DRB Genes Research Articles

Association between HLA-DQA1/DRB1 polymorphism and development of hepatocellular carcinoma during entecavir treatment.

It remains unclear whether there is an association between single-nucleotide polymorphisms (SNPs) and development of hepatocellular carcinoma (HCC) during entecavir (ETV) treatment in nucleos(t)ide analog-naïve patients with chronic hepatitis B virus infection. We investigated the risk factors for HCC, especially host factors, during ETV treatment. A total of 127 Japanese patients undergoing ETV treatment were enrolled in this study. Univariate and multivariate analyses for clinical factors, hepatic fibrosis markers, and SNPs associated with HCC development were analyzed. A total of 10 patients developed HCC during the follow-up period (median duration, 3.3years). The 3-, 5-, and 7-year cumulative rates of HCC development were 4.8%, 10.6%, and 13.6%, respectively. Liver fibrosis (cirrhosis; P=0.0005), age (≥49years; P=0.0048), platelet count (≤115×10/mm3 ; P=0.0007), α-fetoprotein (≥8.0ng/mL; P=0.030), type IV collagen (≥200ng/mL; P=0.043), fibrosis-4 index (≥4.14; P=0.0006), and human leukocyte antigen (HLA)-DQA1/DRB1-SNP (AA genotype; P=0.0092) were significantly associated with HCC development according to the log-rank test. In multivariate analysis, AA genotype in the HLA-DQA1/DRB1 gene (P=0.013; hazard ratio 4.907; 95% confidence interval 1.407-17.113) and cirrhosis (P=0.019; hazard ratio 4.789; 95% confidence interval 1.296-17.689) were significantly associated with HCC development. Our findings suggested that patients with AA genotype in the HLA-DQA1/DRB1 gene or cirrhosis should be carefully followed up as a population potentially at higher risk of HCC during ETV treatment.

Genetic diversity of MHC class II DRB alleles in the marbled polecat, Vormela peregusna, in Bulgaria

The marbled polecat, Vormela peregusna, is a small marten-like mustelid distributed from southeastern Europe to western China. Since the individual numbers of this species have declined, it has been classified as a vulnerable (VU) species in the 2008 IUCN Red List. To better understand the adaptation of V. peregusna, we investigated diversity and selection in the major histocompatibility complex (MHC) class II DRB gene which is reported to be highly polymorphic in many other species. Among 10 V. peregusna individuals from Bulgaria, we detected 9 novel sequences of partial exon 2, including antigen-binding site (ABS) codons, as representatives of DRB allele (Vope-DRBs). Two of the alleles were detected in all individuals analysed, while the others showed limited geographical distributions. Our results provided evidence for positive selection on ABS codons and recombination break points, contributing to maintaining the diversity of Vope-DRBs. In a Bayesian phylogenetic tree, all Vope-DRBs grouped within the mustelid clade, with three of them in the basal group consisted of alleles from Meles, Martes, and Vormela but no Mustela alleles, and the others in the derived group consisted of alleles diverged relatively late in the various mustelid species. The Vope-DRBs showed trans-species polymorphism within the mustelid clade, suggesting that the DRB genes evolved under long-lasting balancing selection.

GENETIC AND INFECTIOUS MARKERS OF INFERTILITY

The report presents the results of own research of the most important factors in the genesis of various forms of sterility. A married couples (n = 50) was examined with regard to possible etiological structure of these forms of pathology — infections of the family herpes (Herpes simplex types 1, 2, Cytomegalovirus hominis (CMV), Epstein–Barr virus (EBV) and genetic distribution for the second class of histocompatibilit (HLA II class): HLA-DRB1, HLA- DQA1 и HLA-DQB1. The authors present own data on genetic typing of the couples for the second class of histocompatibility, which allow to correlate these data with clinical and laboratory associations with infectious agents (infections of the herpes family). The genetic characteristics that determine the reproductive ability of the couple are associated with susceptibility to certain infectious markers. It is shown that the dominant majority of couples determined the HLA- DRB1 gene, which is more frequently associated with infections of the herpes family. Despite the fact that the level of positive PCR results in the group of couples with primary infertility was higher than in couples with secondary sterility (100 and 92%, respectively), maximum titers of specific antibodies to infections of the herpes family, including the frequency of markers of exacerbation, definition of IgM was higher than in the group of couples with primary infertility, and significantly higher than in couples with normal fertility. So, this antigen histocompatibility (HLA-DRB1) is associated with significant frequency of sterility, especially the secondary one, and infectious contamination infections of the family of herpes — HSV 1, 2 types, CMV, EBV in the study group — couples with secondary infertility. Marker HLA-DRB1, the most frequent in coup les with infertility, including idiopathic, associated in this group not only with significant contamination of infectious of the herpes family, but the presence of a chronic infectious process at the stage of reactivation. Conducted clinical and immunological studies clearly demonstrate the need for a comprehensive survey of women with disabilities in the system of immunogenesis with the mandatory assessment of the level of infectious (viral) contamination. Analysis of the results of their clinical and immunological studies allows us to propose an algorithm of complex examination in functional disorders in the system of immunogenesis, including the study of viral load as a necessary component.

