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.     

Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping.

We previously developed a new technique for HLA class II genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class II alleles. However, small fragments or bands located close to each other on polyacrylamide gels sometimes prevent precise analysis of the RFLP bands. Furthermore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, FokI, MboII and Mn1I can discriminate eight alleles of the DQA1 gene. Similarly 19 alleles of the DPB1 gene can be discriminated with Bsp1286I, FokI, DdeI, BsaJI, BssHII, Cfr13I, RsaI, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.     

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.     

Molecular genetic studies of HLA class II alleles in sarcoidosis

Abstract: Previous HLA serological studies showed positive associations of the DR52 antigen, the DR52-associated antigens (DR3, DR5 and DR6) and the DR8 antigen with sarcoidosis. To investigate the HLA alleles that may contribute to the genetic susceptibility to sarcoidosis at the DNA level, HLA-DRB1, -DRB3, -DQA1 and DQB1 genotyping using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was performed in 63 Japanese patients with sarcoidosis. The frequencies of the DR52-associated DRB1 alleles (DRB1*11, DRB1*12 and DRB1*14 except DRB1*1302), DRB1*08, DRB3*0101, DQA1*0501 and DQB1*0301 were significantly increased in patients compared with healthy controls. The significant increase of DRB3*0101, DQA1*0501 and DQB1*0301 could be explained by linkage disequilibrium with the DR52-associated DRB1 alleles. It must be noted that the DR8 haplotype, which does not possess the DRB3 gene, also showed a significant increase in sarcoidosis. These results suggest that the HLA-alleles responsible for the susceptibility to sarcoidosis are located at the HLA-DRB1 locus rather than the HLA-DRB3, -DQA1 and -DQB1 loci. In contrast, DRB1*1302 may confer resistance to the disease.     

HLA-DQB1 genotyping by a modified PCR-RFLP method combined with group-specific primers

Abstract: We previously reported a simple technique for HLA-DQB genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). However, this method has some problems in that some heterozygotes cannot be discriminated from each other. Furthermore, concomitantly amplified product derived from the DQB2 gene by the primers used previously also obstructs precise DQB1 genotyping. To resolve these problems, we have developed two different pairs of specific primers for selective amplification of the DQB1 gene and also used restriction endonucleases which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA-DQB 1 alleles, making reading of RFLP band patterns much easier. The second exon of the DQB1 gene was selectively amplifed by DQwl group-specific primers and/or DQw2,3,4 group-specific primers using genomic DNAs from 70 HLA-homozygous B-cell lines and 50 healthy Japanese. Of the seven DQwl-associated DQB1 alleles, six alleles could be defined by digestion of 6 restriction enzymes, although DQB 1*0602 and DQB 1*0603 could not be discriminated from each other because of unavailability of suitable enzymes. Similarly, all of the six DQw2,3,4-associated DQB1 alleles could be defined by digestion of 5 restriction enzymes. Using this modified PCR-RFLP method, complete DQB1 genotyping of all heterozygotes is possible except for discrimination between DQB 1*0602 and 0603. Thus this method is simpler and more practical for a routine DNA typing than the PCR-SSO method or our previous PCR-RFLP method.     

HLA-DQB1 genotyping by a modified PCR-RFLP method combined with group-specific primers.

We previously reported a simple technique for HLA-DQB genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). However, this method has some problems in that some heterozygotes cannot be discriminated from each other. Furthermore, concomitantly amplified product derived from the DQB2 gene by the primers used previously also obstructs precise DQB1 genotyping. To resolve these problems, we have developed two different pairs of specific primers for selective amplification of the DQB1 gene and also used restriction endonucleases which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA-DQB1 alleles, making reading of RFLP band patterns much easier. The second exon of the DQB1 gene was selectively amplified by DQw1 group-specific primers and/or DQw2,3,4 group-specific primers using genomic DNAs from 70 HLA-homozygous B-cell lines and 50 healthy Japanese. Of the seven DQw1-associated DQB1 alleles, six alleles could be defined by digestion of 6 restriction enzymes, although DQB1*0602 and DQB1*0603 could not be discriminated from each other because of unavailability of suitable enzymes. Similarly, all of the six DQw2,3,4-associated DQB1 alleles could be defined by digestion of 5 restriction enzymes. Using this modified PCR-RFLP method, complete DQB1 genotyping of all heterozygotes is possible except for discrimination between DQB1*0602 and 0603. Thus this method is simpler and more practical for a routine DNA typing than the PCR-SSO method or our previous PCR-RFLP method.     

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.     

Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping

Abstract: We previously developed a new technique for HLA class II genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class II alleles. However, small fragments or bands located close to each other on polyacryl-amide gels sometimes prevent precise analysis of the RFLP bands. Furthermore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, Fokl, MboII and Mnll can discriminate eight alleles of the DQA1 gene. Similarly 19 alleles of the DPB1 gene can be discriminated with Bsp1286I, Fokl, Ddel, BsaJI, BssHII, Cfr13I, Rsal, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.     

