A new B18 Sequence (B∗1802) from Asian individuals

ELSEVIER

A New B18 Sequence (B”1802) from Asian Individuals Ling Lin, Katsushi Tokunaga, Yoshihide Ishikawa, Kouichi Kashiwase, Fumiaki Nakajima, Motoko Nishimura, Shoji Kuwata, Tatsuya Akaza, Kenji Tadokoro, Yoichi Shibata, and Takeo Juji

ABSTRACT: A new HLA-B 18 allele (B* 1802) derived from a Thai individual was sequenced. Comparison of this B18 nucleotide sequence with the published B+ 1801 sequence indicated that this Asian B18 allele has a nucleotide sequence different from that of B* 180 1. Three nucleotide changes were observed in exon 3, in which two substitutions at codon 97, AGG in B+1801 to AAT in the B# 1802, result in an amino acid change from arginine to asparagine. The residue 97Asn has also been described

in some B27 subtypes. A silent mutation was also observed at codon 99, TAC in BY1801 to TAT in the B* 1802. This sequence has been reported in many class I alleles published so far. Moreover, 18 HLA-B 18-positive samples were examined by the PCR-SSO method using specific probes for B* 180 1 and B* 1802. The results demonstrated that three Asian samples possess Be1802 and share HLA-Cw7, DR12, and DQ7. Human Immunology 42, 23-26 (1995)

ABBREVIATIONS one-dimensional isoelectric lD-IEF polymerase chain reaction PCR

SSO

focusing

sequence-specific

oligonucleotide

INTRODUCTION HLA-B18

is a common

antigen

tions, carried by well-defined B 1%DR2 and A30-B 18-DR3 the Japanese

population,

in Caucasian

popula-

haplotypes: HLA-A25f 11. This antigen is rare in

but common

in Southeast

Asian

From the Department of Research (L. L., K.T., Y.I., K.K., M. N., T.A., K.Ta., T.J.), Japanese Red Cross Central Blood Cents, Tokyo; the Department of Transfusion Medicine and lmmunohematology(L. L., S.K., Y.S.), Faculty of Medicine, Univwsity of Tokyo, Tokyo; and the Kanagawa Red Cross Blood Center (F.N.), Yokohama, Japan. The nucleotidesequencedata reported in this article appear in the DDBJ, EMBL, and GenBank nucleotidesequence&abases with the accessionnumbe 025275. The name B*l802 has been officially assigned by the WHO Nomenclature Committeein January 1994. This follows the agreedpohy that, subject to the conditionsstated in the most recent Nomenclature Report (Bodmer et al. 1992), a name will be assigned to a new sequenceas it is identified. The new name will be published in the fohwing WHO Nomenclature Report. Address reprint requests to Dr. L. Lin, Department of Research, Japanese Red Cross Central Blood Center, 4-l -3 1 Hiroo, Shibuya-ku, Tokyo 150, Japan. Received (U) February 14, 1994; acceptedJune 6, 1994.

Human Immunology 42, 23-26 (1995) 0 American Society for Histocompatibility

and Immunogenetics,

1995

6.5% gene frequency in Javanese and 2.5% in Thais [2]. In these populations, HLA-B18 is considered to be carried by a different haplotype than in Caucasian populations 121, A24-B 18-DR12. Although no conclusive evidence for the existence of B18 splits has been presented by serologic analysis, three variants, B18.1, B18.2, and B18v, were identified by onedimensional isoelectric focusing (lD-IEF) on B 18positive panel cells 13, 41. We determined the nucleotide sequence of a B18 allele derived from a Thai individual possessing the combination of A24, B18, and DR12, and compared it with the sequence of the previously described B*1801 Es]. The results demonstrated that this Asian B18 has a nucleotide sequence different from that of B* 180 1 C63. Furthermore, we examined the incidence of this new allele in available B 18-positive samples (including Caucasian and Asian) by using the polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO)

populations:

0198~8859/95/$9.50 0198-8859(94)00073-Y

24

L. Lin et al.

method. The results in Asian populations antigens.

