Genomic diversity and differentiation of a managed island wild boar population.

The evolution of island populations in natural systems is driven by local adaptation and genetic drift. However, evolutionary pathways may be altered by humans in several ways. The wild boar (WB) (Sus scrofa) is an iconic game species occurring in several islands, where it has been strongly managed since prehistoric times. We examined genomic diversity at 49 803 single-nucleotide polymorphisms in 99 Sardinian WBs and compared them with 196 wild specimens from mainland Europe and 105 domestic pigs (DP; 11 breeds). High levels of genetic variation were observed in Sardinia (80.9% of the total number of polymorphisms), which can be only in part associated to recent genetic introgression. Both Principal Component Analysis and Bayesian clustering approach revealed that the Sardinian WB population is highly differentiated from the other European populations (FST=0.126-0.138), and from DP (FST=0.169). Such evidences were mostly unaffected by an uneven sample size, although clustering results in reference populations changed when the number of individuals was standardized. Runs of homozygosity (ROHs) pattern and distribution in Sardinian WB are consistent with a past expansion following a bottleneck (small ROHs) and recent population substructuring (highly homozygous individuals). The observed effect of a non-random selection of Sardinian individuals on diversity, FST and ROH estimates, stressed the importance of sampling design in the study of structured or introgressed populations. Our results support the heterogeneity and distinctiveness of the Sardinian population and prompt further investigations on its origins and conservation status.

Porcine colonization of the Americas: a 60k SNP story.

The pig, Sus scrofa, is a foreign species to the American continent. Although pigs originally introduced in the Americas should be related to those from the Iberian Peninsula and Canary islands, the phylogeny of current creole pigs that now populate the continent is likely to be very complex. Because of the extreme climates that America harbors, these populations also provide a unique example of a fast evolutionary phenomenon of adaptation. Here, we provide a genome wide study of these issues by genotyping, with a 60k SNP chip, 206 village pigs sampled across 14 countries and 183 pigs from outgroup breeds that are potential founders of the American populations, including wild boar, Iberian, international and Chinese breeds. Results show that American village pigs are primarily of European ancestry, although the observed genetic landscape is that of a complex conglomerate. There was no correlation between genetic and geographical distances, neither continent wide nor when analyzing specific areas. Most populations showed a clear admixed structure where the Iberian pig was not necessarily the main component, illustrating how international breeds, but also Chinese pigs, have contributed to extant genetic composition of American village pigs. We also observe that many genes related to the cardiovascular system show an increased differentiation between altiplano and genetically related pigs living near sea level.

Comprehensive and high-resolution typing of swine leukocyte antigen DQA from genomic DNA and determination of 25 new SLA class II haplotypes.

We previously reported the development of genomic-DNA-based high-resolution genotyping methods for SLA-DQB1 and DRB1. Here, we report the successful typing of SLA-DQA using similar methodological principles. We designed a method for comprehensive genotyping of SLA-DQA using intronic sequence information of SLA-DQA exon 2 that we had obtained from 12 animals with different SLA-DQB1 genotypes. We expanded our typing to 76 selected animals with diverse DQB1 and DRB1 genotypes, 140 random animals from 7 pig breeds, and 3 wild boars. This resulted in the identification of 17 DQA alleles with 49 genotypes. Two new alleles were identified from wild boars. Combine with SLA-DQB1, and DRB1 typing results, we identified 34 SLA class II haplotypes including 25 that were previously unreported.

Identification of high utility SNPs for population assignment and traceability purposes in the pig using high-throughput sequencing.

The objectives of this study were to develop breed-specific single nucleotide polymorphisms (SNPs) in five pig breeds sequenced with Illumina's Genome Analyzer and to investigate their usefulness for breed assignment purposes. DNA pools were prepared for Duroc, Landrace, Large White, Pietrain and Wild Boar. The total number of animals used for sequencing was 153. SNP discovery was performed by aligning the filtered reads against Build 7 of the pig genome. A total of 313,964 high confidence SNPs were identified and analysed for the presence of breed-specific SNPs (defined in this context as SNPs for which one of the alleles was detected in only one breed). There were 29,146 putative breed-specific SNPs identified, of which 4441 were included in the PorcineSNP60 beadchip. Upon re-examining the genotypes obtained using the beadchip, 193 SNPs were confirmed as being breed specific. These 193 SNPs were subsequently used to assign an additional 490 individuals from the same breeds, using the sequenced individuals as reference populations. In total, four breed assignment tests were performed. Results showed that for all methods tested 99% of the animals were correctly assigned, with an average probability of assignment of at least 99.2%, indicating the high utility of breed-specific markers for breed assignment and traceability. This study provides a blueprint for the way next-generation sequencing technologies can be used for the identification of breed-specific SNPs, as well as evidence that these SNPs may be a powerful tool for breed assignment and traceability of animal products to their breeds of origin.