OR47 The mystery of the missing DRB1 allele – solved by NGS!!!

The mystery began when HLA typing, of two apparently unrelated individuals at two different institutions, identified the presence of a DRB5∗01 gene without any evidence of an expected linked DRB1∗15, DRB1∗16, or, in rare cases, DRB1∗01 allele. Historic data from Sanger sequence-based typing included DRB1∗14, DRB3∗02:02, DRB5∗01:01, DQB1∗05:03, 06:02 for Individual 1 and DRB1∗04:01; DRB4∗01; DRB5∗01:01; DQB1∗03:01, 06:02 for Individual 2. Since those analyses only assessed exon 2, the apparent absence of a DRB1 allele associated with the DRB5 gene raised the possibility of an entire linked DRB1 gene having gone missing. Both specimens were recently re-examined using next generation sequencing technology. HLA-A, B, C genes were assessed for exons 1–7; DRB1, DRB3/B4/B5, DQA1, DQB1, and DPB1 were assessed for exons 1–4; and DPA1 for exons 2–4. In Individual 1, NGS identified DRB1∗14:54:01 and a DRB1∗15 exon 1 sequence, but without any evidence of DRB1∗15 sequences for exons 2 and 3. Since the exon 4 sequences of DRB1∗14:54:01 and DRB1∗15 are identical, it could not be determined whether the DRB1∗15 exon 4 sequence was present. Individual 2, typed as DRB1∗04:01:01 and, again, a DRB1∗15 exon 1. In the latter case, it was clear that no exon 2, 3, or 4 sequences were present for the DRB1∗15 gene, since exon 4 sequences differ between DRB1∗04:01:01 and DRB1∗15. The table shows the full NGS typing data. The HLA alleles shared by both individuals suggest a putative extended haplotype: A∗25:01:01-C∗12:03:01-B∗18:01:01-DRB1∗15new-DRB5∗01:01:01-DQA1∗01:02:01-DQB1∗06:02:01-DPA1∗01:03:01-DPB1∗23:01:01. NGS typing solved the missing allele mystery through identification of a truncated DRB1∗15 allele comprising an exon 1 sequence but without exons 2, 3, or 4. Similarly, there is DRB4∗03:01N which has an exon 3 sequence of a DRB4∗01:01, but without an exon 2 sequence. These unusual alleles appear to be the results of historic multi-exon deletion events and add to the astounding diversity of the human major histocompatibility complex. Download high-res image (242KB) Download full-size image A.G. Smith: 2. Consultant; Company/Organization; Scisco Genetics Inc. S.E. Pereira: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. C. Pyo: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. W. Nelson: 5. Employee; Company/Organization; Scisco Genetics Inc. M.Z. Askar: 3. Speaker’s Bureau; Company/Organization; Immucor. 4. Scientific/Medical Advisor; Company/Organization; Illumina. D.E. Geraghty: 6. Stock Shareholder; Company/Organization; Scisco Genetics Inc.

