HLA class I sequence-based typing

HLA Class I Sequence-Based Typing Pere Santamaria, Alan L. Lindstrom, Michael T. Boyce-Jacino, Steve H. Myster, Jose J. Barbosa, Anthony J. Faras, and Stephen S. Rich

ABSTRACT: HLA oligogenotyping has been used successfully to characterize most phenotypically undetectable variants of class II genes. Limitations inherent to the class I system have, however, complicated the application of this and other molecular approaches to HLA class I typing. We have previously shown that HLA class II polymorphism can be analyzed by a SBT approach. Here we present a class I-SBT strategy that provides complete sequence information for the two most polymorphic exons of the HLA-A, -B, and -C alleles. HLA class I SBT is based on direct sequencing of PCR-amplified HLA-A, -B, and -C cDNAs and requires a total of six cDNA - P C R sequencing reactions (two per locus) and 13 different oligonucleotides. Each combination of oligonucleotides per reaction results in locus-specific sequence ladders and allows identification of both alleles in heterozygotes. Application of HLA-A, HLA-B, and HLA-C SBT to 26

homozygous and 32 serologically heterozygous samples has resulted in the identification of 24 novel class I nucleotide sequences encoding 17 new major histocompatibility complex class I products. An unexpected high degree of heterogeneity was found at the HLA-C locus with 14 novel sequences. Although there was a good correlation between the serologic phenotypes and SBT results, HLA-C SBT of most HLA-C serologically homozygous samples (heterozygous for HLA-A and/or -B) revealed heterozygozity (six of eight). SBT, the first molecular typing approach that has been generalized to both class I and class II genes, may be of special interest in applications demanding high sensitivity and specificity, such as in paternity testing or in the evaluation of the effects of sequence allelism in the outcome of unrelated bone marrow transplantation. Human Immunology 37, 39-50 (1993)

ABBREVIATIONS

LCL PCR

lymphoblastoid cell line polymerase chain reaction

RFLP SBT

restriction fragment length polymorphism sequence-based typing

INTRODUCTION The H L A class I and class II genes constitute one of the most polymorphic gene systems known [1, 2]. These genes encocre heterodimeric glycoproteins that serve as restricting elements in nominal antigen presentation in the context of self-antigens or as foreign antigens in alloresponses [3, 4]. The cloning and sequencing of class I and class II genes has established that polymorphism at H L A loci is much greater than that identified by serology [5, 6].

From the Institute of Human Genetics (P.S., A.L.L., M.T.B.-J., S.H.M., A.J.F., S.S.R.) and Departments of Medicine (P.S., J.J.B.) and Laboratory Medicine and Pathology (S.S.R.), University of Minnesota, Minneapolis, Minnesota, USA. Address reprint requests to Dr. P. Santamaria (current address) , Department of Microbiology and Infectious Diseases, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. Received tU) November 30, 1992: accepted March 25, 1993. Human Immunology 37, 39-50 (1993) © American Society for Histocompatibility and Immunogenetics, 1993

We have previously reported that H L A class II polymorphism can be unambiguously analyzed by a sequence-based typing (SBT) approach [ 7 - 9 ] . Class II SBT is based on the enzymatic amplification o f reversetranscribed total cellular R N A using locus-specific oligonucleotides followed by direct sequencing of the amplified products using nested oligonucleotides. Complete DRB1, 3, 4, 5, D Q B 1 , and D Q A 1 typing can be performed in less than 24 hours by performing a total of six simultaneous c D N A - P C R reactions. As opposed to oligogenotyping (polymerase chain reaction-sequencespecific oligonucleotide p r o b e [ P C R - S S O P ] ) , SBT only requires a few oligonucleotides (12 for D R - D Q typing) and enables direct determination of the sequences of the alleles expressed at each locus. While the usefulness of obtaining such accurate typing information remains questionable, SBT can be generalized to all H L A loci, 39 0198-8859/93/$6.00

40

regardless of their complexitY or extent of polymorphism. Here we present SBT protocols for all classic HLA class I genes (HLA-A, -B, and -C). HLA class I SBT requires six cDNA-PCR-sequencing reactions, two for each locus, which employ a total of 13 different oligonucleotides. HLA class I SBT has been designed to allow, without relying on sequential runs or on the use of many different oligonucleotides, (a) determination of the nucleotide sequences encoding the two polymorphic domains (a I and o~2) of all alleles at all loci (codons 7-188 for HLA-A and 7-196 for HLA-B and HLA-C); (b) unambiguous typing of any sample, heterozygous or homozygous, without requiring additional typing information; and (c) identification of known and novel class I allelic sequences. MATERIALS AND METHODS

Cell lines. We studied 28 lymphoblastoid cell lines (LCLs) from the Tenth International Histocompatibility Workshop [10] (provided by Dr. Miriam Segall, University of Minnesota) and 31 LCLs corresponding to unrelated individuals who had been previously typed serologically. The cell lines tested are listed in Table 1. HLA-A, -B, and -C transcript amplification. Total cellular RNA was prepared from 5-50 x 106 LCLs by cesium chloride centrifugation [11]. Total cellular RNA, 1-3 Izg, was reverse transcribed with Moloney leukemia virus reverse transcriptase (MLVRT) (200 U; Bethesda Research Laboratories) in 50 mM Tris-HCl, pH 8.3, 75 mM KCI, 10 mM DTT, and 3 mM MgC12, in the presence of the ribonuclease inhibitor RNasin (10 U; Promega), 75 tzM each dNTP, and 10 ng of a locusspecific antisense oligonucleotide (see below and Table 2) in a 20-1zl final volume for 30-45 minutes at 37°C. After the incubation period, the unincorporated cDNA synthesis primers were removed by spin dialysis through Ultrafree-100 columns (Millipore). The retained cDNA molecules were enzymatically amplified by PCR in a 100-~1 final volume reaction containing 5 mM KCI, 10 mM Tris-HCl, pH 8.3, 1.25 mM MgCl2, 0.01% gelatin, 75 mM each dNTP, 0.5 U recombinant Taq polymerase (Cetus Corporation), and 100 ng of each of two amplification primers (Table 2), for 35 cycles (1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C) using a PerkinElmer Cetus Thermocycler [ 12-15].

Direct sequencing of amplified products with Taq polymerase. The reaction mixture (100 Izl) was freed of unincorporated dNTPs and excess of oligonucleotides by spin dialysis through Ultrafree-100 (Millipore) microconcentrators. The retentate was dried down and resuspended in 15 ~l of 1 x Taq sequencing buffer (50 mM Tris-HCl,

P. Santamaria et al.

pH 9, 10 mM MgC12). Nested oligonucleotides were used for sequencing (Table 2). Primers, 80-100 ng, were end-labeled with 10 pmol of ~/-32P-labeled ATP (5000 Ci/mmol, 10 tzCi/~l) and 5 U T4 polynucleotide kinase (Promega) in a 10-~1 final volume. Primers, 10 ng (1 /A), were added to the sequencing mixture without extraction of unincorporated labeled ATP, boiled for 5 minutes, and then left at room temperature for 15 minutes. Eight units of recombinant Taq polymerase (Cetus Corporation) were added to the mixture. Four microliters of the annealed primer-template mixture were later added to 4 tzl of each of the following stop nucleotide mixes (ddG mix: 15 tzM each dGTP, dATP, dCTP, and dTTP; 45 ~M ddGTP; ddA mix: 15 /.~M each dGTP, dATP, dCTP, and dTTP; 600 IzM ddATP; ddT mix: 15 tzM each dGTP, dATP, dCTP, and dTTP; 1200 tzM ddCTP; and ddC mix: 15 tzM each dGTP, dATP, dCTP, and dTTP; 450/.~M ddCTP). The reactions were allowed to proceed for two consecutive periods of 10 minutes at 72°-74°C. After the second cycle, each reaction was chased with 2 ~l of a 7.5 tzM mixture of ATP, GTP, TTP, and CTP, and allowed to proceed for 5 minutes. The reactions were stopped by adding 4 ~l of 95% (vol/ vol) formamide-20 mM EDTA, heated to 80°C for 5 minutes and loaded on a 0.4-mm-thick 6% polyacrylamide-7 M urea gel. Electrophoresis was performed at 2500 V for 2 hours (short run, all reactions) and 3½hours (long run, reactions using Z133 as sequencing primer), respectively, the gel fixed in 5 % (vol/vol) glacial acetic acid-5% (vol/vol) methanol for 15 minutes, dried, and exposed to Kodak X-Omat film for 4-24 hours either at room temperature or at - 70°C with intensifying screen. These conditions allowed interpretation of sequences corresponding to codons 7-188 for HLA-A cDNAs and 7-196 for HLA-B and -C cDNAs. A sequence was considered as novel if different from those published in references 6 and 16-26.

