Reassess known patient, product and therapy related variables of inhibitor risk in combination with measured peptidomic parameters. Used DCs from 24 unrelated health donors in 2 PPAs. In PPA 1, DCs from donors 1–12 were incubated with 5 rtFVIIIs: 1 full-length (FL), 3 B-domain deleted (BDD) and 1 BD-truncated (BDT). In PPA 2, DCs from donors 13–24 were incubated with 1 of 5 tFVIIIs: BDT-rFVIII ± VWF, FL-rFVIII ± VWF, pdFVIII + VWF. After lysis, HLAcII/peptide complexes were purified as DR, DQ and DP bound peptides with monoclonal anti-DR, DQ and DP. Eluted peptidomes were LC-MS/MS sequenced. Peptides were identified and then compared to the reference human proteome and unique sequences of BDD/T-rFVIII and F8 ns-SNPs. All individually sequenced peptides (IPSs) from each donor were assembled as groups of overlapping peptides (GOPs) along FVIII (Fig. 1). The number of GOPs and IPSs from FL-rFVIII were greater in PPA 2 vs 1, likely due to its higher concentration (Fig. 2A). We found a protective effect of glycosylation with non-glycosylated sites at significantly greater risk of being in the bound fraction of potential epitopes (Fig. 2B, a–c). We examined inhibitor development in patients with F8 missense mutations and found a greater risk when in the bound fraction (Fig. 2B, d). We found that BDT-rFVIII + VWF yielded significantly less bound peptides in comparisons with all other tFVIIIs, and that FL-rFVIII yielded significantly more peptides than pdFVIII (Fig. 2C–F). Concentration, glycosylation and VWF co-administration significantly influenced the number of GOPs and IPSs, which both significantly affected the inhibitor risk of F8 missense mutations.Download : Download high-res image (385KB)Download : Download full-size imageDownload : Download high-res image (349KB)Download : Download full-size image

Summary.  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 ≥ 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.

The development of neutralizing antibodies against plasma-derived or recombinant (r) molecules of wildtype (wt) factor (F)VIII protein is the most serious complication of replacement therapy for hemophilia A (hA) patients. Although the pathogenesis of these antibodies, termed inhibitors, is complex and poorly understood, ethnicity, a recently established risk determinant, is clearly involved since African-American (AA) patients experience this complication ~2-fold more often than Caucasians. In a previous study -- in which the FVIII genes (F8) from 137 unrelated healthy people, representing 7 ethnic groups, were resequenced -- we identified 4 common nonsynonymous single nucleotide polymorphisms (nsSNPs) and thereby demonstrated that FVIII is not a monomorphic protein in non-hemophiliacs. Interestingly, 5 of the 6 distinct wt proteins encoded by the allelic combinations or haplotypes (H) of these nsSNPs, designated H1, H2, ..., H5, are expressed by AA’s compared to only 2 in Caucasians (H1 and H2). Because H3, H4 and H5 are 1) partially defined by AA-restricted minor alleles of R484H and M2238V, 2 nsSNPs that substitute amino acids in the A2 and C2 major B-cell inhibitor epitopes, 2) represent the wt FVIII protein in ~27% of AA’s, and 3) differ from the rFVIII proteins used clinically, we designed the PIR study -- the 1st cross-sectional study of AA hA patients -- to determine if pharmacogenetic factors contribute to their greater inhibitor risk. Because the strongest known risk factor for inhibitors is the F8 mutation type, we are resequencing the known functional regions of F8 in the 1st 50 enrolled PIR subjects, out of the 223 total AA hA patients treated at the six participating Region IV South hemophilia treatment centers, to test the plausible alternative hypothesis that the higher inhibitor incidence is due to a different spectrum of molecular abnormalities than has been found in other ethnic subgroups of patients already examined at the DNA sequence level. We have 1) determined the plasma FVIII-activity and -antigen levels, 2) identified the causative mutation in 18 patients, several of which are novel, and 3) are currently performing RT-PCR analyses for detecting the common inversions in introns 1 and 22. Once completed, we will statistically compare the prevalence of AA F8 mutation subtypes to that in hA patients from other ethnic subgroups (detailed in the HAMSTeRS database), and the proportion of AA FVIII deficiencies that are 1) severe, moderate and mild, and 2) CRM-positive and -negative.