Sequence diversity of MHC class-II DRB gene in gazelles (Gazella subgutturosa) raised in Sanliurfa of Turkey

In this study, we aimed to assess the sequence diversity of major histocompatibility complex (MHC) class-II DRB gene at exon 2 in gazelles raised in Sanliurfa Province of Turkey. Twenty DNA samples isolated from gazelles (Gazella subgutturosa) were used for sequencing exon 2 of MHC class-II DRB gene. Target region was amplified by polymerase chain reaction (PCR) and their products were directly sequenced. Nine of these 20 samples yielded unambiguously readable sequences. Three of the nine samples were homozygotes and each showed different sequences. A 262-bp sequence obtained from the three homozygote samples were submitted to GenBank (accession numbers: KC309405, KC309406 and KC309407). Using an allele specific PCR, we detected 10 additional haplotypes. Among 13 haplotypes, 45 nucleotide positions were polymorphic and most of the polymorphic nucleotide positions localized at peptide-binding region (PBR). Rates of nonsynonymous substitutions were significantly higher than synonymous substitutions at PBR. Phylogenetic analysis of the haplotypes showed that 10 haplotypes of the gazelles were clustered together while three were clustered with ovine and bovine haplotypes. The results indicated that at least 13 haplotypes at exon 2 of MHC class-II DRB gene were showing high degree of nucleotide and amino acid diversity, and certain haplotypes of G. subgutturosa were more similar to haplotypes from sheep or cattle than to each other. Rates of synonymous and nonsynonymous substitutions suggested that positive selection was a driving force for diversity at this locus in G. subgutturosa.

Major histocompatibility complex (DRB3) geneexpression pattern indicates differences in BrucellaabortusS19 vaccine induced immune responsein Karan Fries and Sahiwal cattle

Brucella abortus S19 strain vaccination is most effectively used as a tool to control the brucellosis in cattle. To understand the genetic basis of differences in immune responsiveness after immunization in cattle of different genotypes, we assessedthe expression of MHC-DRB3 antigen receptor molecule in six each female calves of Karan Fries crossbreds (KF, Bos indicus x Bos taurus) and Sahiwal (Bos indicus) vaccinated with Brucella S19. Serum and peripheral blood mononuclear cells (PBMC) were isolated from blood collected on 0(before vaccination) and 7, 14 and 28 day of vaccination. Antigenic response was assessed for these days in both the groups using Rose Bengal Plate form Test (RBPT). At 0d, the calves of both groups showed no antigen agglutination, confirming the calves free from the infection.The serum of 7d onward started showing the agglutination with more strong response in later stages specifically in KF, indicating increased immune response against Brucella. Therefore, RBPT can be used as earliest screening (7d onward) for Brucella antigenic reactivity in both cattle groups. The expression of DRB3 gene started with slight upregulation after vaccination,in general, however without any significant differences between two different genetic groups upto14d.The significant (p Lass Than 0.01) higher expression (8 times) of DRB3was observed in KF than Sahiwal at 28d. The study indicated that antigenic reactivity and MHC-DRB3 expression elicited by Brucella S19 vaccination was more prominent in KFduring initial days, which may provide an extra advantage to the host for antigen binding, thereby better immune protection at later stage.

The erythrocyte alloimmunisation in patients with sickle cell anaemia: a systematic review.

Transfusion therapy is a common practice in the treatment of anaemia and can cause erythrocyte alloimmunisation. To systematise data related to erythrocyte alloimmunisation in patients with sickle cell disease (SCD), a bibliographic search was carried out in September 2017 to search for studies in four electronic databases. (i) Referring to the original work, (ii) being cohort or case-control, (iii) having been developed with individuals with SCD and (iv) having evaluated the erythrocyte alloimmunisation. Two reviewers identified the articles for inclusion in the study, extracted the predetermined data and carried out the evaluation of the methodological quality of the work. 21 studies were selected; the studies included data on 20 636 individuals (children and adults), were mostly published in the last 10years, were developed in the United States and had high methodological quality. The occurrence of erythrocyte alloimmunisation ranged from 4·4 to 76%, and there was a higher rate of alloimmunisation against antigens of the Rh system. The risk factors for alloimmunisation were age; gender (female); red blood cell (RBC) units received; presence of ≥1 autoantibodies, TNF-α, interleukin (IL1B), human leukocyte antigens (HLA)-DRB1 gene polymorphisms; first blood transfusion (BT) after 5years of age, transfusion episodic, multiple or during inflammatory events, acute chest syndrome (ACS) and vase-occlusive crisis (VOC); increased percentage of CD41 T memory cells; and positive direct antiglobulin test. Transfusion policies should be developed to protect the patient and his or her health based on the main factors associated with its incidence.

