HLA-DR2 HAPLOTYPIC DIVERSITY IN POPULATIONS OF SOUTH-EAST ASIA,NORTHERN CHINA,MELANESIA AND AUSTRALIAN ABORIGINES USING PCR-RFLP FOR DRB1, DRB5, DQA1 AND DQB1. A NOVEL DRB1 ALLELE: DRB1 * 16022

The polymorphism of the human leucocyte antigen HLA-DR2 and the heterogeneity of HLA-DR2 class II-related haplotypes (HLA-DRB1-DRB5-DQA1-DQB1) were investigated in four populations of east and south-east Asia (SEA) and five Melanesian populations using TaqI restriction fragment length polymorphism (RFLP) analysis, and the polymerase chain reaction (PCR) amplification-based techniques PCR-RFLP and sequence-specific oligonucleotide (SSO) typing. The haplotype DRB1*1502-DRB5*0101-DQA1*0102-DQB1*0601 was common in Malaysians, Javanese, Thursday Islanders, Madang, Goroka and the Australian Aborigines, while DRB1*16021-DRB5*0101-DQA1*0102-DQB1*0502 was common in the Thai and Thursday Islanders. DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602 was present at a high frequency in Northern Chinese, Goroka, Watut and Australian Aborigines. The study describes four rare or unusual haplotypes: HLA-DRB1*1501-DRB5*0101-DQA1*0101-DQB1*0601, DRB1*1502-DRB5*0101-DQA1*0101-DQB1*0502, DRB1*1502-DRB5*0102-DQA1*0102-DQB1*0502 and DRB1*1501-DRB5*0101-DQA1*0101/2-DQB1*0503; the latter two were confirmed by segregation in two Javanese families. A new DR2 allele, initially detected by PCR-RFLP and confirmed by DNA sequencing as DRB1 * 16022 (previously designated DRB1 * 16Madang), was seen in a Madang individual. A new HLA-DR2 TaqI RFLP subtype, locally designated as DR15U, is also described. This RFLP subtype segregated in a Javanese family and correlated with a typically SEA haplotype, DRB1*1502-DRB5*0102-DQA1*0101-DQB1*0501. The allele HLA-DR16Thai, determined by TaqI DRB RFLP, was found by PCR-RFLP and SSO typing to correlate with a unique SEA haplotype, HLA-DRB1*16021-DRB5*0101-DQA1*0102-DQB1*0502, and was observed in the Thai, Malaysian, Thursday Islander, Javanese and Northern Chinese populations.     

An HLA-DR11/DQ3 haplotype with a DRB1*0301 sequence motif in the third hypervariable region of the HLA-DR beta-1 chain: molecular and serological analysis of its generation in a European Caucasian family

The formation of a new human leukocyte antigen (HLA)-DRB1 allele (DRB1*0340) has been detected during the routine testing of a European Caucasian blood and potential stem cell donor and his family. HLA typing of the donor with two polymerase chain reaction - sequence specific oligonucleotides (PCR-SSO) systems yielded inconclusive results. HLA typing of the family members including sequence-based typing of DRB1 in both directions after haplotype-specific amplification showed that the allele had most likely formed by a double crossover event in exon 2 of the DRB1 gene. The HLA haplotype containing the new allele was most probably derived from the father, who was typed as HLA-DRB1*0301,*1101 and DRB3*0101,*0202. The comparison of the sequences of the paternal DRB1 and DRB3 alleles with the exon 2 sequence of the DRB1*0340 showed that it had most likely formed through an uptake of at least the sequence part codons 58–77 of DRB1*0301 (donor) by DRB1*1101 (acceptor). We suppose that the recombination sites are located in the sequences from codons 38–57 and codons 78–88. At the protein level, more than 50% of the alpha-helical structure of the DRB1*1101 chain is replaced by a DRB1*0301-derived sequence with the exchange of several amino acids. Serological typing of the allele showed HLA-DR3. However, one monoclonal anti-DR11 of five DR11-reactive antibodies reacted positive, which might indicate residual immunogenic epitopes of DRB1*1101. HLA alleles that are most similar to HLA-DRB1*0340 are DRB1*030501, *0317, *0329 and *1107 with at least four amino acid differences in exon 2. In conclusion, HLA-DRB1*0340 is a new allele with unique properties compared with other known HLA-DRB alleles with regard to antigenicity, T-cell receptor-binding and peptide-binding possibilities.     

