Clarification of HLA-B serologically ambiguous types by automated DNA sequencing

Abstract: Assignment of HLA-B types can be hampered by ambiguous reactivity of the typing sera resulting in inaccurate HLA-B assignments. In this study, 19 Korean samples exhibiting ambiguous serologic reactivities were characterized by DNA sequencing. Alleles identified from 7 samples were previously undetected in this population (B*1517, B*4101, B*4701, B*5001, and B*5106) and from 9 samples were common alleles in this population (B*4002, B*4003, B*4006, B*1501, B*1401, B*67012, and B*5401). Three samples were putative HLA-B homozygotes. Three major factors causing serologic ambiguity were identified: weak or false negative reactivity of typing sera (52.4%); cross or false positive reactivity of the sera (38.1%); and absence of information on the reaction patterns due to the lack of appropriate sera in the typing kit (e.g. B*4101 encoded molecule) or to the presence of recently characterized molecules (e.g. B*5106 encoded molecule) (9.5%). Overall, sequencing was helpful in clarifying ambiguous serologic reaction patterns improving the HLA typing for the Korean population.     

HLA-B Alleles Associated with the B15 Serologically Defined Antigens

ABSTRACT: Cells expressing HLA molecules in the B15 family were identified by serologic typing in routine testing of volunteer donors of various ethnic backgrounds for a bone marrow registry. DNA sequencing was used to identify HLA-B15 alleles associated with each serologic type and to examine the diversity within the B15 antigen family. Alleles which appeared predominantly in each B15 serologic cluster included: B15 with no defined serologic subdivision (B*1501), B62 (B*1501), B63 (B*1516, B*1517), B75 (B*1502, B*1521), and B76/77 (B*1513). Other B*15 alleles were also found associated with the serotypes and some of these alleles (e.g., B*1501 and B*1516) were found in two or more serologic clusters illustrating the complexity of this family. The B15 unsplit and B75 groups were the most complex exhibiting 16 and 7 alleles, respectively, within each serotype. Five new B*15 alleles (B*1530, B*1531, B*1533, B*1534, B*1535) and 5 other new HLA-B alleles (B*38022, B*3910, B*4010, B*51012, and B*5108) were also identified.     

HLA-B*15 subtypes in Burmese population by sequence-based typing

Human leukocyte antigen (HLA)-B*15 encompasses an increasing number of subtypes of more than 150. Frequency studies and a strong genetic association between HLA subtypes and susceptibility to drug hypersensitivity have been reported in different ethnic populations. To identify HLA-B*15 subtypes in Burmese using sequence-based typing (SBT) method, we selected 65 HLA-B*15 -positive samples from 170 unrelated healthy Burmese who were genotyped HLA-B * by polymerase chain reaction with the sequence-specific primer method. The frequency of HLA-B*15 in Burmese was found to be 38.2%. By the SBT method, results showed 10 alleles of HLA-B*15 subtypes. Four common alleles, B*1502 (45.2%), B*1532 (16.4%), B*1525 (12.3%), and B*1501 (8.2%), were found in 82.1% of HLA-B*15 -positive Burmese. Whereas the B*1501 was the highest in the Caucasians, Koreans, and Japanese, the highest frequency of HLA-B*15 alleles in Burmese was B*1502 (45.2%) that is similar to the frequency found in northeastern Thais and Vietnamese. This study is the first report of HLA-B*15 subtypes in Burmese. These results will provide the basic data in the further study in transplantations, genetic association with diseases, and drug hypersensitivity.     

HLA-B22 diversity including a novel B54 variant (B*5507) in the Korean population.