Genetic diversity of BoLA-DRB3 in South American Zebu cattle populations

BackgroundBovine leukocyte antigens (BoLAs) are used extensively as markers of disease and immunological traits in cattle. However, until now, characterization of BoLA gene polymorphisms in Zebu breeds using high resolution typing methods has been poor. Here, we used a polymerase chain reaction sequence-based typing (PCR-SBT) method to sequence exon 2 of the BoLA class II DRB3 gene from 421 cattle (116 Bolivian Nellore, 110 Bolivian Gir, and 195 Peruvian Nellore-Brahman). Data from 1416 Taurine and Zebu samples were also included in the analysis.ResultsWe identified 46 previously reported alleles and no novel variants. Of note, 1/3 of the alleles were detected only in Zebu cattle. Comparison of the degree of genetic variability at the population and sequence levels with genetic distance in the three above mentioned breeds and nine previously reported breeds revealed that Zebu breeds had a gene diversity score higher than 0.86, a nucleotide diversity score higher than 0.06, and a mean number of pairwise differences greater than 16, being similar to those estimated for other cattle breeds. A neutrality test revealed that only Nellore-Brahman cattle showed the even gene frequency distribution expected under a balanced selection scenario. The FST index and the exact G test showed significant differences across all cattle populations (FST = 0.057; p < 0.001). Neighbor-joining trees and principal component analysis identified two major clusters: one comprising mainly European Taurine breeds and a second comprising Zebu breeds. This is consistent with the historical and geographical origin of these breeds. Some of these differences may be explained by variation of amino acid motifs at antigen-binding sites.ConclusionsThe results presented herein show that the historical divergence between Taurine and Zebu cattle breeds is a result of origin, selection, and adaptation events, which would explain the observed differences in BoLA-DRB3 gene diversity between the two major bovine types. This allelic information will be important for investigating the relationship between the major histocompatibility complex and disease, and contribute to an ongoing effort to catalog bovine MHC allele frequencies according to breed and location.

Evaluating seroprevalence to circumsporozoite protein to estimate exposure to three species of Plasmodium in the Brazilian Amazon

BackgroundBrazil has seen a great decline in malaria and the country is moving towards elimination. However, for eventual elimination, the control program needs efficient tools in order to monitor malaria exposure and transmission. In this study, we aimed to evaluate whether seroprevalence to the circumsporozoite protein (CSP) is a good tool for monitoring the exposure to and/or evaluating the burden and distribution of Plasmodium species in the Brazilian Amazon.MethodsCross-sectional surveys were conducted in a rural area of Porto Velho, Rondônia state. Parasite infection was detected by microscopy and polymerase chain reaction. Antibodies to the sporozoite CSP repeats of Plasmodium vivax, P. falciparum, and P. malariae (PvCS, PfCS, and PmCS) were detected using the enzyme-linked immunosorbent assay technique. Human leukocyte antigen (HLA)-DRB1 and DQB1 genes were typed using Luminex® xMAP® technology.ResultsThe prevalence of immunoglobulin G against P. vivax CSP peptide (62%) was higher than P. falciparum (49%) and P. malariae (46%) CSP peptide. Most of the studied individuals had antibodies to at least one of the three peptides (72%), 34% had antibodies to all three peptides and 28% were non-responders. Although the majority of the population was not infected at the time of the survey, 74.3% of parasite-negative individuals had antibodies to at least one of the CSPs. Importantly, among individuals carrying the haplotypes DRB1*04~DQB1*03, there was a significantly higher frequency of PfCS responders, and DRB1*16~DQB1*03 haplotype for PvCS and PfCS responders. In contrast, HLA-DRB1*01 and HLA-DQB1*05 allelic groups were associated with a lack of antibodies to P. vivax and P. falciparum CSP repeats, and the haplotype DRB1*01~DQB1*05 was also associated with non-responders, including non-responders to P. malariae.ConclusionsOur results show that in low transmission settings, naturally acquired antibody responses against the CSP repeats of P. vivax, P. falciparum, and P. malariae in a single cross-sectional study may not represent a valuable marker for monitoring recent malaria exposure, especially in an area with a high prevalence of P. vivax. Furthermore, HLA class II molecules play an important role in antibody response and require further study with a larger sample size. It will be of interest to consider HLA analysis when using serosurveillance to monitor malaria exposure among genetically diverse populations.

