DQB1 Polymorphisms Research Articles

Paternity assessment in rhesus macaques (Macaca mulatta): multilocus DNA fingerprinting and PCR marker typing.

Establishing kinship relations in primates using modern molecular genetic techniques has enhanced the ability to scrutinize a number of fundamental biological issues. We screened 51 human short tandem repeats (STRs) for cross-species PCR amplification in rhesus macaques (Macaca mulatta) and identified 11 polymorphic loci with heterozygosity rates of at least 0.6. These markers were used for paternity testing in three social groups (M, R, and S) of rhesus macaques from Cayo Santiago, Puerto Rico. Several consecutive birth cohorts were analyzed in which approximately 200 males were tested for paternity against more than 100 mother/ infant pairs. Despite a combined exclusion rate of more than 99.9% in all three groups, some cases could not be solved unequivocally with the STR markers and additional testing of the MHC-associated DQB1 polymorphism. A final decision became possible through multilocus DNA fingerprinting with one or more of the oligonucleotide probes (GATA)4, (CA)8, and (CAC)5. Paternity assessment by multilocus DNA analysis with probe (CAC)5 alone was found to have limitations in rhesus macaques as regards the number of potential sires which might be involved in a given case. Multilocus DNA fingerprinting requires large amounts of DNA, and the ensuing autoradiographic patterns present difficulties in comparisons across gels and even within the same gel across remote lanes. Computer-assisted image analysis was incapable of eliminating this problem. Therefore, a dual approach to DNA typing has been adopted, using STR markers to reduce the number of potential sires to a level where all remaining candidates can be tested by multilocus DNA fingerprinting on a single gel, preferably in lanes adjacent to the mother/infant pair.

HLA-DRB1 and -DQB1 Polymorphism in Liver Recipients: Relationship Between HLA-DQB10302 Allele Frequency and Acute Rejection

ABSTRACT: Polymorphism of HLA-DRB1 and HLA-DQB1 loci was performed in fifty-three orthotopic liver graft recipients as well as in 108 unrelated healthy controls. Nonradioactive SSOPs were used to study PCR-amplified DNA from peripheral blood lymphocytes and biopsied material. The comparison frequency for DQB1 alleles did not reveal any significant differences between the total group of liver recipients and controls. However, when the liver recipients were subgrouped according to their rejection episode manifestations, increased and significant frequencies were observed for HLA-DQB1∗0302 allele in patients showing acute rejection episodes compared to healthy controls or patients without acute rejection. This relationship did not appear influenced by the amino acid β alanine residue in the 57th position. On the other hand, the study of the DRB1 allele frequencies did not show significant differences in any study. These results suggest that HLA-DQB1 genes could be important in the liver graft alloresponses, opening a way to a better understanding of the special tolerance state, normally observed in this type of transplant, leading us to consider the possible HLA-DQB1∗0302 allele effect on tolerance rupture.

HLA class II DRB1, DQA1 and DQB1 polymorphisms in the Polish population from Wielkopolska.

HLA DRB1, DQA1 and DQB1 alleles were determined by DNA PCR-SSO typing in a sample of 99 individuals originating from Wielkopolska (midwestern Poland). A high number of alleles (38 DRB1, 8 DQA1 and 14 DQB1) was detected at each locus, many of them presenting notable frequencies in this population. The three HLA loci are thus characterized by very high heterozygosity levels (93% for DRB1, 85% for DQA1, and 88% for DQB1), which confirms the results found for other European populations. A total of 6 DRB1-DQA1-DQB1 haplotypes are detected with an estimated frequency higher than 5%, namely, DRB1*1501-DQA1*0102-DQB1*0602, DRB1*0701-DQA1*0201-DQB1*0201, DRB1*0101-DQA1*0101-DQB1*0501, DRB1*1101-DQA1*0501-DQB1*0301, DRB1*03011-DQA1*0501-DQB1*0201, and DRB1*1301-DQA1*0103-DQB1*0603. A genetic distance analysis between the Polish and other world populations tested for HLA class II indicates that the Wielkopolska community is close to geographically close, rather than linguistically related populations from Europe. More generally, a good agreement between genetics and geography is found for DRB1 and DQB1 polymorphisms in Europe, suggesting that these two loci are highly informative for assessing historical relationships among humans.