A high-resolution comparative map of porcine chromosome 4 (SSC4).

We used the IMNpRH2(12,000-rad) RH and IMpRH(7,000-rad) panels to integrate 2019 transcriptome (RNA-seq)-generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger-printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high-resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.

Nomenclature for factors of the SLA system, update 2008.

This report summarizes the new swine leukocyte antigen (SLA) allele sequences and haplotypes designated by the SLA Nomenclature Committee of the International Society for Animal Genetics. There have been 74 new SLA alleles, comprising 18 SLA-1 alleles, 11 SLA-2 alleles, six SLA-3 alleles, two SLA-6 alleles, one SLA-DRA allele, 20 SLA-DRB1 alleles, three SLA-DQA alleles and 13 SLA-DQB1 alleles. Twelve new SLA class I and four new class II haplotypes have also been designated. This is the first official update since the 2005 reports on the nomenclature for factors of the SLA class I and II systems. This report also summarizes recent updates to the Immunopolymorphism Database-Major Histocompatibility Complex (IPD-MHC) website (http://www.ebi.ac.uk/ipd/mhc/sla/). All information has now been integrated to the SLA section of the IPD-MHC database, which serves as the repository for maintaining a list of all recognized SLA genes and their allelic sequences.

Genetic induction of tumorigenesis in swine.

The transition from basic to clinical cancer research for a number of experimental therapeutics is hampered by the lack of a genetically malleable, large animal model. To this end, we genetically engineered primary porcine cells to be tumorigenic by expression of proteins known to perturb pathways commonly corrupted in human cancer. Akin to human cells, these porcine cells were quite resistant to transformation, requiring multiple genetic changes. Moreover, the transformed porcine cells produced tumors when returned to the isogenic host animal. The ability to now rapidly and reproducibly genetically induce tumors of sizes similar to those treated clinically in a large mammal similar to humans in many respects will provide a robust cancer model for preclinical studies dependent on generating large tumors.

High-resolution comprehensive radiation hybrid maps of the porcine chromosomes 2p and 9p compared with the human chromosome 11.

We are constructing high-resolution, chromosomal 'test' maps for the entire pig genome using a 12,000-rad WG-RH panel (IMNpRH2(12,000-rad))to provide a scaffold for the rapid assembly of the porcine genome sequence. Here we present an initial, comparative map of human chromosome (HSA) 11 with pig chromosomes (SSC) 2p and 9p. Two sets of RH mapping vectors were used to construct the RH framework (FW) maps for SSC2p and SSC9p. One set of 590 markers, including 131 microsatellites (MSs), 364 genes/ESTs, and 95 BAC end sequences (BESs) was typed on the IMNpRH2(12,000-rad) panel. A second set of 271 markers (28 MSs, 138 genes/ESTs, and 105 BESs) was typed on the IMpRH(7,000-rad) panel. The two data sets were merged into a single data-set of 655 markers of which 206 markers were typed on both panels. Two large linkage groups of 72 and 194 markers were assigned to SSC2p, and two linkage groups of 84 and 168 markers to SSC9p at a two-point LOD score of 10. A total of 126 and 114 FW markers were ordered with a likelihood ratio of 1000:1 to the SSC2p and SSC9p RH(12,000-rad) FW maps, respectively, with an accumulated map distance of 4046.5 cR(12,000 )and 1355.2 cR(7,000 )for SSC2p, and 4244.1 cR(12,000) and 1802.5 cR(7,000) for SSC9p. The kb/cR ratio in the IMNpRH2(12,000-rad) FW maps was 15.8 for SSC2p, and 15.4 for SSC9p, while the ratio in the IMpRH(7,000-rad) FW maps was 47.1 and 36.3, respectively, or an approximately 3.0-fold increase in map resolution in the IMNpRH(12,000-rad) panel over the IMpRH(7,000-rad) panel. The integrated IMNpRH(12,000-rad) andIMpRH(7,000-rad) maps as well as the genetic and BAC FPC maps provide an inclusive comparative map between SSC2p, SSC9p and HSA11 to close potential gaps between contigs prior to sequencing, and to identify regions where potential problems may arise in sequence assembly.

