Major Histocompatibility Complex Typing Research Articles

Functional immune diversity in reindeer reveals a high Arctic population at risk

Climate changes the geographic range of both species as well as pathogens, causing a potential increase in the vulnerability of populations or species with limited genetic diversity. With advances in high throughput sequencing (HTS) technologies, we can now define functional expressed genetic diversity of wild species at a larger scale and identify populations at risk. Previous studies have used genomic DNA to define major histocompatibility complex (MHC) class II diversity in reindeer. Varying numbers of expressed genes found in many ungulates strongly argues for using cDNA in MHC typing strategies to ensure that diversity estimates relate to functional genes. We have used available reindeer genomes to identify candidate genes and established an HTS approach to define expressed MHC class I and class II diversity. To capture a broad diversity we included samples from wild reindeer from Southern Norway, semi-domesticated reindeer from Northern Norway and reindeer from the high Artic archipelago Svalbard. Our data show a medium MHC diversity in semi-domesticated and wild Norwegian mainland reindeer, and low MHC diversity reindeer in Svalbard reindeer. The low immune diversity in Svalbard reindeer provides a potential risk if the pathogenic pressure changes in response to altered environmental conditions due to climate change, or increased human-related activity.

Improved high-throughput MHC typing for non-model species using long-read sequencing.

The major histocompatibility complex (MHC) plays a critical role in the vertebrate immune system. Accurate MHC typing is critical to understanding not only host fitness and disease susceptibility, but also the mechanisms underlying host‐pathogen co‐evolution. However, due to the high degree of gene duplication and diversification of MHC genes, it is often technically challenging to accurately characterise MHC genetic diversity in non‐model species. Here we conducted a systematic review to identify common issues associated with current widely used MHC typing approaches. Then to overcome these challenges, we developed a long‐read based MHC typing method along with a new analysis pipeline. Our approach enables the sequencing of fully phased MHC alleles spanning all key functional domains and the separation of highly similar alleles as well as the removal of technical artefacts such as PCR heteroduplexes and chimeras. Using this approach, we performed population‐scale MHC typing in the Tasmanian devil (Sarcophilus harrisii), revealing previously undiscovered MHC functional diversity in this endangered species. Our new method provides a better solution for addressing research questions that require high MHC typing accuracy. Since the method is not limited by species or the number of genes analysed, it will be applicable for studying not only the MHC but also other complex gene families.

Histocompatibility Complex Status and Mendelian Randomization Analysis in Unsolved Antibody Deficiency.

The pathogenesis in the majority of patients with common variable immunodeficiency (CVID), the most common symptomatic primary immunodeficiency, remains unknown. We aimed to compare the minor and major histocompatibility complex (MHC) markers as well as polygenic scores of common genetic variants between patients with monogenic CVID and without known genetic mutation detected. Monogenic patients were identified in a CVID cohort using whole exome sequencing. Computational full-resolution MHC typing and confirmatory PCR amplicon-based high-resolution typing were performed. Exome-wide polygenic scores were developed using significantly different variants and multi-variant Mendelian randomization (MR) analyses were used to test the causality of significant genetic variants on antibody levels and susceptibility to infectious diseases. Among 83 CVID patients (44.5% females), monogenic defects were found in 40 individuals. Evaluation of the remaining CVID patients without known genetic mutation detected showed 13 and 27 significantly associated MHC-class I and II alleles, respectively. The most significant partial haplotype linked with the unsolved CVID was W*01:01:01-DMA*01:01:01-DMB*01:03:01:02-TAP1*01:01:01 (P < 0.001), where carriers had a late onset of the disease, only infection clinical phenotype, a non-familial form of CVID, post-germinal center defects and a non-progressive form of their disease. Exclusion of monogenic diseases allowed MR analyses to identify significant genetic variants associated with bacterial infections and improved discrepancies observed in MR analyses of previous GWAS studies with low pleiotropy mainly for a lower respiratory infection, bacterial infection and Streptococcal infection. This is the first study on the full-resolution of minor and major MHC typing and polygenic scores on CVID patients and showed that exclusion of monogenic forms of the disease unraveled an independent role of MHC genes and common genetic variants in the pathogenesis of CVID.