MATERIALS

AND

indicated that this new allele exists and is associated with certain HLA

METHODS

Blood samples and preparation of cDNA and genomic DNA. An HLA-Bl%positive sample obtained from a Thai individual TP (HLA-A24, -; B18, 77; Cw7, -; DR12, ->, who was a donor for blood transfusion, was examined for sequencing. Eighteen unrelated B 1% positive samples with different haplotypes (Table 1) were analyzed by PCR-SSO. All of the samples were typed by the standard microcytotoxicity test with local qualified antisera. For the sequence analysis, total cellular RNAs were prepared from peripheral mononuclear cells by the acid guanidinium phenol chloroform method (71. cDNAs were synthesized using M-MLV reverse transcriptase (BRL, Gaithersburg, MD, USA) and random primers. Genomic DNA of 19 B18-positive samples were extracted from frozen leukocytes. Cloning and sepencing. The HLA-B fragment was amplified from the cDNAs by PCR using specific primers for HLA-B (HLA-5P2 and HLA-3B), as described by Ennis et al. {8]. The amplified fragments were cloned using a T-vector cloning system (Invitrogen, San Diego, CA, USA). Ligation and transformation were performed essentially according to the method described in the manufacturer’s instruction manual. Recombinant colonies TABLE

1

were selected and screened by PCR amplification of inserted fragments. Of the 10 clones picked, seven contained B18 inserts, as identified by sequencing a part of exon 2, and five clones were sequenced for each orientation.

Serologic types of HLA-B18-positive B 18 alleles

individuals

PCR-SSO. Genomic DNA was used as the template for PCR amplification with group-specific primers: HLAB 5H (5 ’ -GCCCCGAAGCTTCCTCCTGCTG-3 ‘> [9] and HLAac (5 ‘-CCACTCCACGCACGT-3 ‘). The target sequence between these primers corresponds to the nucleotide sequence no. 15 of exon 1 to no. 573 of exon 3, which includes introns 1 and 2 of the HLA-B gene. A limited number of HLA-B alleles, including B 18, can be amplified with this primer set. SSOs were synthesized by a DNA synthesizer (Applied Biosystems, USA) as follows: P1801 (5’CCTCCAGAGGATGTAC-3’) and P1802 (5’CCTCCAGAATATGTAT-3’). The probe P1801 recognizes the specific sequence in the third exon of B” 180 1 and the probe P 1802 detects the sequence of B*1802 in the same region. PCR products from genomic DNAs were denatured, blotted, and hybridized with P180 1 and P1802 separately, as described previously E101.

RESULTS All exons with the stitutions 19, 20,

were sequenced and the results were compared sequence of B” 180 1 161. Three nucleotide subwere found in exon 3 at nucleotide positions and 26 (Fig. l), of which two substitutions

and their

Serologic types B18 No.

ID

Ethnicitf

A

1 2 3 4

DIH BHP TP su

A A A A

-/ll.l 2124 24/2124

2 7 8 9 10 11 12 13 14 15 16 17 18

MS KP

A C A C A C C C C C C U U C

24/2125 l/24 2125 24126.4 2l213 2124 24126 30126.3 25/2125111.1 213

DJ

KV SS MB Go HM RB FR MC JI NN CP

a A, Asian; C, Caucasian; and U, unknown. ND, not determined.

alleles

C

B

DR

ll7/8N Jl714

18/61 18/15N 18177 lW35

12/13 12/12/12/-

617 7l7l71-

B+ 1802 Be 1802 B+ 1802 Be1801

lO/ND ND ND 7l7110 Jl4/-l5lll715 71li-

18/18/60 18152 18144 18/8 18162 18149 18/35 18/38 18/44 18/55.1 18/38 18/44 18/5 1

12/13 ND ND ND 3/11 1u4.1 11/14 11/13 1u4.1 317 15113 11/13 1614.1 3113

617 ND ND ND 217 7/l/7 l/7 417 2ll! 117 l/3 l/2

Br1801 B*1801 B*1801 Bu1801 B*1801 B*1801 Bx1801 Be1801 B*1801 B*1801 B*1801 B#1801 Be=1801

DQ

B# 1802 from Asian Individuals

25

SO

19 26 EXONB GGTCTCACACCCTCCAGAGGATGTATGGCTGCGACGTGGGGCCGGACGGG -------___--------_------C------------___--_--____ _____--_______----AT___------___--------___-----_-