DNA-based animal models of human disease: from genotype to phenotype.

Biomedical research utilizes animal models to elucidate human disease processes at the cellular and molecular level and for the development of new therapies. Traditionally, mammalian models have been limited to the mouse, primarily because of well characterized genetic lines and the ability to manipulate the genome to directly test hypotheses regarding causal mutations and disease phenotypes. The emerging availability of genome sequences of other mammals (bovine, canine, equine, feline, and porcine) now permits utilization of the mammal in which the phenotype best approximates the human condition. Equally important is the use of somatic cell nuclear cloning (SCNT) coupled with targeted germline manipulation to create animals to resolve the molecular mechanisms of the disease state. Our efforts have focused on the pig, which has emerged as an important biomedical mammalian model due to its closer physiology to humans. The utility of porcine genetically-defined tumour, cardiovascular and neurological disease models is described.

Macrophage function in response to PGE2, L-arginine deprivation, and activation by colony-stimulating factors is dependent on hematopoietic stimulus.

Different macrophage preparations were compared for functional capacity in conditions of high prostaglandin E2 (PGE2) or low L-arginine concentrations. Macrophages derived in vitro from bone marrow progenitor cells (bone marrow-derived macrophages, BMDMs) using colony-stimulating factor 1 (CSF-1) as the myelopoietic stimulus displayed a greater sensitivity to PGE2-induced suppression of tumor necrosis factor alpha (TNF-alpha) secretion than did macrophages derived using granulocyte-macrophage colony-stimulating factor (GM-CSF). Neither BMDM population was inhibited by PGE2 for the direct cytolysis of L929 cells (TNF-alpha sensitive), and only GM-CSF-derived macrophages showed decreased killing of TNF-alpha-resistant K562 targets. Exogenous cAMP inhibited TNF-alpha secretion, but not nitrite secretion, by both BMDM populations. GM-CSF-derived macrophages accumulated less cAMP following PGE2 treatment than did CSF-1-derived macrophages. Removing L-arginine from the medium did not inhibit cytotoxicity or PGE2 secretion, but the listeriacidal activity specific to interferon-gamma plus lipopolysaccharide (LPS)-activated GM-CSF-derived macrophages was blocked by removal of L-arginine. Treatment with CSF-1 or GM-CSF alone did not activate the macrophages, but GM-CSF efficiently primed both BMDM populations for augmented TNF-alpha secretion in response to secondary stimulation using LPS. However, GM-CSF augmented the LPS-induced production of nitrite and PGE2 by CSF-1-derived macrophages only. These results demonstrate the potential for differential macrophage function within inflammatory sites based on the hematopoietic stimulus under which the macrophage is derived and the specific conditions present in the lesion.

Differential immunocompetence of macrophages derived using macrophage or granulocyte-macrophage colony-stimulating factor.

Macrophages derived in vitro from bone marrow progenitors (bone marrow-derived macrophages, BMDMs) using either macrophage colony-stimulating factor (CSF-1) or granulocyte-macrophage colony-stimulating factor (GM-CSF) as the myelopoietic stimulus display differential functional, morphological, and mRNA phenotypes. The data presented here demonstrate further that CSF-1- and GM-CSF-derived BMDMs differ in immunologic capacity. GM-CSF-derived BMDMs, when compared to CSF-1-derived BMDMs, showed greater cytolytic activity against tumor necrosis factor alpha (TNF-alpha)-resistant, but not TNF-alpha-sensitive, tumor targets. In contrast, CSF-1-derived BMDMs produced nitrite in response to lipopolysaccharide (LPS) alone, whereas GM-CSF-derived BMDMs required interferon gamma plus LPS treatment. The two BMDM populations also showed differential sensitivities to LPS for secretion of TNF-alpha and nitrite, but the maximal inducible amounts of these factors and prostaglandin E2 were similar between the BMDM populations. Lastly, GM-CSF-derived but not CSF-1-derived BMDMs showed an L-arginine-dependent listeriacidal activity. These results show that the functional heterogeneity of CSF-1- and GM-CSF-derived macrophages is limited and appears to result largely from differences in the activational signals required by each BMDM population to elicit a given function.

