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DRw8 microvariation: A new DRB1 allele identified in association with DQw7 in American blacks
DRw8 Microvariation: A N e w DRB1 Allele Identified in Association with DQw7 in American Blacks Carolyn Katovich Hurley, Kyung Who Lee, Eric Mickelson, Susan Masewicz, and Armead H. Johnson
ABSTRACT: In the American black population, DRm8 is commonly found in association with DQw7 and an undefined HLA-D specificity. Polymerase chain reaction amplification was used to obtain DRB1 eDNA clones from two unrelated individuals expressing this haploABBREVIATIONS DNV double normalized value HTC homozygous typing cell
type. The DRBI sequence is most similar to a sequence found in cells expressing DRw8, DQw4,Dw8.2 differing at the protein level by a single amino acid substitution at position 86. Human Immunology 31. 109-113 (1991)
MLC PCR
mixed lymphocyte cultures polymerase chain reaction
INTRODUCFION Haplotypes expressing DKw8, a member of the DRw52 haplotype family, are quite heterogeneous encoding DQwl(w6), DQw3(w7), or DQw4 alleles and typing as HLA-Dw8.1, Dw8.2, Dw8.3, or D- (undefined). Each haplotype appears to be race specific in that DRw8, DQw4,Dw8.1 is common in Caucasians; DRw8, DQw4,Dw8.2 is common in Amerindians; and DRw8, DQwl,Dw8.3 is common in Orientals [1, 2]. A fourth haplotype, DRw8,DQw7,Dw8.3, has also been described in the homozygous typing cell (HTC) LUY [1, 3, 4]. Among the DR alleles, DRw8 is unique in that it may have arisen from a gene deletion event which joined the first and second domains of a DRB1 gene with the 3' untranslated region of a DRB3 gene [5]. Thus, the resultant DRw8 haplotypes do not encode DRB3. DKw8 is found in 13% of a panel of 105 unrelated From the Departments of Microbiology and Pediatrics. Georgetown University MedicalSchool, Washington DC, and FredHatehinson Cancer ResearchCenter, Seattle Washington. Address reprint requeststo Dr. Carolyn Hurley, Departmentof Microbiology, Georgetown University MedicalSchool, 3900 Resere'oirRd. NW, Washington, DC 20007. ReceivedOctober11,1990; acceptedDecember11, 1990.
HumanImmunology31,109-113 (1991) © AmericanSocietyfor Histocornpatibifityand lmmunogenedcs,1991
American blacks from the Washington, DC, area. While DRw8 is most frequendy associated with DQw4 in Caucasians, 61% of DRw8-posidve American blacks express DRw8 in association with DQw7(w3). [The remaioder express DQw4 (31%) or DQw6 (8%).] c D N A sequence analysis was used to identify the DRw8 allele associated with this characteristic American black ha#otype. METHODS HLA-D region antigens were defined in primary mixed lymphocyte cultures (MLC) utilizing HTC (Dw8.1: Riekl00, MMI05, ARC, Hambley, 4872, MMR-C, MAD, BM9; Dw8.2: MN79, Olga80; Dw8.1/Dw8.2: DW1109; DwS.3: TAB089, Shim, LUY). Fifty thousand responder cells were combined with 9 x 104 "F-irradiated stimulator cells in triplicate cultures in 96-well Ubottom plates. After 5 days, each culture was pulsed for 18 hr with 1/zCi [3H]thymidine (6.7 Ci/mM) and harvested onto glass fiber filters. Radiolabel incorporation was monitored by liquid scintillation counting. Data were statistically analyzed using the 75th percentile double normalized value (DNV) [1]. 109 0198-8859/91/$3.50
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C.K. Hurley et al.
