Moderation of hemophilia A phenotype by the factor V R506Q mutation

Although many examples of unrelated hemophilia A patients carrying identical point mutations in the factor VIII (FVIII) gene have been reported, the clinical phenotype is not always the same among patients sharing the same molecular defect. Possible explanations for this discrepancy include undetected additional mutations in the FVIII gene or coinheritance of mutations at other genetic loci that modulate FVIII function. We report molecular genetic analysis of potential modifying genes in two sets of unrelated patients carrying common FVIII missense mutations but exhibiting different levels of clinical severity. Both mutations (FVIII R1689C and R2209Q) are associated with severe hemophilia A in some patients and mild/moderate disease in others. The common von Willebrand disease type 2N mutation (R91Q) was excluded as a modifying factor in these groups of patients. However, analysis of the recently described factor V (FV) R506Q mutation (leading to activated protein C resistance) identified a correlation of inheritance of this defect with reduced hemophilia A severity. Two moderately affected hemophilia A patients, each with either of two FVIII gene mutations, were heterozygous for FV R506Q, whereas two severely affected patients and two moderately affected patients were homozygous normal at the FV locus. Our results suggest that coinheritance of the FV R506Q mutation may be an important determinant of clinical phenotype in hemophilia A and that modification of the protein C pathway may offer a new strategy for the treatment of FVIII deficiency.     

Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase

Systemic lupus erythematosus (SLE), the prototypic systemic autoimmune disease, is a debilitating multisystem autoimmune disorder characterized by chronic inflammation and extensive immune dysregulation in multiple organ systems, resulting in significant morbidity and mortality. Here, we present a multidisciplinary approach resulting in the identification of neutrophil cytosolic factor 2 (NCF2) as an important risk factor for SLE and the detailed characterization of its causal variant. We show that NCF2 is strongly associated with increased SLE risk in two independent populations: childhood-onset SLE and adult-onset SLE. The association between NCF2 and SLE can be attributed to a single nonsynonymous coding mutation in exon 12, the effect of which is the substitution of histidine-389 with glutamine (H389Q) in the PB1 domain of the NCF2 protein, with glutamine being the risk allele. Computational modeling suggests that the NCF2 H389Q mutation reduces the binding efficiency of NCF2 with the guanine nucleotide exchange factor Vav1. The model predicts that NCF2/H389 residue interacts with Vav1 residues E509, N510, E556, and G559 in the ZF domain of Vav1. Furthermore, replacing H389 with Q results in 1.5 kcal/mol weaker binding. To examine the effect of the NCF2 H389Q mutation on NADPH oxidase function, site-specific mutations at the 389 position in NCF2 were tested. Results show that an H389Q mutation causes a twofold decrease in reactive oxygen species production induced by the activation of the Vav-dependent Fcγ receptor-elicited NADPH oxidase activity. Our study completes the chain of evidence from genetic association to specific molecular function.     

Characteristics and outcomes of diffuse large B-cell lymphoma presenting in leukaemic phase

Diffuse large B‐cell lymphoma (DLBCL) occasionally presents with circulating malignant cells. The clinical characteristics and long‐term outcomes of these patients have not been described. Twenty‐nine newly diagnosed DLBCL presenting in leukaemic phase were identified between 1996 and 2010, at two institutions. Median age was 48 years, and patients presented with leucocytosis, high lactate dehydrogenase levels, B symptoms, and high International Prognostic Index score. Extra nodal site involvement was observed in all patients and affected the bone marrow (100%), spleen (62%), pleura/lung (41%), liver (21%), bone (17%), bowels (7%) and cerebrospinal fluid (14%). Blood lymphomatous cells co‐expressed CD19, CD20, CD22, CD38, CD45, HLA‐DR and FMC7 in >90%, and kappa or lambda light chain restriction in >50%. Ninety per cent received rituximab and anthracycline‐based chemotherapy. Overall, remission was complete in 54% and partial in 31%; 15% had resistant disease. Median follow‐up was 47 months; 13 (45%) patients remain alive in complete remission. Median progression‐free and overall survivals were 11·5 and 46·7 months, respectively. In summary, patients with DLBCL in leukaemic phase present with high tumour burden and frequent involvement of extra nodal sites. In this uncommon DLBCL subgroup, anthracycline‐based regimens with rituximab are associated with early morbidity and mortality, but yield approximately 50% 4‐year survival.     