Mechanisms generating functionally heterogeneous macrophages: chaos revisited.

Macrophage populations exhibit a wide range of antigenic and functional phenotypes, including cytokine production, response to immunomodulatory stimuli, and clearance of pathogens. The expanding clinical exploitation of recombinant growth factors and cytokines with the potential to regulate the production and function of peripheral macrophage populations necessitates an increased understanding of the mechanisms by which functionally distinct macrophage populations arise as well as the ramifications of macrophage heterogeneity. The present review summarizes recent data which supports multiple mechanisms by which heterogeneous macrophage populations arise: 1) differential signals experienced within diverse tissue microenvironments; 2) developmentally-staged expression of specific functions; 3) clonal variation of myeloid progenitor cells; and 4) alternate hematopoietic stimulation. These data show that the above processes are not mutually exclusive and that each likely contributes to the observed heterogeneity of peripheral macrophage populations.

Effects of N-nitrosodimethylamine on tumor susceptibility.

Dimethylnitrosamine (DMN) exposure altered the activity of the macrophage and natural killer (NK) cell defense mechanisms against the B16F10 melanoma in B6C3F1 adult female mice as assessed by several immunologic assays. Following 14 daily exposures (i.p.) to 1.5, 3.0, or 5.0 mg DMN/kg, mice were injected (i.v.) with B16F10 melanoma. The incidence and number of lung nodules, determined 18 days after challenge, were decreased in the DMN-exposed animals. The initial observation indicated the mice exposed to 3 mg/kg DMN were afforded the greatest protection. However, when mice exposed to the highest dose of DMN were divided into subgroups of mice with or without ascites, there was a degree protection seen in the 5-mg/kg-treated mice without ascites that was comparable to that of the 3-mg/kg group. The development of ascites is an overt toxic effect reflecting damage to the liver and was frequently associated with exposure to 5 mg/kg DMN. Exposure to DMN produced only slight changes in the activity of splenic NK cells as determined by the cytotoxicity of 51Cr-labelled YAC-1 cells. The activity was significantly increased only in mice exposed to 3 mg/kg DMN and only at effector:target (E:T) ratios of 30:1 and 10:1. Interestingly, the activity of the NK cells was significantly decreased at all E:T ratios in mice exposed to 5 mg/kg that developed ascites. The number of peritoneal exudate cells was decreased, albeit nonsignificantly, in a dose-related fashion. The phagocytic activity, as measured by the uptake of fluorescent latex beads, was increased in a dose-related fashion with significance noted at the highest dose of DMN. The role of the macrophage in the increased resistance to the tumor challenge was assessed with bone marrow derived macrophages. The cytostatic activity versus B16F10 tumor cells, as measured by the uptake of 3H-thymidine, was markedly increased in the bone marrow derived macrophages from DMN (5mg/kg) mice when compared to vehicle controls. These results suggest that exposure to DMN alters bone marrow, particularly the differentiation of effector tumoricidal cells, which renders the host more resistant to metastatic tumor formation.

Cytokine regulation of TNF-alpha mRNA and protein production by unprimed macrophages from C57Bl/6 and NZW mice.

It is well known that bacterial lipopolysaccharide (LPS) induces the synthesis of tumor necrosis factor alpha (TNF-alpha) and other inflammatory cytokines by primary monocytes and macrophages and that the Th1 lymphokines, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), augment this response. We investigated the ability of IL-2 and IFN-gamma to induce the production of TNF-alpha mRNA and protein independently of LPS and the modulation of this response by macrophage colony-stimulating factor (M-CSF) and IL-10. We found that IL-2 and IFN-gamma were both able to induce the accumulation of TNF-alpha mRNA, albeit with slower kinetics than LPS, and that they acted synergistically. However, very little TNF bioactivity was secreted by lymphokine-stimulated macrophages unless LPS was also added. This finding underscores the importance of translational effects in the control of TNF production. M-CSF and IL-10 strongly inhibited TNF production at the level of both mRNA and bioactivity but had no effect on the production of IL-6. Bone marrow-derived or thioglycollate-elicited macrophages from the NZW mouse strain, which have been reported to be deficient in their ability to produce TNF, were at least as responsive to LPS or lymphokines as those taken from the C57Bl/6 strain and were similarly affected by M-CSF and IL-10. Therefore, the genetic defect of NZW mice is not a primary deficiency in TNF production.