T A B L E 1 Oligonucleotides utilized as PCR and eDNA sequencing primers Name
Specificity
Strand
DRB.N2 DRB.C 1437 KW1 KW2 KW6 1"3 T7
DRB DR3 DR// DR3 and DQB1 DRB and DQBI DR3 and DQB 1 pBluescript pBluescript
Noncoding Coding Noncoding Coding Noncoding Noncoding
Codons 113-120 (- 12)-(-4) 112-117 39-44 39-44 79-83
RE site Sail Sstl
Nucleotide sequence (5'-Yy GAGTCGACTCACAGA( A,G)CAGACCAGGAGG ACTGATGGTGCTGAGCTCCC CAGACCAGGAGG'I'rGTG CGCTTCGACAGCGACG, TG CACGTCGCTGTCGAAGCG GTAGTI'GTGTCTGCA ATTAACCCTCACTAAAG AATACGACTCACTATAG
• Restrictionenzymesitesin the PCR primersare underlined.
Total R N A was prepared from B-lymphoblastoid cell lines from unrelated American black individuals, 1066 (DRw8,DQw7;DRw12,DQw5) and 1127 (DRw8, DQw7;DR1,DQw5). Class II haplotypes of these individuals have been defined by segregation analysis. cDNAs [6] were subjected to two sets of 15 cycles of Taq D N A polymerase-catalyzed D N A amplification [7] using primers containing conserved sequences found in the leader sequence and second domain of D R 3 (Table 1). The amplified products were purified on low melting agarose and cloned into the pBluescript vector (Stratagene, La Jolla, CA). Sequencing of individual clones was performed by the Sanger dideoxy chain termination method [8] using internal primers (Table 1). The new allele described in these studies has been assigned an allele designation by the W H O Nomenclature Committee in July 1990; the sequence is filed with Genbank under accession no. M34315. RESULTS A N D D I S C U S S I O N N o n e of the five DRw8,DQw7 American black individuals tested typed with HLA-Dw8.1,-Dw8.2, or -Dw8.3 TABLE 2
HTC typing of DRw8,DQw7 individuals~
Cell
Class IItype
1066' 1127' 1431 1064 1065
DRw8,DQw7;DRw12,DQw5 DRw8,DQw7;DR1,DQw5 DRwS,DQw7,DR1,DQw5 DRwS,DQw7;DRwl7,DQw2 DRw8,DQw7;DRwlS,DQw4
DNV TO HTC~ Dw8.1 Dw8.2 Dw8.3 LUY 95 90 88 100 94
83 117 81 99 93
99 67 69 97 89
50 54 44 69 80
• Individualsare unrelated.Haplotypeswere definedby segregationanalysis except for IZl31,for whichan informativefamilywas not available. 6DNV ~ 35 isconsidereda typingresponse.TheDNVsare the averageDNV resultingfromtestingwith8 DwS.1HTC,2 Dw8.2HTC, 3 Dw8.3HTC,and I.UY. •DRB1 characterizedby eDNAsequenceanalysis.
HTCs (Table 2). The latter HTCs included a Caucasian cell, LUY (DRw8,DQw7,Dw8.3). Two levels of response were observed to LUY. Three of the individuals tested (1066, 1127, 1431) gave a lower level of response ( D N V 44-54) compared to the response of two other DRw8-positive cells (1064, 1065). Cells 1064 and 1065 responded at a level equivalent to that observed to the other HTC ( D N V 69-117). Variation in the level of response to H T C LUY among the DRw8, DQw7 American black individuals tested '(cells 1066/1127/ 1431 compared to cells 1064/1065) may indicate additional heterogeneity within this haplotype