CONSENSUS B*1801 B*lBOZ B*2702 B*2703 B*2705 Bt.2707 B*0801

-------___--------A~---------_---------_--------__ ----____-----_-___AT----_-___------_____-----_____ -------___--------A~_--------__---------___------~_-----____-------_C__---C---____-------__-------__-----_____------_C___--C---____-------___-------

P1801

-----__---------__-------C--_--------___-------_-_ -----~__---------_-------C_-_--------___--------------------------~---------------------_-----------------_~-A------_----------__--------_----------

B*3801 B*3901 B.1401 **5301

FIGURE 1 Nucleotide sequences B* 1801, B* 1802, and other related

of a part of exon 3 for HLA-B alleles.

caused

between

one

B*1802

amino

products

asparagine)

(Fig.

also been described

acid

change

at amino

acid position

2). This

asparagine

in the products

B* 180 1 and 97 (arginine

at position of B*2702,

to

The

and B”2705W

results

alleles

of PCR-SSO

Ill,

BX2703,

with

121. Another

specific

probes

P 180 1

3 and Table 1. Within 18 B 18-positive samples, one of which was subjected to the sequence analysis, three samples showed a positive hybridization signal with the P1802 probe and all the remaining samples hybridized with the P1801 probe. These B”1802-positive samples were all from Asian individuals and all share Cw7, DR12, and DQ7 antigens. P1802

are

demonstrated

in Fig.

DISCUSSION HLA-B18 was first recognized at the Fourth International Histocompatibility Workshop I131 and has been well defined with monospecific sera. In the Sixth International Histocompatibility Workshop, two splits of this antigen were suggested by serologic analysis 1141, but no further reports have described the heterogeneity of B 18 in the subsequent International Workshops. lDIEF data supported the existence of B18 splits B 18.1, B 18.2, and B 18v 13, 41. However, there has been no confirmation in terms of DNA sequencing.

FIGURE

2

Amino acid sequences in 01~domain of the prod-

ucts of B*1801, B*1802, and other Shading shows the unique substitution, Be1802 and three subtypes of B27.

FIGURE 3 Typing of B*1801 and B* 1802 by the PCRSSO method. Nos. 1, 3, and 6 are positive for B*1801; nos. 2, 4, and 5 are positive for B”1802; and nos. 7 and 8 are positive controls of Be1801 and B*1802, respectively, which are cloned fragments of B” 1801 and B+1802.

97 has

substitution was a silent mutation at amino acid position 99 (Fig. 2). This sequence was not unique but shared by many class I alleles { 11, 12). B*2705,

and

1234b078

related HLA-B alleles. which appears only in

The present study demonstrated that there is a subtype of B18 in Asian populations. This subtype is different from the B” 1801 product by a single amino acid residue at position 97. An interesting question is whether this change of arginine to asparagine affects the binding of alloantibodies, as this residue is on a P-sheet of the (Y* domain, which is located at the floor of the peptidebinding cleft. In fact, A*2602 product can be distinguished by specific antisera from A”260 1 product: there is only a single amino acid difference between A”2602 and AX260 1 (’ t6Asn vs “‘Asp) on the floor of the peptide-binding cleft 115, 161. The nucleotide sequence covering positions 19-26 in exon 3 of B”1802 is shared with only some B27 subtypes (Fig. l), whereas the sequence of this position in B*1801 is shared by the majority of HLA-B alleles. Therefore, B” 1801 is likely to be the ancestral allele of the B18 group. B”1802 may have originated by a gene conversion-like event between B” 1801 and a B27 subtype. As we reported previously {lo}, different B6 1 alleles are exclusively associated with different haplotypes. B 18 is also carried by different haplotypes in various ethnic groups; e.g., A25-B 18-DR2 and A30-B 18-DR3 in Caucasian populations, A3-B 18-DR13 in African populations, and A24-B18-DR12 in Asian populations 121. Our present study demonstrated that both B’ 180 1 and B” 1802 exist in Asian populations but B* 1802 shows a linkage disequilibrium with Cw7, DR12, and DQ7. Because B 18 is also associated with DR15 in Asian populations 121 and the molecular basis of three B 18 variants

100 110 120 130 140 CONS~SUSGSHTLQRMYGCDVGPDGRLLRGYDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAARVAEQLRAYLEGTCVEWLRRYLENGKETLQRA B.180, .................... ..H..9..............S.__......__._._........................H ..~.._............B..S..............S...._..................................H B.1802 ....

