Cloning and sequence analysis of 14 DRB alleles of the bovine major histocompatibility complex by using the polymerase chain reaction

The genetic diversity in the first domain exon of a bovine class II DRB gene was investigated by PCR amplification and DNA sequencing. Genomic DNA samples representing 14 different class II haplotypes, defined by RFLP analysis, were used. The analysis revealed an extensive polymorphism and 14 alleles at a single locus, designated DRB3, were identified. Multiple amino acid substitutions were found in all pairwise comparisons of alleles; 5 to 21 substitutions in the 83 positions compared. The genetic diversity at the amino acid level found in cattle matches the one previously found in the DRB1 locus in man. The significantly higher frequency of replacement substitutions compared with the frequency of silent substitutions provides strong evidence that there is selection for genetic diversity in the bovine DRB3 first domain exon. A comparison of the DRB polymorphism in man and cattle reveals a striking similarity as regards the location of polymorphic positions in the DRB molecule and the degree of polymorphism at polymorphic positions. The majority of polymorphic positions in both species are found in the proposed antigen recognition site of the class II molecule. In addition, there are eight positions which are polymorphic in both species but have not been assigned to the antigen recognition site. The possible functional significance of the polymorphism of these latter positions is discussed.     

Sequence variation at the major histocompatibility complex DRB loci in beluga (Delphinapterus leucas) and narwhal (Monodon monoceros)

 The variation at loci with similarity to DRB class II major histocompatibility complex loci was assessed in 313 beluga collected from 13 sampling locations across North America, and 11 narwhal collected in the Canadian high Arctic. Variation was assessed by amplification of exon 2, which codes for the peptide binding region, via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Two DRB loci were identified in beluga: DRB1, a polymorphic locus, and, DRB2, a monomorphic locus. Eight alleles representing five distinct lineages (based on sequence similarity) were found at the beluga DRB1 locus. Although the relative number of alleles is low when compared with terrestrial mammals, the amino acid variation found among the lineages is moderate. At the DRB1 locus, the average number of nonsynonymous substitutions per site is greater than the average number of synonymous substitutions per site (0.0806 : 0.0207, respectively;P<0.01). Most of the 31 amino acid substitutions do not conserve the physiochemical properties of the residue, and 21 of these are located at positions implicated as forming pockets responsible for the selective binding of foreign peptide side chains. Only DRB1 variation was examined in 11 narwhal, revealing a low amount of variation. These data are consistent with an important role for the DRB1 locus in the cellular immune response of beluga. In addition, the ratio of nonsynonymous to synonymous substitutions is similar to that among primate alleles, arguing against a reduction in the balancing selection pressure in the marine environment. Two hypotheses may explain the modest amount of Mhc variation when compared with terrestrial mammals: small population sizes at speciation or a reduced neutral substitution rate in cetaceans. Received: 15 July 1997 / Revised: 24 March 1998     

Interdependent MHC-DRB exon-plus-intron evolution in artiodactyls

Exon 2 sequences of an expressed MHC-DRB locus from sheep were examined for polymorphisms in both the antigen-binding regions and the adjacent intronic mixed simple tandem repeat. Twenty-one novel exon 2 Ovar-DRB alleles were identified. Short nucleotide motifs are extensively shared between certain exon 2 regions of Ovar-DRB alleles. The simple repeat variations, the number of different amino acids at usually polymorphic sites, and the number of silent substitutions were reduced in the intraspecies analyses of sheep DRB sequences, compared with those of cattle and goats. It was paradoxical that the abundance of different sheep alleles was similar to that of cattle and goats. This paradox may be explained by postulating a relatively small number of "ancient" alleles, with the present-day Ovar-DRB alleles being generated by reciprocal exchange of nucleotide motifs. At the antigen-binding sites, new combinations of amino acids were maintained in Ovar-DRB alleles by strong positive selection. In sheep--and less pronounced in goats and cattle--the DRB alleles can be divided into two groups. In one group, silent substitutions are increased when compared with the other. This suggests separate evolutionary pathways for certain groups of DRB alleles within a species. The simple repetitive sequences are also discussed with respect to the evolution of DRB alleles.      