group. Identical Dgw8-associated D R B i e D N A sequences were identified from two of the three individuals (1066 and 1127) who responded at a low level to HTC LUY (Fig. 1). The sequence, DRBI*0804, is similar but not identical to the nucleotide sequence of DRB 1"0802 described from DRw8,DQw4,Dw8.2 cells; OLGA [5]; and SPL [9]. The two sequences differ in protein sequence at position 86 (Val/Gly). The DRB1 allele from the DRw8,DQw7,Dw8.3 haplotype (LUY) has not been examined by nucleotide sequencing; however, since the American black haplotype does not type with this HTC, we expect to find differences in the class II alleles encoded by these haplotypes. DRBI*0802 and DRBI*0804 are only one example of several sets of alleles which differ only by a Val/Gly interchange at position 86. This group also includes DRB1 alleles D R B I * 0 4 0 3 / D R B I * 0 4 0 7 [11]; DRBI* 0 4 0 4 / D R B l * 0 4 0 8 [11]; DRB1*1101/DRB1*1104 [12]; DRBl*1301/DRB1*1302 [13]; D R B I * I S 0 1 / DRB 1" 1502 [ 14]; and the less polymorphic D R B-chain alleles DRB3*0201/DRB3*0202 [15]. Interestingly, only valine or glycine is found at position 86 among all D R alleles. The interchange at position 86 ( G T G / G G T ) is not the result of a single nucleotide substitution and may have been generated by two successive point mutations or an inversion and then spread through the D R alleles via gene conversion focused in this conserved
DRw8 Microvariation
ORBI*0804 DRBI*0801 ORBI*0802 DRBI*0803
I 11
1o 20 LY ASP THR ARG PRO ARG PHE LEU GLU TYR SER THR GLY GLU EYS TYR PHE PHE ASH GLY IHR GLU ARG VAL ARG GGG GAC ACC AGA £CA CGT TTC TIG GAG TAC TCT ACG GGI GAG TGT TAT TTC IIC AAT GGG ACG GAG CGG GTG CGG ......... C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................................................. ARG
DRBI*0804 ORBI*0801 DRBI*0802 DRBI*0803
DRBI*0804 DRBI*0801
30 40 50 PHE LEU ASP ARG TYR PHE TYR ASH GLN GLU GLU TYR VAL ARG PHE ASP SER ASP VAL GLY GLU TYR ARG ALA VAL TTC CTG GAC AGA TAC TTC TAT AAC CAA GAG GAG TAC GIG CGC TTC GAC AGC GAC GTG GGG GAG TAC CGG GCG GTG
60 70 THR GLU LEU GLY ARG PRO ASP ALA GLU TYR TRP ASH SER GLH LYS ASP PHE LEU GLU ASP ARG ARG ALA LEU VAL ACG GAG CTG GGG CGG CCT GAT GCC GAG TAC IGG AAC AGC CAG AAG GAC IIC CTG GAA GAC AGG CGG GC¢ CTG GTG AGC
DRBI*0802 DRBI*0803
AGC
SER
;LE
80 90 ASP THR TYR CYS ARG HIS ASN TYR GLY VAL VAL GLU SER PHE THR VAL GLH ARG ARG
DRBI*0804 DRBI*0801 DRBI*0802 DRBI*0803
GAC ACC IAC IG{ AGA CAC AAC IAC GGGGII GIG GAG AGC IlC ACA GTG CAG CGG CGA . . . . . . . . . . GT -gT -GT GLY
FIGURE 1 First domain cDNA sequences and predicted amino acid sequences of DRBl*0804 (1066,1127 (DRw8,DQwT,D-)), DRBI*0801 (MADURA (DRw8, DQw4,DwS.1) [10]), DRBI*0802 (SPL (DRwS, DQw4,Dw8.2) [9]; OLGA (DRw8,DQw4,Dw8.2) [5]), and DRB1*0803 (TAB (DRw8,DQw6,Dw8.3) [5]). A dash indicate's identity with DRBI*0804.