HLA-A, -B, -DRB1, -DQA1, and -DQB1 polymorphism in thais

In this study we examined HLA-A, -B, -DRB1, -DQA1, and -DQB1 gene, allele, and haplotype frequencies in two ethnic Thai populations. We compared these frequencies to the known HLA class I and II allele profiles of non-Thai mainland and insular Southeast (SE) Asians. HLA-A locus gene and allele frequencies, are comparatively homogeneous in both Thai and non-Thai SE Asians. In contrast, HLA-B, -DRB1, -DQA1, and -DQB1 gene and allele frequencies, show more ethnic and geographic variation in SE Asians. Conserved haplotypes, or combinations of linked HLA class I and II alleles were detected in Thais, but at relatively low frequencies. It would appear that ethnic Thais, reflect an admixture of peoples from both the northern mainland and southern island groups of SE Asia.

Extensive HLA class II studies in 58 non-DRB1*15 (DR2) narcoleptic patients with cataplexy.

Narcolepsy is a sleep disorder that has been shown to be tightly associated with HLA DR15 (DR2). In this study, 58 non-DR15 patients with narcolepsy-cataplexy were typed at the HLA DRB1, DQA1 and DQB1 loci. Subjects included both sporadic cases and narcoleptic probands from multiplex families. Additional markers studied in the class II region were the promoters of the DQA1 and DQB1 genes, two CA repeat polymorphisms (DQCAR and DQCARII) located between the DQA1 and DQB1 genes, three CA repeat markers (G51152, T16CAR and G411624R) located between DQB1 and DQB3 and polymorphisms at the DQB2 locus. Twenty-one (36%) of these 58 non-DR15 narcoleptic patients were DQA1*0102 and DQB1*0602, a DQ1 subtype normally associated with DRB1*15 in DR2-positive narcoleptic subjects. Additional microsatellite and DQA1 promoter diversity was found in some of these non-DR15 but DQB1*0602-positive haplotypes but the known allele specific codons of DQA1*0102 and DQB1*0602 were maintained in all 21 cases. The 37 non-DQA1*0102/DQB1*0602 subjects did not share any particular HLA DR or DQ alleles. We conclude that HLA DQA1*0102 and DQB1*0602 are the most likely primary candidate susceptibility genes for narcolepsy in the HLA class II region.

HLA Class II Profile: A Weak Determinant of Factor VIII Inhibitor Development in Severe Haemophilia A

The risk of developing factor VIII inhibitor antibodies in haemophilia A may relate both to factor VIII genotype and genes within the HLA complex known to influence immune response. We investigated a cohort of 176 patients with severe haemophilia A and with either high-level inhibitors (> 10BU/ml) or with no history of an inhibitor, stratified according to the presence or absence of the factor VIII gene intron 22 inversion. HLA DRB1, DQA1 and DQB1 polymorphisms were determined by PCR. HLA frequencies form 137 United Kingdom controls were used for comparison. HLA phenotype frequency differences, expressed as odds ratios with 95% confidence intervals were as follows: HLA-DRB*1501, DQB1*0602 and DQA1*0102 were all increased in frequency in patients with inhibitors, only DQA1*0102 reaching statistical significance (OR 2.7, 1.2-5.9). These alleles form part of an established HLA haplotype. The frequencies of HLA-DRB1*1501, DQB1*0602 and DQA1*0102 were particularly raised in patients with inhibitors and a factor VIII gene intron 22 inversion, although again only DQA1*0102 achieved significance (OR 3.1, 1.0-10.1). The frequency of DRB1*01, DQB1*0501, DQA1*0101 were also increased in inhibitor patients lacking the intron 22 inversion although this failed to achieve statistical significance. This data suggests that HLA class II profile constitutes a weak risk factor for developing inhibitor antibodies to factor VIII. This may be more pronounced in patients with an intron 22 inversion.