Differential expression of novel genes by bone marrow-derived macrophage populations.

In the present study, we have constructed a subtraction cDNA library to identify novel genes induced by IFN-gamma in GM-CSF-derived bone marrow macrophage (m phi). M theta were treated with 50 U/ml IFN-gamma for 40, 70 and 140 min to induce expression of early genes regulated by IFN-gamma, and the M phi were pooled. Poly(A)+RNA was prepared from both unactivated and IFN-gamma-stimulated m theta, and cDNA libraries were constructed in lambda ZAP. Genes expressed in common by both m theta populations were removed by subtraction using biotin-avidin precipitation of hybrid complexes. Further selection was performed by differential screening using cDNA prepared from mRNA of unactivated m phi as a probe, followed by colony hybridization to remove sister clones. Of 17 clones from which sequence information was obtained, two appeared to be identical with the murine genes, C10 (clone GM2B1) and Mac-2 (clone GM2C4) and an additional two clones had high similarity to human cDNAs encoding proteins of unknown function. cDNAs containing sequences which did not match published sequences were used to probe Northern blots prepared from both unstimulated and IFN-gamma-activated GM-CSF- and CSF-1-derived m phi. Five clones (GM1A2, GM1B4, GM1F2, GM2A12 and GM2B8) showed enhanced transcript levels following IFN-gamma treatment of GM-CSF-derived m phi, but demonstrated high constitutive transcript levels in CSF-l-derived m phi. In addition, C10 transcripts were constitutively expressed by GM-CSF-derived m phi, but not by CSF-1-derived m phi, even after activation by IFN-gamma. These data suggest that much of the functional heterogeneity of GM-CSF- and CSF-1-derived m phi resides in the differential expression of early genes specifically induced by IFN-gamma.

Identification of human vascular endothelial cell/monocyte antigenic system using monoclonal antibodies.

Recent studies have suggested that non-major-histocompatibility-encoded antigens expressed preferentially on vascular endothelial cells and/or monocytes may act as immunogens leading to allograft rejection. In order to further characterize the antigens and to investigate the roles of interleukins in the induction of such antigens, three murine monoclonal antibodies (SK2H10, SK3A1, SK2F11) were produced against interferon-gamma-induced human umbilical vein endothelial cells. All three monoclonal antibodies reacted specifically with human endothelial cells cultured from umbilical veins as well as blood monocytes. Several human established cell lines with B, T, and erythroid/myeloid cell properties, as well as cell lines expressing HLA-class I and II antigens (SB, REH, HSB, K562 and HL-60) failed to react with these reagents, indicating that the binding structures on the endothelial/monocyte cell surface are distinct from HLA class I and II antigens. Furthermore, the binding characteristics of the three antibodies to endothelial cells, monocytes, and human monocytic cell line, U937, suggest that they recognize different determinants on the cell surface. Analysis of Ia-positive and Ia-negative clones of U937 demonstrated that the antibodies reacted strongly with the Ia-positive cells lines. The monoclonal SK2H10 reacted preferentially with phorbol esters-induced HL-60 and K562 cell lines; thus indicating that the antigens detected on the monocytes are associated with differentiation. These results indicate that these monoclonal antibodies to endothelial cells have specificities similar to those described for alloantisera defining the vascular endothelial cell/monocyte antigenic system and should facilitate definition of the biochemical and molecular nature of such antigens.

Biochemical characterization of human vascular endothelial cell-monocyte antigens defined by monoclonal antibodies.

Although the clinical relevance of endothelial cell-monocyte (E-M) antigens has been demonstrated in organ graft transplantation, very limited data exist describing the nature of these antigens. The current study presents biochemical characterization of three different surface antigens of endothelial cells and monocytes that are defined by murine monoclonals produced against gamma-interferon-induced human umbilical vein endothelial cells. The antigens gp150, gp48, and gp24 have molecular weights of 150,000, 48,000, and 24,000, respectively, under reducing conditions. The antibody binding sites of gp150 and gp48 are destroyed by pronase and chymotrypsin, indicating that the molecules are at least partly protein in nature. The inability to label the gp48 molecule with 125I using lactoperoxidase suggests that there is little protein structure exposed to the cell surface or that the molecule lacks sufficient cell surface tyrosine residues to enable detection. Immunoprecipitation of the gp24 molecule under nonreducing conditions shows that a molecule with a higher molecular weight ranging from 40,000-70,000 is detected. Although it is possible that this higher-molecular-weight species is a multimer of the 24,000 Mr species, it is also possible that there is another molecule(s) bound to the 24,000 Mr molecule. All three E-M antigens have some carbohydrate nature as evidenced by lectin-binding studies. The possible relevance of these antigens in the rejection of transplanted organ grafts is discussed.