Recombinant factor VIII (r-fVIII) is the most widely-used and effective therapy for hemophilia A (hA). Many patients unfortunately become refractory when the wildtype(wt) protein is seen as foreign and is targeted by functionally neutralizing anti-fVIII antibodies termed inhibitors. While inhibitors occur most often in severe hemophiliacs with complex fVIII mutations and a complete circulating absence of the fVIII protein, recent studies show patients with missense-mutations (mMt) have a higher incidence than previously thought and demonstrate r-fVIII can be immunologically targeted even when differing by only a single amino acid. Because mMt represent the most frequent overall etiology of hA (~39% of cases), common non-hemophilic protein variants of fVIII may represent an important novel modulator of inhibitor development in this setting. To determine the extent of such variation, we resequenced all coding regions of the fVIII loci (F8) in 137 healthy subjects from 7 ethnic groups, including 86 Caucasians and 16 African-Americans (AA). We identified 5 common nonsynonymous single nucleotide polymorphisms (nsSNPs). Surprisingly, whereas 4 were polymorphic in AA only 2 were variable in Caucasians, despite having examined 6x the number AA X-chromosomes. Since recent studies show AA patients have a 2-fold higher inhibitor incidence than Caucasians, thus establishing ethnicity as a risk factor in this complication, we were intrigued to find that minor alleles for 2 nsSNPs are restricted to AA and substitute amino acids in major B-cell inhibitor epitopes located in the A2 and C2 domains. To confirm these findings and accurately define the number and frequency of human haplotypes (H) (eg. distinct combinations in which the alleles of these 5 nsSNPs are linked in vivo) we resequenced F8 in a second study group that included 75 additional healthy AA. Here we show there are at least 7 distinct wt forms of the human fVIII protein by defining 7 haplotypes (H) from the 5 nsSNPs: H1, H2, …, H7. H1 exists in all ethnic groups, is the most common overall form of fVIII and represents 2 of the 3 r-fVIII concentrates used clinically. While H2 is the most common form in AA (44%) and possibly represents the other therapeutic r-fVIII molecule, we found that at least 20% will have AA-restricted fVIII proteins; H4 (4%), H5 (12%) and H7 (4%). Only 2 forms of fVIII were found in Caucasians, H1 (90%) and H2 (10%), in contrast, and neither were ethnically restricted. In summary, when combined with reports of at least 3 other nsSNPs, our findings establish as inaccurate the long held view that fVIII is basically a monomorphic protein in non-hemophiliacs. We hypothesize that greater immunologic barriers exist when r-fVIII is infused into patients with mMt in endogenous fVIII molecules containing one or more minor alleles of these nsSNPs. Moreover, due to the number and frequency of AA-restricted wt fVIII variants, we predict these nsSNPs contribute pharmacogenetically to the higher incidence of inhibitors in this ethnic group. As such, we have established a 6-site multi-center study to test this hypothesis using AA hA patients as the optimal group. So far we have enrolled 34 of 223 total AA hA patients at the participating centers and are obtaining blood samples from each for fVIII:C and fVIII:Ag measurements and F8 mutation detection. Because the allelic basis of hA has not been studied in AA, this mutation scan is essential to rule out the plausible alternative hypothesis that their higher inhibitor incidence is due to a distinct spectrum of molecular abnormalities. Ultimately, all patients with mMt will be tested to determine the presence and titer of anti-fVIII, using both Bethesda and ELISA assays, and the H of their mutant fVIII will be defined. The number of patients with inhibitors and AA-restricted forms (H4, H5 or H7) will then be compared to those with inhibitors but whose mMt are in haplotypes found in r-fVIII molecules (H1 or H2).

