthesis continues to be a rate-limiting step for efficient transduction by the single-stranded AAV (ssAAV) vectors. This is due, in part, to the presence of a cellular chaperone (FK506-binding) protein, FKBP52, phosphorylated forms of which interact with the D-sequence in the inverted terminal repeats of AAV2 genome and inhibit the viral second-strand DNA synthesis. Our previous studies have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP), and at serine/threonine residues by protein phosphatase 5 (PP5) enhances viral second-strand DNA synthesis and consequently, the transgene expression. We have also reported that coinfection with a self-complementary AAV (scAAV)-TC-PTP vector results in up to sixfold increase in the transduction efficiency of conventional ssAAV2 vectors in primary murine hepatocytes in vivo. We reasoned that coinfection with scAAV-TC-PTP and scAAV-PP5 vectors may lead to a further increase in the transduction efficiency of ssAAV2 vectors. We demonstrate here that this strategy does indeed lead to approximately 16-fold increase in the transduction efficiency of conventional ssAAV vectors in primary murine hepatocytes in vivo following tail-vein injections. Neither scAAV2-TC-PTP nor scAAV2-PP5 vectors alone or together had any adverse effect on the hepatocytes. Thus, this coinfection strategy may be useful for achieving expression from recombinant ssAAV2 vectors containing larger genes, such as coagulation factor VIII, which exceed the packaging capacity of scAAV vectors, for the potential gene therapy of hemophilia A.