High resolution molecular typing of HLA class II region in the population of the island of Krk, Croatia

The HLA class II alleles (DRB1, DRB3, DRB5, DQA1, and DQB1) and haplotypic associations were studied in the population of the island of Krk using the PCR-SSOP method and the 12th International Histocompatibility Workshop primers and probes. Allele and haplotypic frequencies were compared with the general Croatian population. Significant differences were observed between the population of the island of Krk and Croatians for: a) three broad specificities at DRB1 locus (DRB1*01, *15, and *07), b) one allele at DRB3 locus (DRB3*0301), c) one allele at DQA1 locus (DQA1*0201), d) one allele at DQB1 locus (DQB1*0303). Four unusual haplotypic associations, which have not yet been described in the Croatian population, DRB1*1301-DQA1*0103-DQB1*0607, DRB1*1302-DQA1*0102-DQB1*0605, DRB1*1305-DQA1*0102-DQB1*0605 and DRB1*1305-DQA1*0103-DQB1*0603 were observed in the population from the island of Krk.     

Ovar-MHC Polymorphism in Malpura and Avikalin Sheep Vaccinated for Peste des Petits Ruminants (PPR) Virus

India harbors a vast diversity of sheep (40 breeds). The study was carried out to assess the genetic diversity of DRB1 and DQA2 locus of the ovar-MHC and their possible association with Peste des petits ruminants (PPR) virus vaccine response in Malpura and Avikalin sheep breeds maintained at an organized institute flock in the semi-arid region of India. Genetic analysis revealed the rich diversity of DRB1 locus with 23 alleles in Malpura and 21 alleles in Avikalin sheep that included 9 new alleles. DQA2 locus also had rich diversity with 19 alleles in Malpura and 20 alleles in Avikalin sheep that included 7 new alleles. At the protein level, high variability alike at the nucleotide level was observed. A marker for footrot susceptibility, DQA2*1101 was absent in both breeds. Genotypic association of DRB1 and DQA2 with PPR vaccine response was statistically non-significant. Vaccine response being a multifactorial (polygenic and influenced by environment) variable, could not show statistically significant association with MHC genotypes in the present study. However, rich genetic diversity of DRB1 and DQA2 gene reflects the importance of this locus for future selection programs.     

HLA－DRB1基因位点多态性的PCR－RFLP分析

设计并建立一套适合国内应用的改良ＰＣＲ－ＲＦＬＲ方法,分５组特异性扩增ＤＮＡ样品,随后进行酶切定型分析,准确检测了编码ＤＲ抗原特异性的ＨＬＡ－ＤＲＢ１基因位点的多态性,该法采用分组扩增,不发生与其它ＤＲＢ位点等位基因的交叉扩增,不仅适合纯合子的区分而且可以清楚准确地检测杂合子样品,已报道过的ＤＲＢ１位点编码的特异性组合都可以通过这个方法得到准确分析。所使用的Ⅱ类限制性内切酶均价格便宜、易购。     

Sequence and PCR-RFLP analysis of 14 novel BoLA-DRB3 alleles

The genetic diversity of the bovine class IIDRB3 locus was investigated by polymerase chain reaction (PCR) amplification and DNA sequencing of the first domain exon. Studying 34 animals of various cattle breeds, 14 previously unrecognized DRB3 alleles were identified. In three alleles, amino acid substitutions were observed that had not been previously found in bovine DRB3, but occurred at the same position in bovine DQB and in the DRB alleles of other mammals. For all newly identified alleles, the restriction fragment length polymorphism (RFLP) patterns of PCR products obtained with the enzymes Rsa I, Bst YI, and Hae III were compared with patterns of 38 previously described alleles. Altogether, eleven novel PCR-RFLP types were defined. Twelve out of the 42 PCR-RFLP types identified so far were not found to be fully informative because they corresponded to more than one allelic sequence. PCR-RFLP may therefore be a rapid and useful method for DRB3 typing in cattle families, but for studies on outbred populations, sequencing and hybridization techniques are required.     

