hla-drb1*01 subtyping by heteroduplex analysis

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Tissue Anrigens 1995: 45: 120-124 Printed in Denmark . All rights reserved Copyright 0 Munksraard 1995 TISSUE ANTIGENS ISSN 0001-2ais HLA-DRB 1 "0 1 subtyping by heteroduplex analysis D. A. Savage, N. A. P. Wood, J. L. Bidwell, K. M. Hui. HLA-DRBl*Ol subtyping by heteroduplex analysis. Tissue Antigens 1995: 45: 120-124. 0 Munksgaard, 1995 Abstract: In this report we describe an alternative method for the identi- fication of the four known HLA-DRBl*Ol alleles, which is based on the generation of unique heteroduplex patterns between the different DRB1*01 alleles and a synthetic DNA heteroduplex generator (DHG) molecule. The method is technically simple, rapid and cost-effective, as it essentially involves only a single polymerase chain reaction (PCR) fol- lowed by polyacrylamide gel electrophoresis. This technique allows the rapid discrimination of the various known HLA-DRBI*OI subtypes, both in homozygous and heterozygous situations. We propose that this tech- nology can potentially be applied to most HLA class I and class I1 I subtyping. A total of four HLA-DRBl*Ol alleles have been defined by DNA sequencing of the polymorphic second exon of HLA-DRBl*Ol genes. These are designated as HLA-DRBl*OlOl, *0102 and "0103 (l), and the recently defined rare DRBl*Ol allele, HLA-DRB 1 *O 104 (2). Cells expressing either HLA-DRBl*OlOl or -DRB1*0102 specificities are clearly D R l positive by serological typing. In con- trast, cells expressing the HLA-DRB 1 *O 103 speci- ficity can only be defined by serological typing in the absence of other DR1 reactivity using selected DR1 sera (3). Cellular methods, using homo- zygous typing cells, type HLA-DRB 1 *O 10 1, *O 102 and '0103 as HLA-Dwl, -Dw20 and -Dw "Bon" respectively (3). HLA-DRB1*0104 is apparently DR1 positive by serology (2), however, the cellular specificity for HLA-DRBI "0104 is at present un- known. Routine identification of HLA-DRBl*Ol sub- types is usually by molecular methods involving either the PCR-sequence-specific primer method (PCR-SSP) (4), or by PCR-sequence-specific oligo- nucleotide (PCR-SSO) typing (5). However, the latter suffers from the disadvantage of being cum- bersome and labour intensive, while PCR-SSP typ- ing, although involving little post-amplification D. A. Savage1, N. A. P. Wood2, J. 1. Bidwell* and K. M. HuV 'Molecular Immunology Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 2Department of Transplantation Sciences, Molecular Research Division, University of Bristol, Bristol Homoeopathic Hospital, Cotham, Bristol, United Kingdom Key words: DRBl'O1 - group-specific amplification - HLA subtyping - polymerase chain reaction - DNA heteroduplex generator Received 15 July, revised, accepted for publication 20 October 1994 processing requires multiple sequence-specific PCR reactions in order to assign an individual subtype In this report we describe the use of a synthetic DHG molecule (otherwise termed universal heter- oduplex generator) to create unique heteroduplex patterns for each of the HLA-DRB 1 *01 ,alleles fol- lowing non-denaturing po!yacrylamide gel electro- phoresis. This technology is based on the fact that the mobility of mismatched heteroduplexes is re- tarded compared to completely matched DNA homoduplexes when subjected to polyacrylamide gel electrophoresis. Depending on the ,number, type and position of the mismatches, different het- eroduplexes are generated which migrate at differ- ent rates, thus giving rise to specific banding pat- terns for each of the HLA-DRBl*Ol alleles. (4). Material and methods Cant rol s DNAs from homozygous typing cells, which were positive for HLA-DRBI*OlOl, *0102 and "0103 specificities respectively, and which formed part of a HLA-DRB typing kit, were purchased from the UKTSSA. Bristol. and used as controls. Control 120

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Page 1: HLA-DRB1*01 subtyping by heteroduplex analysis