Identification of a new HLA-DRB1 allele (DRB1*0318) in three members of a Caucasian Spanish family

This communication reports the identification of a new allele HLA-DRB1*03 in three members of a Caucasian Spanish family. The new allele has been officially named HLA-DRB1*0318 by the World Health Organization Nomenclature Committee. The exon 2 sequence of this new allele is identical to that of DRB1*03011 except for the first nucleotide of codon 45. The nucleotide change (C replacing G) leads to the amino acid substitution of glycine to arginine (GGG-->CGG) at position 45. This position of the beta1 domain shows very little polymorphism among DRB1* alleles (nucleotide changes at this position have only been reported for DRB1*1436 and DRB1*0105) and locates in the vicinity of the highly polymorphic position 47, which is a constituent of the groove's pocket interacting with the amino acid position 7 of the antigen peptide. The familial study showed that the new allele was maternally transmitted into the HLA-A*3002, -B*1801, -Cw*0501, -DRB1*0318, -DRB3*0202, -DQB1*0201 haplotype. Interestingly, the two siblings of the family, which were HLA identical and suffered of insulin-dependent diabetes mellitus (IDDM), were carriers of the two HLA haplotypes (DRB1*03/DQB1*0201 and DRB1*04/DQB1*0302) reported as susceptibility markers to IDDM in Caucasians.     

New DR5 sequences: a novelDRB1*11122 allele identified in Paiwan tribe members of Taiwan and a corrected sequence for the DRB1*1201 allele

We report herein the identification of a new DRB1 allele using sequence-based typing (SBT). This novel allele, HLA-DRB1*11122, was found in an aboriginal individual (SWP71) from the Paiwan tribe in the southern part of Taiwan. This individual was typed by SBT method as having an HLA genotype of HLA-A*24021/24021, HLA-B*4001/4002, HLA-DRB1*11122/15011, HLA-DRB3*0202, and HLA-DRB5*01011. This new allele differs from DRB1*1112 in the polymorphic exon 2 only at codon 34 (CAA-->CAG; both specify glutamine) and from DRB1*1110 in the exon 2 sequence only at codon 32 (CAT-->TAT; H32T). The most likely candidate allele which is found in the aboriginal populations of Taiwan and which may mutate into this new allele is DRB1*11011. DRB1*11122 allele differs from DRB1*11011 allele in the polymorphic exon 2 at both codon 34 (CAA-->CAG) and codon 37 (TAC-->TTC; T37F). This novel HLA-DRB1*11122 allele was also found in another aboriginal individual (SWP90) from the same Paiwan tribe. This SWP90 individual was typed by SBT method as having an HLA genotype of HLA-A*24021/24021, HLA-B*4002/5502, HLA-DRB1*11122/1201, and HLA-DRB3*01011/0202. However, the original DRB1*1201 sequence from HERLUFF was found to be erroneously reported and the corrected sequence from SWP90 is now presented herein.     

Identification of a novel HLA-DRB1 allele,DRB1*0108, by sequence-based DRB typing in two siblings

We report here a novel DRB1 allele identified during sequence-based HLA-DRB typing. This allele was detected during routine HLA typing of a patient and his family prior to bone marrow transplantation. The new allele, DRB1*0108, was found in the patient and in a brother. Molecular cloning and sequencing confirmed that the new DRB1 allele is identical to DRB1*0101 at exon 2 except for a single nucleotide substitution at codon 37 (TauCC-->TauAlphaC), changing the encoded serine to tyrosine. This position of the beta1 domain lies in the floor of the antigen-binding groove and shows the highest polymorphism among DRB1 alleles.     