Evolution of MHC class II SLA‐DRB1 locus in the Croatian wild boar (Sus scrofa) implies duplication and weak signals of positive selection

The wild boar is an ancestor of the domestic pig and an important game species with the widest geographical range of all ungulates. Although a large amount of data are available on major histocompatibility complex (MHC) variability in domestic pigs, only a few studies have been performed on wild boars. Due to their crucial role in appropriate immune responses and extreme polymorphism, MHC genes represent some of the best candidates for studying the processes of adaptive evolution. Here, we present the results on the variability and evolution of the entire MHC class II SLA‐DRB1 locus exon 2 in 133 wild boars from Croatia. Using direct sequencing and cloning methods, we identified 20 SLA‐DRB1 alleles, including eight new variants, with notable divergence. In some individuals, we documented functional locus duplication, and SLA‐DRB1*04:10 was identified as the allele involved in the duplication. The expression of a duplicated locus was confirmed by cloning and sequencing cDNA‐derived amplicons. Based on individual genotypes, we were able to assume that alleles SLA‐DRB1*04:10 and SLA‐DRB1*06:07 are linked as an allelic combination that co‐evolves as a two‐locus haplotype. Our investigation of evolutionary processes at the SLA‐DRB1 locus confirmed the role of intralocus recombination in generating allelic variability, whereas tests of positive selection based on the dN/dS (non‐synonymous/synonymous substitution rate ratio) test revealed atypically weak and ambiguous signals.     

Several regions in the major histocompatibility complex confer risk for anti-CCP-antibody positive rheumatoid arthritis, independent of the DRB1 locus

Recent evidence suggests that additional risk loci for RA are present in the major histocompatibility complex (MHC), independent of the class II HLA-DRB1 locus. We have now tested a total of 1,769 SNPs across 7.5Mb of the MHC located from 6p22.2 (26.03 Mb) to 6p21.32 (33.59 Mb) derived from the Illumina 550K Beadchip (Illumina, San Diego, CA, USA). For an initial analysis in the whole dataset (869 RA CCP + cases, 1,193 controls), the strongest association signal was observed in markers near the HLA-DRB1 locus, with additional evidence for association extending out into the Class I HLA region. To avoid confounding that may arise due to linkage disequilibrium with DRB1 alleles, we analyzed a subset of the data by matching cases and controls by DRB1 genotype (both alleles matched 1:1), yielding a set of 372 cases with 372 controls. This analysis revealed the presence of at least two regions of association with RA in the Class I region, independent of DRB1 genotype. SNP alleles found on the conserved A1-B8-DR3 (8.1) haplotype show the strongest evidence of positive association (P ~ 0.00005) clustered in the region around the HLA-C locus. In addition, we identified risk alleles that are not present on the 8.1 haplotype, with maximal association signals (P ~ 0.001-0.0027) located near the ZNF311 locus. This latter association is enriched in DRB1*0404 individuals. Finally, several additional association signals were found in the extreme centromeric portion of the MHC, in regions containing the DOB1, TAP2, DPB1, and COL11A2 genes. These data emphasize that further analysis of the MHC is likely to reveal genetic risk factors for rheumatoid arthritis that are independent of the DRB1 shared epitope alleles.     