Tissue Anrigens 1995: 45: 120-124 Printed in Denmark . All rights reserved

Copyright 0 Munksraard 1995

T I S S U E A N T I G E N S ISSN 0001-2ais

HLA-DRB 1 "0 1 subtyping by heteroduplex analysis

D. A. Savage, N. A. P. Wood, J. L. Bidwell, K. M. Hui. HLA-DRBl*Ol subtyping by heteroduplex analysis. Tissue Antigens 1995: 45: 120-124. 0 Munksgaard, 1995

Abstract: In this report we describe an alternative method for the identi- fication of the four known HLA-DRBl*Ol alleles, which is based on the generation of unique heteroduplex patterns between the different DRB1*01 alleles and a synthetic DNA heteroduplex generator (DHG) molecule. The method is technically simple, rapid and cost-effective, as it essentially involves only a single polymerase chain reaction (PCR) fol- lowed by polyacrylamide gel electrophoresis. This technique allows the rapid discrimination of the various known HLA-DRBI*OI subtypes, both in homozygous and heterozygous situations. We propose that this tech- nology can potentially be applied to most HLA class I and class I1

I subtyping.

A total of four HLA-DRBl*Ol alleles have been defined by DNA sequencing of the polymorphic second exon of HLA-DRBl*Ol genes. These are designated as HLA-DRBl*OlOl, *0102 and "0103 (l), and the recently defined rare DRBl*Ol allele, HLA-DRB 1 * O 104 (2). Cells expressing either HLA-DRBl*OlOl or -DRB1*0102 specificities are clearly D R l positive by serological typing. In con- trast, cells expressing the HLA-DRB 1 *O 103 speci- ficity can only be defined by serological typing in the absence of other DR1 reactivity using selected DR1 sera (3). Cellular methods, using homo- zygous typing cells, type HLA-DRB 1 *O 10 1, * O 102 and '0103 as HLA-Dwl, -Dw20 and -Dw "Bon" respectively (3). HLA-DRB1*0104 is apparently DR1 positive by serology (2), however, the cellular specificity for HLA-DRBI "0104 is at present un- known.

Routine identification of HLA-DRBl*Ol sub- types is usually by molecular methods involving either the PCR-sequence-specific primer method (PCR-SSP) (4), or by PCR-sequence-specific oligo- nucleotide (PCR-SSO) typing ( 5 ) . However, the latter suffers from the disadvantage of being cum- bersome and labour intensive, while PCR-SSP typ- ing, although involving little post-amplification

D. A. Savage1, N. A. P. Wood2, J. 1. Bidwell* and K. M. HuV 'Molecular Immunology Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 2Department of Transplantation Sciences, Molecular Research Division, University of Bristol, Bristol Homoeopathic Hospital, Cotham, Bristol, United Kingdom

Key words: DRBl'O1 - group-specific amplification - HLA subtyping - polymerase chain reaction - DNA heteroduplex generator

Received 15 July, revised, accepted for publication 20 October 1994

processing requires multiple sequence-specific PCR reactions in order to assign an individual subtype

In this report we describe the use of a synthetic DHG molecule (otherwise termed universal heter- oduplex generator) to create unique heteroduplex patterns for each of the HLA-DRB 1 *01 ,alleles fol- lowing non-denaturing po!yacrylamide gel electro- phoresis. This technology is based on the fact that the mobility of mismatched heteroduplexes is re- tarded compared to completely matched DNA homoduplexes when subjected to polyacrylamide gel electrophoresis. Depending on the ,number, type and position of the mismatches, different het- eroduplexes are generated which migrate at differ- ent rates, thus giving rise to specific banding pat- terns for each of the HLA-DRBl*Ol alleles.

(4).

Material and methods Cant rol s

DNAs from homozygous typing cells, which were positive for HLA-DRBI*OlOl, *0102 and "0103 specificities respectively, and which formed part of a HLA-DRB typing kit, were purchased from the UKTSSA. Bristol. and used as controls. Control

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HLA subtyping by heteroduplex analysis

DNA samples positive for DRB 1 *0104 were ob- tained as gifts from Drs Guignier and Mercier. All possible DRBl*OI heterozygote combinations were created by mixing equal amounts of the re- spective PCR products from the DNAs above prior to heteroduplex formation with the DHG as described below.