Oligonucleotide sequences and cDNA-PCR-sequencing primer combinations for HLA class I SBT. Figure 1 shows the relative positions of each of the oligonucleotides used for the cDNA, PCR, and sequencing steps of each reaction on the mature HLA class I mRNA molecules. The sequences of these primers, the loci they are specific for, the positions (codons) to which they anneal, and the reaction(s) they are used in (reverse transcription, PCR, or sequencing) are indicated in Table 2A. In Table 2B we show the specific combinations of primers that are used for the cDNA/PCR/sequencing reactions for each locus. RESULTS

Sequence-based typing of HLAoA, HLA-B, and HLA-C genes in homozygous typing cells: high degree of sequence

TABLE 1

Cell lines and heterozygous combinations tested

Cell lines

Ethnic group

A

B

C

SA(9001) WJR076(9012) DEM(9007) RML(9016) SCHU(9013) WT8(9017) AMAI(9010) PF405(9088) EJ32B(9085) SAVC(9034) SPOO10(9036) JBUSH(9035) JVM(9039) H0301(9055) WDV(9062) TEM(9057) AMALA(9064) BH(9046) DBB(9052) CALOGERO(9084) BTB(9067) LUY(9070) TAB089(9066) 1340 0048702 MZ070782(9002) KR PC SVD ICJ TF RSH(9021) KT17(9024 CF996(9094) OLGA(9071) K562 0596 17V06940 1101 0166579 0534 17V05991 1049 008136 1276 0105379 1098 0008607 0818 17V07953 1157 1157URD 1283 073841 1213 1213URD 1066 1066URD AR

O C C AI C C C C C C C C C C C C AI C C C C C O C C C C C C C C N O C AI ~ C C C C C C C C C C C C C C C C C C C C C C C

24 2 2 2 3 3 28 1 30/31 3 2 32 2 3 2 25 2 2 2 2 2 2 2 1 1 24 1 1/2 25/2 24/2 11/1 68/30 2/11 3/2 31 -1/2 1/2 1/2 1/2 1/3 1/3 1/3 1/3 1/2 1/2 2/28 2/28 2/3 1/3 2 2 11/28 2/11 1/2 1/2 2/3 2/3 24/1

7 57 57 51 7 7 53 7 18 7 44 38 18 14 38 38 62 13 57 40 27 51 46 8 8 14 8 8/51 44 15/44 8/7 42 62/35 14 62 -8/44 8/44 51/8 51/8 7/8 7/8 7/8 7/8 27/63 27/63 7/27 7/27 7/44 7/44 35/62 35/62 14/35 14/35 8/49 8/49 7/56 7/62 39/51

w7 w7 w6 -w7 w7 w4 w7 w5 w7 w5 -w5 w8 --w9 w6 w6 w2 wl -wl 1a --w2/w8 -----w2 w9/w4 w8 wll ~ -w5 w5 --------w2 wl w3/w5 -w3 ---------

A

B

C

2401 0201 0201 0204 b 0301 0301 6802 0101 b 3002 0301 0201 3201 0201 0301 0201 6601 0201-MN4 0201 0201 0201 0201 0201 0201 0101 0101 2401 0101 0101/0201 0201/2501 0201/2401 0101/1101 b 6802/3001 0206/1101 b 0201/0301 3101 ? 0101/0201 0101/0206 0101/0201 0101/0201 0101/0301 0302/0301 0101/030Y 0101/0301 0101/0201 0101/0201 0201/6801 0201/6801 0201-MN6/0301 0101/0301 0201 0201 1101/6802 1101/0205 0205/0101 0205/0101 0201/0301 0201/0301 2401/0101

0702-MN1 5701 5701 5101 0702-MN1 0702-MN1 5301 0801 1801 0702-MN1 4401-MN3 3801-MN4 1801 1402 3801 b 3801 ~ 1501 1302 5701 4002-MN14 2701-MN12 5101 4601 0801 0801 1402 0801 0801/5101 4402-MN8 1501/4402-MN8 0801/0702-MN1 4201 3501/1501-MN6 1401 1501/3501-MN7 1801/4001 c 0801/4402-MN8 0801/4402-MN8 0801/5101 0801/5101 0702-MN1/0801 0702-MN1/0801 0702-MN1/0801 0702-MN1/0801 / 2702-MN13/63-MN9 2701-MN12/63-MN9 0702-MN1/2701-MN12 0702-MN1/2701-MN12 0702-MN1/4402-MN8 0702-MN1/440x f 3501/1501 3501/1501 140x/350x f 140x/350x f 0801/4901 f 0801/4901 f 0702-MN1/5601 b 0702-MN1/1501 3901b/5101

0702-MN1 0701 0601-MN2 0601-MN3 0702-MN4 0702 c 1401-MN5 0701 0801-MN6 0702-MN4 1101-MN6 1202-MN7 0801-MN6 0801-MN8 1202-MN9 1202-MN9 0301-MN10 0601-MN2 0601-MN2 02022 0102 b 1401-MN12 0102 b 0701 0701 02022/0801-MN8 0701 1401-MN12/0701 1202-MN9/0801-MN6 0302~/0801-MN6 0701/0702-MN4 0601-MN13 1401-MN12/0301-MN10 ~ 080Y 0102b/0302 b 0302b/0801-MN6 0801-MN6/0701 0801-MN6/0702-MN4 0801-MN16/0701 02022/0701 0701/0702-MN4 0702-MN1/0702-MN4 0701/0702-MN4 0701/0702-MN4 02022 0701/0101 0702-MN1/02022 0702-MN1/0102 b 0302f/0801-MN6 e 0702-MN1/0801-MN6 0301-MN10/0801-MN6 0301-MN10/0601-MN5 0801/1401 d 0801/1401 a 0701-MN19 0701-MN19 0702-MN4/0102 b 0702-MN4/0302 b 1401-MN12/0702-MN1

Allele designation followed by MN indicates a new DNA sequence variant for the corresponding allele. New alIelic DNA sequences encoding amino acid substitutions are underlined. C, Caucasian; AI, Amerindian; N, Negroid; O, Oriental; ?, no HLA-A locus sequences could be identified by SBT in this cell line; and --, typing information not available. a The Cwl 1 antigen nomenclature has been changed to Cw8 [16]. b "Novel" sequences (or corrections of known sequences) published while this manuscript was in preparation [6, 16, 26]. Partial sequence information: ' codons 7-118; i codons 7-86; ~codons 83-188;/codons 123-188.

42

P. Santamaria et al.

TABLE 2

Oligonucleotides and reactions for class I SBT A.