Low prevalence of immunogenetic markers of IDDM in adult Koreans with diabetes detected on OGTT

<h2>Abstract</h2> In the Asian populations, it is not uncommon for adult patients with NIDDM to eventually lose <i>β</i>-cell function and develop IDDM. Accepting that IDDM is an autoimmune disease, which occurs on a genetic background, it could be hypothesized that by measuring autoantibody prevalence and HLA DQ gene polymorphism, known important prediagnostic markers of IDDM, the prevalence of adult-onset IDDM in patients with previously undiagnosed NIDDM patients could be estimated. To do this, anti-GAD prevalence and HLA-DQ A1 and DQ B1 polymorphisms after PCR amplification of genomic DNA were analyzed in 121 newly diagnosed diabetic patients of Yonchon cohort and compared to the results with those of 100 matched healthy control subjects. We also compared the results with those of other populations to assess the difference of genotype distribution. The overall prevalence of anti-GAD antibodies was 1.7% (2 of 121) in patients with previously undiagnosed NIDDM, whereas 1 of 100 controls had positive antibodies. Among those who were positive, their titer of antibodies to GAD were not high. No statistically significant differences in the distribution of either mean levels of anti-GAD or DQA1 and DQB1 alleles were found comparing NIDDM patients to controls. Interestingly, the frequency of DQB1*non-Asp-57 and DQA1*Arg-52 alleles in the Korean adult control population was similar to that of US Caucasians (DQB1*non-Asp-57: 0.431 vs. 0.475; DQA1*Arg-52: 0.492 vs. 0.463). The low prevalence of anti-GAD antibodies and HLA-DQA1 and DQB1 susceptibility alleles among recent-onset NIDDM patients, not different compared to controls suggests that diabetes in Korean adults is unlikely to have an autoimmune component to its pathogenesis.

Low prevalence of immunogenetic markers of IDDM in adult Koreans with diabetes detected on OGTT

In the Asian populations, it is not uncommon for adult patients with NIDDM to eventually lose β-cell function and develop IDDM. Accepting that IDDM is an autoimmune disease, which occurs on a genetic background, it could be hypothesized that by measuring autoantibody prevalence and HLA DQ gene polymorphism, known important prediagnostic markers of IDDM, the prevalence of adult-onset IDDM in patients with previously undiagnosed NIDDM patients could be estimated. To do this, anti-GAD prevalence and HLA-DQ A1 and DQ B1 polymorphisms after PCR amplification of genomic DNA were analyzed in 121 newly diagnosed diabetic patients of Yonchon cohort and compared to the results with those of 100 matched healthy control subjects. We also compared the results with those of other populations to assess the difference of genotype distribution. The overall prevalence of anti-GAD antibodies was 1.7% (2 of 121) in patients with previously undiagnosed NIDDM, whereas 1 of 100 controls had positive antibodies. Among those who were positive, their titer of antibodies to GAD were not high. No statistically significant differences in the distribution of either mean levels of anti-GAD or DQA1 and DQB1 alleles were found comparing NIDDM patients to controls. Interestingly, the frequency of DQB1*non-Asp-57 and DQA1*Arg-52 alleles in the Korean adult control population was similar to that of US Caucasians (DQB1*non-Asp-57: 0.431 vs. 0.475; DQA1*Arg-52: 0.492 vs. 0.463). The low prevalence of anti-GAD antibodies and HLA-DQA1 and DQB1 susceptibility alleles among recent-onset NIDDM patients, not different compared to controls suggests that diabetes in Korean adults is unlikely to have an autoimmune component to its pathogenesis.

Linkage disequilibrium between HLA class II (DR, DQ, DP) and antigen processing (LMP, TAP, DM) genes of the major histocompatibility complex.

TAP, LMP and DM genes map within the major histocompatibility complex (MHC) class II region between the DQB1 and DPB1 loci, and are involved in the processing of peptides bound to HLA class I or class II molecules. In order to determine the various linkage disequilibria existing between these genes and HLA class II genes, we have analyzed TAP1, TAP2, LMP2, DMA, DMB, DRB1, DQA1, DQB1 and DPB1 polymorphisms in 162 unrelated healthy Caucasian individuals. Many positive or negative associations were observed between alleles at these loci, such as between DR/DQ and TAP2, DM or LMP, between DP and DMB, and between TAP2 and DM, TAP2 and LMP. Conversely, no linkage disequilibrium was detected between some closely related genes (DR/DQ and TAP1, TAP1 and TAP2, LMP2 and DM), in agreement with the existence of recombination hot spots in this region. Other weak linkage disequilibria are likely to exist in this region. These data allow to define some conserved MHC class II haplotypes including HLA class II and TAP, LMP and DM alleles. Furthermore, the knowledge of such linkage disequilibria is of outstanding importance in order to avoid misinterpretation of the data when studying MHC class II associations with autoimmune diseases.

