DRB1*03 diversity and DRB3 associations in five major population groups in the United States

One hundred sixty-one DRB1*03 positive individuals from each of five U.S. population groups (Caucasoids, African Americans, Asians/Pacific Islanders, Hispanics, and Native Americans) were randomly selected from a database of 82,979 individuals. DRB1*03 alleles were identified by polymerase chain reaction-sequence-specific oligonucleotide probe typing. A total of six DRB1*03 alleles out of 21 known alleles were detected. DRB1*03011 was the predominant DRB1*03 allele in all populations. Caucasoids were found to be the least diversified; only DRB1*03011 was observed. African Americans carried DRB1*03021 at a high frequency. This allele was observed in three other populations. DRB1*0304 was found in Asians/Pacific Islanders and DRB1*0305, DRB1*0307 and a new allele, DRB1*0316, was found in Hispanics. A subset of individuals was also typed for DRB3 alleles. DRB3*0101, DRB3*0202, and DRB3*0301 were detected and seven DRB1-DRB3 haplotypes were defined. Testing of other individuals not included in the DRB1*03 frequency study identified a variation of a common extended haplotype, A1, B8, DR3, which carries DRB1*0304 and two previously unreported DRB1*03 alleles, DRB1*0311 and *0320, are also described.     

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.     

Relative HLA-DRB1*13 allele frequencies and DRB3 associations of unrelated individuals from five US populations

The frequencies of 30 HLA-DRB1*13 alleles and 15 DRB3 alleles were determined for the 5 major U.S. ethnic populations: Caucasians, African Americans, Asian/Pacific Islanders, Hispanics, and Native Americans. A random sampling (163) of DRB1*13-positive individuals from each self-described ethnic group was selected out of a pool of 82,979 unrelated individuals, providing at least an 80% probability of detecting a rare allele that occurred at 1%. These 815 samples were subjected to allele-level SSOP typing and/or DNA sequencing which identified 11 different DRB1*13 alleles. DRB1*1301 and DRB1*1302 were the most common alleles seen in the five major ethnic groups while DRB1*1304 was not detected among Caucasians and DRB1*1305 was not detected among African Americans. DRB1*13 allele diversity was surprisingly more limited among African Americans compared to both Caucasians and Asian/Pacific Islanders. To determine the extent of DRB1*13-DRB3 associations, 504 of these samples expressing only one DRB3-associated DRB1 allele were subjected to PCR-SSOP typing and 14 DRB1*13-DRB3 haplotypes were detected. The distribution revealed that African Americans were significantly different from Caucasians, Asian/Pacific Islanders, and Hispanics. Allele frequency studies such as this further support previous findings that the distribution of HLA types can differ significantly among different ethnic populations.     

Relative frequencies of DRB1*11 alleles and their DRB3 associations in five major population groups in a United States bone marrow registry

One hundred sixty-one individuals from each of five US population groups, Caucasians (CAU), African Americans (AFA), Asians/Pacific Islanders (API), Hispanics (HIS), and Native Americans (NAT), were randomly selected from a volunteer bone marrow registry database consisting of 14,452 HLA-DRB1*11 positive individuals. This sampling provided at least an 80% probability of detecting a rare allele that occurred at 1% in the DRB1*11 positive population. Samples were typed for DRB1*11 alleles by polymerase chain reaction-sequence specific oligonucleotide probe typing (PCR-SSOP). A total of 10 DRB1*11 alleles out of 27 possible alleles were detected. The distribution and diversity of DRB1*11 alleles varied among populations although DRB1*1101 was the predominant DRB1*11 allele in all populations. Caucasians were the least diversified; only four common alleles (DRB1*1101-*1104) were observed. As well as the four common alleles, other groups also carried one or two other less frequent alleles including DRB1*1105 (API), *1106 (API), *1110 (AFA), *1114 (HIS), *1115 (NAT), and *1117 (AFA). A subset (418) of these individuals were also typed for DRB3 alleles. Most (97.6%) showed a strong association of DRB1*11 with DRB3*0202.     