Oligonucleotides

Oligo

Sequence

Anneal

A B C 101 A B C 102 C 103 B 104 A 105 C 106 Z235 A 108 A B C 109 Z 133 B 1579

5'-GGCCCTGACCGAGACCTGGGC-3' 5'-AGATGGCTCCCATCTCAGGGT-3' 5'-AGGGGCTCTGGCAGCCCCTCG-3' 5'-AGGGGCTTCGGCAGCCCCTCA-3' 5'-AGGGGCTTGGGCAGACCCTCA-3 ' 5 ' - G T C A C G T G T G T C I T F G G G T G T - 3' 5'-GACCACAGCTCCGATGACCACA-3' 5'-AGAGATAGCGTGGTGGGTCAT-3 ' 5'-CAGTGGGCTACGTGGACGACA-3 ' 5'-GTCCAGGAGCGCAGGTCCT-3 ' 5'-TCCCCATGCGGCCGCCAGGTC A G T G T G A T C - 3' 5'-GCTCCGATGACCACAACTGCT-3 ' 5'-TAGGACAGCCAGGCCAGCAACA-3 '

- 8/- 1 271-277 270-263 270-263 270-263 183 - 190 297-304 189-195 24-31 128-134 211-221 294-302 287-294

Z286 Z285

B. Reaction

cDNA

1. 2. 3. 4. 5. 6.

ABC102 ABC105 Z235 Z285 C103 Z285

Ori a

Loci

Step

S AS AS AS AS AS AS AS S AS AS

A/B/C A/B/C C B A C B A A/B/C A/B/C A/B/C

PCR RT RT RT RT/PCR PCR RT PCR/SEQ PCR SEQ SEQ

AS AS

B C

RT RT

SBT reactions PCR

ABC109 ABC101 ABC101 ABC109 ABC101 ABC109

+ + + + + +

ATffMgCI2 A105 A108 B104 B104 C106 C103

50°C/1.25 50°C/1.25 55°C/1.25 55°C/1.25 55°C/1.25 55°C/1.25

mM mM mM mM mM mM

Sequence A108 Z133 Z133 B1579 Z133 B1579

Ori, the orientation of the primer (AS, antisense; and S, sense). b AT, annealing temperature in PCR.

heterogeneity at the HLA-C locus. The six class I SBT reactions shown in Fig. 1 represent optimal combinations of primers. These optimal combinations were arrived at after extensive testing in a series of homozygous typing cells. Upon optimization, these SBT reactions were tested on 26 HLA-A, -B, -C homozygous LCLs, including 23 Tenth Histocompatibility Workshop cell lines (Table 1). These experiments were performed to ensure the locus specificity of each of the two reactions per locus, to obtain subtypic information for these cell lines, and to determine the existence of further sequence heterogeneity within known serological specificities at each locus. Each of the class I SBT reactions described here generate locus-specific products. As an example, Fig. 2A shows representative sequence ladders corresponding to HLA-A, HLA-B, and HLA-C cDNAs of a homozygous LCL sequenced with primer Z133. Oligonucleotide primers and primer combinations were designed not only to provide locus specificity, but also to enable interpretation of both allelic sequences in heterozygotes (see below). However, there are very few locus-specific sequences that are shared by all alleles at a single locus and that are substantially different from the sequences

of all alleles at other loci. To avoid dominant selection of specific alleles in heterozygotes, the locus specificity of each class I-SBT reaction is provided by a combination of oligonucleotide primers, rather by the cDNA or PCR or sequencing primers alone. The reverse transcription reaction provides certain locus specificity; however, reverse transcription primers have to be used at a low concentration and must be removed after cDNA synthesis to enable locus-specific amplification. Reactions 1, 4, and 6 provide sequence information from the 5' end of exon 4 through most of exon 3; reactions 2, 3, and 5 provide sequence information for exon 2 and most of exon 3 (overlapping with reactions 1, 4, and 6) (Fig. 1). Except for reaction 1, which uses a locus-specific primer for sequencing (A108), reactions 2 - 6 use sequencing primers annealing to regions conserved for all class I genes (Z133 and B1579). The locus specificity of some primers (e.g., primer A108) was achieved by constructing "hybrid" oligonucleotide sequences (i.e., with 5'-end sequences homologous to one group of alleles and 3'-end sequences homologous to another group). Primer ABC101, annealing to a conserved region of all three class I loci, also binds to an homologous region located 3' from sequencing primer

HLA Class I Sequence-Based Typing

43

EXON3

EXON2

/I

EXON4

I

/

i

HLA-A mRNA

ABC102 I

A~c.og--~

~iII

~ev Transc I

~ lO5

Sequenemg

AIO8

AT05

Rev Transc

~IIIIAI08

PCR

".4-[~--I Z!33 EXON2

/i

i

I

REACTION 2

Sequencing

EXON]

EXON4

I

I

/

HLA-B mRNA

I

Z2~5

I

REACTION i

PCR

Agc]c,.,..

Rev [R~nSC

~IIIB,04

.,:.

~Z133

Sequencing Z286

I ABC~CI~

Bi04

~PV TraQsc

I I REACTION3

I

I

PCR

REAC~:ON 4

Seqdenc!ng EXON2

/L

i

EXON3

EXON4

I

I

/f

HLA-C mRNA

"9~IIc103

I

ABCICI-Ib-

Rev Transe I

C106

DCR

REACTION 5

Sequencing

~Z133

~e,/ Transc I I

ABCIO~÷

~IIIcIo~ ~BISV9

FIGURE 1 HLA class I SBT. Strategies for sequence analysis of exons 2 and 3 of HLA-A, HLA-B, and HLA-C gene transcripts in homozygotes and heterozygotes. Reactions 1, 4, and 6 provide sequence information from the 5' end of exon 4 through most of exon 3; and reactions 2, 3, and 5 provide sequence information for exons 2 and most ofexon 3 (overlapping with reactions 1, 4, and 6). Stippled boxes indicate approximate binding positions of antisense primers for cDNA synthesis. Black boxes correspond to amplification primers and open boxes are sequencing primers. The locus specificity of each reaction is provided by the combination of cDNA-PCRsequencing primers used, rather than by one specific primer. These primer combinations allow equal amplification of both allelic transcripts in heterozygotes, regardless of their respective sequences (see also Table 1).

Z133. If used in reactions 1, 4, and 6 (A108 or B1579 as sequencing primers), primer ABC101 generates a second, shorter amplification product that difficults interpretation o f sequences 5' from this secondary primerbinding site. Thus, reactions 1, 4, and 6 (intended to provide 3'-end information only) must use a conserved 5' amplification primer other than ABC 101 (ABC 109).

P~R

REACTION 6

SeqJe~iclng

Each sequencing primer only generates reproducible, good-quality ladders on products generated by reactions using specific c D N A - P C R primer combinations; not all PCR products generated by different c D N A - P C R primer combinations can be sequenced effectively with the same sequencing primer. This is the reason why each of the two reactions per locus employ different c D N A - P C R primer combinations, as opposed to one set of c D N A - P C R primers for both. Reactions employing sequencing primer Z133 have to be run twice; the long run enables reading the 5'-end sequences of exon 2, which enables defining subtypes for certain serologic specificities (i.e., A*0201 vs A*0206 or B*1401 vs B'1402). For most of the homozygous cell lines tested, each of these six class I SBT reactions generated only one sequence ladder (Table 1 and Fig. 2A). SBT enabled us to define the molecular subtypes of the "broader" serologic types o f these cell lines (i.e., A*0201 as opposed to A2) and to identify novel sequences (Table 1). Experiments with these 26 homozygous LCLs resulted in the identification of two novel HLA-A nucleotide

44

A*0201 T

C'1101-MN6

B'1801

\Q

Codon 91

G~G

G---C

C----C

A C~T

T IC

/G

G---C

D

G

Codon 104 B-Z133

C-Z133

A-Z133

• " - O,I

v - . ,P..