region of the DR # genes. In the three-dimensional model of the class II molecule, residue 86 lies at the end of the/3-chain c~ helix and may point into the peptide bindingsite [16]. Since the size of the valine and glycine side chains differ substantially, this substitution may change the topography of the binding site [17] and, thus, may have some relevance in the positioning of antigenic peptide in the groove of the molecule. Although the relative importance of each class II molecule and each amino acid sequence difference in generating a mixed lymphocyte reaction is not known, the difference in HLA-D typing between DRw8 haplotypes appears to reside, at least in part, in differences in DRB1 allelic products. This is most clearly seen in a comparison of DRw8,DQw4,Dw8.1 and DRw8, DQw4,Dw8.2 haplotypes which appear to differ only in the expressed DRw8 molecules [5, 10]. However, since the first domain of the DRB1 allele encoded by the DRwS,DQw7,D- haplotype in American blacks differs by only a single amino acid at position 86 from the DRw8,DQw4,Dw8.2, differences in the DQ allelic products may also contribute to the relatively robust
stimulation noted in an MLC between cells bearing these haplotypes. The position of the DRBI*0804 allele in a hypothetical evolutionary pathway giving rise to DRw8 is shown in Fig. 2. It has been postulated that DRw8 and DRwl 1 arose from the same precursor [5]. This is supported by the observation that DRB 1"0804 is found in association with DQw7 as is DRwl 1. Comparison of all DRw8associated alleles also places DRBI*0804 at the initial step in the diversification of DRw8. Since all other DRw8 microvariants exhibit GGT (Gly) at residue 86, the simplest explanation is that DRB 1"0804 may be the original allele in the DRw8 pathway which was then altered by a gene conversion event at position 86. In addition, all other members of the DRw52 family share the AGA c0don at position 4 with DRBl*0804 (and DRBl*0802) suggesting that DRBI*0804 and DRBl*0802 may be older than DRBI*0801, which carties a CGA codon at this position. Finally, DRBl*0803 appears to be the most recently derived DRw8 allele since it appears to differ from all other DRw8 alleles at positions 36 and 67. Like other DRw8 haplotypes, the haplotype encoding DRBI®0804 lacks a DRB3 gene when examined by restriction fragment length polymorphism analysis using a DR ~ probe [18]. The position of DRBI*0804 in the evolutionary pathway is consistent with the theory that American blacks carry genes of the oldest human population, African blacks [19, 20]. It is interestingthat the DRw8 microvariants differ in DQ association (Fig. 2). Supporting its origin from a DRw8/DRwl 1 precursor haplotype, DRBIO0804 is as-
112
C.K. Hurley et al.
! DRw 11
DRw8
4, A
GAG GAT TTO GTG
AGA GAG GAT
"rTC GGT ~
GAG AGO TTC
4, DQW4 ~.|OaO~ . ~ . .
GGT
GAA AGC ATC GGT
~DQw§ ~ ~060:1~ I
FIGURE 2 Hypothetical evolutionary scheme for the origin of the DRw8 microvariants. It has been hypothesized that the DRw8 haplotype may have arisen from a gene deletion event which fused portions of the DRB 1 and DRB3 loci [5]. An X indicates a postulated reciprocal recombination event which altered the DR/DQ association.
3.
4. sociated with a DQw7 allele as is DRwl 1. The recombination that generated the DQw4 association then paralleled the divergence of DRBI*0802 and DRBI*0801. Finally, the recombination with a DQw6 allele paralleled the events which resulted in DRBI*0803. It is interesting that DR divergence coincides with changes in D Q association suggesting that class II alleles encoded by a haplotype may be evolving in concert. Thus, the repertoire of class II molecules expressed by an individual may be derived by selective pressure which generates optimal allele combinations on a haplotype. ACKNOWLEDGMENTS This work is supported by the National Institutes of Health Grant no. AI23371. The authors would like to thank Ms. Barbara Ziff for her technical assistance. REFERENCES 1. Mickelson EM, Nisperos B, Layrisse Z, Kim SJ, Thomas ED, Hansen JA: Analysis of the HLA-DRw8 haplotype: Recognition by HTC typing of three distinct antigen complexes in Caucasians, Native Americans, and Orientals, lmmunngenetics 17:399, 1983. 2. Herrera F, Layrisse Z, Carreno B, Qunitero M, Honeyman M, Mickelson EM, Tsuji K: Definition of Dw8.2 by
5.
6.
