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Keywords:

  • alleles;
  • HLA;
  • MHC;
  • polymorphism;
  • SBT;
  • Taiwanese

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. Disclosures
  9. References

HLA allelic polymorphism among different ethnic groups and racial populations is widely observed and the patterns of linkage disequilibrium among various alleles differ significantly among human populations have been shown in many studies. The population composition of Taiwanese comprises four major ethnic groups, namely, Minnan, Hakka, Aborigines and Chinese Mainlanders, and other minority ethnic groups. Thus, the database of our haematopoietic stem cell donor registry comprises volunteer donors bearing HLA alleles and haplotypes with its unique polymorphism and characteristics. In our donors, we have discovered new alleles, rare frequency alleles and Taiwanese conserved alleles and haplotypes. In addition, beyond the HLA system, we found genetic expression of Oriental restricted white blood cell antigen in Taiwanese population.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. Disclosures
  9. References

The major histocompatibility complex (MHC) of human is the HLA (human leukocyte antigen) system which consists of several loci of genes located on the short arm of chromosome 6 at 6p21·3. These loci are classified into classes I–III of the MHC. The central protective immunity functions contributed by HLA molecules in MHC restriction and antigen presentation clearly demonstrate the essential role of these molecules in the performance of immune regulation and self-defence mechanism against environmental pathogens. With a vast category of polymorphism that the HLA system is being unfolded, it is imperative to precisely characterize any new allele encountered along the way of routine HLA typing procedures. Clinically speaking, HLA allelic matching is known to have a great impact on successful outcome in tissue and/or organ transplants. However, when a transplant donor and recipient are identical with respect to their HLA genes, the amino acid differences in minor histocompatibility or other unknown cell surface proteins can cause the grafted tissue to be slowly rejected. Therefore, exploration on minor histocompatibility and unknown cell surface antigens that are able to elicit immune response is equally important in the arena of transplantation medicine.

The population composition of Taiwanese population comprises four major ethnic groups, namely, Minnan, Hakka, Aborigines and Chinese Mainlanders, and other minority ethnic groups. Thus, the database of the Tzu Chi haematopoietic stem cell donor registry comprises volunteer donors carrying HLA alleles and haplotypes with its unique polymorphism and characteristics. In addition, using serological methodology, we also detected an Oriental restricted antigen in Taiwanese. In this report, Taiwanese novel HLA alleles and Taiwanese restricted HLA haplotype and non-HLA Oriental antigen found in Taiwanese are presented.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. Disclosures
  9. References

Peripheral blood sample of the donors was collected in acid citrate dextrose (ACD) anticoagulant. Formal written consents were signed by the donors before blood collection were performed. ACD whole blood was stored at −80°C until use. Genomic DNA was extracted using QIAamp DNA Blood Mini Kit according to the manufacturer’s instructions (Qiagen, GmbH, Hilden, Germany). Genomic DNA typing of HLA-A, -B, -C and -DRB1 loci were first performed using the Dynal Reli SSOP (sequence-specific oligonucleotide probe) HLA-A, -B, -C and -DRB1 Typing Kits (Dynal Biotech, Bromborough, Wirral, UK). For sequence-based typing (SBT), exons 2 and 3 for class I and exon 2 for class II were sequenced to verify rare or novel alleles as described previously [1–5]. Serological investigation of WBC surface antigen was performed according to the standard NIH microlymphocytotoxicity test in Terasaki microtitre wells using either eosin dye exclusion or fluorescence staining methods or both. The reactions were graded where a true positive was a 6 (51–80% cell death in test well) or 8 (81–100% cell death in test well).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. Disclosures
  9. References

