Comprehensive typing of DR52 (DRB3)-associated DRB1 and DRB3 alleles by PCR-RFLP

Abstract: The DR52-associated DRB1 and DRB3 alleles were resolved by PCR-RFLP. Second exon was amplified using four primer pairs (groups 1–4) for DRB1 and a pair for DRB3 alleles. Except for three endonucleases, all others had either none or only one site for a specific amplified product. Group 1 primers amplify 10 DRB1 alleles (DRB1*0302, 1101, 1302, 1303, 1305, 1307, 1402, 1403, 1407 and 1409). All but one pair, DRB1*1402 from 1409, could be resolved using seven endonucleases (ApaI, SacII, FokI, AvaII, BsaAI, BsrBI and SfaNI). Group 2 consisted of four alleles (DRB1*1201, 1202, 1404 and 1411) that can be resolved along with co-amplified DRB1*0804 and 0806 using five endonucleases (AvaII, SacII, FokI, HaeII and RsaI). Group 3 primers amplify 15 DRB1 alleles (DRB1*0301, 0303, 1102, 1103, 1104, 1107, 1301, 1304, 1306, 1308, 1401, 1405, 1406, 1408 and 14-New), which can be resolved using nine enzymes (KpnI, AvaII, FokI, SacII, HaeII, BsrBI, SfaNI, DdeI and RsaI). BsrBI, a new endonuclease, can resolve DRB1*1301 from 1306 and the previously unresolved allele DRB1*1103 from 1104. DRB1*1410, co-amplified with DR4 group-specific primers, is resolved with PstI which cleaves all DR4 alleles but not DRB1*1410. All four DRB3 alleles (DRB3*0101, 0201, 0202 and 0301) and their heterozygotes are resolved using two endonucleases, RsaI and HphI. Thirty-four DR52-associated alleles and their heterozygotes can be unambiguously resolved, except for DRB1*1402 from 1409. Thus, PCR-RFLP remains an effective method for high-resolution HLA-DR typing. Furthermore, PCR-RFLP can complement the evolving PCR-SSP method for allele-specific typing using a minimal number of restriction endonucleases.     

Direct sequencing of SSP-PCR-amplified cDNA to identify new alleles in the DR52-associated DRB1 group: identification of DRB1*1115, DRB1*1117 and DRB1*1319

Abstract: Low and high resolution sequence specific oligonucleotide probe hybridization patterns were used to design an approach to direct sequencing of allele specific amplified cDNA. Several PCR amplifications were used to derive overlapping sequence fragments to define complete first domain sequences for a single allele. This method has been used to characterize three new DRB1 alleles in the DR52 family, DRB1*1115, DRB1* 1117, and DRB1*1319. All three alleles carry polymorphisms previously observed in other DRB alleles and underscore the importance of utilizing a directed sequencing approach for obtaining unambiguous typing results in matching for bone marrow transplantation between unrelated donor and recipient.     

HLA-DRw52-associated DRB1 alleles: Identification using polymerase chain reaction-amplified DNA, sequence-specific oligonucleotide probes, and a chemiluminescent detection system

Polymerase chain reaction (PCR) primers were designed to specifically amplify exon 2 of the DRw52-associated DRB1 alleles for subsequent typing by sequence-specific oligonucleotide probe (SSOP) hybridization and chemiluminescent detection. The DRw52 DRB1 group, encoding 22 of the 44 W.H.O. designated DRB1 allelic products, was divided by differential PCR with two polymorphism-directed forward primers. Based on a polymorphism at codon 13, these forward primers separate the DRw52-associated alleles into subsets; one comprised of the alleles of DR3/DRw11/DRw6 and the other of DRw8/DRw12/DRB1*1404. The DRB1 alleles in the latter subset were then defined by SSOP hybridization to the amplified DNA. The preferential amplification also resulted in SSOP definition of 15 alleles in the DR3/DRw11/DRw6 subset but some DRw11/DRw13 heterozygous allelic combinations were still unresolved. Two reverse PCR primers specific for the polymorphism at codon 86 were used to obtain amplified material to which SSOP reactivity provided definitive identification of the ambiguous heterozygotes.     

