Our study was designed to test the most likely candidate genes given their physiologic function. as well as 104 brother pairs discordant for inhibitor status. Using an Illumina iSelect platform, 13 331 single-nucleotide polymorphisms from 1081 genes, primarily immune response and immune modifier genes, were typed. Each cohort was analyzed separately with results combined using a meta-analytic technique. After adjustment for potential confounders, 53 single-nucleotide polymorphisms were found to be significant predictors of inhibitor status using the criteria of odds ratios in the same direction in all cohorts or allowing for a 20% interval around an odds ratio = 1 in 1 of the 3 and significant in at least 2. Of the Fadrozole 53 markers, 13 had meta .001. Eight of the 53 were significant predictors among the discordant pairs. Results support the complexity of the immune response and encourage further research with the goal of understanding the pathways involved. Introduction The treatment of hemophilia has improved significantly over the years, but the development of Abs that neutralize the effect of the infused factor remains a serious obstacle for patients and care givers. The reasons that only a fraction of patients, 10%-15% overall and typically 20%-30%1 among those with severe disease, develop Abs remain obscure, but there are several observations indicating that genetic factors are of major importance. The most extensively studied is the type of causative gene mutation.2 The highest risk has been associated with null mutationsthose considered to result in no protein production, thereby keeping the immune system naive to the deficient factor. In particular, large deletions involving multiple domains confer high risk and yet, similar to that seen with other high-risk mutation types, there are families containing multiple siblings with this mutation who remain inhibitor free.3 Independent of the type of causative mutation, the infused factor will be endocytosed in the APCs and proteolytically degraded to smaller peptides that will be presented on the cell surface by the HLA class II molecules to the Th cells. This interaction is fundamental for the immune response to occur, and without HLA class II molecules with the ability to present the immunogenic peptides to the T cells, no immune response will take place. It is therefore not surprising that associations with HLA class II alleles such as DRB*1501 and DQB*0602 have been reported.4 A higher concordance of inhibitor status than expected between siblings and ethnic Fadrozole variations4,5 suggest that other genetic markers may be decisive in the determination of whether the immune response occurs. Indeed, genetic markers have been reported, independent of the type of mutation, such as single nucleotide polymorphisms (SNPs) in the genes coding for mutation typing Standard methods for the analyses of the gene mutation were used16 in HIGS and MIBS. In HGDS, the presence or absence of an inversion mutation (inversion/no inversion) was determined for 58% of the HGDS cohort.17 The remaining HGDS samples were mutation typed using the methods of Oldenburg.16 The following gene mutations were categorized as high risk: inversions, large deletions, nonsense, small deletions/insertions (outside A-runs), missense (Arg593Cys, Tyr2105Cys, Arg2150His, Arg2163His, Trp2229Cys, Pro2300Leu, and Asn2286Lys), and splice site (at conserved nucleotides at position + or ?1 and 2). Those considered low risk were: small deletions/insertions (within A-runs), splice site (at position + or ?3 or more remote), missense (other regions), or other mutation types based on data from the Hemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS) database (http://hadb.org.uk), a resource site for study of FVIII genetic variation, and unpublished data from the Bonn Center in Germany. Genotyping An Illumina iSelect platform was used to genotype 14 626 SNPs (supplemental Appendix 1, available on the Web site; see the Supplemental Materials link at the top of the online article) from a set of 1081 genes. The genes (chiefly immune response and immune modifier genes) and cytokines, cytokine receptors, chemokines, chemokine receptors, immune and inflammatory pathway genes, and HLA genes were selected from a literature review of inflammatory and immune genes and pathway public databases. SNPs were selected from a region spanning 5 kb upstream and 1 kb downstream of the target genes using data for Yoruba (YRI) from Nigeria and CEPH Europeans from Utah (CEU) Fadrozole from the International HapMap Project (http://www.hapmap.org). We first selected all known or putative functional coding region (nonsynonymous, insertion, deletions, frameshift) and regulatory SNPs. Functional status was vetted using information from the National Center for Biotechnology Information (NCBI), PupaSuite (http://pupasuite.bioinfo.cipf.es), and SNPEffect (http://snpeffect.vib.be/index.php). SNPs that are reported to affect amino acid Fadrozole composition or to occur in splice sites, exonic splicing enhancer, exonic splicing silencer, or regulatory regions were selected Rabbit Polyclonal to KAPCG as long as they met minimum Illumina platform restrictions. Using these.

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