DNA sequences of HLA-A*11:31, -A*11:60, and -A*11:90 in Taiwanese

The sequence of A*11:31 is identical to A*11:25 in exon 2 but differs in exon 3 by one nucleotide (nt) substitution at nt position 527 A[RIGHTWARDS ARROW]T, resulting an amino acid change at residue 152 E[RIGHTWARDS ARROW]V. Compared to the sequence of A*11:01:01, the predominant subtype of A*11:01 in Taiwanese, A*11:31 has three nt differences in exon 3: 527 C[RIGHTWARDS ARROW]T, 538 C[RIGHTWARDS ARROW]T, and 539 A[RIGHTWARDS ARROW]T, translating to two amino acid variations at residues 152 A[RIGHTWARDS ARROW]V and 156 Q[RIGHTWARDS ARROW]L. Given the fact that the substituted sequence of A*11:31 is identical to HLA-A*02:01:01, A*02:06 and A*02:07 (the three most common alleles of A*02 in Taiwanese) at a maximum DNA segment starting from nt 507 to nt 558, we speculate that the new allele A*11:31 was derived in a gene conversion event where A*11:01:01 received at least the segment of sequence beginning at nt starting from 507 to nt 558 from one of the following alleles: A*02:01:01, A*02:06 or A*02:07. The most probable HLA-A, -B and -DRB1 haplotypes in association with A*11:31 may be deduced as A*11:31-B*48-DRB1*15:01:01 based on linkage disequilibrium analysis.

The sequence of A*11:60 is identical to A*11:03 in exon 2 but differs from A*11:03 in exon 3 by one nt substitution at position 527, causing an amino acid change at residue 152 E[RIGHTWARDS ARROW]V. In comparison with HLA-A*11:01:01, allele A*11:60 has two nt differences in exon 3 at nt 524 A[RIGHTWARDS ARROW]G and nt 527 C[RIGHTWARDS ARROW]T leading to two amino acid variations at residues 151 H[RIGHTWARDS ARROW]R and 152 A[RIGHTWARDS ARROW]V. We assume the most probable HLA-A, -B, -C and -DRB1 haplotype associated with the segregation of the allele A*11:60 is A*11:60- B*40:01- C*07:02-DRB1*09:01 based on the family study and the haplotype database that we have established from our previous investigations on Taiwanese population [6–8].

The sequence of A*11:90 is identical to the sequence of A*11:01:01 in exon 2 but differs from A*11:01:01 in exon 3 by two nt substitutions at codon 163 C[RIGHTWARDS ARROW]G and 488 G[RIGHTWARDS ARROW]A, resulting an amino acid change at residue163 R[RIGHTWARDS ARROW]E. In comparison with the sequence of A*11:02:01, A*11:90 has one nt difference at residue 55 A[RIGHTWARDS ARROW]G in exon 2 resulting an amino acid substitution at residue 19 K[RIGHTWARDS ARROW]E and two nt variations at 487 C[RIGHTWARDS ARROW]G and 488 G[RIGHTWARDS ARROW]A in exon 3 resulting an amino acid replacement at residue 163 R[RIGHTWARDS ARROW]E. We think A*11:90 was probably derived from a DNA recombination event where A*80:01 acted as the sequence donor donating the fragment sequence between nt 487 and nt 497 to the recipient sequence A*11:01:01. This assumption was based on our observation that the sequence of A*11:90 is identical to A*11:01:01 in exons 2 and 3 except the segment of the sequence from nt 487 to nt 497, which is identical to A*80:01. The HLA-A, -B and -DRB1 haplotype in association with A*11:90 may be reasonably deduced as A*11:90-B*40:02-DRB1*11:01 taking into the account that the HLA typing of the cell HC22801 listed on the IMGT/HLA database (http://www.ebi.ac.uk/imgt/hla/ethnicity.html [9]) which also carries the allele A*11:90.

DNA sequences of HLA-B*15:189, -B*27:40, -B*40:137, -B*40:158, and -B*52:14 in Taiwanese

Our SBT analysis revealed the sequence of B*15:189, in exons 2 and 3, is identical to B*15:01:01:01 except the segment of the nt from nt 246 to nt 411. The mismatched segment is identical to a segment of HLA-B*39:01:01. This notion led us to assume the origin of B*15:189 was most likely resulted from a sequence recombination event where B*39:01:01 acted as the segment donor and B*15:01:01:01 as the recipient.