IS0

160

170

B*2702

....

..~.._.............~.D........._....S...._._........................E

...................

8’2703 9*2705

.... ....

..N.._.............H.D..............S...............................E ..~.._.............H.D..............S...._..........................S

................... ...................

8.2707 9*0*0, 9*,80* 9.390,

.~..__...........HN............................................__..E .... ..~.._............HN..........._.............................D._.......__..._.....D_.E .... .............. ............. .BR.._..HN.~ .............. ............. .BR_._..HN.~

9*140,

....

9*5301

..

................... .. .............. .............. ...........

.S...._....._...............T......__C” .S..........................T......__C”

..~................N.~........._....S...._............_..S....____..........H .,~.......L.........H..S......._._....S...._..........................L

180 ........... ...........

...................

26

L. Lin et al.

distinguished ther

studies

HLA-B

by lD-IEF should

18 allelic

[3, 47 are still

be done

in order

unknown,

fur-

to elucidate

the

diversity.

8. Ennis

PD, Zemmour J, Salter RD, Parham P: Rapid cloning of HLA-A, B cDNA by using the polymerase chain reaction: frequency and nature of errors produced in amplification. Proc Nat1 Acad Sci USA 87:2833, 1991.

9. Lin L, Watanabe Y, Tokunaga K, Kuwata S, Kohsaka T,

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2. Imanishi T, Akaza T, Kimura A, Tokunaga K, Gojobori T: Reference tables: allele and haplotype frequencies for HLA and complement loci in various ethnic groups. In Tsuji K, Aizawa M, Sasazuki T (eds): HLA 199 1, vol 1. London, Oxford University Press, 1993, p 1506.

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3. Yang SY: Population analysis of class I HLA antigens by one-dimensional isoelectric focusing gel electrophoresis: workshop summary report. In Bupont B (ed): Immunobiology of HLA, vol 1: Histocompatibility Testing 1987. New York, Springer-Verlag, 1987, p 309. 4. Kaidoh T, Yang SY, Yashiki S, Sonoda S, Choo SY, Wong KH, Mervart H, Reekers P, Sachs JA, Yoshida TO: Analysis of HLA class I antigens by one-dimensional isoelectric focusing: summary report. In Tsuji K, Aizawa M, Sasazuki T (eds): HLA 1991, vol 1. London, Oxford University Press, 1993, p 824. 5. Parham P, Lomen CE, Lawlor DA, Ways JP, Holmes N, Coppin HL, Salter RD, Wan AM, Ennis PD: Nature of polymorphism in HLA-A, -B, and -C molecules. Proc Nat1 Acad Sci USA 85:4005, 1988. 6. Zemmour J, Parham P: HLA class I nucleotide 1991. Immunogenetics 33:310, 1991.

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AC, Terasaki PI: 13. Albert ED, Mickey MR, McNicholas Seven new HL-A specificities and their distribution in three races. In Terasaki PI (ed): Histocompatibility Testing 1970. Copenhagen, Munksgaard, 1970, p 221. F 14. Bodmer J: The A B C of HLA. In Kissmeyer-Nielsen (ed): Histocompatibility Testing 1975. Copenhagen, Munksgaard, 1975, p 43. 15. Ishikawa Y, Tokunaga K, Lin L, Imanishi T, Saitou S, Kimura A, Kashiwase K, Akaza T, Tadokoro K, Juji T: Sequences of four splits of HLA-A10 group: implications for serological cross-reactivities and their evolution. Hum Immunol 39:220. WH, Belich MP, Benjamin 16. Madrigal JA, Hildebrand RJ, Little AM, Zemmour J, Ennis PD, Ward FE, PetzlErler ML, du Toit ED, Parham P: Structural diversity in the HLA-A10 family of alleles: correlations with serology. Tissue Antigens 41:72, 1993.