Diversity in the HLA-B22 group was investigated in the Korean population using PCR-SSOP and DNA sequencing analyses. Allelic typing of the B22 gene was performed by gene amplification of the polymorphic exons 2 and 3 of the HLA-B genes from 91 B22 positive individuals followed by a hybridization assay using 63 digoxigenin-labelled probes. Five different SSOP patterns including an unexpected pattern were identified and correlated well with the observed serologic types and with data obtained from DNA sequencing analyses. Novel allele, B*5507, was identified from two unrelated individuals who exhibited standard B54 serologic reactivity but an unexpected SSOP pattern. The DNA sequence of B*5507 is identical to B*5502 in exons 2 and 3 except for a single nucleotide substitution at codon 45 (GAG-->GGG) altering glutamic acid to glycine. Among the already known B molecules, this substitution has been observed only in the B54 molecule encoded by B*5401 allele. This is the evidence that Gly-45 is a crucial site forming the B54 serologic epitope. Interestingly, both alleles (B*5401 and B*5507) exhibit strong association with Cw*0102. Along with previous data, B22 appears to be a very diverse group in the Korean population consisting of at least seven different alleles. B*5401, B*5502, and B*5601 are the most frequent alleles. B*5507, B*5501, B*5504, and B*5604 appear at lower frequencies. Data obtained from this study will be useful in hematopoietic stem cell donor searches as well as in determination of a typing strategy for the HLA-B22 types in this population.     

The relationship between HLA-B45 and B* 5002 in the five major U.S. population groups

The antigen encoded by B*5002 differs in sequence from that encoded by B*5001 only at amino acid residue 167 (consensus tryptophan vs. serine) which results in B45 serologic reactivity. To search for B*5002, the frequencies of alleles encoding the serologically defined B45 antigen were determined by sequence-based typing in 5 major U.S. populations: Caucasians, African Americans, Asians/Pacific Islanders, Hispanics, and Native Americans. The percent of serologically defined B45-positive individuals in the 5 populations ranged from 0.7-9.0%. Thirty-two B45-positive individuals were randomly chosen, when available, for sequence-based typing from each ethnic group from a database of 82,979 consecutively typed unrelated individuals. The B*5002 allele was most prevalent in Hispanic (22%) and Caucasian (9%) individuals, while conspicuously absent in African Americans. In addition, a new allele associated with the B45 antigenic specificity, B*4502, has been identified from an African American individual of Middle Eastern descent. In light of the continuing need to reconcile differences between relationships determined by the sequence homologies among alleles and relationships based on the serologic determinants carried by allelic products when determining the level of HLA match for hematopoietic stem cell transplantation, it is suggested that B*5002 be recognized individually from other B*50 alleles when reporting HLA-B typings for clinical purposes.     

Sequencing-based typing of HLA-B*51 alleles and the significant association of HLA-B*5101 and -B*5108 with Behçet's disease in Greek patients

Beh?et's disease (BD) is widely known to be strongly associated with human leukocyte antigen (HLA) B51 in many different ethnic groups.Recently, HLA-B51 allele typing of Greek BD patients was performed to study the distribution of B*5101-B*5107 alleles in this Greek population, the B51 antigen strongly associated with BD was found to be predominantly encoded by allele B*5101. As it is now known that the B51 antigen can be encoded by 21 alleles, B*5101-B*5121, we performed HLA-B*51 allele genotyping among 58 Greek patients with BD. After serological HLA typing, typing of HLA-B*51 alleles was performed using the polymerase chain reaction-sequencing-based typing (PCR-SBT) method. The frequency of the B51 antigen was found to be significantly higher in the patient group as compared with the control group (75.9% of patients vs 22.0% of controls. In the genotyping of B51 alleles, 34 out of 44 B51-positive patients possessed B*5101, 13 out of the 44 carried B*5108. In contrast, all of the 9 B51-positive normal controls carried B*5101. This study revealed a strong association between Greeks with BD, both B*5101, B*5108, provided important insights into the molecular mechanism underlying the association between HLA status, this disease.     