We found the allele B*27:40 has a single nt change at position 527 T[RIGHTWARDS ARROW]A resulting an amino acid alteration at residue 152 V[RIGHTWARDS ARROW]E in comparison with B*2708. The most probable three-locus (HLA-A, -B and -DRB1) haplotype carrying B*27:40 is probably A*02:01-B*27:40-DRB1*12:02 based on our observation on five unrelated Taiwanese individuals carrying the same B*27:40 allele.

The sequence of B*40:137 has three nt changes from B*40:21 at nt 353 C[RIGHTWARDS ARROW]T, nt 355 C[RIGHTWARDS ARROW]A and nt 369 C[RIGHTWARDS ARROW]T resulting two amino acid replacement at residue 94 T[RIGHTWARDS ARROW]I and residue 95 L[RIGHTWARDS ARROW]I. We speculate the generation of B*40:137 was probably as a result of a recombination event involving B*40:01:01 and B*15:25:01 based on the observation that the sequence of B*40:137 is identical to B*40:01:01 in exons 2 and 3 except the sequence segment starting from nt 97 to nt 408, which is identical to B*15:25:01.

The sequence of B*40:158 differs from B*40:01:01 with five nt substitutes at nt 463 C[RIGHTWARDS ARROW]A, nt 477 C[RIGHTWARDS ARROW]G, nt 499 T[RIGHTWARDS ARROW]A, nt 512 T[RIGHTWARDS ARROW]G and nt 527 T[RIGHTWARDS ARROW]A causing five amino acid exchanges at residues 140 Y[RIGHTWARDS ARROW]S, 155 R[RIGHTWARDS ARROW]S, 168 S[RIGHTWARDS ARROW]T, 171 L[RIGHTWARDS ARROW]W and 179 V[RIGHTWARDS ARROW]E. A family study on HLA was conducted and the haplotype in association with B*40:158 was determined as A*11:01-B*40:158-C*07:02-DRB1*09:01. We assume the generation of B*40:158 was probably due to sequence recombination between B*40:01:01 and either one of the following three commonly detected Taiwanese alleles: B*46:01, B*15:02:01, and B*15:01:01 [8,10]. This speculation is based on our observation that the sequence of B*40:158 is identical to B*40:01:01, except the DNA segment starting from nt 419 to nt 527, which is identical to the three above mentioned alleles in Taiwanese.

We found the sequence of B*52:14, is identical to that of B*52:01:01 in exon 2 but differs from B*52:01:01 in exon 3 at nt positions 419 A[RIGHTWARDS ARROW]T and 435 A[RIGHTWARDS ARROW]G. Alteration of these two nt resulted an amino acid exchange at residue 116 Y[RIGHTWARDS ARROW]F and a silent substitution at residue 121 K[RIGHTWARDS ARROW]K. We speculate the most probable HLA-A, -B, -C, -DRB1 and DQB1 haplotypes carrying B*52:14 on our bone marrow donor is A*24:02-B*52:14-C*12:02-DRB1*10:01-DQB1*0:501 based on linkage disequilibrium of alleles.

DNA sequences of HLA-DRB1*14:01:03, -DRB1*04:61, and -DRB1*03:31

Our SBT study indicated that, in exon 2, the DNA sequence of DRB1*14:01:03 is identical to the sequence of DRB1*14:01:01 except a silent nt substitute at residue 192 G[RIGHTWARDS ARROW] A. In exon 3, we found DRB1*14:01:03 has a sequence identical to DRB1*14:54 but differs from DRB1*14:01:01 with a nt replacement at residue 421, T[RIGHTWARDS ARROW]C, analogous to the reported nt substitution pattern between DRB1*14:01:01 and DRB1*14:54, translating an amino acid replacement from Y[RIGHTWARDS ARROW]H of the mature HLA molecule. The HLA-A, -B and -DRB1 haplotype deduced is HLA-A*02:01-B*13:01-DRB1*14:01:03. The fact that the DRB1*14:01:03 is restricted to three unrelated Taiwanese is unusual and suggests a close genetic tie between the three donors.