Diversidad de haplotipos del complejo principal de histocompatibilidad en equinos de la raza Árabe de la República Argentina

La diversidad de los genes del complejo principal de histocompatibilidad (MHC) es clave en su función en la presentación de antígenos en el sistema inmune. La tipificación indirecta basada en microsatélites (STR) del MHC aporta información de la variabilidad genética y de la estructura poblacional y es una estrategia para conocer procesos selectivos y evolutivos. Con el fin de caracterizar una población de caballos de raza Árabe, se identificaron los haplotipos del MHC sobre la base de tres microsatélites (UM011, DRB2-STR2 y COR112) abarcando una región de ~1Mpb. El ADN genómico se extrajo de sangre y pelo de 30 caballos de cinco haras de la provincia de Buenos Aires y los STRs se amplificaron con cebadores fluorescentes para tipificar en secuenciador automático. Seestimaron parámetros poblacionales y de diversidad y la detección de haplotipos se realizó mediante análisis de segregación y con el programa de reconstrucción de haplotipos PHASE. Se reconocieron 24 haplotipos; entre ellos, 12 se verificaron por segregación en los registros de pedigree del StudBook. Los resultados obtenidos demostraron la existencia de ligamiento con alelos conocidos de genes de clase II del equine leukocyte antigen (ELA) y la cantidad de haplotipos identificada permitió expandir la valoración de la diversidad del MHC equino. Esta metodología constituye una herramienta de utilidad y un método alternativo para la tipificación del MHC en linajes de caballos y en estudios poblacionales relacionados con la inmunidad.

A quick and robust MHC typing method for free-ranging and captive primate species

Gene products of the major histocompatibility complex (MHC) of human and non-human primates play a crucial role in adaptive immunity, and most of the relevant genes not only show a high degree of variability (polymorphism) but also copy number variation (CNV) is observed. Due to this diversity, MHC proteins influence the capability of individuals to cope with various pathogens. MHC and/or MHC-linked gene products such as odorant receptor genes are thought to influence mate choice and reproductive success. Therefore, MHC typing of wild and captive primate populations is considered to be useful in conservation biology, which is, however, often hampered by the need of invasive and time-consuming methods. All intact Mhc-DRB genes in primates appear to possess a complex and highly divergent microsatellite, DRB-STR. A panel of 154 pedigreed olive baboons (Papio anubis) was examined for their DRB content by DRB-STR analysis of genomic DNA. Using the same methodology on DNA of feces samples, DRB variability of a silvery gibbon population (Hylobates moloch) (N = 24), an endangered species, could successfully be studied. In both species, length determination of the DRB-STR resulted in the definition of unique genotyping patterns that appeared to be specific for a certain chromosome. Moreover, the different STR lengths were shown to segregate with the allelic variation of the respective gene. The results obtained expand data gained previously on DRB-STR typing in macaques, great apes, and humans and strengthen the conclusion that this protocol is applicable in molecular ecology, conservation biology, and colony management, especially of endangered primate species.

Isolation and characterisation of 11 MHC-linked microsatellite loci in the Tasmanian devil (Sarcophilus harrisii)

The Tasmanian devil is under threat of extinction due to a deadly contagious cancer, devil facial tumour disease (DFTD). DFTD tumour cells can overcome histocompatibility barriers and be transferred between unrelated individuals as allografts. The low level of genetic variability at major histocompatibility complex (MHC) loci is believed to have contributed to the high transmissibility of DFTD across the devil population. Copy number variations and high sequence similarity between MHC genes in the devil has made MHC typing by sequencing laborious and problematic. A more efficient approach is required in order to facilitate large-scale MHC typing. Here we report the development of a suite of polymorphic microsatellite markers that are located close to devil MHC Class I or II genes. Two to six alleles were identified at each locus, with the expected heterozygosity ranging from 0.142 to 0.696.

Definition of Mafa-A and -B haplotypes in pedigreed cynomolgus macaques (Macaca fascicularis)

The major histocompatibility complex (MHC) class I B gene/allelic repertoire was investigated in a pedigreed population of cynomolgus macaques of mixed Indonesian/Malaysian origin. The Mafa-B alleles detected in this cohort are mostly specific for a given geographic area, and only a small number of alleles appears to be shared with other populations. This suggests the fast evolution of Mafa-B alleles due to adaptation to new environments. In contrast to humans, the B locus in Old World monkeys displays extensive copy number variation. The Mafa-B and previously defined -A gene combinations segregate in families and thus allowed the definition of extended haplotypes. In many cases it was possible to assign a particular Mafa-I allele to one of these Mafa-A/B haplotypes as well. The presence of a large number of stable haplotypes in this cohort of animals, which was pedigreed for up to eight generations, looks promising for developing discriminative MHC typing tools that are less cumbersome. Furthermore, the discovery of 53 unreported Mafa-B sequences expands the lexicon of alleles significantly, and may help in understanding the complex organisation of the macaque B region.

