Whole-exome sequencing to identify genetic risk variants underlying inhibitor development in severe hemophilia A patients.

The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most problematic and costly complication of FVIII replacement therapy that affects up to 30% of previously untreated patients with severe hemophilia A. The development of inhibitors is a multifactorial complication involving environmental and genetic factors. Among the latter, F8 gene mutations, ethnicity, family history of inhibitors, and polymorphisms affecting genes involved in the immune response have been previously investigated. To identify novel genetic elements underling the risk of inhibitor development in patients with severe hemophilia A, we applied whole-exome sequencing (WES) and data analysis in a selected group of 26 Italian patients with (n = 17) and without (n = 9) inhibitors. WES revealed several rare, damaging variants in immunoregulatory genes as novel candidate mutations. A case-control association analysis using Cochran-Armitage and Fisher's exact statistical tests identified 1364 statistically significant variants. Hierarchical clustering of these genetic variants showed 2 distinct patterns of homozygous variants with a protective or harmful role in inhibitor development. When looking solely at coding variants, a total of 28 nonsynonymous variants were identified and replicated in 53 inhibitor-positive and 174 inhibitor-negative Italian severe hemophilia A patients using a TaqMan genotyping assay. The genotyping results revealed 10 variants showing estimated odds ratios in the same direction as in the discovery phase and confirmed the association of the rs3754689 missense variant (OR 0.58; 95% CI 0.36-0.94; P = .028) in a highly conserved haplotype region surrounding the LCT locus on chromosome 2q21 with inhibitor development.

Why do inhibitors develop? Principles of and factors influencing the risk for inhibitor development in haemophilia.

The formation of inhibitory alloantibodies is, in the postinfectious era, the most severe and costly complication of replacement therapy in patients with haemophilia. The complexity of the immune response to the infused factor becomes more and more obvious as knowledge in the area increases. Antibodies develop as a result of a complex multi-factorial interaction between antigen-presenting cells, T- and B-lymphocytes. Genetic susceptibility of cell surface molecules, such as the major histocompatibility complex, the T-cell receptor and cytokine receptors, as well as various immunomodulatory molecules have a major impact on the outcome. In addition, environmental factors probably influence the risk of inhibitor development. The current concept of inhibitor development is reviewed. A better understanding of the pathophysiological mechanisms involved will facilitate improvement of therapy in the future, and hopefully provide an opportunity to prevent this complication of treatment.

Genetic diagnosis of haemophilia and other inherited bleeding disorders.

Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand's disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500,000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand's disease and of rare bleeding disorders.

Effect of factor VIII concentrate on leucocyte cytokine receptor expression in vitro: relevance to inhibitor formation and tolerance induction.

Inhibitor formation in haemophilia patients receiving factor VIII (FVIII) concentrate is a serious problem requiring tolerance induction therapy. Inhibitor antibody formation is dependent on interactions between leucocyte cytokines with their corresponding receptors. To investigate this we studied the effect of FVIII on cytokine receptor expression using multiparameter flow cytometry and a whole blood stimulation assay. Upregulation of many cytokine receptors was inhibited by plasma-derived FVIII (pdFVIII) in a dose-dependent manner on T cells, B cells and monocytes although interleukin (IL)-4Ralpha and IL-7Ralpha were upregulated on T cells. The decrease in cytokine receptor upregulation on B cells in the presence of pdFVIII, may result in reduced antibody production. Inhibition of CD132 in the presence of pdFVIII may result in immune tolerance in some recipients of pdFVIII. The immunomodulatory effects of pdFVIII were dose and batch dependent, some being more inhibitory than others. The inhibitory effects of prednisolone with pdFVIII, on cytokine receptor upregulation, were additive. Cytokine receptor expression was not altered in the presence of human recombinant FVIII (rFVIII) concentrate. These findings may explain the reports of less frequent inhibitor antibody formation in some recipients of pdFVIII concentrates. The use of pdFVIII, particularly the more inhibitory batches, may be more suitable than rFVIII for tolerance induction protocols. A clinical study needs to be undertaken to determine the significance of these in vitro findings.