Genetic diversity of MHC class II DRB alleles in the continental and Japanese populations of the sable Martes zibellina (Mustelidae, Carnivora, Mammalia)

The sable (Martes zibellina) is a medium-sized mustelid inhabiting forest environments in Siberia, northern China, the Korean Peninsula, and Hokkaido Island, Japan. To further understand the molecular evolution of the major histocompatibility complex (MHC), we sequenced part of exon 2 in MHC class II DRB genes, including codons encoding the antigen binding site, from 33 individuals from continental Eurasia and Japan. We identified 16 MHC class II DRB alleles (Mazi-DRBs), some of which were geographically restricted and others broadly distributed, and eight putative pseudogenes. A single-breakpoint recombination analysis detected a recombination site in the middle of exon 2. A mixed effects model of evolution analysis identified five amino acid sites presumably under positive selection. These sites were all located in the region 3′ to the recombination site, suggesting that positive selection and recombination could be committed to the diversity of the M. zibellina DRB gene. In a Bayesian phylogenetic tree, all Mazi-DRBs and the presumed pseudogenes grouped within a Mustelidae clade. The Mazi-DRBs showed trans-species polymorphism, with some alleles most closely related to alleles from other mustelid species. This result suggests that the sable DRBs have evolved under long-lasting balancing selection.

Genetic Diversity of Bovine Major Histocompatibility Complex Class II DRB3 locus in cattle breeds from Asia compared to those from Africa and America.

Genetic polymorphisms and diversity of BoLA-DRB3.2 are essential because of DRB3 gene's function in innate immunity and its association with infectious diseases resistance or tolerance in cattle. The present study was aimed at assessing the level of genetic diversity of DRB3 in the exon 2 (BoLA-DRB3.2) region in African, American and Asian cattle breeds. Amplification of exon 2 in 174 cattle revealed 15 haplotypes. The breeds with the highest number of haplotypes were Brangus (10), Sokoto Gudali (10) and Dajal (9), while the lowest number of haplotypes were found in Holstein and Sahiwal with 4 haplotypes each. Medium Joining network obtained from haplotypic data showed that all haplotypes condensed around a centric area and each sequence (except in H-3, H-51 and H-106) representing almost a specific haplotype. The BoLA-DRB3.2 sequence analyses revealed a non-significant higher rate of non-synonymous (dN) compared to synonymous substitutions (dS). The ratio of dN/dS substitution across the breeds were observed to be greater than one suggesting that variation at the antigen-binding sites is under positive selection; thus increasing the chances of these breeds to respond to wide array of pathogenic attacks. An analysis of molecular variance revealed that 94.01 and 5.99% of the genetic variation was attributable to differences within and among populations, respectively. Generally, results obtained suggest that within breed genetic variation across breeds is higher than between breeds. This genetic information will be important for investigating the relationship between BoLADRB3.2 and diseases in various cattle breeds studied with attendant implication on designing breeding programs that will aim at selecting individual cattle that carry resistant alleles.

Characterization of classical major histocompatibility complex (MHC) class II genes in northern pig-tailed macaques (Macaca leonina)

The northern pig-tailed macaque (Macaca leonina) has been identified as an independent species from the pig-tailed macaque group. The species is a promising animal model for HIV/AIDS pathogenesis and vaccine studies due to susceptibility to HIV-1. However, the major histocompatibility complex (MHC) genetics in northern pig-tailed macaques remains poorly understood. We have previously studied the MHC class I genes in northern pig-tailed macaques and identified 39 novel alleles. Here, we describe the MHC class II alleles in all six classical loci (DPA, DPB, DQA, DQB, DRA, and DRB) from northern pig-tailed macaques using a sequence-based typing method for the first time. A total of 60 MHC-II alleles were identified of which 27 were shared by other macaque species. Additionally, northern pig-tailed macaques expressed a single DRA and multiple DRB genes similar to the expression in humans and other macaque species. Polymorphism and positive selection were detected, and phylogenetic analysis suggested the presence of a common ancestor in human and northern pig-tailed macaque MHC class II allelic lineages at the DQA, DQB, and DRB loci. The characterization of full-length MHC class II alleles in this study significantly improves understanding of the immunogenetics of northern pig-tailed macaques and provides the groundwork for future animal model studies.