Polymorphism of HLA-DRw52-associated DRB1 genes as defined by sequence-specific oligonucleotide probe hybridization and sequencing

We have used group-specific DNA amplification and sequence-specific oligonucleotide probe (SSOP) hybridization to study DRB1 sequence polymorphisms associated with DR3, DRw11(5), DRw12(5), DRw13(w6), DRw14(w6) and DRw8 alleles. Group-specific amplification of DRw52-associated DRB1 alleles was achieved using a 5' amplification primer designed to hybridize with a first hypervariable region (HVR) sequence common to all known alleles in this group, together with a 3' intron primer. Prospective SSOP typing of DR3, DRw11, DRw12, DRw13, DRw14 and DRw8 alleles was performed in 318 individuals, including 124 patients, 46 family members and 148 unrelated marrow donors. Among the 395 DRw52-associated DRB1 alleles tested in our study, a subtype corresponding to the previously defined alleles DRB1*0301-2 (DR3), DRB1*1101-4 (DR5), DRB1*1201-2 (DR5), DRB1*1301-5 (DRw6), DRB1*1401-2 and 1404 (DRw6), and DRB1*0801-4 (DRw8) could be assigned in all but 6 individuals (1.9%) tested. In addition to the 22 known alleles, we identified two new DRw6-associated alleles, DRB1*13.MW(1) and DRB1*14.GB(1). DRB1*13.MW typed serologically as DRw13 and was identical to DRB1*1301 except at codon 71 where AGG encodes arginine instead of GAG encoding glutamic acid. DRB1*14.GB represents a DRB1*1402 variant whose sequence at codon 86 encodes valine (GTG) instead of glycine (GGT). These results demonstrate that SSOP methods represent an efficient and precise approach for typing DRB1 alleles and for identifying potential novel variants previously unrecognized by conventional typing methods.     

HLA-DR51 expression failure caused by a two-base deletion at exon 2 of a DRB5 null allele (DRB5*0110N) in a Spanish gypsy family

Here we describe a new HLA class II null allele at the DRB5 gene. Serologic HLA typing of a Spanish gypsy family rendered the following paternal haplotype: A2-Cblk-B52-Bw4-DR15-DQ5. However, DNA typing demonstrated the presence of a DRB5 gene in the haplotype DRB1*1502-DRB5*0102-DQB1*05031. Complete DRB5 cDNA sequencing revealed a DRB5*0102 allele with a deletion of two nucleotides at exon 2 (239-240) in codon 80. This change generates a frame shift leading to a stop codon at position 86, and could explain the lack of DR51 protein at the cell surface. This is the second DRB5 null allele described together with DRB5*0108N, raising the number of HLA alleles with an expression disorder.     

Limited diversity of HLA-DRB1*02 alleles and DRB1-DRB5 haplotype associations in four United States population groups

At least 60 DRB1*02 positive individuals from each of four US population groups found within a hematopoietic stem cell volunteer donor registry - Caucasoids, African Americans, Asians/Pacific Islanders, and Hispanics - were randomly selected from a database of 82,979 individuals. DRB1*02 alleles were identified by DNA sequencing. A total of five of 23 known DRB1*02 alleles were detected. DRB1*15011 was the predominant DRB1*02 allele in Caucasoids and Hispanics. The most common DRB1*02 allele observed in African Americans was DRB1*1503, and DRB1*15021 in Asians/Pacific Islanders. Caucasoids were found to be the least diversified; only DRB1*15011 and DRB1*16011 were observed. A subset of individuals was also typed for DRB5 alleles by DNA sequencing. DRB5*01011, DRB5*0102, DRB5*0103, DRB5*0108N and DRB5*0202 were detected and nine DRB1-DRB5 haplotypes defined.     

Distribution of HLA class II alleles among Scandinavian patients with systemic lupus erythematosus (SLE): An increased risk of SLE among non[DRB1*03,DQA1*0501,DQB1*0201] Class II homozygotes?

HLA-DRB1, -DRB3, -DQA1 and -DQB1 alleles were determined by DNA typing in 51 Scandinavian patients with systemic lupus erythematosus (SLE) and 129 controls. DRB1*03,DRB3*0101,DQA1*0501,DQB1*0201 were significantly increased in the patient group, with relative risks (RR) of 2.80, 3.07, 3.55 and 2.12, respectively. These alleles are in strong linkage disequilibrium, and their possible relative contributions in predisposition to SLE are difficult to distinguish. The strongest association was found for DQA1*0501, which is in linkage disequilibrium with DRB1*03 as well as DRB1*11,12 (DR5). An increased frequency of DRB1*11,12 was observed (RR = 1.89, ns). No association with DRB1*15,16 (DR2) was found. The patients had a higher frequency of HLA class II homozygosity than the controls (RR = 5.05, p = 0.0005). When compared to the low-risk group (nonDRB1*03 class II heterozygotes), the cases homozygous for DRB1*03,DQA1*0501,DQB1*0201, known to be in linkage disequilibrium with the complement allele C4A*Q0, had the highest relative risk of developing SLE (RR = 16.39, p = 0.0002). However non[DRB1*03,DQA1*0501,DQB1*0201] class II homozygotes had a higher relative risk (RR = 4.68, p = 0.0147) than DRB1*03,DQA1*0501,DQB1*0201 heterozygotes, known to carry the C4A*Q0 allele (RR = 2.72, p = 0.0088). This may suggest that HLA class II molecules are directly involved in susceptibility to SLE.     