Relative HLA-DRB1*04 allele frequencies in five United States populations found in a hematopoietic stem cell volunteer donor registry and seven new DRB1*04 alleles

The frequencies of 29 HLA-DRB1*04 alleles were determined for five major U.S. populations found within a hematopoietic stem cell volunteer donor registry. One hundred sixty-one DRB1*04 positive individuals from each of the self-described groups, Caucasians, African-Americans, Asian/Pacific Islanders, Hispanics, and Native Americans, were randomly chosen from a database of 82,979 unrelated persons. Subjected to polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) typing, these 805 individuals carried a total of ten different DRB1*04 alleles, ranging from DRB1*0401 to DRB1*0411 with DRB1*0409 conspicuously absent from all five groups. The distribution of DRB1*04 alleles varied among the groups, with DRB1*0401 being predominant in Caucasians, African-Americans, and Native Americans. DRB1*0404 and DRB1*0407 were the two most commonly observed alleles in Hispanics, whereas DRB1*0405 and DRB1*04031 were most common in Asian/Pacific Islanders. The remaining 18 DRB1*04 alleles known at the time of the study were not observed. Although not observed in the frequency study, seven previously unreported DRB1*04 alleles are also described.     

Diversity of alleles encoding HLA-B40: relative frequencies in united states populations and description of five novel alleles

The frequency of each B*40 allele was determined by DNA sequencing in four major United States populations: Caucasians, African Americans, Asians/Pacific Islanders, and Hispanics. Thirty-two individuals from each ethnic group, who were previously described serologically as B40, B60, or B61, were randomly selected out of a pool of 82,979 unrelated individuals for allele characterization. Out of nine different B*40 alleles identified in this study, B*4001 and B*4002 were the two most frequent B*40 alleles in all the population groups. B*4001 was the primary B*40 allele seen in Caucasians (83%) and African Americans (76%), while B*4002 was found in the majority of Hispanics (62%). The distributions of both alleles were comparable in the Asian/Pacific Islander population. These two alleles were the only B*40 alleles detected in Caucasians while four to five additional B*40 alleles were seen in the other population groups. The other B*40 alleles detected in this study included: B*4003 and B*4010 in Asian/Pacific Islanders; B*4012 and B*4016 in African Americans; and B*4004, B*4006, and B*4027 in Hispanics. Analysis revealed significant differences between Hispanics and all other groups as well as between African Americans and Asian/Pacific Islanders. This report also describes five novel B*40 alleles: B*4019, B*4020, B*4024, B*4027, and B*4028.     

High-resolution HLA typing for the DRB3/4/5 genes by sequence-based typing

The high degree of polymorphism of the HLA genes at the nucleotide sequence level has proven sequence-based typing a major typing strategy. For DRB1 the allelic variability is predominantly present in the second exon and by DNA sequencing of exon 2 all hitherto known DRB1 alleles can be detected. For the associated genes DRB3, DRB4 and DRB5 the situation is slightly different. Allelic differences are not limited to exon 2 and the sequence of exon 3 and sometimes exon 4 is needed for complete subtyping. Oligonucleotides to amplify the exons needed for subtyping of DRB3, DRB4 and DRB5 were designed. Gene-specific products were generated to make simultaneous detection of alleles in heterozygous combinations possible. In this way 238 individuals were fully typed for their DRB3, 4 and 5 subtypes. Additional samples were typed for only one of the genes. All samples had been previously typed by PCR-SSP. Concordant typing results were obtained for all individuals tested. The DRB3 alleles typed for included *0101, *0201, *0202 and *0301, for DRB4 they were *01011, *0102 and *0103 and for DRB5 *0101, *0102, *0103, *0105, *0201, *0202 and *0203. All alleles were easily detected by the protocol described except for DRB5*0201. Sequencing of exon 3 and 4 of the DRB5*0201 allele showed this allele to be a sequencing error and the sequences obtained were identical to the exon 2, 3 and 4 sequences of DRB5*0202. Two new alleles were identified in the samples studied, DRB4*0105 and DRB3*0207. Sequence based typing has been recognized as a valuable tool for HLA typing of DRB1, DQB1 and DPB1 since several years. It is shown to be a superior typing method as well in the detection of the different DRB3, 4 and 5 subtypes.     