Analysis of extended HLA haplotypes in multiple sclerosis and narcolepsy families confirms a predisposing effect for the class I region in Tasmanian MS patients

Human leucocyte antigen (HLA)-DRB1*15 is associated with predisposition to multiple sclerosis (MS), although conjecture surrounds the possible involvement of an alternate risk locus in the class I region of the HLA complex. We have shown previously that an alternate MS risk allele(s) may be encompassed by the telomerically extended DRB1*15 haplotype, and here, we have attempted to map the putative variant. Thirteen microsatellite markers encompassing a 6.79-megabase (D6S2236-G51152) region, and the DRB1 and DQB1 genes, were genotyped in 166 MS simplex families and 104 control families from the Australian State of Tasmania and 153 narcolepsy simplex families (trios) from the USA. Complementary approaches were used to investigate residual predisposing effects of microsatellite alleles comprising the extended DRB1*15 haplotype taking into account the strong predisposing effect of DRB1*15: (1) Disease association of the extended DRB1*15 haplotype was compared for MS and narcolepsy families--predisposing effects were observed for extended class I microsatellite marker alleles in MS families, but not narcolepsy families; (2) disease association of the extended DRB1*15 haplotype was investigated after conditioning MS and control haplotypes on the absence of DRB1*15--a significant predisposing effect was observed for a 627-kb haplotype (D6S258 allele 8-MOGCA allele 4; MOG, myelin oligodendrocyte glycoprotein) spanning the extended class I region. MOGCA allele 4 displayed the strongest predisposing effect in DRB1*15-conditioned haplotypes (p = 0.0006; OR 2.83 [1.54-5.19]). Together, these data confirm that an alternate MS risk locus exists in the extended class I region in Tasmanian MS patients independent of DRB1*15.     

HLA-DRB1 alleles in four Amerindian populations from Argentina and Paraguay

The major histocompatibility complex (MHC) is one of the biological systems of major polymorphisms. The study of HLA class II variability has allowed the identification of several alleles that are characteristic to Amerindian populations, and it is an excellent tool to define the relations and biological affinities among them. In this work, we analyzed the allelic distribution of the HLA-DRB1 class II locus in four Amerindian populations: Mapuche (n = 34) and Tehuelche (n = 23) from the Patagonian region of Argentina, and Wichi SV (n = 24) and Lengua (n = 17) from the Argentinean and Paraguayan Chaco regions, respectively. In all of these groups, relatively high frequencies of Amerindian HLA-DRB1 alleles were observed (DRB1*0403, DRB1*0407, DRB1*0411, DRB1*0417, DRB1*0802, DRB1*0901, DRB1*1402, DRB1*1406 and DRB1*1602). However, we also detected the presence of non-Amerindian variants in Mapuche (35%) and Tehuelche (22%). We compared our data with those obtained in six indigenous groups of the Argentinean Chaco region and in a sample from Buenos Aires City. The genetic distance dendrogram showed a clear-cut division between the Patagonian and Chaco populations, which formed two different clusters. In spite of their linguistic differences, it can be inferred that the biological affinities observed are in concordance with the geographic distributions and interethnic relations established among the groups studied.     

Search for schizophrenia susceptibility variants at the HLA-DRB1 locus among a British population

Schizophrenia is a complex mental disorder with unknown aetiology. Both candidate gene and genome-wide association (GWA) studies suggest that the human leukocyte antigen (HLA) system may play a part in development of the illness, but the causal HLA variant(s) remain(s) unclear. Previous studies showed that the DRB1*0101 and DRB1*13 alleles might be associated with a high risk of schizophrenia. Therefore, the present study was undertaken to test their association with the disease by genotyping seven DRB1-tagging single nucleotide polymorphisms (SNPs) in a British population. The results showed that, of the previously reported variants that were associated with schizophrenia, the DRB1*1303 allele was the only one marginally associated with a protective effect on the illness in our sample set (χ ²?=?4.138, P?=?0.042, odds ratio (OR)?=?0.42, 95 % confidence interval (CI) 0.27–0.66). Interestingly, a significant association was found for rs424232 (χ ²?=?9.404, P?=?0.002, OR?=?0.69, 95 % CI 0.54–0.88), which is a tag SNP for the DRB1*1303 allele and located near to the NOTCH4 gene that is a schizophrenia susceptibility locus confirmed by GWA studies. Analysis with the Haploview program demonstrated that rs424232 was in complete linkage disequilibrium with rs3130297 and rs3131296 present in the NOTCH4 locus. While we have failed to confirm association of the candidate alleles in the DRB1 gene with a high risk of schizophrenia, the present work suggests that the association signal detected in the HLA class II locus may extend a relatively long distance, and more work is needed in order to identify the true causal variants within this region or nearby.     

