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Neutralizing antibodies (inhibitors) to replacement factor VIII (FVIII, either plasma derived or recombinant) impair the effective management of hemophilia A. Individuals with hemophilia A due to major deletions of the FVIII gene (F8) lack antigenically cross-reactive material in their plasma ("CRM-negative"), and the prevalence of inhibitors in these individuals may be as high as 90%. Conversely, individuals with hemophilia A caused by F8 missense mutations are CRM-positive, and their overall prevalence of inhibitors is <10% (ref. 2). Individuals with the F8 intron 22 inversion (found in ∼50% of individuals with severe hemophilia A) have been grouped with the former on the basis of their genetic defect and CRM-negative status. However, only ∼20% of these individuals develop inhibitors. Here we demonstrate that the levels of F8 mRNA and intracellular FVIII protein in B lymphoblastoid cells and liver biopsies from individuals with the intron 22 inversion are comparable to those in healthy controls. These results support the hypothesis that most individuals with the intron 22 inversion are tolerized to FVIII and thus do not develop inhibitors. Furthermore, we developed a new pharmacogenetic algorithm that permits the stratification of inhibitor risk for individuals and subpopulations by predicting the immunogenicity of replacement FVIII using, as input, the number of putative T cell epitopes in the infused protein and the competence of major histocompatibility complex class II molecules to present such epitopes. This algorithm showed statistically significant accuracy in predicting the presence of inhibitors in 25 unrelated individuals with the intron 22 inversion.

The possibility of alloimmunization in patients receiving protein replacement therapy depends on (at least) three risk factors, which are necessary concomitantly but insufficient alone. The first is the degree of structural difference between the therapeutic protein and the patient's own endogenous protein, if expressed. Such differences depend on the nature of the disease mutation and the pre-mutation endogenous protein structure as well as on post-translational changes and sequence-engineered alterations in the therapeutic protein. Genetic variations in the recipients' immune systems comprise the second set of risk determinants for deleterious immune responses. For example, the limited repertoire of MHC class II isomers encoded by a given person's collection of HLA genes may or may not be able to present a 'foreign' peptide(s) produced from the therapeutic protein - following its internalization and proteolytic processing - on the surface of their antigen-presenting cells (APCs). The third (and least characterized) variable is the presence or absence of immunologic 'danger signals' during the display of foreign-peptide/MHC-complexes on APCs. A choice between existing therapeutic products or the manufacture of new proteins, which may be less immunogenic in some patients or patient populations, may require prior definition of the first two of these variables. This leads then to the possibility of developing personalized therapies for disorders due to genetic deficiencies in endogenous proteins, such as haemophilia A and B. [Correction made after online publication 11 July 2011: several critical corrections have been made to the abstract]., (© 2011 Blackwell Publishing Ltd.)

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., (2009 Massachusetts Medical Society)

Plasma factor VIII coagulant activity (FVIII:C) level is a highly heritable quantitative trait that is strongly correlated with thrombosis risk. Polymorphisms within only 1 gene, the ABO blood-group locus, have been unequivocally demonstrated to contribute to the broad population variability observed for this trait. Because less than 2.5% of the structural FVIII gene (F8) has been examined previously, we resequenced all known functional regions in 222 potentially distinct alleles from 137 unrelated nonhemophilic individuals representing 7 racial groups. Eighteen of the 47 variants identified, including 17 single-nucleotide polymorphisms (SNPs), were previously unknown. As the degree of linkage disequilibrium across F8 was weak overall, we used measured-genotype association analysis to evaluate the influence of each polymorphism on the FVIII:C levels in 398 subjects from 21 pedigrees known as the Genetic Analysis of Idiopathic Thrombophilia project (GAIT). Our results suggested that 92714C>G, a nonsynonymous SNP encoding the B-domain substitution D1241E, was significantly associated with FVIII:C level. After accounting for important covariates, including age and ABO genotype, the association persisted with each C-allele additively increasing the FVIII:C level by 14.3 IU dL(-1) (P = .016). Nevertheless, because the alleles of 56010G>A, a SNP within the 3' splice junction of intron 7, are strongly associated with 92714C>G in GAIT, additional studies are required to determine whether D1241E is itself a functional variant.

Background: High-normal and elevated plasma FIX activity (FIX:C) levels are associated with increased risk for venous- and possibly arterial-thrombosis., Objective: Because the broad normal range for FIX:C involves a substantial unknown genetic component, we sought to identify quantitative-trait loci (QTLs) for this medically important hemostasis trait., Methods: We performed a genome-wide screen and a resequencing-based variation scan of the known functional regions of every distinct FIX gene (F9) in the genetic analysis of idiopathic thrombophilia project (GAIT), a collection of 398 Spanish-Caucasians from 21 pedigrees., Results: We found no evidence for linkage (LOD scores <1.5) despite genotyping more than 540 uniformly-spaced microsatellites. We identified 27 candidate F9 polymorphisms, including three in cis-elements responsible for the increase in FIX:C that occurs with aging, but found no significant genotype-specific differences in mean FIX:C levels (P-values > or = 0.11) despite evaluating every polymorphism in GAIT by marginal multicovariate measured-genotype association analysis., Conclusions: The heritable component of interindividual FIX:C variability likely involves a collection of QTLs with modest effects that may reside in genes other than F9. Nevertheless, because the alleles of these 27 polymorphisms exhibited a low overall degree of linkage disequilibrium, we are currently defining their haplotypes to interrogate several highly-conserved non-exonic sequences and other F9 segments not examined here.

Background: The information content of a continuous variable exceeds that of its categorical counterpart. The parameterization of a model may diminish the benefit of using a continuous variable. We explored the use of continuous versus discrete environment in variance components based analyses examining gene x environment interaction in the electrophysiological phenotypes from the Collaborative Study on the Genetics of Alcoholism., Results: The parameterization using the continuous environment produced a greater number of significant gene x environment interactions and lower AICs (Akaike's information criterion). In these cases, the genetic variance increased with increasing cigarette pack-years, the continuous environment of interest. This did not, however, result in enhanced LOD scores when linkage analyses incorporated the gene x continuous environment interaction., Conclusion: Alternative parameterizations may better represent the functional relationship between the continuous environment and the genetic variance.

We discuss analyses of Genetic Analysis Workshop 14 data from the Collaborative Study on the Genetics of Alcoholism (COGA) as well as from a simulated complex disease, Kofendrerd personality disorder (KPD), with both genetic and phenotypic heterogeneity. Both data sets included numerous related phenotypes in addition to disease definitions. All analyses either chose from the given selection of phenotypes or defined new ones, including traits that may not have been related to alcoholism or KPD. Some contributors evaluated the genetic components of the trait. Many investigated genome-wide linkage and/or association, using microsatellites and/or single-nucleotide polymorphism (SNP) chip data. Here we will focus on methodological issues that the investigators faced. Their results depended on phenotype selection, whether continuous or discrete, the covariates included, and ethnicity of the study population. For SNP chip data, members of our group detected no difference in results for Affymetrix or Illumina chips, although higher marker density for association studies appeared to be advantageous. Overall, there were some observations that different chromosomal segments, i.e., physical locations on the p-arm, q-arm, or middle segment, might lead to possible differences in type I error rates. This finding and others highlight the importance of empirical determination of P-values to determine significance.