7.
primary and secondary mixed lymphocyte cultures. Hum Immunol 12:9, 1985. Layrisse Z, Carreno B, Svejgaard A, Platz P, Ryder LP, Kristensen T: First level testing ofDw8 HTC. In Albert ED, Baur MP, Mayr WR (eds): Histocompatability Testing 1984. New York, Springer-Verlag, 1984. Flomenberg N: Assignment of T-cell-defined (TCD) HLA class II specificides for the reference panel of Blymphoblastoid cell lines. In Dupont B (ed): lmmunobiology of HLA, vol I. Histocompatibility Testing 1987. New York, Springer-Verlag, 1989. Gorski J: The HLA-DRw8 lineage was generated by a deletion in the DR B region followed by first domain diversification. J Immunol 142:4041, 1989. Gubler U, Hoffman B: A simple and very effective method for generating eDNA libraries. Gene 25:263, 1983. Saiki R, Gelfand D, Stoffel S, Scharf S, Higuchi R, Horn G, Mullis K, Erlich H: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487, 1988.
8. Sanger F, Nicklen S, Coulson A: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463, 1977. 9. Jonsson AK,. Andersson L, Rask L: A cellular and functional split in the DRw8 haplotype is due to a single amino acid replacement (DR~r~7"~P~7). Immunogenetics 29:308, 1989. 10. BeUJ, Denney D, Foster L, Belt T, Todd J, McDevitt H: Allelic variation in the DR subregion of the human major histocompatibility complex. Proc Natl Acad Sci USA 84:6234, 1987. 11. Lanchbury J, Hall M, Welsh K, Panayi G: Sequence analysis of HLA-DR4B1 subtypes: Additional first dbmain
DRw8 Microvariation
variability is detected by oligonucleotide hydridization and nucleotide sequencing. Hum lmmuno127:136,1990. 12. Gregersen PK, Kao H, Nunez-Roldan A, Hurley CK, Kerr R, Silver J: Recombination sites in the HLA class II region are haplotype dependent. J lmmunol 141:1365, 1988. 13. Gorski J, Eckels DD, Tiercy JM, Ucla C, Mach B: Sequence analysis of the DRw13/3 chain genes: The Dw19 specificity may be encoded by the DRB3 locus. In Dupont B (ed): Immunobiology of HLA, vol 2. New York, Springer-Verlag, 1989. 14. Lee B, Rust N, McMichael A, McDevitt HO: HLA-DR2 subtypes form an additional supertypic family of DR/3 alleles. Proc Natl Acad Sci USA 84:4591, 1987. 15. Didier D, Schiffenbauer J, Shuman S, Abruzzini L, GorskiJ, Watling D, Tieber V, Schwartz B: Characterization of two distinct DR beta chain alleles at the betam locus of the DR5 haplotype: betam alleles are highly conserved. J Immunol 137:2627, 1986.
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16. Brown JH, Jardetsky T, Saper MA, Smmraoui B, Bjorkman P, Wiley DC: A hypothetical model of the foreign antigen binding site of class II tfistocompadbi|ity molecules. Nature 332:845, 1988. 17. Garrett TPJ, Saper MA, Biorkman PJ. Stromir~gcr J L Wiley DC: Specificity pockets for the side chains of peptide antigens in HLA-Aw68. Nature 342:692, 1989. 18. Hurley CK, Gregersen PK, GorskiJ, Steiner N, Robbins FM, Hartzman R, Johnson AH, Silver J: The DR3 (wl8),DQw4 haplotype differs from DR3(wlT),DQw2 haplotypes at multiple class II loci, Hum lmmuno125:37, 1989. 19. Cavalli-Sforza LL, Piazza A, Menozzi P, Mountain J: Reconstruction of human evolution: Bringing together genetic, archaeological, and linguistic data. Proc Natl Acad Sci USA 85:6002, 1988. 20. Simons E: Human origins. Science 245:1543, 1989.