HLA DRB1 polymorphism and risk of HIV/tuberculosis co-infection.

In the research data on the study of interconnection between polymorphism of HLA DRB1 genes and the risk of HIV infection and co-infection and HIV/tuberculosis are presented.The cohort of observation was HIV-infected individuals with different clinical stages aged from 24 to 58 years, average age being 38 years. 90 patients (89.2%) received HAART.There were 32 (31.4%) HIV-infected patients with manifestations of tuberculosis, 70 (68.6%) HIV patients without manifestations of tu­ber­culosis. Among the cohort of HIV-infected patients with manifestations of tuberculosis, pulmonary forms of tuberculosis was in 32 (100%), of which extrapulmonary forms of tuberculosis were in 12 (37.5%) patients. The majority of cases of tuberculosis occurred in HIV-infected patients in the IV clinical stage. Among HIV-infected patients, the following alleles of the HLA gene DRB1 *01 (10%) *04 (10%) *07 (13%), *11 (15%), *13 (13%), *15 (10%) occurred more often, HLA DRB1 *03 *08 *09 *10, *12, *14, *16, * 17, *18 were rarely found (1-7%). Similar distributions of DRB1 genes were noted among residents of the region with the dominance of HLA DRB1 *01 *04 *07 *11 *13 and *15. With the presence of alleles of the locus DRB1 *13, DRB1 *03 and DRB1 *08, the greatest probability of co-infection of tuberculosis was observed in HIV-infected patients. The risk of tuberculosis is by 2 times higher with a significant difference in HIV-infected patients with DRB1 *13 locus alleles, DRB1 *03 – by 4.4 times and DRB1 *08 – by 5.4 times. Significant chances of tuberculosis occurrence are higher – by 2.6 times in HIV-infected patients with DRB1 *13 locus alleles, DRB1 *03 – by 5.1 times and DRB1 *08 – by 6.4 times. Thus, the variability of the HLA DRB1 locus alleles may play a predisposing or protective role in the occurrence of tuberculosis in HIV-infected patients.

P052 A rare DRB1 allele with exon 2–5 deletions

A 4-month old SCID patient was worked up in HLA lab for hematopoietic stem cell transplant. DNA was extracted from blood samples for the patient and both parents. HLA typing for A, B, C, DRB1 and DQB1 was initially performed using a combination of high resolution rSSO and high resolution SSP. Family segregation analysis suggests that the child has inherited mother’s “c” haplotype with the exception of DRB1. The child was typed as DRB1∗01:02 homozygous and the mother was typed as DRB1∗01:01 homozygous. In addition, mother sample appears to have an uncommon association of DRB1∗01:01-DQB1∗06:02. Testing of DRB345 locus reveals that the mother also has an unusual DRB5∗01 associated with DRB1∗01:01 homozygosity. To rule out the possibility of potential rSSO failure of 2nd DRB1 allele detection (possibly due to initial PCR failure caused by primer binding complementary region mutations), we tried high resolution DRB1∗15 and DRB1∗16 SSP. Both gels reveal no positive reaction bands for all lanes other than internal controls. A send out NGS based DRB1 test targeting Exons 2 and 3 also agrees with our own typing and so is our own NGS based HLA typing targeting Exon 2, 3 and partial Exon 4 of DRB1 gene. Buccal swab samples from both the patient and the mother confirm HLA findings derived from DNA of blood origin. Initial conclusion was drawn that the patient and the mother likely carry a DRB1∗”Blank” which cannot be detected by current testing platforms. Finally, with the help from R&D department of GenDx, full-length DRB1 gene was amplified. A small size amplicon was noticed from both the patient and the mother DNA samples. NGS sequencing of the amplicons reveals the presence of a truncated DRB1 allele missing Exons 2–5 and the left over regions suggested likelihood of DRB1∗15 origin. Download : Download high-res image (145KB) Download : Download full-size image

P261 On call drama: DR-DQ associations strike back!