O0

O0

IOI0

04~O 0

tOO')

O0

ZlS

Z133

A108 C. 149 AT

93

B1579 91

A T

B

..................................* "

A

C. 148 I , :

~ : : ! C . 91

C. 149

AT C C

A

TC

GT

TA

C. 159

101

99

C. 161

A 101

C. 161

F I G U R E 2 (A) Locus-specific amplification of HLA-A, HLA-B, and HLA-C transcripts. Sequence ladders between codons 91 and 104 of HLA-A, -B, and -C alleles of a cell line homozygous for A*0201, B'1801, and C*0801-MN6 are shown. Lanes are read from left to right as G-A-T-C. The three panels of this figure correspond, from left to right, to SBT reactions 2, 3, and 5, respectively. Positions where each of these three HLA-A, -B, and -C alleles differ in sequences are indicated on the side of each panel to show the locus specificity of each reaction (i.e., G at bottom of HLA-A and HLA-B ladders is substituted for a C at the bottom of the HLA-C ladder). All sequencing reactions shown were done using the Z-133 primer. (B) Generation of locus-specific ladders in heterozygotes. Examples of overlapping ladders generated by each SBT reaction ( 1 - 6 from left to right). Positions with two bands (due to sequence differences between the two alleles expressed at that locus) are indicated on the right-hand side of each panel, and the alleles to which each of these bands correspond are shown at the top. The numbers at the bottom and top of each panel indicate codon numbers corresponding to the first and last base of the ladders shown. Lanes are read from left to right as G-A-T-C. Sequencing primers used are indicated directly above each panel.

.

,,,

1|ol

-Ira

I~

. . . . .

. . . .

....

....

. . . .

. . . .

""

¢3-----¢3

~'"

¢3-*-*-¢3

. . . . .

. . . . .

42---2

Q-

. . . . . . . . .

-"

. . . . . . . . . .

. . . . . . . . .

.....

. . . . . . . . . .

- ~

-Iml

(."1=O=

O.I=O=

.

T-

~-

. . . . . . . .

. . . . . . . .

.

. . . . . . .

T-2

T-A

~-&

~ . . . . .

42 . . . . .

P-2

J~

• . . . . .

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

• . . . . . . . . . . . . . . . . .

c . . . . . . . . . .

~ . . . . . . . . . .

2 . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . .

6601

•

N

IRJ

IOI7

-mm

BDm

-ira6

-roll

o.o?o=

c40001

+o=l?

-imll

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

OF

c'P

C . . . . . . . . . . . . . . . . . . . . . . . .

J~ . . . . . .

. . . . . . .

T . . . . . . . . . . . . . . . . .

& . . . . .

j~ . . . . .

• . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

42 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

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

A-

- --T

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

..

..

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

. • - . . . . . . . . . . . . .

2-2-

&-A

¢3-O

¢3-O

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

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

. . . . . . . . . . .

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

~ . . . . . . . . . . . . . . . . . . . . . . .

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

~ . . . . . . . . . . . . . . . . . . . . . . .

-T-

c . . . . . . . . . . .

c . . . . . . . . . .

C . . . . . . . . . . .

C . . . . . . . . . .

¢ . . . . . . . . . . .

. . . . . . . . . . .

-'P-

T-

T -

-'P-

c-

. . . . . . . .

. . . . . . .

C . . . . . . . .

. . . . . . . .

- -a

. . . . . . . .

- -¢3 . . . . . . . .

- -Q

- -¢3 . . . . . . . .

. . . . . . . .

- -¢3 . . . . . . . .

- .0

T-T-C3

-'r-

T-

c . . . . . . . . . . . . . . .

C . . . . . . . . . . . . . . .

. . . . . . . .

C . . . . . . . .

-¢3 . . . . . . . .

- -0

. . . . . . . .

- -O . . . . . . . .

G . . . . . . . .

- -~

T +-

-T-

C . . . . . . . . . . . . . . .

C . . . . . . . . . . .

c . . . . . . . . . .

C . . . . . . . . . .

C . . . . . . . . . . .

T-

c . . . . . . . . . . . . . . .

e . . . . . . . . . . . . . . . . . . . . . . . .

196

"1+ . . . . . . . . . . . . . . . . .

? . . . . . . . . . . . . . . . . .

42 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

---~--

. . . . . .

. . . . . . . 0-

- - -2-

C++--2---2

--C8---~

- - -<~-

- -0~-

. . . . . .

.0

--+

- .0

C--+

c--+

~--~-

~..~-

~-

. . . . . . . .

A . . . . . . . . . .

2 . . . . . . . . . . .

A . . . . . . . . . . . . .

A . . . . . . . . . . . . .

. . . . . .

C . . . . . . .

. . . . . . .

~ . . . . . . . . . . . . . .

,II+. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

42 . . . . . . . . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . .

=' . . . . . . . . . . . . . .

T . . . . . . . . . . . . . .

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

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

(~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .

C . . . . . . .

5" . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . .

-e

C . . . . . . . . . . . . . . . . . . . . .

-T-C

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

T--G

T-

-.

. . . . . . . . .

~I

. . . . . . . . . . .

¢3 . . . . . . . . . . .

T . . . . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

..

..

. . . . . . . . . . .

- . . . . . . . . . .

- . . . . . . . . . .

..

..

2 . . . . . . . . . . .

2 . . . . . . . . . . .

A . . . . . . . . . .

2 . . . . . . . . . .

.42 . . . . . . . . . . .

. . . . . . . . . . .

~

~

. . . . . . . . . . .

~ . . . . . . . . . . .

G . . . . . . . . . . .

~ . . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

~ . . . . . . . . . . .

d

- . . . . . . . . . . . . . .

- . . . . . . . . . . . . . .

.& . . . . . . . . . . .

42 . . . . . .

~ . . . . . .

C . . . . . . . . . . . . . . . . . . . . . . .

A

A . . . . . . . . . .

TO

A . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . .

.,It

- ~

- ~

•

~ . . . . . . . . . . . . . . . . . .

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

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

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

T . . . . . . . . . . . .

T--G

T--G

A . . . . . . . . . . . . . . . . . .

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

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

. . . . . . . . .

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

-¢3 . . . . . . . . . . . . . . . . . . . . . . . . . . . .

'P-*~

T--O

A . . . . . . . . . . . . . . . . . .

2 . . . . . . . . . . . . . . . . . .

J), . . . . . . . . . . . . . . . . . .

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

2 . . . . . . . . . . . . . . . . . . . . . . .

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

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

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

c . . . . . . . . C-

. . . . . . . . . ~ . . . . . . . . .

¢3

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

. . . . . .

. . . . . .

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

T--.

T - < I

T-

T--@

"[' . . . . . . . . . . . .

? . . . . . . . . . . . .

'1" . . . . . . . . . . . .

0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C-

. . . . . . . . ~1 . . . . . . . . .

~-

~ . . . . . . . . .

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

- -2 . . . . . . . . . . . . . . . . .

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Co--.

. . . . . . . . . .

¢3 . . . . . . . . .

¢3 . . . . . . . . .

- -¢3 . . . . . . . . . . . . . . . .

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

¢3-A-

-o-2--

¢3-A--

- ¢ 3 - A - - - - 0 3 - - . 0

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

- -¢3 . . . . . . --<1

T-~C-

¢-42-1~C--T-~

C - C - 0 ~ - ~

¢3 . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . .

T . . . . . . . . .