Polymorphism of human HLA-DRB1 antigens generated by genetic exchange between DR2 (DRB1*15011) and DR6 (DRB1*1405) alleles: a novel DRB1 allele (DRB1*1437) identified in a Paiwan tribe member of Taiwan

We report herein the identification of a new DRB1 allele using sequence-based typing (SBT). This novel allele, HLA-DRB1*1437, was found in an aboriginal individual from the Paiwan tribe in the southern part of Taiwan. This individual was typed by SBT method as having an HLA genotype of HLA-A*02011/0203, HLA-B*15011/3901, HLA-DRB1*11011/1437, HLA-DRB3*0202/0202, and HLA-DPB1*0501/1301. This new allele differs from DRB1*1309 in the 5'-end nucleotide sequence of polymorphic exon 2 at codon 16 (CAT-->CAA; H16Q), codon 37 (AAC-->TTC; R37F), codon 47 (TTC-->TAC; F47Y), and codon 58 (GCC-->GCT; both specify alanine). By sequence comparison, it was found that this new allele has a 5'-end sequence (from amino acid residues 7 to 66) identical to that found in the DRB1*1405 allele and a 3'-end sequence (from amino acid residues 58 to 94) identical to that found in the DRB1*15011 allele. Both DRB1*1405 and DRB1*15011 alleles have been identified among the Paiwan members (Note).     

A novel HLA-DRB1 sequence, DRB1*11272

We describe the complete exon 2 sequence of a novel HLA-DRB1 allele, DRB1*11272. This allele differs from the DRB1*11271 allele by a synonymous mutation in codon 77 where an AAT is replaced with AAC, both encode for the amino acid asparagine. The same motif at codon 77 has also been found in DRB1*1107, DRB1*1333, DRB1*0422 and in most DRB1*03 alleles. A partial exon 2 sequence of this allele has previously been deposited in the EMBL Sequence Database under the accession number AF186407.     

Analysis of the complete cDNA sequences of HLA-DRB1 alleles with group-specific amplification primers in the Chinese Han population

Currently for the majority of HLA-DRB1 alleles the focus has been mainly on exon 2 and complete cDNA sequences of HLA-DRB1 alleles are rare. In this study, we analyzed the complete coding sequences of partial alleles of HLA-DRB1 locus. The cDNA was amplified by polymerase chain reaction using the group-specific primers located in the 5'- and 3'-untranslated regions to obtain the complete coding sequences. The amplification products were sequenced using an ABI BigDye? Terminator Cycle Sequencing kit. The HLA-DRB1 allele phylogenetic tree was analyzed by dnaman software. Full-length cDNA sequences of 22 HLA-DRB1 alleles were obtained in this study. HLA-DRB1*08:09, DRB1 *12:02:01, and DRB1*13:12 alleles were first reported for complete coding sequences. The sequences of exon 1 of HLA-DRB1*04:06:01, DRB1*08:03:02, and DRB1 *14:07:01 were newly presented. The complete coding sequences of HLA-DRB1 *01:01:01, DRB1*03:01:01:01, DRB1*04:01:01, DRB1*04:05:01, DRB1*07:01:01: 01, DRB1*09:01:02, DRB1*10:01:01, DRB1*11:01:01, DRB1*12:01:01, DRB1*13: 01:01, DRB1*13:02:01, DRB1*14:04, DRB1*14:54, DRB1*15:01:01:01, DRB1*15: 02:01, and DRB1*16:02:01 were identical to those previously reported. Forty polymorphic positions in complete coding sequences outside exon 2 of these HLA-DRB1 alleles were confirmed. According to the phylogenetic tree of full-length coding sequence, the HLA-DRB1 allele was classified into seven major allelic lineages. In conclusion, a protocol for HLA-DRB1 cDNA amplification and sequencing was improved and the data may help to determine the polymorphism of coding sequences outside exon 2.     