A 47-year-old Vietnamese man with prior failed renal allograft is selected for evaluation of a kidney from a deceased African American donor. Flow crossmatch with current and archived patient serum are negative. The antibody screen (SA Luminex assay) reveals Class I and II sensitizations but no DSA. Upon further analysis, however, there is concern for lab error based on well-established DR-DQ associations. The donor’s HLA typing includes DR7, DR8, DQ7, DQ8 – an unexpected pattern. DR7 is typically inherited with either DQ2 or DQ9; DR8 with DQ4, DQ6, or DQ7. Inheritance of both DR7 and DQ8 calls into question the validity of the results. This is particularly concerning given the patient’s very high level of sensitization to DQ2 (MFI 18780). Donor typing is repeated by SSP, confirming the initial rSSO results. Repeat cross match is negative. Transplantation proceeds. This case highlights unexpected HLA associations that can arise due to rare crossover events during meiosis. The probability of inheriting DQ8 along with DR7 is low, based on the close proximity of genes DRB1 and DQB1 within chromosome 6. According to allelefrequencies.net, the frequency of a haplotype containing DR7-DQ8 within an African American population is about 0.22%. We estimate the probability of meiotic crossover between DRB1 and DQB1 as 0.03%. While we do not have parental HLA typing for the deceased donor, he likely inherited DQ8 from a parent carrying DR4. Regardless of the origin of the donor’s DQ8, this case also illustrates the importance of questioning uncommon results, and performing confirmation with alternate testing modalities when such results present themselves.

Human Leukocyte Antigen (HLA)-DRB1*15:01 and HLA-DRB5*01:01 Present Complementary Peptide Repertoires.

Human leukocyte antigen (HLA)-DR15 is a haplotype associated with multiple sclerosis. It contains the two DRB* genes DRB1*1501 (DR2b) and DRB5*0101 (DR2a). The reported anchor motif of the corresponding HLA-DR molecules was determined in 1994 based on a small number of peptide ligands and binding assays. DR2a could display a set of peptides complementary to that presented by DR2b or, alternatively, a similar peptide repertoire but recognized in a different manner by T cells. It is known that DR2a and DR2b share some peptide ligands, although the degree of similarity of their associated peptidomes remains unclear. In addition, the contribution of each molecule to the global peptide repertoire presented by the HLA-DR15 haplotype has not been evaluated. We used mass spectrometry to analyze the peptide pools bound to DR2a and DR2b, identifying 169 and 555 unique peptide ligands of DR2a and DR2b, respectively. The analysis of these sets of peptides allowed the refinement of the corresponding binding motifs revealing novel anchor residues that had been overlooked in previous analyses. Moreover, the number of shared ligands between both molecules was low, indicating that DR2a and DR2b present complementary peptide repertoires to T cells. Finally, our analysis suggests that, quantitatively, both molecules contribute to the peptide repertoire presented by cells expressing the HLA-DR15 haplotype.

Associations between gastric dilatation-volvulus in Great Danes and specific alleles of the canine immune-system genes DLA88, DRB1, and TLR5.

OBJECTIVE To determine whether specific alleles of candidate genes of the major histocompatibility complex (MHC) and innate immune system were associated with gastric dilatation-volvulus (GDV) in Great Danes. ANIMALS 42 healthy Great Danes (control group) and 39 Great Danes with ≥ 1 GDV episode. PROCEDURES Variable regions of the 2 most polymorphic MHC genes (DLA88 and DRB1) were amplified and sequenced from the dogs in each group. Similarly, regions of 3 genes associated with the innate immune system (TLR5, NOD2, and ATG16L1), which have been linked to inflammatory bowel disease, were amplified and sequenced. Alleles were evaluated for associations with GDV, controlling for age and dog family. RESULTS Specific alleles of genes DLA88, DRB1, and TLR5 were significantly associated with GDV. One allele of each gene had an OR > 2 in the unadjusted univariate analyses and retained a hazard ratio > 2 after controlling for temperament, age, and familial association in the multivariate analysis. CONCLUSIONS AND CLINICAL RELEVANCE The 3 GDV-associated alleles identified in this study may serve as diagnostic markers for identification of Great Danes at risk for GDV. Additional research is needed to determine whether other dog breeds have the same genetic associations. These findings also provided a new target for research into the etiology of, and potential treatments for, GDV in dogs.

