A Scan of Pleiotropic Immune Mediated Disease Genes Identifies Novel Determinants of Baseline FVIII Inhibitor Status in Hemophilia-A.

Hemophilia-A (HA) is caused by heterogeneous loss-of-function factor (F)VIII gene (F8)-mutations and deficiencies in plasma-FVIII-activity that impair intrinsic-pathway-mediated coagulation-amplification. The standard-of-care for severe-HA-patients is regular infusions of therapeutic-FVIII-proteins (tFVIIIs) but ~30% develop neutralizing-tFVIII-antibodies called "FVIII-inhibitors (FEIs)" and become refractory. We used the PATH study and ImmunoChip to scan immune-mediated-disease (IMD)-genes for novel and/or replicated genomic-sequence-variations associated with baseline-FEI-status while accounting for non-independence of data due to genetic-relatedness and F8-mutational-heterogeneity. The baseline-FEI-status of 450 North American PATH subjects-206 with black-African-ancestry and 244 with white-European-ancestry-was the dependent variable. The F8-mutation-data and a genetic-relatedness matrix were incorporated into a binary linear-mixed model of genetic association with baseline-FEI-status. We adopted a gene-centric-association-strategy to scan, as candidates, pleiotropic-IMD-genes implicated in the development of either ³2 autoimmune-/autoinflammatory-disorders (AADs) or ³1 AAD and FEIs. Baseline-FEI-status was significantly associated with SNPs assigned to NOS2A (rs117382854; p=3.2E-6) and B3GNT2 (rs10176009; p=5.1E-6), which have functions in anti-microbial-/-tumoral-immunity. Among IMD-genes implicated in FEI-risk previously, we identified strong associations with CTLA4 assigned SNPs (p=2.2E-5). The F8-mutation-effect underlies ~15% of the total heritability for baseline-FEI-status. Additive genetic heritability and SNPs in IMD-genes account for >50% of the patient-specific variability in baseline-FEI-status. Race is a significant determinant independent of F8-mutation-effects and non-F8-genetics.

The intron-22-inverted F8 locus permits factor VIII synthesis: explanation for low inhibitor risk and a role for pharmacogenomics.

Intron-22-inversion patients express the entire Factor VIII (FVIII)-amino-acid sequence intracellularly as 2 non-secreted polypeptides and have a positive "intracellular (I)-FVIII-CRM" status. Mutations conferring a positive I-FVIII-CRM status are associated with low inhibitor risk and are pharmacogenetically relevant because inhibitor risk may be affected by the nature of the therapeutic FVIII-protein (tFVIII), the affinity of any tFVIII-derived foreign peptide (tFVIII-fp) for any HLA class-II isomer (HLA-II) comprising individual major histocompatibility complex (MHC) repertoires, and the stability of any tFVIII-fp/HLA-II complex. We hypothesize that mutations conferring a completely or substantially negative I-FVIII-CRM status are pharmacogenetically irrelevant because inhibitor risk is high with any tFVIII and individual MHC repertoire.

Inhibitors of factor VIII in black patients with hemophilia.

BACKGROUND: Black patients with hemophilia A (factor VIII deficiency) are twice as likely as white patients to produce inhibitors against factor VIII proteins given as replacement therapy. There are six wild-type factor VIII proteins, designated H1 through H6, but only two (H1 and H2) match the recombinant factor VIII products used clinically. H1 and H2 are found in all racial groups and are the only factor VIII proteins found in the white population to date. H3, H4, and H5 have been found only in blacks. We hypothesized that mismatched factor VIII transfusions contribute to the high incidence of inhibitors among black patients. METHODS: We sequenced the factor VIII gene (F8) in black patients with hemophilia A to identify causative mutations and the background haplotypes on which they reside. Results from previous Bethesda assays and information on the baseline severity of hemophilia, age at enrollment, and biologic relationships among study patients were obtained from review of the patients' medical charts. We used multivariable logistic regression to control for these potential confounders while testing for associations between F8 haplotype and the development of inhibitors. RESULTS: Of the 78 black patients with hemophilia enrolled, 24% had an H3 or H4 background haplotype. The prevalence of inhibitors was higher among patients with either of these haplotypes than among patients with haplotype H1 or H2 (odds ratio, 3.6; 95% confidence interval, 1.1 to 12.3; P=0.04), despite a similar spectrum of hemophilic mutations and degree of severity of illness in these two subgroups. CONCLUSIONS: These preliminary results suggest that mismatched factor VIII replacement therapy may be a risk factor for the development of anti-factor VIII alloantibodies.

Products for clotting factor replacement in developing countries.

Developing countries provide clotting factor replacement for hemophilia patients using one or more of the following strategies. (1) Production of domestic plasma and cryoprecipitate provides some self-sufficiency but depends heavily on the presence of an excellent blood transfusion service. The risk of transmitting donor infections is not completely obviated even with good serologic testing. (2) Domestic plasma fractionation has been achieved in a handful of emerging countries but can be expensive. Patients may distrust businessmen of their own country and may be dissatisfied with the style of product available from domestic plants. (3) Contract fractionation has been successful when the donor country is able to provide adequate amounts of well-tested plasma. Its cost is not always lower than that of imported concentrate but a degree of national self-sufficiency is retained. (4) Importation of concentrate allows a wide choice of products. The selection should focus not only on price but also on product safety, which depends upon well-validated donor testing and viral inactivation. High levels of purification are not of great importance. Successful national hemophilia programs also address organization of care, sensible dosing, and equitable distribution of resources to gain the maximum benefit from scarce resources.

Advances in care of children with hemophilia.

Care for children with severe hemophilia has moved from pediatric hospital wards and rehabilitation services to the home, school, and community. Advances in hemophilia are due largely to the development of specialized hemophilia treatment centers, which created a system of comprehensive care and focused healthcare efforts on prevention and education. Parallel advances in coagulation resulted in identification of clotting factors VIII and IX, elucidation of the protein molecular and biochemical structures and functions, sequencing of their respective genes and transfer of the human genes for production of proteins by recombinant technology, and development of gene therapy. The tragedy of the human immunodeficiency virus and hepatitis C raised awareness in patients as well as healthcare providers of the vulnerability of blood products to viral contamination and spurred progress in science leading to viral inactivation of purified proteins. Concomitantly, physicians treating bleeding episodes in the clinic investigated pharmacokinetics and pharmacoeconomics of various strategies of clotting factor replacement. The observation that trough factor levels as low as 1 to 2% were adequate to prevent most bleeding episodes led to current prophylactic regimens that allow boys to participate fully in school and community activities while factor concentrate is infused at home on a regular schedule. Currently, children with hemophilia look forward to a normal life expectancy and excellent health-related quality of life. Physician and community partnerships through research and advocacy societies have accelerated clinical advancements as well as extension of treatment to developing countries. The future of hemophilia promises a cure with gene therapy. Given the past accomplishments in hemophilia, a long-term solution to replacement of the genetically deficient protein lies on the horizon.