Serologic ambiguity and allelic frequency of the HLA-B40 family in the Korean population

The most frequently identified HLA-B type in Koreans is HLA-B40 (13.4%). Due to the lack of mono-specific alloantisera and cross reactivity of sera used as typing reagents, discrimination between the serologic splits of B40, B60 and B61, has been a problem in tissue typing laboratories. In this study, an efficient PCR-SSP typing system was established to distinguish B60 and B61 and to assess the difficulty in serologic assignment for these types. The SSP system was also used to elucidate the frequency of B40 alleles (B*4001-B*4008) encoding B40 molecules in the Korean population. Eighty eight unrelated individuals identified serologically as B40 positive were selected from 358 consecutive volunteers from the unrelated bone marrow registry. Seven sets of PCR that amplify exons 2 and 3 of the HLA-B gene using 10 sequence specific primers (SSP) were used for discrimination between B60 and B61, and for B40 allelic typing. A clear discrimination of B60 and B61 was possible in all samples including 48 serologically ambiguous samples (B60 – 14/48; B61 – 34/48) and 5 potentially B40 homozygous samples (B60/B61 heterozygotes – 4/5; B60 homozygote - 1/5). Therefore, the use of a focused SSP approach enhances serologic definition of HLA types in routine clinical testing. In allelic typing, all B60 samples (26) appeared to be B*4001, but B61 samples revealed more heterogeneity (B*4002 – 36/58, B*4003 – 4/58, B*4006 – 18/58). In addition, B*4003 seemed to be closely associated with the A24-Cw3-DRB1*02 haplotype (3/4). The characterization of allele frequency as well as haplotypic association will be helpful in determination of the optimal size of the volunteer marrow donor pool in the Korean population.     

Allelic and haplotypic diversity of HLA-A, -B, -C, -DRB1, and -DQB1 genes in the Korean population

High-resolution human leukocyte antigen (HLA) typing exposes the unique patterns of HLA allele and haplotype frequencies in each population. In this study, HLA-A, -B, -C, -DRB1, and -DQB1 genotypes were analyzed in 485 apparently unrelated healthy Korean individuals. A total of 20 HLA-A, 43 HLA-B, 21 HLA-C, 31 HLA-DRB1, and 14 HLA-DQB1 alleles were identified. Eleven alleles (A*0201, A*1101, A*2402, A*3303, B*1501, Cw*0102, Cw*0302, Cw*0303, DQB1*0301, DQB1*0302, and DQB1*0303) were found in more than 10% of the population. In each serologic group, a maximum of three alleles were found with several exceptions (A2, B62, DR4, DR14, and DQ6). In each serologic group exhibiting multiple alleles, two major alleles were present at 62-96% (i.e. A*0201 and A*0206 comprise 85% of A2-positive alleles). Multiple-locus haplotypes estimated by the maximum likelihood method revealed 51 A-C, 43 C-B, 52 B-DRB1, 34 DRB1-DQB1, 48 A-C-B, 42 C-B-DRB1, 46 B-DRB1-DQB1, and 30 A-C-B-DRB1-DQB1 haplotypes with frequencies of more than 0.5%. In spite of their high polymorphism in B and DRB1, identification of relatively small numbers of two-locus (B-C and DRB1-DQB1) haplotypes suggested strong associations of those two loci, respectively. Five-locus haplotypes defined by high-resolution DNA typing correlated well with previously identified serology-based haplotypes in the population. The five most frequent haplotypes were: A*3303-Cw*1403-B*4403-DRB1*1302-DQB1*0604 (4.2%), A*3303-Cw*0701/6-B*4403-DRB1*0701-DQB1*0201/2 (3.0%), A*3303-Cw*0302-B*5801-DRB1*1302-DQB1*0609 (3.0%), A*2402-Cw*0702-B*0702-DRB1*0101-DQB1*0501 (2.9%), and A*3001-Cw*0602-B*1302-DRB1*0701-DQB1*0201/2 (2.7%). Several sets of allele level haplotypes that could not be discriminated by routine HLA-A, -B, and -DRB1 low-resolution typing originated from allelic diversity of A2, B61, DR4, and DR8 serologic groups. Information obtained in this study will be useful for medical and forensic applications as well as in anthropology.     