The relative frequencies of HLA-A*10 alleles in five major United States ethnic populations

The frequency of each A*10 allele was determined in 5 major United States ethnic populations randomly selected from a pool containing 82,979 unrelated individuals. The phenotype frequency of A10 was 10.5% in Caucasians, 14.0% in African-Americans, 21.1% in Asians/Pacific Islanders, 10.6% in Hispanics, and 9.8% in Native Americans. Fifty-nine individuals who had at least one A10 antigen were randomly chosen from each ethnic group for polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) typing. Thirteen of sixteen known A10 alleles were identified in this pool. The most common alleles observed were: A*2601 in Caucasians (55%), Hispanics (58%), and Native Americans (45%); A*3402 in African-Americans (34%); and A*3401 in Asians/Pacific Islanders (61%). The African-American and Asian/Pacific Islander populations differ from all other populations in the distribution of A*10 alleles, particularly, A*2601, A*3401, and A*3402.     

Diversity within the DRB1*08 allele family in four populations from a United States hematopoietic stem cell donor database and characterization of five novel DRB1*08 alleles

The frequencies of DRB1*08 alleles within four major United States populations found within a hematopoietic stem cell volunteer donor database were determined by DNA sequencing of over 60 DRB1*08 positive individuals from each group. Seven of 30 known DRB1*08 alleles were identified within this study population (080101, 080201, 080302, 080401, 0806, 0807, and 0811). Each ethnic group was characterized by a different highly prevalent allele: DRB1*080101 in Caucasians; DRB1*080401 in African-Americans; DRB1*080302 in Asians; and DRB1*080201 in Hispanics. The alleles DRB1*080101, DRB1*080201, and DRB1*080401 were present in all four populations. This report also describes five novel DRB1*08 alleles uncovered during routine human leukocyte antigen typing.     

Identification of three novel alleles: DRB3*0110, DRB1*1140, and DRB1*140102

Three novel human leukocyte antigen class II alleles (DRB3*0110, DRB1*1140, and DRB1*140102) are described here. The three novel alleles were initially detected as previously unidentified SSO hybridization patterns using CANTYPE((R)) reverse hybridization assay. Sequences were determined by cloning/sequencing. DRB3*0110 allele is identical to DRB3*010101, except for a single nucleotide substitution (CGC-->AGC) changing codon 39 from Arg to Ser. This polymorphism has not, until now, been identified in DRB allele. Thus, this is an unusual mutation as the codon 39 is a fairly conserved region. The new DRB1*1140 is identical to DRB1*1116, except for a single nucleotide substitution at codon 67 from ATC (encoding for isoleucine) to TTC (encoding for phenylalanine). This polymorphism is commonly found in DRB1*11 alleles. Compared with DRB1*140101, DRB1*140102 contains a single silent nucleotide substitution (TAT-->TAC, both encoding for tyrosine) at codon 78. This polymorphism is commonly found in DRB1*14 alleles. The three new DRB alleles may have been generated by a point mutation event. The DRB3*0110 and DRB1*140102 were identified in Caucasoid individuals. The ethnic origin of the subject carrying the DRB1*1140 allele is Egyptian. The DRB1*140102 was detected in two unrelated individuals; the DRB3*0110 and DRB1*1140 were only identified once, in a total population of 80,000.     

HLA-A*28 allele frequencies in the five major U.S. ethnic groups

The frequency of each A*28 allele was determined by PCR-SSOP typing in 5 major U.S. ethnic populations: Caucasians, African Americans, Asians/Pacific Islanders, Hispanics, and Native Americans. The percent of serologically defined A28-positive individuals in the 5 populations ranged from 2.7-17.9%. Fifty-nine individuals who were previously serologically typed as A28, A68 or A69 were randomly chosen for allele-level typing from each ethnic group from a database of 82,979 consecutively typed unrelated individuals. The most common A*28 allele for Caucasians, Asians/Pacific Islanders, Hispanics, and Native Americans was A*68012, while A*6802 was found in the majority of African Americans. Only four and three A*28 alleles were seen in Caucasians and African Americans, respectively, while five to six A*28 alleles were seen in the other population groups. The A*6804 and A*6806 alleles were not observed in any of the five ethnic groups.     