: ~ = - - . .~. . . . . . . . o--c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . .

13 . . . . . . . . . . . . . . . . . . . . . . . . . . . .

-0

- -¢3 . . . . . . T . . . . . . . . . . . . . . . . . . T-0C-T-C . . . . . . . . . . . . . . . . . . . . . . . . . . . '1"--(i . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . .

. . . . . . . .

-A . . . . . . . .

- -¢3 . . . . . .

- -0

- -e

T . . . . . . . . .

T-¢3

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

-J~ . . . . . . . .

- -¢3 . . . . . .

--¢3

~ -

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

- -A-

- -(~--

- - -co-

--(:0-

- - -CO-

- -CI~-

- -C~-

- - -CO-

- - -C~I-

- - ~ -

. . . . . .

- ~ - 2 - - - - C O - - - G

"~-2--

--¢3-2-

¢3-2--

~-A-

2--

@-A--

. -A-

¢3-&-

¢3-J~*.--

- - -A-¢3

T - - C - - - - A - - 2 - - A

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

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

2

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

~-

T---A

&-¢3

-A-

T . . . . . . . . c . . . . . . ¢3 . . . . . . . . . . . . . . . . . .

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

j~ . . . . . . . . . . . . . . . . . . . . . .

~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c-

A . . . . . . . . . . . . . . . . . . . . . .

42 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

. . . . . . .

J ~. . . . . . . . . . . . . . . . . . . . . .

j~ . . . . . . . . . . . . .

p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C-

- - -A-

- C - - - - A - - A -

-¢3 . . . . . . .

T - - c - - - - A - - 2 - - A

T-

T-

-~P- -c-

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

..-.=_:: . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

• . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

T . . . . . . . . . . . . . . . .

~P

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

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

¢3-¢3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

¢3-¢3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

J~ . . . . . . . . . . . . . .

-'r

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

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

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

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

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

¢3.411 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G-G

-~1-(I

0-(I

G-~

-~.¢3

+3-¢3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C . . . . . . . . . .

. -"

~ . . . . . . .

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

-G-

¢3-<1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

-O+0

2 . . . . . . . . . . . . .

. . . . . . . . . . .

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

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

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

+c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

¢

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

- ~

j ~

-=OG

-~1~

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

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

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

A - A . . . . . . . . . . . .

A - 2

A-A

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

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

A . . . . . . . . . . . . . .

A+A

~t-

A-

2-

+16+++~¢3

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

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

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

¢3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A - & . . . . . . . . . . . .

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

A . . . . . . . . . . . . . . . . . . .

& - * 2

. . . . . . . . . . .

. . . . . . . . . . .

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

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

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

A-~I*

A-J~

A-&

A-A

-

~ - - 1 ~ -

~

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

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

C . . . . . . . . . . .

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

. . . . .

-~I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

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

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. - • . . . . . . .

2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

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

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

/~ . . . . . . . . . . . . . . . . . . .

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

. --A

- . . . . . . . . . . . . .

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

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

. . . . . . . .

- . . . . . . . . . . . . .

+ + - . . . . . . . . . . . . .

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

Cl . . . . . . . . . . . . . .

. . . . . . . .

¢3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

e . . . . . . . . . . . . . . . . . . . . . . . .

, . . . . . .

2 . . . . . .

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

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

~

• . . . . . .

42 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C . . . . . . . . . . . . . . . . . . . . . . . .

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

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

. . . . . . . .

c-A----o

~ . . . . . 0

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

OF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

qk~ . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . .

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

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

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

~

O~

--c

-~m-o T--&

F I G U R E 3 Nucleotide sequences of novel class I alleles• Novel sequences are designed with the official World Health Organization nomenclature [5] corresponding to the most closely related allele known followed by M N and an arbitrary number. The sequence shown at the top is the consensus sequence used by Z e m m o u r and Parham [6], and sequences were considered as novel if different from those published earlier [6, 16-26]• Nucleotide substitutions are indicated i n i t a l i c s o r a s d o t s (to indicate homology with the consensus sequence). Sequences from codons 7- 1 96 are shown•

o/,601-1mB

c*1601-~1112

~.* 1= o2

o.OeOl

~ o e o 2 - t m 8

-iml

o.o?o=

o . o ? o l - l m ~

~ o l o l - l m l l

C40eo2

O.0101-mS~

~t0101-mll0

BIB

1SO2

66~

•

*lm£=

•

n*27

. . . . . . . .

. . . . . . . .

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

+

¢ . . . . . . .

42 . . . . . . .

42 . . . . . . .

J~03ol-O=II *

Ira1

.....

42 . . . . . . . . .

~

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c . . . . . . . . . . . . . . . . . . . . . . . .

c

. . . . . .

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

~

- -c

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

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

42.

. . . . . . . . . .

T . . . . . . . . . .

.

. . . . . . . . . . .

CO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

42 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

~ . . . . . . . . . . . . . . . . . . . . . . . .

-Im6

. . . . . . .

c-

& . . . . . . . . . . . . . . .

CO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c . . . . . . .

C . . . . . . .

42 . . . . . . .

42 . . . . . . .

c . . . . . . .

C . . . . . . .

42 . . . . . . .

o . 1 6 o l o m

T . . . . . . . . . .

" . . . . . . . . .

. . . . . . . . . .

42 . . . . . . . c . . . . . . .

J~IO=ol

A . . . . . . . . . . . . . . . . . . .

. . . . . .

. . . . . .

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

~ . . . . . .

~-

~-

CC. . . . . . . . .

~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . .

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

c . . . . . . . . . . . . . . . . . . . . .

42 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

2'-

. . . . . . . . . . . . . T . . . . . . . . . . . . .

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

42 . . . . . . .

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

~-

~-----T T .....

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

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

. . . . .

T .....

~ . . . . .

42- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

42 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . .

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

lso2

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

j~ . . . . . . . . . . . . . . . . . . . . . . . .

~, . . . . . . . . . . . . . . . . . . . . . . . .

~ .

~

~

~

~ . . . . . . . . . . . . . . . .

-c-

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

J~ . . . . . . . . . . . . . . .

N I ~

- ~ 1

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~

A-

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

& . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . .

. . . . . . . . . . .

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

0~O=

2 . . . . . . . . . . . .

~ . . . . . . . . . . . .

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

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

& . . . . . . . . . . . . . . . . . . .

• . . . . . . . . .

c-11o1-II11~

•

2 . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . .

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

2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

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

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

T-2

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

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

cl1~O='lal

•

. . . . . . . . .

2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

olin?

~

T . . . . . . .

"r . . . . . . .

J~i•-

~

.......... ¢3 . . . . . . . . . a-. A-C . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . <1 . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -. . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . 1:1-¢3 . . . . • -. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

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

2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . .

O.01

O.I=O=

~ . . . . . . .

A . . . . . . . . . . . . . . .

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

-~).II

2-~

A-c

• . . . . . . . . .

2 . . . . . . . . . . . . . . .

2

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

oI

.

¢3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

¢3 . . . . . .

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

. . . . . . . . . .

.

o . 0 1 0 1 - 1 m

-Ira

.

o . 0 0 0 1 - 1 m

O.07O=

.

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

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

- - - -~"

-1,1

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

. . . . . . . . . .

-m119

.

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

•

~'.0701

.

~P . . . . . . . .

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

. . . . . . . . . .

o'o111-ImI~

.

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

. . . . . . . . . .

O'O7O2-11211

.

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

o . o 6 0 ~ - I m

o Im o Im

. . . . . . . . . . .

O . 0 1 0 L - I ~

i

.

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

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

11

Ira?

.

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

O.O=0Z-lmI0

•

• • 1601 • + 11O=

3601

•

.