MULTIPLEX ARMS-PCR-RFLP METHOD FOR HIGH-RESOLUTION TYPING OF HLA-DRB1

A reliable method for high-resolution HLA-DRB1 typing using the combination of group-specific amplification and RFLP analysis is described. Group-specific PCR amplification (multiplex ARMS-PCR) was carried out under the same conditions for all groups using seven different primer pairs divided into four groups: (1) DR1 and DR10; (2) DR2, DR7 and DR9; (3) DR3, DR5, DR6 and DR8, and (4) DR4. The subsequent polyacrylamide gel electrophoresis was used to determine the group(s) contained in each sample. DR1, DR2/7, DR3/5/6/8, DR4, DRB1*0901 and DRB1 * 1001 could be distinguished easily using this system. Computer analysis of the various restriction enzyme cleavage sites was carried out on 105 DRB1 allele sequences. It was shown that all DRB1 alleles, except for five allele pairs and some alleles possessing silent mutations, could be distinguished with commonly available restriction endonucleases. Computer analyses on the discrimination of the heterozygous and homozygous combinations were also carried out. Although some heterozygous combinations could not be distinguished with single digestion, double digestion using two restriction enzymes could distinguish most of such heterozygotes. The results of the typing of 100 Japanese individuals using this method showed good agreement with those obtained by other methods.     

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.     

人类白细胞抗原-DRB1第3外显子单链测序方法的建立及其多态性分析

目的 建立人类白细胞抗原(human leukocyte antigen,HLA)-DRB1第3外显子单链测序方法,并分析其多态性.方法 采用组特异性引物扩增HLA-DRB1第2和3外显子序列,扩增产物经双酶切后进行双向测序分析,采用Assign 3.5 SBT分析软件指定等位基因型.结果 PCR产物经直接测序后得到清晰的序列冬,并向IMGT/HLA数据库提交了HLA-DRB1*08:09和DRB1*12:02:01第3外显子全部序列.在检测的25个等位基因中,HLA-DRB1第3外显子全长序列中存在27个单核苷酸多态性位点,占第3外显子序列碱基总数的9.56%.建立的方法可有效区分HLA-DRB1*14∶01∶01/14∶54歧义标本,证实中国人群中存在HLA-DRB1*14∶01∶01.结论 本实验建立的HLA-DRB1第3外显子测序方法是可行的,HLA-DRB1第3外显子存在多态性.     

Additional complexity within the HLA-D region: sequence analysis of two new DRw13-DQw7 haplotypes.

HLA-DRB1 is by far the most polymorphic locus within the HLA-D region with now well over 40 alleles. Nearly one fourth of these alleles are subtypes of DRw6, and these are in most cases undetectable by routine typing procedures. In this paper we present the molecular characterization of two new Caucasian DRw13-DQw7 haplotypes by DNA sequencing of the polymorphic first domain exons of DRB1 and DRB3 loci. The first haplotype, DRB1*1301-DRB3*0101-DQB1*0301, has arisen by a recombination between locus DRB1 from a DRw13-DQw6 haplotype and DQA1 from a DR4-DQw7 haplotype, as determined by DNA sequencing, DQ oligotyping, and restriction fragment length polymorphism typing. The second haplotype, DRB1*1305-DQB1*0301, is characterized by the novel DRB1*1305 allele differing from DRB1*1301 by three amino acids. It probably arose by a gene conversion event between a DRw13-DQw6 allele and DRB1*1101. This allele represents a DRw11/DRw13 hybrid DR molecule with a DRw13 serological epitope in the second hypervariable region and a Dw5 cellular epitope in the third hypervariable region. As determined by sequencing of locus DRB3, this allele is associated with DRw52b. Our molecular analysis of the complex HLA-DRw13 group now allows unambiguous DNA typing of all five DRw13 alleles with seven oligonucleotides, a significant improvement in the context of organ transplantation.     

HLA-DR generic typing by AFLP.

We have described a practical and inexpensive method whereby any individual can be typed and assigned to any of the 14 generic HLA-DR types: DR1, DRw15, DRw16, DRw17, DRw18, DR4, DRw11, DRw12, DRw13, DRw14, DR7, DRw8, DR9 and DRw10. Previous methods to type these specificities include - among others - serology, conventional RFLP, PCR-oligonucleotide typing, and a PCR-RFLP method useful for typing homozygous individuals. In the method reported here, DNA is amplified with a set of group-specific primers and then restricted with a number of endonucleases. Six group-specific pairs of primers have been chosen to avoid cross-amplification with other DRB alleles, including DRB3, DRB4 and DRB5 alleles, and to anneal at uniform temperature: 61 degrees C. Endonucleases were chosen to generate unique patterns of easily recognizable bands that led to unequivocal assignments of HLA-DR generic types in heterozygous as well as homozygous individuals. This technique involves two steps: 1) Amplification of DNA with six different pairs of primers where DR1, DR4 and DRw10 types can be assigned at once, and 2) Endonuclease digests of amplified DNA to assign DR7, DRw8, DR9, DRw11, DRw12, DRw13, DRw14, DRw15, DRw16, DRw17 and DRw18 types. Individuals carrying any combination of all alleles published so far can be typed by this method. The ease, low cost and reliability of this method are discussed.     