Major histocompatibility complex genotyping with massively parallel pyrosequencing

Major histocompatibility complex (MHC) genetics dictate adaptive cellular immune responses, making robust MHC genotyping methods essential for studies of infectious disease, vaccine development and transplantation. Nonhuman primates provide essential preclinical models for these areas of biomedical research. Unfortunately, given the unparalleled complexity of macaque MHCs, existing methodologies are inadequate for MHC typing of these key model animals. Here we use pyrosequencing of complementary DNA-PCR amplicons as a general approach to determine comprehensive MHC class I genotypes in nonhuman primates. More than 500 unique MHC class I sequences were resolved by sequence-based typing of rhesus, cynomolgus and pig-tailed macaques, nearly half of which have not been reported previously. The remarkable sensitivity of this approach in macaques demonstrates that pyrosequencing is viable for ultra-high-throughput MHC genotyping of primates, including humans.

Genetic variation in BoLA microsatellite loci in Portuguese cattle breeds

Major histocompatibility complex (MHC) typing based on microsatellites can be a valuable approach to understanding the selective processes occurring at linked or physically close MHC genes and can provide important information on variability and relationships of populations. Using microsatellites within or in close proximity with bovine lymphocyte antigen (BoLA) genes, we investigated the polymorphisms in the bovine MHC, known as the BoLA, in eight Portuguese cattle breeds. Additional data from non-BoLA microsatellite loci were also used to compare the variability between these regions. Diversity was higher in BoLA than in non-BoLA microsatellites, as could be observed by the number of alleles, allelic richness and observed heterozygosity. Brava de Lide, a breed selected for aggressiveness and nobility, presented the lowest values of observed heterozygosity and allelic richness in both markers. Results from neutrality tests showed few statistically significant differences between the observed Hardy-Weinberg homozygosity (F) and the expected homozygosity (F(E)), indicating the apparent neutrality of the BoLA microsatellites within the analysed breeds. Nevertheless, we detected a trend of lower values of observed homozygosity compared with the expected one. We also detected some differences in the levels of allelic variability among the four BoLA microsatellites. Our data showed a higher number of alleles at the BoLA-DRB3 locus than at the BoLA-DRBP1 locus. These differences could be related to their physical position in the chromosome and may reflect functional requirements for diversity.

Major histocompatibility complex typing of dogs from Russia shows further dog leukocyte antigen diversity

Blood samples from 85 Russian dogs and wolves were collected as dried blood spots on paper and transported to the UK by mail. We obtained partial or complete three-locus canine major histocompatibility complex [dog leukocyte antigen (DLA)] class II haplotypes on 81 of these samples. Six new alleles were identified: three DLA-DRB1 and three DLA-DQB1. These alleles occurred in haplotypic combinations not previously seen in other European dogs. One haplotype appeared to lack a DQB1 allele. Two of the new haplotypes segregated through a family of dogs that was investigated.

Why are systemic glioblastoma metastases rare? Systemic and cerebral growth of mouse glioblastoma