Polymorphism and distribution of HLA-DR2 alleles and haplotypes in a Greek population

Abstract: HLA-DR2 serological subtyping has indicated that the DR16 serotype appears at a higher frequency relative to the DR15 serotype in the Greek population, differing from the distribution observed in most other Caucasian groups. In this study, we have analyzed by the PCR-SSP technique a DR2-positive group of unrelated Greek individuals selected from our normal control panel for the different DRB1, DRB5, DQB1 and DQA1 DR2-associated alleles present. Six of the 50 individuals analyzed were homo-zygous for DR2, contributing a total of 56 haplotypes for DR2. The observed frequencies of the DR2-related DRB1 alleles were as follows: 58.9% for the DRB1*1601, 7.1% for the DRB1*1602, 25.0% for the DRBl*1501 and 7.1% for the DRB1*1502 allele. The rare allele DRB1*1605 was detected in one heterozygous sample and its presence was definitively established by DNA sequencing. The alleles *1503, *1504, *1505, *1603 and *1604 were not detected. Three DRB5 alleles were identified: DRB5*0202 (67.8%), DRB5*0101 (25.0%) and DRB5*0102 (7.1%). Ten different DRB1/DQB1/ DQA1 DR2-associated haplotypes were denned. The most frequently observed haplotype was DRBl*1601-DQBl*0502-DQAl*0102 (relative frequency =57%) followed by DRB1*1501-DQB1*0602-DQA1*0102 (relative fre-quency=14.3%). In conclusion, the refined analysis of the DR2-associated DRB1 alleles in the Greek population revealed the prevalence of the DRB1*1601 allele. The rare allele DRB1*1605 was demonstrated once. A considerable variety of different DR2-related DR/DQ haplotypes was detected and the overall haplotypic frequencies in the Greek population are distributed differently compared to those reported for most other Caucasian populations.     

Identification of four new DR52-associated DRJB1 alleles: DRB1*1424, *1425, *1323 and *1324

Four previously unreported DR52-associated DRB1 alleles have been characterized through DNA sequencing, contributing to the diversity of the HLA system. DRB1*1424 is nearly identical to DRB1*1402 in the second exon, except that it contains the "I—A" motif found at codons 67–71 common to the DRB1*15 alleles. DRB1*1425 contains the "A—H" motif, found in DRB1*1401 at codons 57–60, in a sequence otherwise identical with the second exon of DRB1*1307. Compared with DRB1*11012, DRB1*1323 contains three predicted amino acid changes at codons 58 (ala→glu), 67 (phe→ile), and 71 (arg→glu). The sequence of DRB1*1324 is identical to exon 2 of DRB1*1103, except that DRB1*1324 does not contain the GAG at codon 58 characteristic of the DRB1*11 alleles. These new alleles may have arisen through gene conversion, and they contribute to the complexity of the DR6 family.     

Identification and analysis of MHC class II DRB1 (Patr-DRB1) alleles in chimpanzees

The MHC-DRB1 gene is known to display the most extensive allelic polymorphisms among MHC class II genes. We attempted the selective identification of chimpanzee (Pan troglodytes) DRB1 (Patr-DRB1) alleles using the polymerase chain reaction (PCR) technique in three steps: first, we performed Patr-DRB1*02 lineage-specific 8-kb PCR for *02 lineage detection in each chimpanzee; second, we performed 620-bp PCR for amplification of full-length exon 2; and finally, we carried out an insert check using the pattern of microsatellite repeat length variability. In the genomic DNA of 23 chimpanzees, nine Patr-DRB1 alleles containing two new alleles were detected. Our approach provides a relatively effective method of identifying Patr-DRB1 alleles in individual chimpanzees and should also contribute to our understanding of the features of MHC molecules in non-human primates.     