The sequence of DRB1*04:61 is identical to DRB1*04:08 except the two nt variations at positions 260 C[RIGHTWARDS ARROW]A and 261 C[RIGHTWARDS ARROW]G. Nucleotide mutations caused an amino acid substitution A[RIGHTWARDS ARROW]E at residue 58 as compared to DRB1*04:08 allele. DRB1*04:61, in addition, is homologous with DRB1*11:01:01 for a maximum fragment from nt 228 to nt 285 and for a minimum fragment from nt 256 to nt 261.

The allele of DRB1*03:31 differs from DRB1*03:06 and DRB1*03:25 by one nt at positions 196 and 227, respectively. Nucleotide mutations caused amino acid substitutions N[RIGHTWARDS ARROW]Y at residue 37 and F[RIGHTWARDS ARROW]Y at residue 47, as compared with amino acid sequence encoded by the DRB1*03:01:01 allele. We hypothesize that the origin of DRB1*03:31 is more likely an end product of a gene conversion encompassing nt fragment at least from codon 37 to codon 47. The conversion probably took place with DRB1*03:01:01 as the sequence recipient and DRB1*04:05:01 or DRB1*08:03:02 as the donor of the sequence.

Serological identification of an Oriental non-HLA antigen in Taiwanese

Using the complement-mediated lymphocytotoxicity (LCT) assay, we discovered an antibody, in a transfused Caucasian woman, recognizing an Oriental restricted antigen, named CY which does not appear to be associated with the HLA system. The serum was initially found to react strongly with T and B lymphocytes isolated from one of three HLA-A, -B and -DRB1 identical siblings of a family with Oriental origin. This result alone suggests that the antigen detected by the serum is not related to the HLA antigens. Three other Oriental family studies further confirmed this finding. Further, the possibility of minor histocompatibility antigens and red blood cell antigens were also ruled out accordingly. The antigen CY was found in Oriental populations of Taiwanese, Chinese, Japanese, Vietnamese and Thai origin. This result suggests that some people in these five ethnic groups may be closely related. From a total of 1050 random Taiwanese Chinese donors tested, it was revealed that the frequency of CY antigen was about 3·9% in Taiwanese population. However, for 60 Black, 200 East Indian, and 1000 random Caucasian donors, there was an absence of any donor with antigen CY.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. Disclosures
  9. References

We have detected several previously unrecognized alleles in HLA-A, -B and -DRB1 loci in Taiwanese population by molecular typing method. These new alleles are unusual alleles in Taiwanese. Their frequencies are estimated about one in 20 000 to one in 30 000 of donors tested, based on our SSOP typing experiences. Many haplotypes in association with some of these unusual alleles are presented in this study. These information are useful in terms of searching for unrelated donors for haematopoietic stem cells transplantation purposes and for strategic planning for donor recruitment. Additionally, we detected lymphocyte antigen with specificity beyond the HLA system and the minor histocompatibility antigen in Oriental population using serological method. While molecular typing of genes has significantly improved the accuracy of HLA allele typing, serology continue to play an essential role in the detection of antigen not recognized by DNA typing techniques.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. Disclosures
  9. References

We are indebted to all volunteer donors who willingly join the Taiwan Buddhist Tzu Chi Marrow Donor Registry and gave consents for our research project. Their unselfishness and effort to help needy patients are most respected. We would like to give sincere thanks to Dharma Master Cheng Yen, founder of the Buddhist Compassion Relief Tzu Chi Foundation, for the continuing supports and kind encouragements both intellectually and spiritually. Furthermore, the generosity and camaraderie that our colleagues bestow on us are also greatly and deeply appreciated.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. Disclosures
  9. References