ABSTRACT: In the American black population, DRm8 is commonly found in association with DQw7 and an undefined HLA-D specificity. Polymerase chain reaction amplification was used to obtain DRB1 eDNA clones from two unrelated individuals expressing this haploABBREVIATIONS DNV double normalized value HTC homozygous typing cell
type. The DRBI sequence is most similar to a sequence found in cells expressing DRw8, DQw4,Dw8.2 differing at the protein level by a single amino acid substitution at position 86. Human Immunology 31. 109-113 (1991)
MLC PCR
mixed lymphocyte cultures polymerase chain reaction
INTRODUCFION Haplotypes expressing DKw8, a member of the DRw52 haplotype family, are quite heterogeneous encoding DQwl(w6), DQw3(w7), or DQw4 alleles and typing as HLA-Dw8.1, Dw8.2, Dw8.3, or D- (undefined). Each haplotype appears to be race specific in that DRw8, DQw4,Dw8.1 is common in Caucasians; DRw8, DQw4,Dw8.2 is common in Amerindians; and DRw8, DQwl,Dw8.3 is common in Orientals [1, 2]. A fourth haplotype, DRw8,DQw7,Dw8.3, has also been described in the homozygous typing cell (HTC) LUY [1, 3, 4]. Among the DR alleles, DRw8 is unique in that it may have arisen from a gene deletion event which joined the first and second domains of a DRB1 gene with the 3' untranslated region of a DRB3 gene [5]. Thus, the resultant DRw8 haplotypes do not encode DRB3. DKw8 is found in 13% of a panel of 105 unrelated From the Departments of Microbiology and Pediatrics. Georgetown University MedicalSchool, Washington DC, and FredHatehinson Cancer ResearchCenter, Seattle Washington. Address reprint requeststo Dr. Carolyn Hurley, Departmentof Microbiology, Georgetown University MedicalSchool, 3900 Resere'oirRd. NW, Washington, DC 20007. ReceivedOctober11,1990; acceptedDecember11, 1990.
HumanImmunology31,109-113 (1991) © AmericanSocietyfor Histocornpatibifityand lmmunogenedcs,1991
American blacks from the Washington, DC, area. While DRw8 is most frequendy associated with DQw4 in Caucasians, 61% of DRw8-posidve American blacks express DRw8 in association with DQw7(w3). [The remaioder express DQw4 (31%) or DQw6 (8%).] c D N A sequence analysis was used to identify the DRw8 allele associated with this characteristic American black ha#otype. METHODS HLA-D region antigens were defined in primary mixed lymphocyte cultures (MLC) utilizing HTC (Dw8.1: Riekl00, MMI05, ARC, Hambley, 4872, MMR-C, MAD, BM9; Dw8.2: MN79, Olga80; Dw8.1/Dw8.2: DW1109; DwS.3: TAB089, Shim, LUY). Fifty thousand responder cells were combined with 9 x 104 "F-irradiated stimulator cells in triplicate cultures in 96-well Ubottom plates. After 5 days, each culture was pulsed for 18 hr with 1/zCi [3H]thymidine (6.7 Ci/mM) and harvested onto glass fiber filters. Radiolabel incorporation was monitored by liquid scintillation counting. Data were statistically analyzed using the 75th percentile double normalized value (DNV) [1]. 109 0198-8859/91/$3.50
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C.K. Hurley et al.
T A B L E 1 Oligonucleotides utilized as PCR and eDNA sequencing primers Name
Specificity
Strand
DRB.N2 DRB.C 1437 KW1 KW2 KW6 1"3 T7
DRB DR3 DR// DR3 and DQB1 DRB and DQBI DR3 and DQB 1 pBluescript pBluescript
Noncoding Coding Noncoding Coding Noncoding Noncoding
Codons 113-120 (- 12)-(-4) 112-117 39-44 39-44 79-83
RE site Sail Sstl
Nucleotide sequence (5'-Yy GAGTCGACTCACAGA( A,G)CAGACCAGGAGG ACTGATGGTGCTGAGCTCCC CAGACCAGGAGG'I'rGTG CGCTTCGACAGCGACG, TG CACGTCGCTGTCGAAGCG GTAGTI'GTGTCTGCA ATTAACCCTCACTAAAG AATACGACTCACTATAG
• Restrictionenzymesitesin the PCR primersare underlined.