Genetic drift vs. natural selection in a long-term small isolated population: major histocompatibility complex class II variation in the Gulf of California endemic porpoise (Phocoena sinus)

Although many studies confirm long-term small isolated populations (e.g. island endemics) commonly sustain low neutral genetic variation as a result of genetic drift, it is less clear how selection on adaptive or detrimental genes interplay with random forces. We investigated sequence variation at two major histocompatibility complex (Mhc) class II loci on a porpoise endemic to the upper Gulf of California, México (Phocoena sinus, or vaquita). Its unique declining population is estimated around 500 individuals. Single-strand conformation polymorphism analysis revealed one putative functional allele fixed at the locus DQB (n = 25). At the DRB locus, we found two presumed functional alleles (n = 29), differing by a single nonsynonymous nucleotide substitution that could increase the stability at the dimer interface of alphabeta-heterodimers on heterozygous individuals. Identical trans-specific DQB1 and DRB1 alleles were identified between P. sinus and its closest relative, the Burmeister's porpoise (Phocoena spinipinnis). Comparison with studies on four island endemic mammals suggests fixation of one allele, due to genetic drift, commonly occurs at the DQA or DQB loci (effectively neutral). Similarly, deleterious alleles of small effect are also effectively neutral and can become fixed; a high frequency of anatomical malformations on vaquita gave empirical support to this prediction. In contrast, retention of low but functional polymorphism at the DRB locus was consistent with higher selection intensity. These observations indicated natural selection could maintain (and likely also purge) some crucial alleles even in the face of strong and prolonged genetic drift and inbreeding, suggesting long-term small populations should display low inbreeding depression. Low levels of Mhc variation warn about a high susceptibility to novel pathogens and diseases in vaquita.     

Genetic variation at the MHC DRB1 locus is similar across Gunnison's prairie dog (Cynomys gunnisoni) colonies regardless of plague history

Yersinia pestis was introduced to North America around 1900 and leads to nearly 100% mortality in prairie dog (Cynomys spp.) colonies during epizootic events, which suggests this pathogen may exert a strong selective force. We characterized genetic diversity at an MHC class II locus (DRB1) in Gunnison's prairie dog (C. gunnisoni) and quantified population genetic structure at the DRB1 versus 12 microsatellite loci in three large Arizona colonies. Two colonies, Seligman (SE) and Espee Ranch (ES), have experienced multiple plague‐related die‐offs in recent years, whereas plague has never been documented at Aubrey Valley (AV). We found fairly low allelic diversity at the DRB1 locus, with one allele (DRB1*01) at high frequency (0.67–0.87) in all colonies. Two other DRB1 alleles appear to be trans‐species polymorphisms shared with the black‐tailed prairie dog (C. ludovicianus), indicating that these alleles have been maintained across evolutionary time frames. Estimates of genetic differentiation were generally lower at the MHC locus (F_ST = 0.033) than at microsatellite markers (F_ST = 0.098). The reduced differentiation at DRB1 may indicate that selection has been important for shaping variation at MHC loci, regardless of the presence or absence of plague in recent decades. However, genetic drift has probably also influenced the DRB1 locus because its level of differentiation was not different from that of microsatellites in an F_ST outlier analysis. We then compared specific MHC alleles to plague survivorship in 60 C. gunnisoni that had been experimentally infected with Y. pestis. We found that survival was greater in individuals that carried at least one copy of the most common allele (DRB1*01) compared to those that did not (60% vs. 20%). Although the sample sizes of these two groups were unbalanced, this result suggests the possibility that this MHC class II locus, or a nearby linked gene, could play a role in plague survival.     