Background Systemic metastasis of glioblastoma multiforme (GBM) in the form of bulk tumor is rare. This could be because of patient death before clinically detectable systemic metastasis, impediments to systemic egress, or the inability of GBM to grow outside the central nervous system (CNS). In the present paper, we tested this last hypothesis. Methods The delayed brain tumor (DBT) cell was characterized with respect to in vitro and in vivo morphology, growth rate, anchorage-independent growth, glial fibrillary acidic protein expression and cytogenetic analysis, and major histocompatibility complex (MHC) typing. We then assayed implantation-induced intracerebral and systemic GBM growth using 3 rodent models with increasing relative immunologic differences between implanted DBT cells and hosts (Balb/c mice, an isograft, MHC I H2, class type D; C3H mice, an allograft; Wistar rats, a xenograft). Results After implantation in the brain, DBT cells generated tumors that were similar to human GBM. Intracerebral DBT implantation as an isograft or allograft produced only intracranial tumors, whereas intracerebral and systemic implantation as a xenograft produced no tumors. Systemic isograft implantation yielded only systemic tumors. Systemic implantation as allografts produced only transient subcutaneous masses. Conclusions Delayed brain tumor cells implanted outside the CNS formed tumors unless there was a significant difference between the immunotype of the implanted cells and host. These results support the hypothesis that the rarity of systemic GBM tumors lies in the presence of physical barriers and/or systemic hurdles that prevent their timely growth. These results also demonstrate that GBMs are antigenic, although not immunogenic, with their syngeneic host. Therefore, GBM may be amenable to targeted immunotherapy given successful artificial priming of the immune system.

Denaturing gradient gel electrophoresis and its use in the detection of major histocompatibility complex polymorphism

The major histocompatibility complex (MHC) has been studied extensively in humans and in mice and many methods are available for MHC typing of these well-characterized species. Studies of MHC variation in other species are ever increasing and researchers can choose one of a number of approaches for MHC typing of their species of interest. DNA sequencing is regarded as the 'gold standard' and it is frequently used for MHC typing. However, DNA sequencing is impractical when many individuals must be typed. Denaturing gradient gel electrophoresis (DGGE) offers a flexible and sensitive method for identifying and characterizing MHC alleles in any vertebrate species. This article reviews the theory and the practice of DGGE and examines the use of DGGE for MHC identification in various species. DGGE is compared to other similar techniques for MHC typing, such as single-stranded conformational polymorphism and reference strand-mediated conformational analysis. The advantages, problems, pitfalls and limitations of DGGE are considered and future perspectives on the use of DGGE for MHC typing are discussed.

DNA Typing Revealing High HLA-Cw Polymorphism Completes Availability of Major Histocompatibility Complex Loci in Forensic Medicine

Studies of human population genetics in Hungary have revealed relevant heterogeneity in the major histocompatibility complex. In the present studies, two isolated ethnic groups were chosen: people living in the Káli Basin westward from the Danube River, and those living in Opusztaszer, a village eastward from Danube, who are known as native ancient Hungarians. Blood samples were collected from 70 people in the Káli Basin and from 45 people in Opusztaszer. The frequency of HLA-Cw alleles was determined by serology as well as by DNA typing in 46 and 32 samples of the two populations, respectively, and in 44 randomly selected subjects of Hungarian origin. Compared with a random population of cadaver donors (the deaths having resulted mostly from accidents or, in a smaller number, strokes or heart infarcts) and voluntary bone marrow donors (typed in the last 10 years) recruited from all parts of Hungary and representing the mixed Hungarian population, remarkable differences were found in haplotype and allele frequencies. HLA-A, -B, -Cw typing was performed by serology and, in the case of the HLA-Cw locus, by polymerase chain reaction (PCR)-SSP and/or PCR-SSOP techniques, as well. The PCR-SSO oligotyping procedure allowed the identification of 32 Cw alleles in contrast with the 9 serologically detectable types. Because of the combination of low antigen expression and the lack of specific serologic reagents of good quality, no HLA-Cw antigens were detectable in 41%, and only one was detected in 48%, of the investigated individuals by standard serologic typing. With PCR-SSO typing, however, 97% of the investigated individuals proved to be heterozygous for HLA-Cw alleles. The two isolated populations differed from each other, from mixed Hungarian and other Caucasian populations in HLA-Cw* allele frequencies, as well as in haplotype distribution. This newly recognized polymorphism at the HLA-Cw locus completes the availability of major histocompatibility complex typing in forensic science and practice.

Major histocompatibility complex and tumor necrosis factor-alpha polymorphisms in pigeon breeder's disease.