In-vitro-derived bone marrow macrophages. Expression of Ia antigens during macrophage differentiation.

Cultures of bone marrow stem cells, grown in the presence of L-cell-conditioned medium, were harvested on successive days to determine the expression of Ia antigens and the acquisition of Ir4 gene-regulated antigen presentation. I-A subregion antigen expression was detected by an indirect radiobinding assay (RBA) as early as day 3 and reached maximal binding at day 7, before declining with additional time in culture. Indirect immunofluorescence (IIF) demonstrated 20% Ia+ cells on day 3 of culture and peaked at 60% on day 7 before declining with continued incubation. A mixed lymphocyte reaction (MLR) across an I region difference was used to assess the kinetics of bone-marrow-derived macrophage ( BMDM ) Ia expression. Maximal stimulation occurred with BMDM stimulator cells obtained from 5-9-day cultures. To investigate the acquisition of Ir gene function, BMDM harvested after various days in culture were used to reconstitute the T cell proliferative response to poly Glu60Ala30Tyr10 (GAT). The ability of BMDM to present GAT was detected after day 5, increased to maximal levels on day 7, and then declined with weak proliferative responses obtained using day 12 BMDM . The presentation of GAT by BMDM was inhibited by monoclonal anti-Ia. 17 antibody. Thus, the acquisition of Ir gene function by BMDM was found to parallel the expression of Ia molecules. Additional experiments were performed to determine whether treatment of BMDM at day 7 with lymphokines or beta-interferon could extend Ia antigen expression. Whereas treatment of day 7 BMDM cultures with Con-A-stimulated rat splenocyte supernatants extended and augmented Ia expression for an additional three days, beta-interferon treatment did not result in augmentation or extension of Ia antigen expression.

Clonal analysis of gene expression by PCR.

A method for determining the heterogeneity of gene expression from isolated macrophage colonies is described. Clones derived using specific growth factors were isolated by soft agar cloning and used as sources of mRNA for analysis. Gene expression was monitored by the reverse transcription polymerase chain reaction method to permit a number of specific genes to be simultaneously amplified. The technique described here involves the use of internal primers in addition to the usual two external primers typically used in gene amplification. Such internal primers greatly increased the accuracy and precision of amplification by adding an extra constraint such that the gene of interest was reliably amplified while decreasing background noise. This method permits gene expression to be monitored in cell colonies, thereby allowing the extent of phenotypic heterogeneity in developing macrophages (or other cell types) to be determined. This technique may also be useful in gene expression studies involving other cell types under the influence of specific growth factors.

Relationship of major histocompatibility complex class II genes to inhibitor antibody formation in hemophilia A.

Approximately 14% of transfused hemophiliacs develop an anti-factor VIII inhibitory antibody which specifically neutralizes factor VIII procoagulant activity. In this study an association of the major histocompatibility complex (MHC) with inhibitor antibody formation was evaluated by restriction fragment length polymorphism (RFLP) analysis using BamHI, EcoRI, HindIII, PstI, PvuII and TaqI digested genomic DNA probed with DP beta, DQ alpha, DQ beta and DR beta class II MHC gene probes. The RFLP patterns for 16 non-inhibitor and 11 inhibitor hemophiliac patients were analyzed. These 24 enzyme:probe combinations generated 231 fragments. Fifteen (15) fragments associated with the inhibitor phenotype; odds ratios ranged from 5.1 to 45 and lower bounds of 95% confidence intervals were greater than 1.000 for all 15 fragments. Five (5) fragments associated with non-inhibitors, with odds ratios ranging from 6.4 to 51.7. This report establishes a MHC related genetic basis for the inhibitor phenotype. No statistically significant differences in the distribution of serologically defined HLA-DR phenotypes were observed between the inhibitor and non-inhibitor groups.