PCR amplification

PCR mixtures (100 pl) were set up consisting of the following; 50 mM KCI, 10 mM Tris-HC1, pH 8.3, 1.5 mM MgC12, 0.001% gelatin, 200 pM of each deoxynucleotide triphosphate, 0.2 pM of each of the DRB1*01 sequence-specific primers

3’) and DRBAMP-B (5’CCGCTGCACTGTGA- AGCTCT 3’) (9, 1 unit of AmpliTaq T‘(Perkin- Elmer Cetus) and 0.25 pg of genomic DNA. The mixtures were heated to 96°C for 3 min and then subjected to 30 cycles of 96°C for 1 rnin; ramp to 60°C over I min followed by 60°C for 30 sec; 72°C for 1 min, followed by 5 min at 72°C using a Perkin Elmer 480 thermal cycler.

DRBAMP-1 (S’TTCTTGTGGCAGCTTAAGTT

Construction of the DHG and heteroduplex formation

Two synthetic oligonycleo tides (“longmers”) with 13 bp overlaps at their 3’ ends [longmer 1 (137 bases)5’TTCTTGTGGCAGCTTAAGTTTGAA TGTCATTTCTTCAATGGGACGGAGCGGG TGCGGTTGCTGGAAAGATGCATCTATAA CCAAGAGGAGTCCGTGCGCTTCGACAGC GACGTGGGGGAGTACCGGGCGGTGACG GA 3’, and longmer 2 (131 bases) S‘CCGCTGC ACTGTGAAGCTCTCACACCCCGTAGTTG TGTCTGCAGGGTGTCCACCGCGGCCCGC CTCTGCCAGGAGGTCCTTCTGGCTGTTCC AGTACTCGGCATCAGGCCGCCCCAGCTC CGTCACCGCCC 3‘1 were synthesized on an ABI 391A DNA synthesizer and used to construct a 255 bp DHG essentially as previously described (6). Figure I shows a portion of the nucleotide se- quence from exon 2 of HLA-DRBl*Ol alleles as compared to the same region from the deduced se-

7 0

Figure Heteroduplex patterns obtained between each of the four HLA-DRBI*OI PCR products and the DHG. Track (1) DRB1*0101, (2) DRB1*0102, (3) DRBI*0103 and (4) DRBI* 0104. M=Molecular weight marker (pUC BM21 digested with Hpa 11, Dra I and HiridIII). h=homoduplex; hd=heteroduplex; S=putative SSCP. The mobilities of the heteroduplex bands (ex- pressed as apparent molecular weights) are as follows; DRBI* 0101 (404 bp and 358 bp), DRB1*0102 (349 bp and 330 bp), DRBI*0103 (608 bp and 422 bp) and DRB1*0104 (386 bp and 368 bp).

quence of the DHG moleule. A total of six nucleo- tide deletions were strategically located within the DHG molecule in order to promote heteroduplex formation (Figure 1).

The DHG was PCR-amplified as described above using 10 p1 of a dilution of stock DHG DNA. This stock DHG dilution was empirically determined prior to use. Fifteen pl of PCR product (261 bp) from genomic DNAs was mixed with 15 p1 of PCR-amplified DHG DNA, heated to 96°C for 5 min and then slowly cooled to 37°C over 30 min in order to generate heteroduplexes. The mix- tures were electrophoresed on a 15% polyacryl-

an - - DRB1’0101 CTC CTG GAG CAG ACG CGG GCC GCO GTG GAC ACC TAC TGC AGA CAC AAC TAC t3GG GTT GGT

-C- -TG DRB1’0102 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ --_ --_ --- -_- --- ..-T _ _ _ _ _ _ _ _ _ _ _ _ __- --- --A 0-c a- -__ --_ _ _ _ __- _ _ _ _ _ _ _ -_ --- __- __- --- --- --- --- --- DRB1*0103 A-- ---