SEQUENCE OF A NEW HLA-DR ALLELE,DRB5*0106

We report here the exon 2 sequence of a new HLA-DRB5 allele, DRB5*0106, that was identified in two volunteer bone marrow donors from the Swiss national registry. This new allele differs from DRB5*0101 by five amino acids at positions 67, 70, 71, 85 and 86. It is associated with DRB1*1501 and DQB1*0602. This unusual DRB1*1501–DRB5*0106 association increases the complexity of the DR2 group, although it appears to be very rare, at least in our population. HLA-DRB5*0106 can be readily detected upon DR generic oligotyping, provided the two probes that mark the major DRB5 subtypes, DRB5*0101 and DRB5*02, respectively, are included in the assay.     

HLA-DRB11124, the missing link between DRB11101 and DRB11115?

The DRB1^*1115 allele differs from DRB1^*1101 in two nucleotides at codons 37 and 38. Since this peculiar motif is also found in the HLA-DRB5^*0101 allele we speculated that a double recombination event could have brought the motif to a DRB1^*1101 allele. Interestingly, we observed in the course of the 1995 German cell exchange study a novel DRB1^*11 variant. We typed blood sample DZA95-7c as DRB1^*01 and DRB1^*11 by low resolution, ligation based DNA typing and as DRB1^*0101 and DRB1^*1124 by sequencing based typing. The nucleotide sequence of exon 2 of HLA-DRB1^*1124 was identical with HLA-DRB1^*1115 except nucleotide 112 at codon 38 which is the same as in HLA-DRB1^*1101 alleles. Thus the generation of the HLA-DRB1^*1115 allele can be envisaged as a series of point mutations. The HLA-DRB1^*1101 allele is converted to the HLA-DRB1^*1124 allele by a T to G exchange at position 109. A G to T mutation at position 112 finally led to the DRB1^*1115 allele. The HLA-DRB1^*1124 allele is rare we have not found it in more than 150 DRB1^*11 positive unrelated individuals typed by nucleotide sequencing.     

Description of a novel HLA-DRB1 allele,DRB1*1207

A new DRB1 allele has been identified during routine HLA-DRB1 generic typing with sequence specific oligonucleotides. The new allele was confirmed by PCR-restriction fragment length polymorphism analysis and subsequently sequenced. This showed the novel allele, named HLA-DRB1*1207, was similar in sequence to DRB1*12011, except for codon 69 of exon 2.     

Identification of a new HLA-DRB1 allele in three members of an Italian family

Abstract A new human leucocyte antigen (HLA)-DRB1 allele, HLA-DRB1*1149, has been identified in three members of an Italian family during routine sequence based typing. This new allele differs from HLA-DRB1*110101 only for a single nucleotide substitution at position 113 of exon 2 resulting in an amino acid change from Valine (GTG) to Alanine (GCG) at codon 38.     

DRB1*14 diversity and DRB3 associations in four major population groups in the United States

At least 59 DRB1*14 positive individuals from each of four U.S. population groups, Caucasoids, African Americans, Asians/Pacific Islanders, and Hispanics, were randomly selected from a database of 82,979 individuals. DRB1*14 alleles were identified by DNA sequence analysis using intron-specific primers to obtain complete exon 2 sequences. Only 23% of the known DRB1*14 alleles were detected. DRB1*14011 was the predominant DRB1*14 allele in three populations while Hispanics carried DRB1*1402 and DRB1*1406 more frequently. Asians/Pacific Islanders were the most diversified carrying seven alleles. DRB3*0101, DRB3*02021 and DRB3*0210 were detected in a subset of individuals typed for this locus and 15 DRB1-DRB3 haplotypes were defined. This study completes the exon 2 sequences of previously identified alleles, DRB1*1405-*1408, including the identification of two silent codon 90 variants of DRB1*1407. In addition, two new DRB1*14 alleles, DRB1*1441 and DRB1*1442, are described.     