Pigeon breeders disease (PBD) is caused by the exposure of a susceptible host to avian antigens. However, genetic factors determining individual predisposition are unknown. In this work, polymorphisms of the major histocompatibility complex (MHC) class II alleles and tumor necrosis factor alpha (TNF-alpha) promoter were evaluated in 44 patients with PBD, 99 healthy unrelated controls (HC), and 50 exposed but asymptomatic subjects (EAS). MHC typing was performed by PCR-specific sequence oligonucleotide analysis, and TNF-alpha polymorphism at -238 and -308 positions by amplification refractory mutation system-PCR. PBD patients showed a significant increase of the alleles HLA-DRB1*1305 (p < 0.001, OR = 15.4, 95% CI = 3.18-102.6 [HC], and OR = 17.05, 95% CI = 2.25-357.8 [EAS]) and HLA-DQB1*0501 (p < 0.05, OR = 2.93, 95% CI = 1.21-7.15 [HC], and OR = 2.96, 95% CI = 1.0-9.14 [EAS]). A decrease of HLA-DRB1*0802 was also noticed in patients when compared with both control groups (p < 0.05). Haplotype analysis revealed an increase of DRB1*1305-DQB1*0301 and a decrease of DRB1*0802-DQB1*0402. PBD patients had an increased frequency of TNF-2(-)(308) compared with both control groups (p < 0.05). Patients exhibiting the TNF-2(-)(308) allele were younger (33.9 +/- 14.6 versus 44.2 +/- 10.4 yr; p < 0.05), and displayed more lymphocytes in their bronchoalveolar lavages (88.0 +/- 12.1 versus 68.9 +/- 17.2; p < 0.05). These results suggest that genetic factors located within the MHC region contribute to the development of PBD.

Class II major histocompatibility complex typing across the ethnic barrier in pemphigoid gestationis. A study in Mexicans

Pemphigoid gestationis (PG), also called herpes gestationis, is a rare autoimmune disease of pregnancy or puerperium (estimated 1 out of 50,000 pregnancies among Caucasians). A previous series has demonstrated an association of PG with human leukocyte antigen (HLA)-DR3 or HLA-DR4 haplotypes. While these haplotypes are most commonly found in individuals of European ancestry, they have also been found in African-American patients affected with PG. PG has rarely been reported in other ethnic groups, and the HLA association in non-Europeans has not been examined. We have characterized eight patients of Mexican ancestry who have PG by clinical, histologic, and immunofluorescence criteria. Class I and class II major histocompatibility complex (MHC) antigens were studied by standard microlymphocytotoxicity assays. Class II MHC antigens were further studied by polymerase chain reaction (PCR) amplification of HLA-DRB1, DQA, and DQB genes and allele-specific oligonucleotide hybridization. For comparison purposes, we used results obtained from a group of 100 ethnically matched healthy individuals. We found that all eight patients had the HLA-DR3/DR4 phenotype; all HLA-DR3 haplotypes were HLA-DRB1*0301, DQA1*0501, and DQB1*0201, whereas half of the HLA-DR4 haplotypes were from the DRB1*0401 subtype and the other half were DRB1 *0407. These results suggest that, in Mexicans, the genetic susceptibility for the development of PG is strongly influenced by the genetic admixture of Caucasian origin, and the role of class II MHC antigens in the pathophysiology of this disease is confirmed.

HLA-DR, -DQA1 and -DQB1 associations in Australian multiple sclerosis patients.

Molecular genotyping for the major histocompatibility complex (MHC) class II loci, HLA-DRB1, -DQB1 and -DQA1, in 100 patients with relapsing/remitting multiple sclerosis (MS) demonstrated an association with the HLA-DR2, DQw6-associated alleles DRB1*1501, DQB1*0602 and DQA1*0102, thereby extending this finding among MS patients in several countries to an Australian population. Analysis by the relative predispositional effect (RPE) method provided no evidence for a second susceptibility allele at either DQA1 or DQB1. However, our data and that of others suggest a negative association with DQA1*0101. Associations were found with DQB1 alleles sharing sequence homology with DQB1*0602, with DQB1 alleles encoding leucine at residue 26 (Leu 26), with DQA1 alleles encoding glutamine at residue 34 (Gln 34) and with Leu 26 plus Gln 34 alleles, but each was shown by two-loci linkage analysis to be secondary to the DRB1*1501, DQB1*0602, DQA1*0102 association. The recently reported negative association with DQA1 alleles encoding phenylalanine at amino acid 25, leucine at amino acid 69 and arginine at amino acid 52 was not found in this study, although there was a trend towards reduced phenylalanine at amino acid 25. The determination at a molecular level of an explanation for the world-wide association with these alleles remains elusive despite major advances in MHC typing.