Molecular analysis of HLA-B35 alleles and their relationship to HLA-B15 alleles

The HLA-B35 serotype is one of the largest allelic groups of HLA class I molecules and includes four isotypes. Of the four, the B35 variant isoform is relatively rare and is the most acidic form. DNA sequencing of die rare isoforms revealed three alleles, B*1522, B*3511, and B*3517. A phylogenetic tree of HLA-B15- and HLA-B35-related alleles for the exon 2 and 3 nucleotide sequences showed that exon 2 of B* 1522 clusters with B35 alleles whereas exon 3 clusters with B15 alleles. Branches of the tree suggest that the serodeterminants of B35, B62, B63, and B70 may reside in the αl domain, encoded by exon 2. The B*1520 and B*1522 genes, which type as B62 and B35, respectively, are hybrid molecules alternatively using exon 2 and exon 3 sequences of B*3501 and B*1501. A comparison of intron 2 sequences for B*3501, B*1501 and B*1522 suggests that the recombination site may have been in the region at the 3' end of intron 2. Despite being flanked by two highly polymorphic exons (exons 2 and 3), intron 2 is relatively well conserved in the B-locus, and it is characterized by seven to eight tandem repeats of the CGGGG pentanucleotide. A high degree of sequence homology and repetitive sequences are essential for a significant frequency of recombination. In this report, we reveal more about the complex evolutionary history of the HLA-B alleles     

HLA-B*15 subtypes distribution in Han population in Beijing,China, as compared with those of other populations

To identify HLA-B*15 subtypes distribution in Han population in Beijing, People’s Republic of China, 826 unrelated healthy individuals were typed using the polymerase chain reaction-sequence-based typing method. Within the 246 HLA-B*15 positive individuals, 29 HLA-B*15 alleles were identified, the most predominant of which is B*1501 (40.07%), followed by B*1502 (12.87%), B*1511 (12.87%), B*1518 (9.19%) and B*1532 (3.31%). The distribution of HLA-B*15 subtype frequencies was compared between the Beijing Han, eight other Chinese ethnic minorities and six Chinese populations covering the mainland of China, Taiwan, Hong Kong and Singapore. A neighbor-joining phylogenetic tree was constructed and revealed that the Beijing Han population clustered into the northern populations group and had a closer relationship with northern Han and Hui than with southern Han or other ethnic minorities. These results thus provide useful information that can be used in anthropology, selection for bone marrow transplantation as well as in disease-association study, such as in carbamazepine (CBZ)-induced Stevens–Johnson syndrome and toxic epidermal necrolysis.     

Molecular diversity of the HLA-C locus in unrelated marrow transplantation

Abstract: The diversity of HLA-C exon-2 alleles in 56 HLA-A, B, DRB and DQB1-matched patient-unrelated marrow donor pairs was examined by non-cloning polymerase chain reaction-based sequencing of genomic DNA. This method allows simultaneous analysis of both alleles in heterozygous samples. All Cw5-positive individuals encoded a sequence which differed from the published Cw*0501 sequence at position 61. Among 82 samples assigned a single antigen by serologic testing, 64 (78%) were heterozygous for two distinct alleles when tested by sequencing. Cw*1202, 1601 and 15 were identified in samples for which no phenotype could be assigned (C "blank"). Finally, 7 of the 56 HLA-A, B, DRB, DQB1-matched pairs (12.5%) were mismatched for one or both HLA-C alleles. We conclude that sequence-based methods constitute the optimal strategies for typing HLA-C alleles in the unrelated marrow transplant population.     

Identification of two new alleles in a single Korean individual,HLA-B*1568 and HLA-DRB1*1208

We have identified a new HLA-B*15 allele and a new HLA-DRB1*12 allele, named B*1568 and DRB1*1208, respectively. The alleles were identified using a combination of sequence specific primers, reverse line sequence specific oligonucleotide probing and sequence-based typing. Both alleles were identified in a single individual of Korean origin. HLA-B*1568 appears to be an HLA-B*4801/B*1507 hybrid combining the exon 2 sequence of B*4801 and the exon 3 and 4 sequences of B*1507. Exon 2 of DRB1*1208 was most similar to DRB1*1201 or 1206, with a single mismatch at nucleotide position 165 (A to C). At the protein level, this substitution results in a phenylalanine substitution at position 26 that creates an identical amino acid sequence to DRB3*0202 between amino acid positions 17 and 36.     