Two novel DRB alleles detected by PCR-SSO and confirmed by sequencing: DRB1*0317 and DRB3*0210

We here describe two new DRB alleles (DRB1*0317 and DRB3*0210) which were detected by polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO) and confirmed by direct sequencing. The exon 2 sequence of DRB1*0317 is the result of an intergenic recombination event yielding a hybrid allele between 5'DRB1 and 3' DRB3 sequences with the recombination breakpoint located between codons 39-51. The new DRB3*0210 allele is closest related to DRB3*02021 except for a single nucleotide position at codon 51. Here, the sequence AGG at codon 51 which was a group-specific motif for all DRB3*02 alleles described previously, is replaced by the DRB consensus sequence ACG.     

Six new DR52-associated DRB1 alleles, three of DR8, two of DR11, and one of DR6, reflect a variety of mechanisms which generate polymorphism in the MHC

We have sequenced DNA from six new DR52-associated DRB1 alleles initially detected by PCR/SSOP analysis. Three DR8 associated alleles differed from previously known alleles by single nucleotide substitutions. DRB1*0807 and DRB1*0811 both vary from DRB1*08021 at codon 57 resulting in two different amino acids at this residue. DRB1 *0807 was identified in samples of Brazilian origin while *0811 was identified among samples from the Tlingit Native American population of Southeast Alaska. DRB1*0814, identified in a family of Chinese origin, differed from DRB1*08032 at codon 12 at both the nucleotide and the amino acid level. In addition, two alleles of DR11, DRB1*1113 and *1119, were each detected in Caucasian individuals. DRB1*1113 differs from other DR11 alleles at codons 37, 67, 70 and 74, while DRB1*1119 differs from *1101 by a single nucleotide substitution at codon 67. Finally, DRB1*1418 was detected in a sample from an Asian or Pacific Islander and shares sequences with several other DR52-associated DRB 1 alleles. These six DRB 1 alleles appear to have been generated by either gene conversion events, DRB1*1113 and * 1418, or by point mutations, DRB1*0814, *0807, *0811 and *1119, although the single nucleotide substitutions found in the latter three alleles are also present in at least one other DRB1 allele and, therefore, could have been the product of gene conversions.     

Identification of two novel HLA-DRB1*11 alleles (DRB1*110404 and DRB1*1161) in two Italian cord blood units

We describe the isolation and characterization of two novel HLA-DRB1*11 alleles, officially named DRB1*1161 and 110404. These two new variants were both identified in two Caucasoid individuals. The exon 2 sequence of DRB1*1161 is identical to that of DRB1*110101 except at codon 41, where a nucleotide substitution (GAC>AAC) is responsible for an amino-acidic change from Asp to Asn. The exon 2 sequence of the second novel allele described here, DRB1*110404, differs from that of DRB1*110401 only at codon 34 where the nucleotidic change CAA>CAG gives rise to a silent mutation.     

DRB3 alleles with variations in the annealing sites of commonly used amplification primers

New HLA alleles are often identified initially from observing uncommon patterns found in low-resolution typing performed via polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP). Recently, the HLA-DR oligotyping analysis of two Caucasian, one Caucasian/American Indian and two African American individuals resulted in the identification of three novel DRB3 alleles. Using DRB-specific primer sets commonly employed in amplification-based typing, all four individuals were originally characterized as DRB3 negative. Direct sequencing identified DRB3*0104 (variation at codon 8, TCG instead of TTG), and DRB3*0101202 (variation at intron (-13), G instead of C). One individual appeared to carry a DR52-associated DRB1 allele without an associated DRB3 allele. Lack of conservation at the junction of intron 1 and exon 2 of the DRB3 gene suggests that commonly used DRB-specific primer sets may need to be modified.     