Polymorphism at the ovine major histocompatibility complex class II loci

Southern hybridization analysis of the ovine major histocompatibility complex (MHC) ( MhcOvar ) class II region, using sheep-specific probes for the DQA1, DQA2, DQB and DRA loci, has revealed extensive polymorphism. DQA1 and DQAP had eight and 16 alleles respectively, DQB had six and DRA had three alleles. Little information was derived from the DRB locus owing to extensive cross-hybridization between the DRB probe and the DQB locus. Differences in allele frequency between breeds were revealed. At the DQA1 locus a null allele (DQA1-N) was observed with a frequency of between 27% and 45%, making this the most common DQA1 allele in all breeds examined. The frequency of DQA1-N homozygotes was between 11% and 18%, raising questions as to the functional significance of the DQA1 gene. Linkage analysis between the DQA1, DQA2, DQB and DRA loci did not reveal any recombination.     

Low diversity in the major histocompatibility complex class II DRB1 gene of the Spanish ibex, Capra pyrenaica

During the last two centuries, the Spanish ibex (Capra pyrenaica) has shown a significant demographic decline as a result of the progressive destruction of its natural habitat, disease epidemics, and uncontrolled hunting. Partial sequencing of the class II MHC DRB1 gene revealed that the Spanish ibex has remarkably low levels of genetic variation at this locus, with only six different DRB1 alleles and an observed heterozygosity of 0.429-0.579. The rates of nonsynonymous vs synonymous substitutions were significantly different in the peptide-binding region (dN/dS=5.347, P=0.002), a feature that indicates that the DRB1 gene is under positive selection. A phylogenetic analysis of the Spanish ibex and a set of domestic goat DRB1 alleles revealed that the reported sequences represent four major allelic lineages. The limited allelic repertoire of the DRB1 gene in the Spanish ibex is likely the direct result of the recent history of population bottlenecks and marked demographic decline of this species. A genetic survey of 13 microsatellite loci was consistent with this idea. The Spanish ibex subspecies C. p. hispanica and C. p. victoriae consistently showed considerably lower levels of microsatellite heterozygosity (Ho=0.184-0.231) and allelic diversity (mean number of alleles per locus=2-2.4) than those reported in other wild ruminants. This study demonstrates the significance of both natural selection and the demographic history of populations in determining patterns of genetic variation at MHC loci. In addition, our results emphasize the importance of locally adapted populations for the preservation of genetic diversity.     

Recent divergence of the<Emphasis Type="Italic"> HLA-DRB1*04</Emphasis> allelic lineage from the<Emphasis Type="Italic"> DRB1*0701</Emphasis> lineage after the separation of the human and chimpanzee species

Conventional phylogenetic trees for the human leukocyte antigen (HLA)-DRB1 alleles constructed by the neighbor-joining (Saitou and Nei 1987) and UPGMA (Sneath and Sokal 1973) methods using nucleotide sequences of the DRB1 alleles suggest that DRB1*0701 may have diverged from other DRB1 alleles before the separation of the human and chimpanzee species, because of a large number of nucleotide changes in DRB1*0701 compared with any of the other DRB1 alleles. Here we show new evidence that the haplotypes centering on DRB1*0701 and DRB1*04 alleles are the most homologous. This suggests that these haplotypes have derived from the common ancestral haplotype, and that they have likely retained complete linkage disequilibrium even after the divergence of the DRB1*0701 and DRB1*04 allelic lineages. Together with the corresponding haplotype carrying chimpanzee DRB1*0701, which has a high sequence homology to HLA-DRB1*0701, these haplotypes reveal that: (1) the DRB1*04 allelic lineage may have been generated from the DRB1*0701 lineage after the separation of the human and chimpanzee species; (2) the DRB1*04 allelic lineage possibly has a higher substitution rate of DRB1 compared with pseudogene and neutral region; (3) there could be a significant difference in the substitution rate of DRB1 between the DRB1*0701 and DRB1*04 allelic lineages. Based on the difference between the present and previous results, we would like to propose that phylogenetic studies using not only nucleotide sequences of the DRB1 alleles but also haplotypes centering on the alleles should be conducted for understanding detailed phylogenetic relationships of the DRB1 alleles.     