2 . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.*.~.~-............................ : . . ~ . . ~ ~ ~ + - + * ;

& . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

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

BIII:I

.

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

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

=~

.

T . . . . . . .

c . . . . . . . . .

c . . . . . . . . .

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

-IBIS

•

.

¢3 . . . . . . . . . . . . . . . . . . . . . .

-II~ Iml

""~

.0

I~0~O= • • 1101

~-

.

¢3 . . . . . . . . . . . . . . . . . . . . . .

"~

.0

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

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

~1-~"

J~O~01

.....

O.1601

c---A

.

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

"¢3"'P . . . . . . . . . . . . . . . . . . . . . . . . . . . .

"~"

"&

¢3--Ja

.0--A

.0

A-C

-A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

¢3" "~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

~ 0 + o 2 - 1 m

.....

. . . . . . . . . .

. . . . . . . . .

. . . . . . . . .

o . 0 1 0 1 - 1 m

c,16oi-imi~

-0-

c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . .

. . . . . . . . . .

....

42tl01111"11•141

-I~S

:~.'...'7.-~_~-.:-L..~_

..... T - - - 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c-A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . & . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . .

-Ira1

o=

?

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

m

o . o • 0 1 - 1 m l

01~0=

- ~ 1 ~

~'~G7

O.0~

] " U I I ~

010"0

01

- I ~

-IBII

0,0101

m

O.0612

O=

•

lira

I m

01 "1~110

66O=

.

-1~

0106

,101

•

iml

-Ira?

-Ira1=

•

I*~SO2

• ~

•

~ o = 0 1 - m • o~o= ~

2 . ....

46

P. Santamaria et al.

1

I0

At0201

-F

30

20

GSHSMRYFYTSVSRPGRGEP

RFIAVUYVDD

40

50

TQFVRPDSDA~PRMEPRJiP

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

70

60

WI~Q~GPEYW

D R E ~ F K A H

90

Q . . . . . . . . . . . . . . . . . .

G-

-F .................................

Q ..................

G--RKV-

A~0201-MN6

-F .................................

Q ..................

G--R-V---

Bt3501

-'-

AM

T-

-Y-

-S ................

Bfl501

---

AM

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

A ...................

IS-TN

T-

-Y-

-S .......................

B*350I-MN7

---

AM

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

A ...................

IS-TN

T-

-Y-

-S ................

R~15Ol-b~6

---

AM

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

IS-TN

T-

-Y-

-S

B~5701

---

AM

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

G- -R-M--S

B'63-I~9

-'-

AM

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

G- -R-M-

T .................

C~0301

-C"

A .........

H ..................

CTO3Ol-b~qlO

-C-

A .........

H .......................

C~0601

-D-

A ............

C*0601-MNI3

---

C~O601-b~3

---

EA

D

E---O

V-RK

S-

- H - V D - G

- -

S"

-H-VD-G

8--H-VD-G

N--I--TN

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

......

- -

LRGYYN(~EAG~HTLQRMYG

A*0201-MN4

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

-RK'V-

80 A ~ R E N L R N

....

-S

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

V . . . . .

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

V-M-F-

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

V .....

II .....

II .....

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

-'-Y

.....

I

ALR

..........

- - - Y

. . . . .

I

ALR

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

II-V'--

.....

KY-PQ

.....

VB

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

G ......

V .......

.....

KY - RQ

.....

VB

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

G ......

V .......

.....

KY-RQ

- "A-

-V-

- -K

.........

A .................................

G ......

V .......

.....

KY-RQ

- -A-

-V-

- -K

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

A .........

O . . . . . .

V

V-

- -K

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

S ....................

H . . . . . . . . . .

~ . . . . . . . . . . . .

.......

......

Y-RQ

. . . . .

II ..... R--II D

.....

......

W-

I7

. . . . .

C*0601-MIq2

-D-

A ............

S ....................

O ......

V .......

.....

KY-RQ

- -A-

-V-

- -K

D

......

C'0702

-D-

A ............

S ....................

G ......

V .......

.....

KY-RQ

- -A-

-VS

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

D

........

- -A-

-V~

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

D

..........

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

D

........

C*0701

......

Y-RQ

C*O702-b~ql

-~9-

A . . . . . . . . . . . .

G . . . . . .

V . . . . . . . . . . . .

K'Y

- -A-

-V~

C'0801

-'-

A .......................

Q .........

G ......

V .............

KY-RQ

.....

VS

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

C~0801-blN6

---

A .......................

Q .........

G ......

V .............

KY-RQ

.....

V-

- -K

G ......

V .............

KY-RQ

.....

%/B ...................

(2 . . . . . . . . .

G . . . . . .

V . . . . . . . . . . . . .

K Y - R Q

S . . . . . . . . . . . . . . . . . . . .

C~0801-MNI6

---

A .................................

C'0801-MN8

---

A . . . . . . . . . . . . . . . . . . . . . . .

C*1202

- RQ

- -

I . . . . .

W"

" 8-

S -

.................... W-

" -

---

A .................................

G ......

V .............

KY-RQ

-VB

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

C*1202-b~/9

---

A .................................

O ......

V .............

KY-RQ

" -A-

-V8

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

W-

- -

C~1202-bK~7

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

G ......

V .............

KY-RQ

- -A-

-V8

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

W-

- -

C'1401

-S ...................................

G ......

V .............

KY-RQ

.....

C*1401-b~ql2

-S ...................................

G ......

C*1401-b~5

-S ...................................

G-

I00

ii0

120

CDV~PDGRLL

..... - -A-

150

160

- -K

170

180

QITQRKWEAA

R%~0~LRAYL

....

H ............

K ..........

M--

-T'KH

.....

H ..............................

T-A

....

....

H ............

K ..........

M--

-T-KH

.....

H ..............................

T-A

....

S-W-F

....

H ............

K ..........

M--

-T-KH

.....

H ..............................

T-A

....

B*IS01

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

H--S

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

S ....................

BT3501-MN7

--L

H'-S

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

S ...............................

B~I501-MN6

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

H--S

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

S ....................

B~5701

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

H--S

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

S ...............................

B*63-b~9

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

H'D

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

S ....................

C*0301 C*0301-MNI0

.........

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

H . . . . . . . . . . . . . . . . . . . .

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

S

C*0601

-L

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

C*0601-bR~I3

-L

. . . . . . . . .

C*0601-~3

- -L

C'0601-MN2 C*0702 C*0701

-L "

N-F

- -L

. . . .

K . . . . . . .

L . . . . . . . . .

K . . . . . . . . .

- -W

. . . . . . . . . . . . . . . . . . . . . S

E . . . . . . . . . .

E . . . . . . . . .

G-E}{

....

G . . . . . . . . .

EH

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

V . . . . . . V-

-

EH .........

V ......

RR

V-

-

. . . . . . . . .

-

"

- -L .........

C~1401-/~W5

L . . . . . . . . .

I~ . . . . . . . . . .

L ......

C*0801-~6

C~1401-b~12

E . . . . . . . . . .

V-

L . . . .

C~1202-M~/9

L .................................

V-

L . . . .

C*1202-bIN'7

E ..........

E ..........

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C*1401

L .....................................

Eli . . . . . . . . .

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

-L

V--

L .................................

E-

. . . . . . . . . . .

- -L

L ..............................

A-DE

L ....

. . . . . . . . . . .

- -L

......

V ......

L .....................................

F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eli . . . . . . . . .

. . . . . . . . .

C*1202

E---W

......

S ..............................

- -L

C~0801-MN8

--AV

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

- -L

C~0801-~16

HHPISDHEAT M"

. . . . . . . . . . .

C'0801

C*0702-~qi

......