Due to its position in the coagulation cascade, factor X (fX), a circulating zymogen is activated during hemostatic challenges to its serine protease fXa first via the extrinsic pathway's tissue factor/factor VIIa complex and next by the intrinsic Xase complex of factors Ixa and VIIIa on phospholipids membranes. Among the congenital factor abnormalities manifesting as hemorrhagic predispositions, fX deficiency states, which are often autosomal recessive and associated with consanguinity, are among the rarest. Since concurrent studies on the unrelated Stuart and Prower kindreds in the southeastern United States and London (England), respectively, led to the discovery of fX, it is also known as the Stuart-Prower factor. Only 59 distinct fX gene mutations have been described worldwide since its discovery in the 1950s. We report a novel mutation within the encoding structural locus that we have designated FX-Augusta. The phenotype and genotype of the proband, a 14 year-old African-American boy, were studied and we are currently in the process of obtaining samples from the patient's parents and siblings. The proband has experienced severe bleeding throughout his life with his first episode occurring three days after birth following circumcision. He experiences ~30 bleeds per year, mainly into joints and muscles, which occur spontaneously or following minor trauma. Although the patient appears to be mildly mentally retarded a formal workup has not occurred. On laboratory testing, a severely prolonged PT of 137.4 seconds and aPTT of 112.8 seconds were observed. While clotting-based fX activity (fX:C) levels were consistently <1% when using fX-deficient plasma and the PT-reagent, chromogenic factor X activity levels were ~5%. In light of these findings, we suspect the patient may have a CRM-reduced, or less-likely a CRM-positive, deficiency. However, the patient's fX antigen level (fX:Ag) has not yet been measured. Molecular analysis of the patient's fX gene by direct automated sequencing of PCR-amplicons representing ~200-bp of the promoter, all exonic and flanking intronic regions and ~200-bp of the flanking 3′-genomic DNA, disclosed a novel 5-bp deletion mutation (ATGCC) in exon 8 (del[1350–1354]; 1 is the transcription start site) that disrupts codons 477 and 478, and results in a frameshift. The patient is homozygous for this deletion and an additional 8-bp insertion mutation (TGCCGCCA) located downstream in the 3′-flanking genomic DNA between nucleotides 28376 and 28377 (GenBank #: AF503510). Previously, no insertions and only 4 small deletions have been reported for this gene. Interestingly, the wildtype fX mRNA has a 14-base 3′-UTR as the AUUAAA hexanucleotide polyadenylation element in the fX gene is located 1-bp upstream of the stop codon. Because the resultant frameshift predicts the use of an alternative stop codon 75 nucleotides downstream, 3′ of the in vivo polyadenylation site, this mutation may result in mature cytoplasmic transcripts that lack in-frame stop codons. To our knowledge this has not been reported. Since non-sense mediated decay has not been described for mutations that necessitate the use of downstream stop codons, if we are correct, translation of the mutant mRNAs would not terminate normally and the ribosome would translate the poly(A)-tail into a C-terminal poly-lysine stretch. We hypothesize that the mutant fX pre-mRNAs will be cleaved and polyadenylated immediately 3′ of nucleotide 28346, the same site utilized in vivo during 3′-end processing of the wildtype pre-mRNAs for the following reasons. First, there are no downstream signals capable of supporting 3′-end formation. Specifically, an examination of >1kb of the flanking 3′-genomic DNA failed to identify consensus AAUAAA polyadenylation signals or variants of this highly conserved element. Second, the native fX 3′-end signals appear to be “strong” or efficiently utilized, as no alternative polyadenylation was found in a comprehensive review of the fX cDNAs in the NCBI database, which included 6 full-length cDNAs and >20 3′-ESTs containing polyA-stretches on the 3′-end of high-quality fX mRNA sequence. Finally, neither the causative 5-bp deletion nor the 8-bp insertion disrupts these native 3′-end signals. Nevertheless, the molecular studies underway, including fX:Ag measurements, 3′-end characterization of the mutant mRNA via 3′-RACE and western blotting, should help to distinguish between the possible molecular mechanisms.