HLA-DRB1*1101: a significant risk factor for sarcoidosis in blacks and whites

Sarcoidosis is a granulomatous disorder of unknown etiology, associated with an accumulation of CD4+ T cells and a TH1 immune response. Since previous studies of HLA associations with sarcoidosis were limited by serologic or low-resolution molecular identification, we performed high-resolution typing for the HLA-DPB1, HLA-DQB1, HLA-DRB1, and HLA-DRB3 loci and the presence of the DRB4 or DRB5 locus, to define HLA class II associations with sarcoidosis. A Case Control Etiologic Study of Sarcoidosis (ACCESS) enrolled biopsy-confirmed cases (736 total) from 10 centers in the United States. Seven hundred six (706) controls were case matched for age, race, sex, and geographic area. We studied the first 474 ACCESS patients and case-matched controls. The HLA-DRB1 alleles were differentially distributed between cases and controls (P<.0001). The HLA-DRB1*1101 allele was associated (P<.01) with sarcoidosis in blacks and whites and had a population attributable risk of 16% in blacks and 9% in whites. HLA-DRB1-F(47) was the amino acid residue most associated with sarcoidosis and independently associated with sarcoidosis in whites. The HLA-DPB1 locus also contributed to susceptibility for sarcoidosis and, in contrast to chronic beryllium disease, a non-E(69)-containing allele, HLA-DPB1*0101, conveyed most of the risk. Although significant differences were observed in the distribution of HLA class II alleles between blacks and whites, only HLA-DRB1*1501 was differentially associated with sarcoidosis (P<.003). In addition to being susceptibility markers, HLA class II alleles may be markers for different phenotypes of sarcoidosis (DRB1*0401 for eye in blacks and whites, DRB3 for bone marrow in blacks, and DPB1*0101 for hypercalcemia in whites). These studies confirm a genetic predisposition for sarcoidosis and present evidence for the allelic variation at the HLA-DRB1 locus as a major contributor.     

Sex influences on the penetrance of HLA shared-epitope genotypes for rheumatoid arthritis.

The association between rheumatoid arthritis (RA) and HLA DRB1 alleles may arise through linkage disequilibrium with a disease locus or the direct involvement of HLA alleles in RA. In support of the latter possibility, the shared-epitope hypothesis has been postulated, stating that conformationally similar DR beta chains encoded by several DRB1 alleles confer disease susceptibility. To examine these alternative hypotheses of marker-disease association and to investigate gender differences in RA susceptibility, we analyzed the distributions of PCR-based DRB1 genotypes of 309 Caucasian RA patients and 283 Caucasian controls. Initially, the marker-association-segregation chi 2 method was used to evaluate evidence for linkage disequilibrium and the direct involvement of markers DR4 Dw4, DR4 Dw14, and DR1 in RA susceptibility. Additional shared-epitope models that grouped DRB1 alleles into five classes (*0401, *0404/*0102, *0405/*0408/*0101, *1001, and all others) and postulated relationships between genotypes and RA susceptibility were also fitted to observed genotypic distributions by the method of minimal chi 2. For females, a linkage-disequilibrium model provided a good fit to the data, as did a shared-epitope model with RA most penetrant among individuals with the *0401,*0401 genotype. For males, the best model indicated highest RA penetrance among shared-epitope compound heterozygotes. Clinically, male RA patients had more subcutaneous nodules and greater use of slowly acting antirheumatic drugs, while female RA patients had earlier disease onset. This study therefore suggests that sex-related factors influence the RA penetrance associated with DRB1 shared-epitope genotypes and that DRB1 effects on RA prognosis and pathogenesis should be considered separately for men and women.     

MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.