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. . . . . . . . . . . . . . . . . . . . . . . . . . S-

RADpPKTHVT

L .............................. E---W

F-

. . . . . . . . . . .

-L ...........

- -L

N . . . . . . . . . . . . . .

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

YLENGKETLQ

........

190

S-W-F

S ...............................

R

W-FD

S-W-F

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

R

.........

....

H--S

~

W-F-

"48 ................... -V-

....

-L .........

RSWTAADTAA

KY-RQ KY-RQ

A~0201-MN4

Bw3501

KDYIALNEDL

140

V ............. -V .............

VIS ...................

A~0201

A*0201-MN6

RGYDQYAYDG

130

- -E-

- -A-

- -W . . . . . . . . . . . . . . . . . . . . . . . . . .

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

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E . . . . . . . . . .

L-

N-F

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

T . . . . . . . . . . . . . . . . . . . . . . . .

K . . . . .

~-I

V . . . . . .

N-F

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

E- - -R ....................

K .....

EH .........

V-

K .............

. . . . . . . . .

L . . . . . . -

-

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

A-

- -Q . . . . . . . . . . . . . . . . . . . . . . . . . .

Eli . . . . . . . . .

V-

-

. . . . . . . . . .

E-

- -R

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

EH

. . . . . . . . .

V-

-

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-

- -W

. . . . . . . . . . . . . . . . . . . . . . . . . . ~

. . . . . . . . .

V ......

N-F

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

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

K . . . . . . . . . . . . .

K . . . . .

- -L

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

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-

- -W

. . . . . . . . . . . . . . . . . . . . . . . . . . Eli . . . . . . . . .

V-

-

- -L

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

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-

- -W

. . . . . . . . . . . . . . . . . . . . . . . . . . Eli . . . . . . . . .

V-

-

- -L

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

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E-

- -R

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

. . . . . . . . .

V . . . . . .

- -L

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

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-

- -R

. . . . . . . . . . . . . . . . . . . . . . . . . . EH

. . . . . . . . .

V-

-

-L

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

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-

- -R

. . . . . . . . . . . . . . . . . . . . . . . . . . EH

. . . . . . . . .

V-

-

FIGURE 4 Novel class I amino acid sequences. New amino acid sequences are compared with known homologous alleles.

sequences (both encoding new amino acid sequences) and seven novel HLA-B sequences (three encoding new amino acid sequences) (Table 1 and Figs. 3 and 4). With a few exceptions, the SBT typings correlated with the HLA-A and HLA-B serologic phenotypes of these LCLs. For LCLs PF405 and OLGA, the SBT results did not correlate with the serologic typings. LCL-PF405 had been serologically typed as B7 homozygous but SBT revealed a B*0801 sequence. LCL-OLGA (from an Amerindian) had been typed as B62 homozygous, but

S . . . .

~

SBT indicated presence of two sequences: B* 1501 (B62 serotype) and a novel B ' 3 5 sequence (B*3501-MN7). Interestingly, the four amino acid substitutions of this new B'35 allele resulted in an c~l-domain sequence identical to that of a B* 1501 allele, thus providing a plausible explanation for the serologic results (Fig. 4). Alternatively, this allele may be serologically silent, as previously observed for a related sequence (B'3506) [23]. As opposed to HLA-A and HLA-B SBT, HLA-C SBT revealed that most of these homozygous LCLs expressed HLA-C alleles with sequences different from those published earlier [6, 17]; 14 novel HLA-C alleles ( 12 encoding new amino acid sequences) were identified (Table 1 and Figs. 3 and 4). In spite of this, there was also a good

48

HLA-C typing had usually mistaken heterozygous samples as homozygous. Six of eight HLA-C serologically homozygous LCLs were heterozygous, as defined by SBT; only three of nine HLA-C heterozygous LCLs (by SBT) were also typed as heterozygous by serology (Table 1). As observed above in homozygotes, however, there was a good correlation between the HLA-C type(s) identified by serology and the allelic types identified by SBT. Cell line RSH (Cw2 by serology and C*0601-MN13 by SBT) was an exception. SBT of some heterozygous LCLs generated ladders which, upon comparison to known sequences [6, 16-28] including those identified above in homozygous LCLs, appeared to correspond to new allelic sequences. A sequence ladder was interpreted as novel in heterozygotes if (a) it was found in more than one cell line (B*4402MN8, B*63-MN9); or (b) it paired with a second allele with a sequence that matched perfectly a known sequence (A*0201-MN6, B*1501-MN6, B*3501-MN7, and C*0801-MN16); and (c) only if these results could be reproduced in a repeated SBT experiment. Interestingly, the sequences C*0102 and C*0302, found in five different heterozygous cell lines including a C*0102/ C*0302 heterozygote and initially considered as "novel," have been recently described by Zemmour and Parham [17]. DISCUSSION We have previously reported that HLA class II polymorphism can be analyzed by a BT strategy [7-9]. Highly accurate HLA-DRB, -DQB, and -DQA SBT can be performed by running a total of six cDNA-PCR-sequencing reactions using a limited number ofoligonucleotides. Here we have generalized this typing approach to all the classic HLA class I genes. Including DPB1 and DPA1 (unpublished data), complete HLA SBT requires a total of 15 reactions exclusively employing 28 different oligonucleotides. SBT is thus the first molecular typing approach that has been generalized to all polymorphic class II and class I genes. Application of molecular approaches to class 1 typing has been complicated by a series of limitations imposed by the complexity of the HLA class I system; the high degree of polymorphism at each class I locus is scattered over 2 exons, sequence information is only available for part of the existing alleles, and there is high interlocus homology with few locus-specific regions shared by all alleles at each locus [6]. While HLA oligogenotyping has been used successfully to characterize most phenotypically silent allelic variants of class II genes [12-14, 27, 28], it has not yet been fully developed for HLA class I typing. One-dimensional isoelectric focusing is an effective method for identifying serologically unde-

P. Santamaria et al.

tectable variants or subtypes of HLA-A and HLA-B antigens [10]. However, accurate interpretation of band patterns requires serologic information and it involves cell culture and complex biochemical manipulations which have precluded its use in routine HLA typing. Thus, serology remains as the method of choice for clinical class I typing, even though it only provides partial typing information, especially for HLA-C, and relies on availability of relatively large amounts of specific antisera. SBT not only overcomes most of these limitations but also provides high sensitivity and specificity. Of special note is our identification of 24 novel class I alleles by SBT analysis of 58 samples. Most interesting, however, is the unexpected high degree of heterogeneity found at the HLA-C locus with 14 new sequences. This brings the total number of known HLA-C alleles from 26 [17] to 40 and indicates that HLA-C is at least as polymorphic as the HLA-A locus (39 known sequences) [6]. This high level of diversification suggests that HLA-C molecules may be as functional as HLA-A and HLA-B, as has been recently proposed [17]. It may be argued that some of the novel sequences reported here may correspond to sequencing artifacts or Taq polymerase incorporation errors. We believe this to be unlikely for several reasons. First, direct sequencing of the PCR products greatly reduces detectable errors in Taq incorporation. Second, our interpretation of novel sequences was possible by testing a large number of cell lines. Ladders corresponding to known sequences provided good positive controls for SBT and potentially novel sequences were only called as such if found in more than one cell line and if the same sequences could be reproduced in repeated experiments from new RNA samples. SBT can be used to define new sequences even in heterozygotes as indicated by the fact that several sequences, initially considered as "novel" (see Table 1), were independently reported by others while this manuscript was in preparation. However, SBT has limitations of its own. For instance, the current SBT technology uses RNA rather than DNA, a preferable starting material in applications where RNA is not available in necessary amounts (i.e., in forensic applications). As discussed before [7], the lower number of circulating mononuclear cells in leukemic patients (resulting in lower RNA yields) may also constitute a limitation of RNA SBT. Finally, with SBT (as well as with other current typing methods) one may not be able to assign individual sequences of overlapping ladders in all heterozygotes carrying two alleles of unknown sequences. While in its present developmental stage SBT is not yet a practical method for routine HLA typing, where large numbers of samples have to be tested with a short