HLA-DR,DQ nucleotide sequence polymorphisms in five Melanesian populations

HLA-DRB1 nucleotide sequence polymorphisms have been examined in 304 Melanesians from the Papua New Guinean coast (Madang), islands (Rabaul) and highlands (Goroka), and from New Caledonia and Fiji. A total of 20 HLA-DRB1 alleles were detected by oligonucleotide hybridizations of exon 2 HLA-DRB1 polymerase chain reaction products, in a typing protocol designed to detect all 42 officially-designated HLA-DRB1 alleles. DRB1*1502 and 1101 alleles were the most common alleles in coastal and island Melanesians, while DRB1*1501, 1502 and 1408 predominated in Papua New Guinean highlanders. Undefined mixed lymphocyte reaction determinants in earlier studies of Melanesians could be accounted for in the present study as DRB1*0410, 1407 and 1408 in Papua New Guinean highlanders and as DRB1*1104 and 1602 in coastal people. Nucleotide sequence polymorphisms at HLA-DQA1, -DQB1, -DRB3 and -DRB5 were also determined for estimating HLA-DR,DQ allelic disequilibrium relationships; unusual haplotypes in Melanesians included DBR1*1502, DRB5*0101 and DRB1*0410, DQB1*0402. Previous claims of limited heterogeneity in the HLA-DR allele repertoire in Melanesians are now seen to reflect limitations of early typing reagents rather than any dramatic restriction in HLA-DR allelic diversity.     

HLA-DRB1*1218新等位基因克隆测序鉴定及内含子序列分析

目的 鉴定中国人群人类白细胞抗原(human leukocyte antigen,HLA)DRB1基因,分析新等位基因第1和2内含子序列信息.方法 采用聚合酶链反应-序列特异寡核苷酸探针反向杂交法(polymerase chain reaction-sequence specific oligonucleotide probes,PCR-SSOP)对广东地区随机正常人群进行HLA常规基因分型,发现1个与HLA-DRB1*120201相近的未知基因,对先证者DNA应用组特异性引物扩增HLA-DRB1位点第2外显子,PCR产物经克隆到质粒载体中以获得单链,对克隆所得产物进行HLA-DRB1基因的第2外显子及第1和第2内含子双向测序分析.并与DRB1*120201基因序列的第2外显子和DRB1*03010101等位基因内含子相比较.结果 发现该个体的一个HLA-DRB1*080302基因被确认,而另一个HLA-DRB1基因为新等位基因,其序列被GenBank接受(编号为FJ481086).新等位基因与最相近的DRB1*120201相比,在第2外显子上有1个核苷酸的不同,即第262位G→C(密码子59 GAG→CAG,氨基酸59 Glu→Gln).DRB1*1218与DRB1*03010101等位基因第2内含子序列完全相同,而与DRB1*03010101等位基因第1内含子序列相比较有12个碱基不同.结论 发现并鉴定一个新的HLA等位基因,经世界卫生组织HLA因子命名委员会正式命名为HLA-DRB1*1218.     

Peptide-binding characteristics of the novel allele DRB1*0112 are probably identical to DRB1*0101

So far 11 different amino acid variants of the HLA-DRB1*01 family have been reported. We here describe the identification of a new HLA-DRB1*01 allele in a healthy female Caucasian. The allele was detected by sequencing-based typing during high-resolution typing of a potential unrelated donor from the North German Bone Marrow Registry (NKR). Compared with DRB1*010101, to which it is closest, the new variant is characterized by a new replacement mutation (G-->T) at nucleotide position 202 of exon 2, resulting in the amino acid substitution Arg-->Leu at position 72. Because this amino acid position is not involved in peptide binding or T-cell interaction, it is likely to represent a permissive mismatch to the more common HLA-DRB1*0101 allele.     

