High-resolution HLA typing for the DQB1 gene by sequence-based typing

Abstract: The ideal high-resolution typing strategy for polymorphic genes is sequence-based typing. SBT of genomic DNA has been developed for the HLA class H genes DRB1, DRB3/4/5 and DPB1. For the DQB1 gene the sequence-based typing method was shown to cause a number of problems. To resolve those problems, different primers to amplify and sequence exon 2 of DQB1 were designed and tested. With several primer combinations, preferential amplification was observed in individuals heterozygous for DQB1*02/*03 and DQB1*02/*04. The preference was for DQB1*02 in many instances but could also be demonstrated for DQB1*03 or *04 and resulted occasionally in allelic drop-out. The best primer combination was selected and successfully used to type individuals heterozygous for DQB1*02, *03 and*04. To distinguish DQB1*0201 and *0202, primers for amplification and sequencing of exon 3 were developed and correct subtyping was obtained. The ambiguous typing DQB1*0301/*0302 and DQB1*0303/*0304 was resolved by allele-specifk amplification and sequencing. A total of 258 individuals were fully typed for their DQB1 subtypes. All samples had been previously typed by PCR-SSP and serology. Concordant typing results were obtained for all individuals tested. The DQB1 alleles detected included *0501, *0502, *0503, *0601, *0602, *0603, *0604, *0609, *0201, *0202, *0301, *0302, *0303, *0304, *0401 and *0402. Sequence-based typing of the DQB1 gene proved a reliable typing strategy for assignment of the different DQB1 alleles after intensive selection of primers and test conditions.     

HLA-DQB1 allele typing by a new PCR-RFLP method: Correlation with a PCR-SSO method

We have developed a new PCR-RFLP method for HLA-DQB1 typing. This method was easy to follow, requiring only one DQB1 generic amplification and 5 endonucleases to assign 14 out of 15 HLA-DQB1 alleles. In addition, we determined that by using one generic amplification and two enzymes (Sau96 I and Hae III) it was possible to type the generic specificities: DQw2, DQw4, DQw5, DQw6, and DQw7, DQw8-9, providing a practical alternative for serological HLA-DQ generic typing. We also performed a side-by-side correlation with a PCR-SSO typing method and found an almost 100% concordance between the methods. The limitations of these methods were: 1) the PCR-RFLP method did not allow the differentiation between the HLA-DQB1*0602 and *0603 alleles; 2) the PCR-SSO method gave crosshybridization signals in the detection of *0302 or *0303 alleles. Our results suggested that both methods, PCR-RFLP and PCR-SSO, are useful alternatives for HLA-DQB1 typing.     

Sequence-based typing of HLA class II DQB1

Due to the expanding number of known HLA class II DQB1 alleles, high-resolution oligotyping is becoming ineffective, therefore a sequence-based typing (SBT) strategy was developed to provide rapid and definitive typing of HLA-DQB1. HLA-DQB1*02, *03, *04, *05, and *06 alleles were individually amplified by polymerase chain reaction (PCR) using exon 2 group-specific primers. Forward and reverse PCR primers were tailed with M13 universal and M13 reverse sequences, respectively. Subsequent bi-directional cycle-sequencing was carried out using Cy5.5-labeled M13 universal primer and Cy5.0-labeled M13 reverse primer. Automated sequencing was performed in 30 min using a Visible Genetics, Inc. (VGI) MicroGene Clipper Sequencer. Full concordance was observed between this SBT method and oligotyping among 151 individuals.     

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.     

A new HLA–DQB 1*0306 allele sharing motifs from DQB 1*03032 and DQB1*04 sequences

We have discovered a new HLA-DQB 1 allele in a Japanese family, MAT. In the family the new allele segregates in three generations and demonstrates the positive association with DRB 1*0901. We observed a novel RFLP pattern in the course of examining the modified PCR-RFLP method for HLA-DQB 1 genotyping. The PCR-SSOP analysis also showed a new hybridized pattern. Sequence analysis of the allele indicates that it was generated by a gene conversion-like event between the HLADQB 1*03032 and one of DQB 1*04 contemporary alleles. This new allelic product did not react with all of allosera and monoclonal antibodies against DQ1, DQ2, DQ3, DQ4 and DQ7. The HLA molecule encoded by the allele is not defined by serology. This new allele was officially recognized and named DQB 1*0306 by the WHO Nomenclature Committee in November 1995.     