Further molecular diversity in the HLA-B15 group

In order to further clarify the diversity of the HLA-B15 antigens and the correspondence of serological types with alleles in Asians, we screened various B15 serological splits by means of a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method. Subsequently, the genes encoding various B15 variants were sequenced. Two novel alleles, B*1528 and B*1529, were identified: the nucleotide sequence of the former contained a single-base substitution at position 263 in exon 2 as compared to that of the B*1501 allele, which results in an amino acid change at position 64 in the α1 domain, and the nucleotide sequence of the latter differs from that of B*1518 by a single-base substitution at position 272 of exon 2 which results in an amino acid change at position 67 of the α1 domain. One new allele, B*1521, described recently in Australian Aborigines was also identified in Asians in the present study. Moreover, the results of sequencing demonstrated that Asian HLA-B62, B70, and B77 antigens are encoded by B*1501, B*1518, and B*1513, respectively. Two splits of B75 antigens, B75V (TS-1) and B15N, which have been proposed to exist in the Japanese population were encoded by B*1511 and B*1502, respectively. Most of the B15 alleles detected in the present study showed positive associations with other locus antigens. Especially, B*1502 was strongly associated with Cw8, while B*1521 was strongly associated with A34 and Cw6.     

Novel HLA-B alleles, B*8201, B*3515 and B*5106, add to the complexity of serologic identification of HLA types

Three class I alleles, B*8201, B*3515 and B*5106, have been described using DNA and cDNA sequencing. The B*8201 allele is most structurally related to B*5602, differing from it by 14 nucleotide substitutions resulting in 5 amino acid differences. The other two alleles, B*3515 and B*5106, differ from their most closely related HLA-B alleles by 2–3 nucleotide substitutions resulting in 1–2 amino acid substitutions, respectively. The majority of nucleotide substitutions marking these new alleles are observed in other HLA-B alleles suggesting that gene conversion and/or reciprocal recombination have created this diversity. All of the amino acid substitutions are predicted to alter the antigen binding site of the HLA-B molecule. The newly defined HLA-B allelic products were originally defined by their unusual serologic reactivity patterns. The B*8201 allelic product is serologically typed as a B "blank" or as a variant of B22 or B45. These patterns and the serologic reactivity of the other newly described allelic products are consistent with the protein sequence homology among specific HLA-B molecules. While serology remains a powerful tool for detecting HLA diversity, alleles generated by events resulting in the sharing of HLA sequence polymorphisms among alleles at a locus will continue to create complexity in the interpretation of typing results.     

Molecular and serological characterization of HLA-B71 in association with different class I haplotypes or in different ethnic groups

Abstract: The HLA-B70 antigen is among the most common antigens present in African Americans; however, monospecific serologic reagents defining B70 and its subtypes, B71 and B72, are rare. We have recently reported the molecular characterization of a B71 allele (B*1510) from an African American individual carrying the haplotype HLA-A30, Cw3, B71(w6). In order to better define the degree of polymorphism of molecules carrying the B71 serological specificity in the human population, we have used serology, cDNA sequencing, and PCR/SSOP typing to characterize B71 alleles from additional individuals from different ethnic populations and carrying different class I haplotypes. All carried either B*1510 or B*1518 alleles. Other HLA-B alleles isolated from these individuals (B*5001, B*4901, B*3501, B*3701) were identical to previously reported sequences except for a novel B41 allele (B*4102) identified in one Hispanic individual. This allele has concurrently been identified by Rufer and colleagues in Caucasian individuals. The B*4102 allele differs from B*4101 at codons 95 (Leu/Trp) and 97 (Ser/Arg). In addition, the B*4102 allele differs from B*4101 by two silent substitutions at codons 94 (ACC/ACT) and 99 (TAC/TAT). Since the polymorphic sequence present in B*4102 is also present in other HLA-B alleles (e.g., B*2707, B*4002, B*0702), it may represent a gene conversion cassette. The allelic diversity at the class I loci and the scarcity of monospecific alloantisera support the importance of the application of molecular based methods to identify HLA class I alleles in matching unrelated donor/recipient pairs for bone marrow transplantation.     