Complete cDNA sequences of the HLA‐DRB1*0402 and DRB1*11041 alleles1

Since the development of the polymerase chain reaction, most HLA class II allele sequencing has been exclusively focused on the highly polymorphic exon 2. We present here the full cDNA sequences of two HLA‐DRB1 alleles, DRB1*0402 and DRB1*11041, both of which were previously only available as partial sequences. HLA‐DRB1*11041 was found to be completely homologous to DRB1*11011 in exons 1, 3, 4, 5 and 6 and HLA‐DRB1*0402 was found to be identical to DRB1*04011 in exons 1, 3, 4, 5 and 6.     

Characterization of a novel DRB1*14 allele (DRB1*1449) in the Han-Chinese population

We report here a novel human leukocyte antigen (HLA) allele, DRB1*1449, in the Han-Chinese population. The nature of the new allele was confirmed by the sequencing-based typing (SBT) method. Genomic DNA and six subclones containing DNA fragment of DRB1 exon 2 were sequenced in both forward and reverse directions. The exon 2 nucleotide sequence of DRB1*1449 is closely related to DRB1*1432 allele based on sequence homology. It has four nucleotide (nt) substitutions at positions 71, 196, 244 and 245 in exon 2, which lead to changes of amino acid sequences. The serological assignment of DRB1*1449 is DR14 based on the serological HLA typing result. This novel allele might be a result of recombination between DRB1*1402 and DRB1*140101 like alleles, which are very common among Chinese population.     

New DRB1* alleles (HLA-DRB1*1135, DRB1*1430 and DRB1*1433) and a confirmatory sequence (DRB1*1133)

Three new DRB1 alleles (DRB1*1135, DRB1*1430 and DRB1*1433) and a confirmatory sequence (DRB1*1133) have been identified after following up unusual or novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns during routine typing of the DRB1*03,*08,*11,*12,*13 and *14 allele groups. Of the new alleles found and described in this paper, two alleles were initially detected by the PCR-RFLP method which produced unexpected restriction polymorphism (DRB1*1133 and DRB1*1135) while the remaining two were found after following up rare allele typings from this technique (DRB1*1430 and DRB1*1433).     

Group-specific amplification of cDNA from DRB1 genes. Complete coding sequences of partially defined alleles and identification of the new alleles DRB1*040602, DRB1*111102, DRB1*080103, and DRB1*0113

We present here the complete coding sequences, previously unavailable, of the DRB1 alleles DRB1*030102, *0306, *040701, *0408, *1327, *1356, *1411, *1446, *1503, *1504, *0806, *0813, and *0818. For cDNA isolation, new group-specific primers located at the 5′UT and 3′UT regions were used to carry out allele-specific amplification and a convenient method for determining full-length sequences for DRB1 alleles. Complete coding sequencing of samples previously typed as DRB1*0406, DRB1*080101, and DRB1*1111 revealed new alleles with noncoding nucleotide changes at exons 1 and 3. In addition, we found a novel allele, DRB1*0113, whose second exon carries a sequence motif characteristic of DRB1*07 alleles. The predicted class II haplotypic associations of all alleles are reported and discussed.     

Identification of two novel HLA-DRB1 alleles, DRB1*0903 and DRB1*1145

Two new HLA-DRB1 alleles were identified by sequencing-based typing in the oral submucous fibrosis and buccal cancer patients of Taiwan. They have been officially named HLA-DRB1*0903 and DRB1*1145 by the World Health Organization Nomenclature Committee. The complete exon 2 sequence of DRB1*0903 was identical to that of the DRB1*090102 but differed by nucleotides of position 207-210 and 216 (AGAC, C replacing GCGG, and G). The DRB1*1145 was identical to the DRB1*110101 except for three nucleotide substitutions at codon 199, 220, and 221 (A, CT replacing T, and GC). Two complete exon 2 sequences of those new alleles had been deposited in the EMBL Sequence Database under accession number AY465114 and AY465115, respectively.