Postoperative hypothermia and patient outcomes after major elective non‐cardiac surgery

Using a multicentre adult patient database from Australia and New Zealand, we obtained the lowest and highest temperature in the first 24 h after admission to the intensive care unit after elective non‐cardiac surgery. Hypothermia was defined as core temperature < 36 °C; transient hypothermia as a temperature < 36 °C that was corrected within 24 h, and persistent hypothermia as hypothermia not corrected within 24 h. We studied 50 689 patients. Hypothermia occurred in 23 165 (46%) patients, was transient in 22 810 (45%), and was persistent in 608 (1.2%) patients. On multivariate analysis, neither transient (OR = 1.07, 95% CI 0.96–1.20) nor persistent (OR = 1.50. 95% CI 0.96–2.33) hypothermia was independently associated with increased hospital mortality.     

γ-Aminobutyric Acid Regulates Both the Survival and Replication of Human β-Cells

γ-Aminobutyric acid (GABA) has been shown to inhibit apoptosis of rodent β-cells in vitro. In this study, we show that activation of GABA_A receptors (GABA_A-Rs) or GABA_B-Rs significantly inhibits oxidative stress–related β-cell apoptosis and preserves pancreatic β-cells in streptozotocin-rendered hyperglycemic mice. Moreover, treatment with GABA, or a GABA_A-R– or GABA_B-R–specific agonist, inhibited human β-cell apoptosis following islet transplantation into NOD/scid mice. Accordingly, activation of GABA_A-Rs and/or GABA_B-Rs may be a useful adjunct therapy for human islet transplantation. GABA-R agonists also promoted β-cell replication in hyperglycemic mice. While a number of agents can promote rodent β-cell replication, most fail to provide similar activities with human β-cells. In this study, we show that GABA administration promotes β-cell replication and functional recovery in human islets following implantation into NOD/scid mice. Human β-cell replication was induced by both GABA_A-R and GABA_B-R activation. Hence, GABA regulates both the survival and replication of human β-cells. These actions, together with the anti-inflammatory properties of GABA, suggest that modulation of peripheral GABA-Rs may represent a promising new therapeutic strategy for improving β-cell survival following human islet transplantation and increasing β-cells in patients with diabetes.A central focus of research in the type 1 diabetes (T1D) field is to develop ways to safely improve β-cell survival and function and promote their replication. The addition of γ-aminobutyric acid (GABA) or the GABA_B receptor (GABA_B-R)–specific agonist baclofen to culture media has been shown to inhibit β-cell apoptosis in cultured rodent cell lines and islets (1,2). It remains to be determined whether GABA treatment can inhibit mouse β-cell apoptosis in vivo or, more importantly, whether it can protect human β-cells from stress-induced apoptosis. If GABA can inhibit human β-cell apoptosis, elucidating whether this effect is mediated through the G-protein–coupled GABA_B-Rs, and/or the chloride channel GABA_A-Rs will enable more specific drug targeting.GABA can promote neurogenesis and neuronal proliferation and is a neuronal survival factor (3–8). GABA has also been shown to promote rodent β-cell replication (1,2). Those studies, however, differentially pointed to GABA_A-Rs or GABA_B-Rs as modulators of GABA’s effects, making it important to clarify whether one or both types of GABA receptors modulate rodent β-cell replication. While a number of mitogens and growth factors can promote rodent β-cell replication, most fail to promote human β-cell replication (reviewed in refs. 9,10). Therefore, a key question is whether GABA can promote human β-cell replication. Even a small amount of GABA-induced human β-cell replication may be clinically useful by lowering insulin requirements and reducing the risk for long-term complications in T1D patients (11).