Significant association of HLA-B and HLA-DRB1 alleles with cleft lip with or without cleft palate

Nucleotide sequence level typing of HLA-B, -DRB1, and -DPB1 alleles was performed on Japanese patients with cleft lip with or without cleft palate (CL/P). Two HLA-B alleles, B*1501 and B*5101, showed a significant positive association with CL/P. The increase of B*1501 was evident in female patients (OR=3.6, Pc=0.003), whereas the increase of B*5101 was evident in male patients (OR=3.7, Pc < 0.001). One HLA-DRB1 allele, HLA-DRB1*0802 also showed an increase in CL/P patients. Conversely, HLA-B*4403 and DRB1*1302 were not observed in the patient group (Pc=0.01 and Pc=0.02, respectively). No HLA-DPB1 alleles showed significant association with CL/P. Thus, the present study indicates that HLA alleles, or closely linked loci, may be involved in the pathogenesis of CL/P.     

Allele-specific HLA-B*15 typing by PCR-SSP and its application to four distinct ethnic populations

Abstract: We present a set of primer mixes for the allele-specific typing of the HLA-B*15 group by PCR-SSP. The set comprises 46 primer mixes which are designed to unequivocally resolve all but two of the 666 possible combinations of the B*15 alleles, B*1501–37 (B*1536 sequence unavailable). A core subset of 34 of the 46 mixes can be used alone to give a high resolution B*15 typing set. This allows for the identification of each B*15 allele when present as the only B*15 allele and the majority of the possible B*15 homozygotic combinations. The method was validated using reference DNA samples and the B*15 allele frequency in 4 distinct ethnic populations was investigated. The results show that these populations contain predominantly mutually exclusive sets of B*15 alleles.     

HLA-B27 polymorphism in Mumbai, Western India

Human leucocyte antigen (HLA)-B27 encompasses an increasing number of subtypes that show diverse racial/ethnic prevalence in the world. One thousand-one-hundred and seventy unrelated individuals from Mumbai, Maharashtra, Western India were typed for HLA-B27 antigen by serological methods. HLA-B27 positivity was confirmed by polymerase chain reaction using sequence specific primers. High-resolution typing using sequence specific primers for HLA-B27 alleles (B*2701 - B*2721) was carried out in 70 HLA-B27-positive individuals. The frequency of B27 ranged between 1.48 and 9.6% among the caste groups studied. HLA-B27 subtyping identified B*2702 (1.43%), B*2704 (14.29%), B*2705 (70%), B*2707 (12.86%) and B*2718 (1.43%), respectively. The findings illustrate substantial genetic variation and heterogeneity within population groups from India. Extensive subtyping in other Indian caste groups will be necessary to resolve the evolutionary implications of HLA-B27 subtypes and their relationship to disease association in the Indian context.     

Two new alleles of HLA-B22 group in Japanese

Five different B22 split antigens, B54, B55.1, B55.2, B56 and B22N, have been reported in Japanese. Genomic DNAs were analyzed by PCR-SSCP and sequencing methods for Japanese samples serologically positive for each split antigen. In the SSCP analysis of exon 2, B55.1, B55.2 and B56 showed identical patterns, whereas B54 and B22N showed distinct patterns. In the SSCP analysis of exon 3, B54 and B55.1 showed identical patterns, whereas B55.2, B56 and B22N showed distinct patterns. Sequence analysis further showed that B54, B55.1 and B56 were encoded by B^*5401, B^*5502 and B^*5601, respectively. The other two rare antigens, B55.2 and B22N, were found to be encoded by new alleles. The exon 2 amino acid sequences encoded by B55.2 and B22N alleles were identical to those of B55- and B56-group alleles, although that of B22N included a synonymous substitution. The exon 3 sequence of B55.2 was unique among HLA-B alleles. The exon 3 sequence of B22N was identical to those of B^*1501 and B^*4601, but distinct from those of other B22-group alleles.