P770 - Holistic approach in the study of population structure based on the analysis of HLA-DRB1, DRB3, DRB4, DRB5, DQA1, DQB1 Polymorphisms (island of KRK, croatia)

The population structure of six rural communities of the northern Adriatic island of Krk, Croatia, was studied utilising PCR methodology and non-radioactive oligonucleotide hybridisation for the analysis of HLA-DRB1, DRB3, DRB4, DQA1, DQB1 polymorphism. Allele frequencies, measures of genetic kinship (R matrix) and genetic distances (E2) were computed, after which a model-free approach (distance matrix correlations) was employed. The studied rural populations revealed high levels of reproductive isolation (endogamy rate of 84.19) and clusterisation into three groups, the explanation of which can be found in known ethnohistorical events (3 migrational waves) and the results of current linguistic distance analyses (HMS range of 0.305 to 0.894). Quadratic assignment procedures indicated highly statistically significant and positive correlations of matrices (genetic vs. geographic distances, r=0.8048 ; genetic distances vs. migrational kinship, r=0.7556 ; genetic vs. linguistic distances, r=0.8577 ; geographic distances vs. migrational kinship, r=0.6388). The results indicate ethno-historical and socio-cultural events, rather than geographical distances, to be the primary determinants of anthropogenetic structure of the studied island population groups. The contribution of HLA polymorphisms analysis to holistic anthropological apprehension of human (micro)evolutionary processes in contemporary European populations is discussed.

Polymorphism of HLA-DRB, -DQA1, and -DQB1 in Rheumatoid Arthritis in Asian Indians: Association with DRB1∗0405 and DRB1∗1001

We investigated the DRB, DQA1, and DQB1 polymorphism and haplotypes in sporadic and familial RA subjects of Asian Indian origin by PCR oligotyping using biotinylated SSOPs. Molecular subtyping of DRB1 ∗04 in RA patients showed strongest association with highest relative risk with DRB1 ∗0405, followed by DRB 1 ∗0401. A significant decreased frequency of DRB1 ∗1502 was observed in patients compared to controls ( X 2 = 4.5). Among other alleles, DRB1 ∗1001 was found to be significantly increased. A total of 73.3% of patients carried the shared sequence of the third HVR (67–74) of DRB1 domain compared to its presence in only 37.6% of controls. A significant number of patients carried DR4 haplotypes on DQB1 ∗0302 (58%) as against DQB1 ∗0301 which was present only on 10.5% of the haplotypes. When compared to controls, the difference was significant for the latter allele only. Few unique DRDQ haplotypes were observed in Asian Indians. Among DR-DQ haplotypes, DRB1 ∗0401-DQB1 ∗0302 gave the highest risk whereas DRB1 ∗0403-DQB1 ∗0301 was negatively associated. Alleles with negative charge at position 70 confer protection or are negatively associated with RA whereas among the associated alleles, glycine at position 86 resulted in higher risk than those with valine at this position. A heterogenous association of DQB1 alleles with DR4 subtypes, influencing susceptibility to RA, suggests the DQB locus is not primarily associated with RA and susceptibility lies in the sequence 67–74 of the DRB1 loci.

An analysis of HLA class II gene polymorphism in British and Greek idiopathic membranous nephropathy patients.

Fifty-two British and 29 Greek idiopathic membranous nephropathy (IMN) patients were analysed for DRB, DQA1, DQB1 and DPB1 gene polymorphism using second exon amplification and sequence-specific oligonucleotides (SSO). In addition 100 British and 92 Greek controls were analysed. A highly significant increased frequency of the DRB1*0301 allele was found in IMN patients from Britain (80%), when compared to controls (27%, OR 10.6, P = 0.000004). A lower frequency of DRB1*0301 was observed in Greek IMN patients (33%), but this was just significant before correction, when compared to Greek controls (15%, OR 3, P = 0.02). The DRB3 allele most often associated with DRB1*0301 was DRB3*0101 (OR 4.2, P = 0.00025) in British patients and DRB3*0201/2 (OR 11, P = 0.006) in Greek patients. In Greek IMN patients a decrease in DR16 was found (OR 0.08, P = 0.004), and the overall incidence of DR2 was significantly lowered when both sets of IMN patients were combined (OR 0.21, chi 2 17.6, P = 0.00013). The incidence of DQA1*0501 was raised in both Greek (96% vs. 66%, OR 9.7, chi 2 6.9, P = 0.009) and British IMN (85% vs. 45%, OR 7.4, chi 2 20, P = 0.00007) patients. This gives some support to a proposal for a major role for this allele in IMN.(ABSTRACT TRUNCATED AT 250 WORDS)

HLA class II gene polymorphism in Tunisians.