HLA-DRB1 and -DRB3 allele frequencies and haplotypic associations in Koreans

We have investigated the frequencies of human leukocyte antigen-DRB1 (HLA-DRB1) and -DRB3 alleles and DRB1-DRB3 haplotypic associations in 800 Koreans. DRB1 genotyping was done using polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) and PCR-single strand conformation polymorphism (SSCP) methods. DRB3 genotyping was done on 447 samples carrying DRB3-associated DRB1 alleles (DRB1*03, *11, *12, *13, and *14) using PCR-SSCP method. The allele frequencies of DRB3*0101, DRB3*0202, and DRB3*0301 were 0.073, 0.136, and 0.120, respectively, and we found one case of a probable new allele (DRB3*01new, 0.001). DRB1-DRB3 haplotypes with frequency (HF) > 0.005 exhibited strong associations between DRB3*0101 and DRB1*1201, *1301, and *1403; between DRB3*0301 and DRB1*1202 and *1302; between DRB3*0202 and DRB1*0301, *1101, *1401, *1405, and *1406 alleles. Most of the DRB1 alleles with frequency > 0.005 were exclusively associated with particular DRB3 alleles with relative linkage disequilibrium values of 1.0, except for DRB1*1201, *1202 and *1301; the rare presence (HF < 0.005) of DRB3*0202 associations were observed for these DRB1 alleles. We also investigated and presented rare DRB1-DRB3 associations in additional 6000 Koreans. Comparison with other ethnic groups revealed that DRB1*0301 and *1301 related DRB1-DRB3 haplotypes vary among different populations, in that Koreans and other Asian populations show less diversity compared with Caucasoids or African Americans.     

New HLA-DRB1* nucleotide sequence of Italian origin: HLA-DRB1*13022

High-resolution polymerase chain reaction using sequence-specific primer typing of the HLA-DRB1 gene of an Italian patient waiting for unrelated bone marrow transplantation revealed a new allelic variant of HLA-DRB1*13. Sequencing the exon 2 of DRB1* gene demonstrated a G-->C transition at the nucleotide 216 resulting in a silent mutation at codon 72: CGG-->CGC. The closest sequence was the HLA-DRB1*1302 and the new allele was named HLA-DRB1*13022. This variant was carried by the haplotype HLA-A*24; Cw*0702; B*39; DRB1*13022; DRB3*0301; DQA1*0102; DQB1*0604 as demonstrated by a family study.     

A novel HLA-DRB1 allele,DRB1*0707

We report herein the identification of a new HLA-DRB1 allele, DRB1*0707. This new allele was seen in a volunteer bone marrow donor (ID#118069) belonging to the German bone marrow donor registry (DKMS). HLA-DRB1*0707 was detected while performing HLA-DRB1 high resolution typing by sequence based typing. This novel allele differs from DRB1*070101 by a single nucleotide substitution at position 163 (C-->T) in exon 2.     

PCR-RFLP typing detects new HLA-DRB1 alleles: DRB1*13022, DRB1*1336 and DRB1*1435

It is difficult to resolve all heterozygous combinations of the HLA-DRB1*03, *08, *11, *12, *13 and *14 allele group in a one-step generic HLA-DRB1 typing system. Therefore, it is common to employ a secondary technique utilizing group-specific primers to amplify this group of alleles separately from the other HLA-DRB1, -DRB3, -DRB4 and -DRB5 alleles. This paper describes a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method for broad typing of the HLA-DRB1*03, *08, *11, *12, *13 and *14 alleles which, as well as being time-efficient and cost-effective, has so far allowed the detection of 10 new alleles. The new alleles were identified after following up unusual or novel PCR-RFLP patterns. Of the 10 novel alleles found so far with this method, seven have been described previously while three, DRB1*13022, DRB1*1336 and DRB1*1435, are presented here.