Identification of the HLA-Cw*0774 and DQB1*060105 alleles

Cw*0774 differs from Cw*070201 by one nucleotide within the coding sequence of exons 2–4. DQB1*060105 differs from DQB1*060101 by one nucleotide within the coding sequence of exons 2–3.     

用PCR－RFLP方法研究新疆地区汉族HLA－DQA1和－DQB1基因多态性

应用ＰＣＲ－ＲＦＬＰ技术，对新疆地区汉族健康群体进行了ＨＬＡ－ＤＱＡ１（４９人）和－ＤＱＢ１（４７人）基因分型。在ＤＱＡ１８个等位基因中，ＤＱＡ１０３０１的基因频率最高（３２．５６％），０４０１最低（１．０２％）。在ＤＱＢ１１６个等位基因中，ＤＱＢ１０２０１（２０．２１％）、０３０１（１５．９６％）、０３０３（１４．８９％）为最常见；没有观察到０５０３２、０５０４和０６０５。与河北固安县及江浙沪地区汉族群体进行比较，ＤＱＡ１基因未发现存在差异。而ＤＢ１０６０２（３．１９％）与固安汉族（Ｐｃ＝０．０１４４）和上海汉族（Ｐｃ＝０．０１４０）有显著差异，ＤＱＢ１０５０３（８．５２％）与上海汉族有显著差异（Ｐｃ＝０．０２１６）。     

Identification of a novel HLA-DQB1 allele, DQB1*0314, by sequence-based typing in the Korean population

The polymorphism of HLA class II genes is largely confined to the exon 2 region. Sequence analysis of exon 2 of the DQB1 gene revealed the novel polymorphism in the Korean population. The new DQB1 allele, DQB1*0314, was differed from DQB1*0304 only at codon 46 (GAG-->GGG), corresponding to non-synonymous amino acid change (Glu-->Gly).     

Two new HLA-DQB alleles routinely identified by sequence-based typing: DQB1*030103 and DQB1*0505

Here, we report the identification of two novel human leukocyte antigen-DQB1 alleles, DQB1*030103 and DQB1*0505, found by routine typing using commercial kits.     

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.     

Identification of two novel HLA class II alleles, DQB1*0311 and DQB1*0620

Two novel HLA class II alleles have been identified in routine typing of a kidney transplant patient and a cord blood unit from the Australian Cord Blood Bank in Sydney. Sequence analysis of exon 2 of the DQB1 genes revealed the novel polymorphism. A substitution of A to C at nucleotide position 136 has been identified for the DQB1*0311 allele when compared to the closest-matched allele, DQB1*030201. An identical substitution has also been identified for the DQB1*0620 allele when compared to the closest-matched allele, DQB1*0602. The substitution results in an amino acid change from methionine to leucine at position 46 implicating different specificity and affinity of antigen binding.     

DQB1*0202 and the new DQB1*0203 allele: a fourth pair of DQB1 alleles differing only at codon 57

Amino acid 57 of DQ β chains is of functional importance as it influences peptide binding, is part of B and T cell epitopes, and is associated with susceptibility and resistance to insulin-dependent diabetes mellitus and humoral immunodeficiencies. Polymorphism of codon 57 is conserved in primates and in HLA class II B genes implying that balancing selection operates on this residue. Previously, three DQB1 allele pairs have been described, that only differ at residue 57. In an African-American Black individual with the HLA pheno-type A23, 30; B58, 63; Cw6; DR18, 12; DR52; DQ5, 2, we found a fourth example of this dimorphism; the new DQB1*0203 allele, that was identical to DQB1*0202 except for codon 57, which encodes aspartic acid and alanine respectively in the two alleles. The class II haplotype carrying the new allele was deduced to be DRB1*0302, DRB3*0101, DQA1*05011, DQB1*0203     