The polymorphism of HLA class II genes (HLA-DRB, DQB, DPB) was investigated in 101 Tunisians using polymerase chain reaction. (PCR) amplification and reverse dot blot (RDB) hybridization. Allele and haplotype frequencies, as well as DRB1-DQB1 linkage disequilibria, were calculated. A total of 26 DRB1 alleles were detected and the most prevalent variant was DRB1*0301 with an allelic frequency at 21.87%. In the DR1 group, DRB1*0102 was most frequent than DRB1*0101. In the DR4 group, DRB1*0403 was the most common allele and was associated with DQB1*0402. Interestingly this DRB1-DQB1 association has not been observed in other populations. With regard to the DR8 group, DRB1*0804 was the unique variant detected, whereas with the DR13 specificity, the most common variant was DRB1*1303 in Algerians also. Although the DQB1 polymorphism analysis showed an allelic distribution very close to that observed in caucasoids, many DRB1-DQB1 associations which have not been reported in studies of other populations, were described. Finally at the DPB1 locus DPB1*1701 and *1301 allele frequencies distinguish clearly this Tunisian sample from a French caucasoïd panel of 83 subjects. In conclusion, a specific distribution of HLA components in terms of gene and haplotype frequencies characterizises this Tunisian population. This specific pattern may reflect the great ethnic diversity of this community. All these informations may be helpful in the future for HLA and disease association studies.

Maintenance of DQB1 polymorphisms in primates.

To understand the evolution of the class II major histocompatibility complex (MHC) DQB1 locus in primates, the second exons of seven DQB1 alleles from five non-human primate species were amplified by polymerase chain reaction. Comparisons of these and other primate sequences show that no between-species diversity is greater than within-species diversity, suggesting maintenance of DQB1 alleles through the history of Old-World primates. There is a preponderance of nonsynonymous nucleotide substitutions at antigen-binding-site codons; this pattern is in marked contrast to what is seen at the closely related, presumably nonfunctional DQB2 gene. The results support the hypothesis that DQB1 polymorphism is maintained by overdominant selection relating to antigen presentation.

Complete analysis of HLA-DQB1 polymorphism and DR-DQ linkage disequilibrium by oligonucleotide typing

HLA class II polymorphism is functionally important in the control of immune responses, in transplantation immunology, and in the susceptibility to autoimmune disease. HLA-DQA1 and -DQB1 genes exhibit a larger degree of allelic polymorphism than usually recognized by routine serology. We have therefore performed an extensive analysis of DQB1 polymorphism by oligotyping. A set of 12 oligo probes was hybridized on polymerase chain reaction - amplified DNA, thus allowing the detection of 12 DQB1 alleles, as demonstrated in homozygous as well as in heterozygous individuals. This highly sensitive detection system is particularly relevant within the DQw1 specificity where the 7 allelic sequences can easily be identified. The DQ-DR linkage disequilibrium was analyzed by oligotyping of 80 Caucasoid heterozygous individuals (160 haplotypes), and very tight associations were observed between DRB1 and DQB1 alleles. Five DRB1 alleles, DR-BON, DR4/Dw4 or Dw14, DR7, DRw8.3, and DRw11, however, can be associated with different DQB1 alleles. Moreover the DRB1 oligotyping analysis performed on 20 randomly chosen DRw8 Caucasoid individuals showed a high prevalence of the DRB1 *0801-DQB1 *0402 haplotype. By combining the analysis of allelic variations at DRB1, DRB3, and DQB1 loci, we can detect 33 different DR-DQ combinations in our panel of Caucasoid individuals. We now apply DQB1 oligotyping on a routine basis for optimal matching of unrelated donors for bone narrow transplantation.