HLA class I (A, B, C) and class II (DRB1, DQA1, DQB1, DPB1) alleles and haplotypes in the Han from southern China

In this study, polymerase chain reaction-sequence-specific oligonucleotide prode (SSOP) typing results for the human leukocyte antigen (HLA) class I (A, B, and C) and class II (DRB1, DQA1, DQB1, and DPB1) loci in 264 individuals of the Han ethnic group from the Canton region of southern China are presented. The data are examined at the allele, genotype, and haplotype level. Common alleles at each of the loci are in keeping with those observed in similar populations, while the high-resolution typing methods used give additional details about allele frequency distributions not shown in previous studies. Twenty distinct alleles are seen at HLA-A in this population. The locus is dominated by the A*1101 allele, which is found here at a frequency of 0.266. The next three most common alleles, A*2402, A*3303, and A*0203, are each seen at frequencies of greater than 10%, and together, these four alleles account for roughly two-thirds of the total for HLA-A in this population. Fifty alleles are observed for HLA-B, 21 of which are singleton copies. The most common HLA-B alleles are B*4001 (f= 0.144), B*4601 (f= 0.119), B*5801 (f= 0.089), B*1301 (f= 0.068), B*1502 (f= 0.073), and B*3802 (f= 0.070). At the HLA-C locus, there are a total of 20 alleles. Four alleles (Cw*0702, Cw*0102, Cw*0801, and Cw*0304) are found at frequencies of greater than 10%, and together, these alleles comprise over 60% of the total. Overall, the class II loci are somewhat less diverse than class I. Twenty-eight distinct alleles are seen at DRB1, and the most common three, DRB1*0901, *1202, and *1501, are each seen at frequencies of greater than 10%. The DR4 lineage also shows extensive expansion in this population, with seven subtypes, representing one quarter of the diversity at this locus. Eight alleles are observed at DQA1; DQA1*0301 and 0102 are the most common alleles, with frequencies over 20%. The DQB1 locus is dominated by four alleles of the 03 lineage, which make up nearly half of the total. The two most common DQB1 alleles in this population are DQB1*0301 (f= 0.242) and DQB1*0303 (f= 0.15). Eighteen alleles are observed at DPB1; DPB1*0501 is the most common allele, with a frequency of 37%. The class I allele frequency distributions, expressed in terms of Watterson's (homozygosity) F-statistic, are all within expectations under neutrality, while there is evidence for balancing selection at DRB1, DQA1, and DQB1. Departures from Hardy-Weinberg expectations are observed for HLA-C and DRB1 in this population. Strong individual haplotypic associations are seen for all pairs of loci, and many of these occur at frequencies greater than 5%. In the class I region, several examples of HLA-B and -C loci in complete or near complete linkage disequilibrium (LD) are present, and the two most common, B*4601-Cw*0102 and B*5801-Cw*0302 account for more than 20% of the B-C haplotypes. Similarly, at class II, nearly all of the most common DR-DQ haplotypes are in nearly complete LD. The most common DRB1-DQB1 haplotypes are DRB1*0901-DQB1*0303 (f= 0.144) and DRB1*1202-DQB1*0301 (f= 0.131). The most common four locus class I and class II combined haplotypes are A*3303-B*5801-DRB1*0301-DPB1*0401 (f= 0.028) and A*0207-B*4601-DRB1*0901-DPB1*0501 (f= 0.026). The presentation of complete DNA typing for the class I loci and haplotype analysis in a large sample such as this can provide insights into the population history of the region and give useful data for HLA matching in transplantation and disease association studies in the Chinese population.     

HLA-DQA1, DQB1 and DRB1 alleles associated with Takayasu arteritis in the Chinese Han population

Takayasu arteritis (TA) is a chronic large-vessel vasculitis of unknown etiology. Human leukocyte antigen (HLA) alleles play an important role in the development of TA. Sequence specific primer-polymerase chain reaction was used to detect 10 alleles of the HLA-DQA1 gene and 13 alleles of the HLA-DQB1, -DRB1 gene. A significant increase in the frequencies of DRB1¹07 (Pc < 0.01, OR = 3.44, CI: 2.15–5.52) was observed among TA patients compared with the control group. The significantly increased frequencies of the haplotype DQA1¹03:01-DQB1¹03:01-DRB1¹07 (Corrected P-values < 0.01) were observed in TA patients. But in the analysis of clinical manifestations, there are no significant associations with the HLA-DRB1¹07 allele.     

Complete coding sequences and haplotypic associations of HLA-B*0707, -B*1524, -B*4405, -B*4802, -DRB1*0409, -DRB1*0411, -DRB1*1115, -DRB1*1305, and the novel allele -DRB1*0709. Group-specific amplification of cDNA from DRB1 alleles associated to DRB3 and DRB4

We present here the characterization of the complete coding sequences, previously unavailable, of the human leukocyte antigen (HLA) alleles B*0707, B*1524, B*4405, B*4802, DRB1*0409, DRB1*0411, DRB1*1115, DRB1*1305, and that of a new allele, DRB1*0709. For the isolation of cDNA from the DRB1 gene, we designed a novel set of polymerase chain reaction (PCR) primers that makes it possible to amplify separately the groups of DRB1 alleles associated to each of the DRB3 and DRB4 loci. The primary structures, functional features, evolutionary relationships, haplotypic associations, and population distributions of each of the nine HLA-B and -DRB1 alleles reported here are reviewed.     