DQB1 intron 3 sequences provide an insight into the evolution of DQB1 alleles and form the basis for intron-based sequencing

The goal of this project was to obtain sequences of intron 3 of DQB1 in order to develop a sequencing-based typing protocol that provides a complete DQB1 exon 3 sequence. Current protocols do not provide complete sequences of exon 3, thus not allowing to differentiate common and well-documented alleles DQB1*0301 and *0319 and resolve some common trans-ambiguities with group-specific sequencing primer (GSSP) sequencing using positions 641 and 650. Samples homozygous for the most common DQB1 alleles were used to obtain intron 3 sequences, which were used to design intron-based primers for exon 3 amplification. The protocol was extensively validated; no allele dropouts were observed. The presented protocol allows differential typing of DQB1*0301 and *0319 and resolves some common trans-ambiguities.     

Sequence-specific priming and exonuclease-released fluorescence detection of HLA-DQB1 alleles

Molecular typing of HLA DQB1 alleles, employing sequence-specific primers (SSP) for PCR amplification, was used to test a novel method that eliminates the requirement for subsequent gel electrophoresis or additional hybridization steps by directly detecting positive reactions. We have evaluated the performance of this fluorescence-based oligonucleotide probe assay to assign the most common DQB1 alleles on DNA from 14 homozygous cell lines and in a blind study of 50 diabetic patient samples that had been previously typed at the DQB1 locus using SSOP and conventional SSP-based approaches. We used a panel of 14 DQB1 SSP primer pairs, internal control primers, and a combination of 4 fluorescent oligonucleotide probes to detect 14 alleles or groups of alleles and controls. We can reliably detect single-base allelic differences, observe 100% concordance with the results obtained using both of the standard methods, and are able to further subtype several alleles that are not easily distinguished using SSOP (e.g. DQB1 *0401/0402 and DQB1 *0302/ 0303). Sequence-specific priming and exonuclease-released fluorescence (SSPERF) detection is technically simple and can be performed in less than 2 hours, including DNA extraction, PCR amplification, data analysis and allele identification. This method is particularly useful for the analysis of large numbers of samples, for which high throughput is critical and for which gel-based approaches are difficult to perform. This technique may also be useful for small-scale class I and class II molecular typing in clinically oriented laboratories.     

Characterization of a novel HLA-DQB1 allele, DQB1*020102

A novel human leukocyte antigen (HLA)-DQB1 allele, DQB1*020102, was detected in a 28-year-old woman of Han ethnic in Guangzhou, China. Compared with HLA-DQB1*020101 and HLA-DQB1*0202, they differed in only one nucleotide at the position 167 (C to T) of exon 2, which was a highly conserved position. This is a synonymous mutation, which does not cause any change in the amino acid sequence of mature protein.     

DQB1*0323 is the result of a gene conversion event between a DQB1*06 and a DQB1*030303 allele

A novel human leukocyte antigen-DQB1 allele, DQB1*0323, was identified in a volunteer hematopoietic stem cell donor. DQB1*0323 differs from the closely related allele DQB1*030303 in five nucleotide positions.     

Sequence-based HLA high-resolution retyping of a bone marrow donor/recipient pair reveals the novel HLA allele DQB1*0322

In this paper, we characterize the novel human leukocyte antigen (HLA)-DQB1*0322. We found this novel allele in a hematopoietic stem cell donor. The donor and the recipient were high-resolution HLA retyped using sequence-based typing. Both, the female patient and her donor were previously typed HLA identical, which was confirmed with the exception of the novel DQB1 allele. The novel allele is characterized by a nucleotide exchange 'G' to 'A' at position 485 in exon 3. This affected codon 130-arginine (CGG), which is replaced by glutamine (CAG) in the new allele DQB1*0322. The transplant was performed because of an acute myeloid leukemia at first remission.