HLA Class I Sequence-Based Typing

turn-around time, d e v e l o p m e n t o f computerized robotic workstations using capillary electrophoretic systems may bring SBT closer to the clinical laboratories in the future. In this regard, SBT offers a series o f advantages over other typing methods. With SBT, complete H L A typing can be p e r f o r m e d by using only one method, as opposed to different methods for different loci and purposes. Furthermore, SBT employs a limited n u m b e r ofoligonucleotides, which is independent of the number of alleles at each locus, in a limited number of reactions that are run simultaneously. Finally, SBT provides the most detailed typing information that is currently possible and does not require additional typing information (i.e., serology l. While the practical usefulness of obtaining highly accurate sequence information is questionable for certain applications, others, such as paternity testing or typing of phenotypically identical unrelated bone marrow transplant pairs to evaluate the effects of sequence allelism in outcome, could benefit considerably from SBT.

ACKNOWLEDGMENTS

We thank Dr. Fritz Bach for his continuous support and helpful suggestions in the course of this work. We also thank Drs. Harriet Noreen, Nancy Reinsmoen, and Miriam Segall for their support and for providing cell lines. Cinthia Lewis is acknowledged for excellent technical assistance. This work was supported by a General Clinical Research Center N I H (National Institutes of Health) grant (RR00400) and the American Diabetes Association. P.S. was supported by a postdoctoral fellowship from the Juvenile Diabetes Foundation International.

REFERENCES 1. Parham P, Lomen C, Lawlor D, Ways J, Holmes N, Coppin H, Salter R, Wan A, Ennis P: Nature of polymorphism in HLA-A, -B, and -C molecules. Proc Natl Acad Sci USA 85:4005, 1988. 2. Bach F: Class II genes and products: the HLA-D region. Immunol Today 6:89, 1985. 3. Zinkernagel R, Doherty P: Restriction of in vitro T cellmediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 248:701, 1974. 4. Erb P, Feldman M: The role ofmacrophages in thegeneration ofT-helper cells. II. The genetic control of the macrophage T cell interaction for helper cell interaction with soluble antigens. J Exp Med 142:460, 1975. 5. Bodmer J, Marsh S, Albert E, Bodmer W, Dupont B, Erlich H, Mach B, Mayr W, Parham P, Sasazuki T, Schreuder GT, Strominger J, Svejgaard A, Terasaki P: Nomenclature for factors of the HLA system, 1991. Tissue Antigens 39:161, 1992.

49

6. ZemmourJ, Parham P: HLA class I nucleotide sequences, 1992. Tissue Antigens 40:221, 1992. 7. Santamaria P, Boyce-Jacino M, Lindstrom A, Barbosa J, Faras A, Rich S: HLA class II "typing": direct sequencing of DRB, DQB, and DQA genes. Hum Immunol 33:69~ 1992. 8. Santamaria P, Boyce-Jacino M, Lindstrom A, Barbosa J, Faras A, Rich S: Detection of novel sequence heterogeneity and haplotypic diversity of HLA class II genes. Immunogenetics 33:374, 1991. 9. Santamaria P, Noreen H, Lindstrom A, BarbosaJ, Faras A, Segall M, Rich S: DRw52-group haplotypes are frequent acceptors of DRw15-Dw2 DQ genes in DQA1-DRB1 recombination. Immunogenetics 36:56, 1992. 10. Yang SY: Population analysis of class I HLA antigens by one-dimensional isoelectric focusing gel electrophoresis: workshop summary report. In Dupont B (ed): Immunobiology of HLA. New York, Springer-Verlag, 1989. 11. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294, 1979. 12. Mullis K, Faloona F: Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335, 1987. 13. Saiki R, Scharff S, Faloona F, Mullis K, Horn G, Erlich H, Arnheim N: Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350, 1985. 14. Saiki R, Bugawan T, Horn G, Mullis K, Erlich H: Analysis of enzymatically amplified beta-globin and HLA-DQce DNA with allele-specific oligonucleotide probes. Nature 324:163, 1986. 15. Sharf S, Horn G, Erlich H: Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233:1076, 1986. 16. Zemmour J, Gumperz J, Hildebrand W, Ward F, Marsh S, Williams R, Parham P: The molecular basis for reactivity of anti-Cw I and anti-Cw3 alloantisera with HLA-B46 haplotypes. Tissue Antigens 39:249, 1992. 17. Zemmour J, Parham P: Distinctive polymorphism at the HLA-C locus: implications for the expression of HLA-C. J Exp Med 176:937, 1992. 18. Watkins D, McAdam S, Liu X, Strang C, Milford E, Levine C, Garber T, Dogon A, Lord C, Ghim S, Troup G, Hughes A, Letvin N: New recombinant HLA-B alleles in a tribe of South American Amerindians indicate rapid evolution of MHC class I loci. Nature 357:329, 1992. 19. Little A, Parham P: The HLA-Bw75 subtype of B15: molecular characterization and comparison with crossreacting antigens. Tissue Antigens 38:186, 1991. 20. Little A, Madrigal J, Parham P: Molecular definition of an elusive third HLA-A9 molecule: HLA-A9.3. Immunogenetics 35:41, 1992. 21. Hildebrand W, Madrigal J, Little A, Parham P: HLA-

50

P. Santamaria et al.

Bw22: a family of molecules with identity to HLA-B7 in the ~1 helix. J Immunol 148:1155, 1992.

driven diversification of HLA-A2 antigens. Immunogenetics 35:344, 1992.

22. Zemmour J, Little A, Schendel D, Parham P: The HLAA,B "negative" mutant cell line CIR expresses a novel HLA-B35 allele, which also has a point mutation in the translation initiation codon. J Immunol 148:1941, 1992.

26. DomenaJ,Johnston-Dow L, Parham P: The B'4002 allele encodes the B61 antigen: B40* is identical to B61. Tissue Antigens 40:254, 1992.

23. Belich M, Madrigal J, Hildebrand W, Zemmour J, Williams R, Luz R, Petzl-Erler M, Parham P: Unusual HLA-B alleles in two tribes of Brazilian Indians. Nature 357:326, 1992. 24. Madrigal J, Belich M, Benjamin R, Little A, Hildebrand W, Mann D, Parham P: Molecular definition ofa polymorphic antigen (LA45) of free HLA-A and -B heavy chains found on the surfaces of activated B and T cells. J Exp Med 174:1085, 1991. 25. Castano A, Lopez de Castro J: Structure of the HLAA*0211 IA2.5) subtype: further evidence for selection-

27. Erlich H, Bugawan T: DNA typing, evolution and relationship to disease susceptibility. In Erlich HA (ed): PCR Techniques. New York, Stockton, 1989. 28. Tiercy J-M, Morel C, Freidel AC, Zwahlen F, Gebuhrer L, Betuel H, Jeannet M, Mach B: Selection of unrelated donors for bone marrow transplantation is improved by HLA class II genotyping with oligonucleotide hybridization. Proc Natl Acad Sci USA 88:7121, 1991. 29. Mayr W, Contu L, Kirnbauer M, Mervart H: Antigen society #17 report (Cw5 and Cw8). In Dupont B (ed): Immunobiology of HLA, vol 1. New York, SpringerVerlag, 1989, pp 222.