Class II HLA allele polymorphism: DRB1, DQB1 and DPB1 alleles and haplotypes in the New Zealand Maori population

Class II alleles of interest to transplantation comprise the DRB1, DQB1 and DPB1 loci. Sequence-based typing was used to determine the class II allelic variability present in New Zealand Maori, a population with close genetic ties to Polynesia and known anthropological and linguistic connections to mainland Asia. The most common DRB1 alleles identified were DRB1*1201, DRB1*110101, DRB1*0403 and DRB1*080302, with frequencies of 21.5%, 14%, 11.25% and 9.25%, respectively. Standard linkages between the DRB1 locus and the DRB3, 4 and 5 loci were maintained, with no novel patterns identified. The most common DQB1 alleles identified were DQB1*030101, DQB1*060101, DQB1*020101, DQB1*0602 and DQB1*050201, with frequencies of 29.5%, 8%, 7.8%, 6.4% and 6.2%, respectively. The most common DPB1 alleles identified were DPB1*0501, DPB1*040101 and DPB1*020102, with frequencies of 40.2%, 28.89% and 15.83%, respectively. A total of 80 estimated DRB1-DQB1 two-locus haplotypes were detected. DRB1*1201-DQB1*030101 was the most frequent (15.40%) haplotype, followed by DRB1*110101-DQB1*030101 (9.97%), DRB1*0403-DQB1*030201 (7.37%) and DRB1*080302-DQB1*060101 (5.96%). The allelic variation determined is being used in further analysis of the requirement for bone marrow transplantation in the New Zealand Maori population and has implications for optimal ethnic donor distribution on the New Zealand Bone Marrow Donor Registry, anthropological studies and disease association.     

Hardy-Weinberg testing for HLA class II (DRB1, DQA1, DQB1, and DPB1) loci in 26 human ethnic groups

Testing the fit of population data to Hardy-Weinberg proportions is crucial in the validation of many current approaches in population genetic studies. In this paper, we tested fit to Hardy-Weinberg proportions using exact approaches for both the overall and individual heterozygote genotype data of four HLA Class II loci: DRB1, DQA1, DQB1, and DPB1, from 26 human populations. Eighty of 99 overall tests fit the Hardy-Weinberg expectation (73% for DRB1, 89% for DQA1, 81% for DQB1 and 81% for DPB1). Deviations from Hardy-Weinberg proportions were both locus and group specific. Although we could not rule out other mechanisms at work, the individual test results indicated that the departure was possibly partly due to recent admixture. Evidence for selection and other sources of deviation are also discussed.     

Diversity is demonstrated in class I HLA-A and HLA-B alleles in Cameroon, Africa: description of HLA-A*03012, *2612, *3006 and HLA-B*1403, *4016, *4703

To examine the genetic diversity in west Africa, class I HLA-A and HLA-B alleles of 92 unrelated individuals from two areas in the Cameroon, the capital Yaoundé and the village of Etoa, were identified by direct automated DNA sequencing of exons 2 and 3 of the HLA-B locus alleles and sequence-specific oligonucleotide probe (SSOP) and/or sequencing of the HLA-A locus alleles. HLA-A*2301 (18.7%), A*2902 (10.4%), B*5301 (10.9%), and B*5802 (10.9%) were the most frequently detected alleles, present in at least 10% of the population. A total of 30 HLA-A locus and 33 HLA-B locus alleles, including six novel alleles, were detected. The novel alleles were HLA-A*03012, A*2612, A*3006 and HLA-B*1403, B*4016, and B*4703. HLA-B*4703 contains a novel amino acid sequence that is a combination of the first 5 amino acids of the Bw6 epitope and the last 2 residues of the Bw4 epitope. The addition of 6 alleles to the ever-expanding number of known class I HLA alleles supports our hypothesis that extensive genetic diversity, including previously undescribed alleles, would be observed in this African population. In the Yaoundé population, the allele frequency distribution at the HLA-A locus is consistent with distributions indicative of balancing selection. Extensive HLA-A-B haplotypes were observed in this population suggesting that only a fraction of the Cameroon HLA-A-B haplotype diversity has been observed.