A cell-based high-throughput screening assay system for inhibitor compounds of antigen presentation by HLA class II molecule

A number of autoimmune diseases are associated with the genotypes of human leukocyte antigen class II (HLA), some of which present peptides derived from self-proteins, resulting in clonal expansion of self-reactive T cells. Therefore, selective inhibition of self-peptide loading onto such disease-associated HLA could ameliorate the diseases. To effectively identify such compounds, in this study, we established, for the first time, a cell- and 96-well microplate-based high-throughput screening system for inhibitors of antigen presentation. A panel of DRB1 genes plus DRA*01:01 gene were expressed in HEK293T cells and in 3T3 cells, and their binding with biotinylated known self-antigen peptides was measured by flow cytometry. HLA-DR1 (DRB1*01:01) and DR15 (DRB1*15:01) showed a high affinity with myelin basic protein peptide (MBP83-98). Therefore, in 96-well plate wells, MBP83-99 was allowed to bind to DR1 or DR15 on 3T3 cells in competition with a test compound, and the HLA-bound peptide was detected by streptavidin-conjugated β-galactosidase, thereby identifying inhibitor compounds for rheumatoid arthritis or multiple sclerosis. Our assay system has a potential for broad applications, including designing peptide vaccines.

Non-additive and epistatic effects of HLA polymorphisms contributing to risk of adult glioma.

Although genome-wide association studies have identified several susceptibility loci for adult glioma, little is known regarding the potential contribution of genetic variation in the human leukocyte antigen (HLA) region to glioma risk. HLA associations have been reported for various malignancies, with many studies investigating selected candidate HLA polymorphisms. However, no systematic analysis has been conducted in glioma patients, and no investigation into potential non-additive effects has been described. We conducted comprehensive genetic analyses of HLA variants among 1746 adult glioma patients and 2312 controls of European-ancestry from the GliomaScan Consortium. Genotype data were generated with the Illumina 660-Quad array, and we imputed HLA alleles using a reference panel of 5225 individuals in the Type 1 Diabetes Genetics Consortium who underwent high-resolution HLA typing via next-generation sequencing. Case-control comparisons were adjusted for population stratification using ancestry-informative principal components. Because alleles in different loci across the HLA region are linked, we created multigene haplotypes consisting of the genes DRB1, DQA1, and DQB1. Although none of the haplotypes were associated with glioma in additive models, inclusion of a dominance term significantly improved the model for multigene haplotype HLA-DRB1*1501-DQA1*0102-DQB1*0602 (P = 0.002). Heterozygous carriers of the haplotype had an increased risk of glioma [odds ratio (OR) 1.23; 95% confidence interval (CI) 1.01-1.49], while homozygous carriers were at decreased risk compared with non-carriers (OR 0.64; 95% CI 0.40-1.01). Our results suggest that the DRB1*1501-DQA1*0102-DQB1*0602 haplotype may contribute to the risk of glioma in a non-additive manner, with the positive dominance effect partly explained by an epistatic interaction with HLA-DRB1*0401-DQA1*0301-DQB1*0301.

Low genetic variation in the MHC class II DRB gene and MHC-linked microsatellites in endangered island populations of the leopard cat (Prionailurus bengalensis) in Japan.

Isolated populations of the leopard cat (Prionailurus bengalensis) on Tsushima and Iriomote islands in Japan are classified as subspecies P. b. euptilurus and P. b. iriomotensis, respectively. Because both populations have decreased to roughly 100, an understanding of their genetic diversity is essential for conservation. We genotyped MHC class II DRB exon 2 and MHC-linked microsatellite loci to evaluate the diversity of MHC genes in the Tsushima and Iriomote cat populations. We detected ten and four DRB alleles in these populations, respectively. A phylogenetic analysis showed DRB alleles from both populations to be closely related to those in other felid DRB lineages, indicating trans-species polymorphism. The MHC-linked microsatellites were more polymorphic in the Tsushima than in the Iriomote population. The MHC diversity of both leopard cat populations is much lower than in the domestic cat populations on these islands, probably due to inbreeding associated with founder effects, geographical isolation, or genetic drift. Our results predict low resistance of the two endangered populations to new pathogens introduced to the islands.