~-1.0 104 _ _ _ _ _ _ - _ _ _ _ - _ _ _ _ _ _ - - _ - _ _ _ _ _ _ - _ -AT _-- _ _ _ _ _ _ _ _ _ _-- _-- -_ - --- -TG

DliO _ _ _ _ _ _ **- _ _ _ _ _ _ _ _ _ -__ _ _ _ _-_ -__ _-- .*- _ _ _ _ _ _ --_ -_- --- --- --* *-- Figure 1. Nucleotide sequence alignment of a region of exon 2 from HLA-DRBI*OI alleles as compared to the same region from the deduced sequence of the DHG. The deduced nucleotide sequence of the DHG is otherwise identical to all DRBI*Ol alleles within the PCR-amplified region of exon 3. Numbers above the nucleotide sequences refer to codon numbers ( I ) . Dashes indicate nucleotide identities, and *indicates a base deletion.

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Savage et al.

amide minigel for 4.5 h at 100 V, stained with ethi- dium bromide, and visualized under UV illumi- nation essentially as previously described (7).

Results

Figure 2 shows unique heteroduplex patterns gen- erated by the DHG for each of the four HLA- DRBl*Ol alleles. Figure 3 shows the heteroduplex patterns generated by the DHG for all possible heterozygote combinations of HLA-DRB1*01 al- leles. These heteroduplex patterns consist of the summation of the respective individual heterodu- plex patterns. However, in all DRB1*01 combi- nations, with the exception of DRB1*0101 and *0102, additional bands were also observed (Fig- ure 3, bands indicated as*). In order to study the nature of these additional bands, we mixed to- gether the PCR products of all heterozygote com- binations of DRB1*01 alleles and allowed heterod- uplex formation in the absence of the DHG. The results showed these additional bands to be trans heteroduplexes (Figure 4). These trans heterodu- plexes are formed between the coding strand of one DRBl*01 allele and the non-coding strand of the other DRBl*Ol allele. Other additional bands are also observed near to the gel slots for each of the DRBl*01 alleles, both in homozygous and het-

Figure 3. Heteroduplex patterns obtained between all possible pairwise combinations of DRBI*OI PCR products and the DHG. Track ( I ) DRB1*0101 and *0102, ( 2 ) DRB1*0101 and * 0103, (3) DRB1*0101 and *0104, (4) DRBl*0102 and *0103, (5) DRBI*0102 and *0104, and (6) DRBI*0103 and *0104. M=Molecular weight marker. *indicates additional bands which would not be expected from the summation of the re- spective individual DRBI*OI alleles.

Figure 4. Heteroduplex patterns obtained between all possible pairwise combinations of DRB1*01 PCR products in the ab- sence of the DHG. Track (1) DRB1*0101 and *0102, (2) DRB1*0101 and *0103 (3) DRB1*0101 and *0104, (4) DRB1* 0102 and *0103, (5) DRB1*0102 and *0104, and (6) DRBI* 0103 and *0104. M=Molecular weight marker. T=Trans hete- roduplexes.

erozygous situations (Figures 2-5). Based on our other DHG-heteroduplex studies, these bands are probably the result of single stranded confor- mational polymorphism (SSCP) (unpublished ob- servations).

A total of eight DNA samples of known DRB 1 * 01 type (four DRBl*Ol homozygotes; two DRB1* 0 10 1 , DRB 1 ‘0 103 heterozygo tes; and two DRB 1 * 0 103, DRB 1 *030 1 heterozygotes) were re-typed by heteroduplex analysis and the results showed 100% concordance (Figure 5).

Discussion It is well established that accurate HLA typing is clinically imporant for both organ transplant sur- vival and HLA-disease association studies. How- ever, because HLA alleles share different combi- nations of sequence motifs, identification of the various class I and I1 subtypes usually requires multiple PCR reactions or large batteries of oligo- nucleotide probes.