Distribution of MHC class II alleles in primary systemic vasculitis

Previous studies have shown a number of different associations between major histocompatibility complex (MHC) alleles and primary systemic vasculitis. Disease heterogeneity and the lack of specificity of certain MHC typing techniques may have contributed to the lack of consistency in those studies. We therefore studied a relatively homogeneous group of 94 patients with Wegener's granulomatosis, microscopic polyangiitis, or renal-limited vasculitis using molecular techniques that allow more precise assignment of MHC genotype. DNA was prepared from peripheral blood and DRB1 genotype determined by Taq restriction fragment length polymorphism. DQB1 and DPB1 genotype were assigned by polymerase chain reaction amplification followed by probing with allele-specific oligonucleotides. Specificity of associated anti-neutrophil cytoplasm antibodies (ANCA) was determined where possible by solid phase immunoassays using purified proteinase 3 (PR3) and myeloperoxidase (MPO). After correction for multiple comparisons there were no significant differences in the distribution of DRB1, DQB1 and DPB1 alleles between a local control group (N = 90 for DRB1, N = 50 for DQB1 and DPB1) and the patient group as a whole (N = 94) or two a priori defined subgroups (anti-PR3 positive, N = 35; anti-MPO positive, N = 22). We have therefore found no significant association between primary systemic vasculitis and any MHC class II allele. This, together with the fact that previous smaller studies have shown no consistent association, suggests that any such association is very weak, if it exists at all.

Social rank and disease susceptibility in pigs

Two experiments were carried out to investigate the inter-individual variation in immune reactivity and disease susceptibility of group housed pigs of different social status. The social status of the individual pig was determined by the outcome of social ranking fights and food competition tests. On Day 75 after the start of both experiments, all pigs were challenged with 0.5 ml of an Aujeszky disease virus (ADV) in each nostril. Data combined from both experiments showed that mortality and/or morbidity after the ADV challenge was highest among subordinates. In both experiments, a lymphocyte proliferation assay, using purified ADV as an antigenic stimulus, showed that dominant pigs had significantly higher counts per minute than subdominant and subordinate pigs. Kendall's partial correlations showed that morbidity had been associated with high values in haematological and clinicochemical blood parameters and not with social status of the individual pig. In each experiment, maternal derived antibodies against the ADV and the antibody level after the ADV challenge hardly differed between pigs of different social status. Major histocompatibility complex typing of class I and II by iso-electro focusing of all pigs in Experiment 2 showed that not all haplotypes were distributed equally among dominant, subdominant and subordinate pigs. The present work shows that there are large individual differences in immune reactivity and disease susceptibility which appear to be related to the social status of the individual pig in a stable social structure.

Production, immunoanalysis and selection of monoclonal antibodies for studies of polymorphism of bovine MHC class I antigens.

Two monoclonal antibodies (MoAbs) were raised against bovine lymphocyte antigens of the major histocompatibility complex (MHC) for studies of the polymorphism of MHC class I antigens of African cattle. Immunoanalysis of lymphoid tissues and molecular weight data were used to characterize the epitopes seen by the MoAbs. Competitive binding assays indicated specificity of the MoAbs for two distinct epitopes. Application of the MoAbs for MHC typing of a cattle population showed that the epitopes were co-expressed on the target MHC antigen but occurred independently on non-target MHC antigens. It is concluded that, to obtain MoAbs for studies of MHC polymorphism, it is important to conduct large-scale analysis using immunoassays and population studies.

Genetic Variability in Shr (Shrsr), Shrsp and Wky Strains

Genetic background for SHR (SHRSR) and SHRSP and WKY lines were screened by using biochemical genetic markers and class I major histocompatibility complex (RT1) typing. There were many kinds of SHR (SHRSR), SHRSP and WKY inbred lines. The genetic variabilities within SHR (SHRSR) or SHRSP strains were small, but those within WKY strains were not so small. When SHR lines were compared with WKY lines, the allele distribution in SHR lines was different from that in WKY lines at 3-13 loci. Three genetic markers, the Es-3b, Es-4a and PT1k, were identified as specific markers of SHR (SHRSR) and SHRSP. WKY/Izm, WKY/Hos, and WKY/Jim also carried the PT1k as well as SHR lines, but WKY/N and WKY/NCrj had the RT1l haplotype.