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-DR generic typing by AFLP

Abstract: We have described a practical and inexpensive method whereby any individual can be typed and assigned to any of the 14 generic HLA-DR types: DR1, DRwl5, DRwl6, DRwl7, DRwl8, DR4, DRwll, DRwl2, DRwl3, DRwl4, DR7, DRw8, DR9 and DRw10. Previous methods to type these specificities include - among others - serology, conventional RFLP, PCR-oligonucleotide typing, and a PCR-RFLP method useful for typing homozygous individuals. In the method reported here, DNA is amplified with a set of group-specific primers and then restricted with a number of endonucleases. Six group-specific pairs of primers have been chosen to avoid cross-amplification with other DRB alleles, including DRB3, DRB4 and DRB5 alleles, and to anneal at uniform temperature: 61deg;C. Endonucleases were chosen to generate unique patterns of easily recognizable bands that led to unequivocal assignments of HLA-DR generic types in heterozygous as well as homozygous individuals. This technique involves two steps: 1) Amplification of DNA with six different pairs of primers where DR1, DR4 and DRw10 types can be assigned at once, and 2) Endonuclease digests of amplified DNA to assign DR7, DRw8, DR9, DRw11, DRw12, DRw13, DRw14, DRw15, DRw16, DRw17 and DRw18 types. Individuals carrying any combination of all alleles published so far can be typed by this method. The ease, low cost and reliability of this method are discussed.     

HLA-DRB1,3,4,5 and -DQB1 allele frequencies and HLA-DR/DQ linkage disequilibrium of 231 German caucasoid patients and their corresponding 821 potential unrelated stem cell transplants

Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient pairs. Highly accurate HLA typing data were achieved by PCR-SSOP and a combination of group specific PCR-SSP and subsequent sequencing-based typing of nearly the whole second exon of each locus. The alleles DRB1*07, *09, and *10 were analyzed by PCR-reverse dot blot hybridization instead of sequencing. Additionally, DRB1 homozygosity was verified by temperature gradient gel electrophoresis. The identified 2104 HLA-DRB1 and HLA-DQB1 alleles as well as data on HLA-DRB3, -DRB4, and -DRB5 alleles were applied to a statistical program and absolute and relative delta values of DR/DQ linkages were calculated. The achieved data on the HLA-DRB1 allele distribution and on DR/DQ associations in terms of subtypes significantly ensure the typing reliability, since rare allele combinations will result in further investigations. Furthermore, detailed data on the DR/DQ allele associations allow estimations of the number of HLA-A, -B, and -DR matched unrelated stem cell donors necessary for the identification of DRB and DQB subtype identical donors.     

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.     

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.     

Serology, restriction fragment length polymorphism, and sequence analysis of a unique HLA class II antigen, DR5x6.

We analyzed a new class II HLA haplotype, which we have designated DR5x6, by serology, restriction fragment length polymorphism (RFLP), and sequence analysis. As the name DR5x6 implies, the antigen is serologically closely related to both DR5 and DRw6. RFLP analysis of this haplotype suggests a close similarity with DRw11 haplotypes. The DNA sequences encoded by the second exon of its DRB1, DRB3, and DQB1 genes were also determined. Comparison of these sequences with those of alleles at these loci in other haplotypes suggests that this haplotype could have evolved from a DRw11 ancestor haplotype (DRw11-DRw52b (Dw25)-DQw7) by means of: (a) a gene conversion at the DRB1 locus involving DRw8 (Dw8.3) as the sequence donor, plus a point mutation or a gene conversion involving DR4-Dw4; and (b) a recombination event by which this haplotype would have acquired the DRw5a (Dw24) allele at the DRB3 locus.     

HLA genotyping of colorectal carcinoma in the Chinese population.

The HLA-DR genotypes of 61 primary colorectal carcinomas obtained from patients of Chinese origin were determined by using DNA-RFLP. No increase or decrease of a particular HLA genotype could be ascertained with the disease, although we detected an antigen frequency of 29.5% for the serologically ill-defined DR"X3" specificity. We identified and sequenced HLA-DRB1 and DRB3 genes from the DR"X3" haplotype. The DR"X3" DRB1 gene was found to be identical to DRB1*1201 (DR5[w12]). A unique observation is its unusual linkage with DRB3*0101 (DRw52a) or DRB3*0301 (DRw52c) instead of the usual linkage with DRB3*0201/2 (DRw52b). These associations are rare in whites and blacks.