An alternative approach to HLA typing involves the use of DNA heteroduplex analysis. Recently, “PCR-fingerprinting’’ using DNA heteroduplex analysis has been applied to HLA-DRB matching for the rapid selection of HLA-matched unrelated

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HLA subtyping by heteroduplex analysis

eroduplex bands (Figures 3 and 4). Thus, DHG technology can be applied to the rapid identifi- cation of the various known HLA-DRB 1 *O I sub- types, both in DRB 1 *O 1 homozygous and DRB 1 * 0 1 heterozygous situations. Also, the technique can be applied to DRB 1 *Ol-positive individuals carry- ing a non-DRBl*Ol allele, since only DRB1*01 alleles are amplified using the primers described in this study (5) . This is illustrated in Figure 5 (tracks 5 and 6) by the two individuals who were positive for both DRBl"0103 and DRB1*0301.

The method essentially involves a single PCR amplification followed by polyacrylamide minigel electrophoresis. This is in contrast to PCR-SSP which would require a total of five separate PCR reactions (4), and PCR-SSO which would require five different oligonucleotide probes for the posi- tive identification of the four known DRB1*01 sub types in both homozygous and heterozygous situations (5) . Further advantages of the DHG technique are that it is technically simple, cost-ef- fective and applicable to both small and large-scale subtyping. Also, novel DRB1*01 alleles may be de- tected by heteroduplex analysis using the DHG molecule described in this report, since different patterns may be generated by as yet undefined DRBI*Ol alleles.

In conjunction with group-specific amplifi- cation, the application of DHG molecules in hete- roduplex analysis lends itself to subtyping for both HLA class I and class I1 alleles, since the DHG molecules can be specifically designed for particu- lar group-specific subtyping. In addition, this tech- nology can theoretically be automated by for ex- ample attaching a fluorescent label onto the 5' end of one of the PCR primers. Heteroduplex frag- ments could then be detected directly as they passed through a polyacrylamide gel and the hete- roduplex patterns compared to standard patterns stored in a data-base.

Following HLA-DR low-resolution typing using either PCR-SSP (13) or PCR-SSO typing (14), we propose that DRB1*01 subtyping can be per- formed by heteroduplex analysis as described in this report. Furthermore, we are at present ex- tending this study by constructing DHG molecules for subtyping of other HLA groups.

Figure 5. Heteroduplex patterns obtained between PCR prod- ucts from samples of known DRBl*Ol type and the DHG. Track (1) DRB1*0101 homozygous, (2) DRBI*0101 and *0103, (3) DRB1*0101 homozygous, (4) DRB1*0102 homozygous, ( 5 ) DRB1*0103 (and DRB1*0301), ( 6 ) DRB1*0103 (and DRB1* 0301). (7) DRB1*0101 and *0103, and (8) DRB1*0103 homo- zygous. M=Ivlolecular weight marker.

bone marrow donors (8). It has also been reported that heteroduplex analysis can be applied to the identification of HLA-DRB3 alleles (9), HLA- DR4 alleles (lo), and also HLA-DPB1 alleles ( I 1). These methods rely on the formation of either cis and/or trnns heteroduplexes derived from the PCR products themselves (7).

A refinement of this technology involves the use of synthetic DHGs to promote heteroduplex for- mation between the DHG and the PCR product (12). The resulting heteroduplexes are then re- solved using non-denaturing polyacrylamide gel electrophoresis. DHGs are DNA sequences that mimic a genomic DNA sequence, but which con- tain "controlled" nucleotide substitutions, de- letions, or insertions at nucleotide positions op- posite to and contiguous with known polymorphic sites within the genomic DNA. The DHG and gen- omic DNA sequences are amplified with the same PCR primers, and are hybridized together in equal proportion post-PCR by heating and slow cooling in order to generate heteroduplexes.

In this report we show that a single DHG can be used to generate unique heteroduplex banding patterns for each of the four known HLA- DRB1*01 subtypes (Figure 2). Furthermore, HLA-DRB 1 *O 1 heterozygote combinations con- sist of the summation of the respective DRBl*Ol heteroduplex patterns as well as specific trans het-

Acknowledgements

This work was supported by the Singapore Na- tional Science and Technology Board.. We thank Drs E Guignier (Dijon, France) and B. Mercier (Brest, France) for their generous gifts of HLA- DRB1*0104 DNAs.

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Address: D. A. Savage Molecular Immunology Laboratory Institute of Molecular and Cell Biology The National University of Singapore 10 Kent Ridge Crescent Sinsapore 05 I I Republic of Singapore

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