Total R N A was prepared from B-lymphoblastoid cell lines from unrelated American black individuals, 1066 (DRw8,DQw7;DRw12,DQw5) and 1127 (DRw8, DQw7;DR1,DQw5). Class II haplotypes of these individuals have been defined by segregation analysis. cDNAs [6] were subjected to two sets of 15 cycles of Taq D N A polymerase-catalyzed D N A amplification [7] using primers containing conserved sequences found in the leader sequence and second domain of D R 3 (Table 1). The amplified products were purified on low melting agarose and cloned into the pBluescript vector (Stratagene, La Jolla, CA). Sequencing of individual clones was performed by the Sanger dideoxy chain termination method [8] using internal primers (Table 1). The new allele described in these studies has been assigned an allele designation by the W H O Nomenclature Committee in July 1990; the sequence is filed with Genbank under accession no. M34315. RESULTS A N D D I S C U S S I O N N o n e of the five DRw8,DQw7 American black individuals tested typed with HLA-Dw8.1,-Dw8.2, or -Dw8.3 TABLE 2
HTC typing of DRw8,DQw7 individuals~
Cell
Class IItype
1066' 1127' 1431 1064 1065
DRw8,DQw7;DRw12,DQw5 DRw8,DQw7;DR1,DQw5 DRwS,DQw7,DR1,DQw5 DRwS,DQw7;DRwl7,DQw2 DRw8,DQw7;DRwlS,DQw4
DNV TO HTC~ Dw8.1 Dw8.2 Dw8.3 LUY 95 90 88 100 94
83 117 81 99 93
99 67 69 97 89
50 54 44 69 80
• Individualsare unrelated.Haplotypeswere definedby segregationanalysis except for IZl31,for whichan informativefamilywas not available. 6DNV ~ 35 isconsidereda typingresponse.TheDNVsare the averageDNV resultingfromtestingwith8 DwS.1HTC,2 Dw8.2HTC, 3 Dw8.3HTC,and I.UY. •DRB1 characterizedby eDNAsequenceanalysis.
HTCs (Table 2). The latter HTCs included a Caucasian cell, LUY (DRw8,DQw7,Dw8.3). Two levels of response were observed to LUY. Three of the individuals tested (1066, 1127, 1431) gave a lower level of response ( D N V 44-54) compared to the response of two other DRw8-positive cells (1064, 1065). Cells 1064 and 1065 responded at a level equivalent to that observed to the other HTC ( D N V 69-117). Variation in the level of response to H T C LUY among the DRw8, DQw7 American black individuals tested '(cells 1066/1127/ 1431 compared to cells 1064/1065) may indicate additional heterogeneity within this haplotype group. Identical Dgw8-associated D R B i e D N A sequences were identified from two of the three individuals (1066 and 1127) who responded at a low level to HTC LUY (Fig. 1). The sequence, DRBI*0804, is similar but not identical to the nucleotide sequence of DRB 1"0802 described from DRw8,DQw4,Dw8.2 cells; OLGA [5]; and SPL [9]. The two sequences differ in protein sequence at position 86 (Val/Gly). The DRB1 allele from the DRw8,DQw7,Dw8.3 haplotype (LUY) has not been examined by nucleotide sequencing; however, since the American black haplotype does not type with this HTC, we expect to find differences in the class II alleles encoded by these haplotypes. DRBI*0802 and DRBI*0804 are only one example of several sets of alleles which differ only by a Val/Gly interchange at position 86. This group also includes DRB1 alleles D R B I * 0 4 0 3 / D R B I * 0 4 0 7 [11]; DRBI* 0 4 0 4 / D R B l * 0 4 0 8 [11]; DRB1*1101/DRB1*1104 [12]; DRBl*1301/DRB1*1302 [13]; D R B I * I S 0 1 / DRB 1" 1502 [ 14]; and the less polymorphic D R B-chain alleles DRB3*0201/DRB3*0202 [15]. Interestingly, only valine or glycine is found at position 86 among all D R alleles. The interchange at position 86 ( G T G / G G T ) is not the result of a single nucleotide substitution and may have been generated by two successive point mutations or an inversion and then spread through the D R alleles via gene conversion focused in this conserved
DRw8 Microvariation
ORBI*0804 DRBI*0801 ORBI*0802 DRBI*0803
I 11
1o 20 LY ASP THR ARG PRO ARG PHE LEU GLU TYR SER THR GLY GLU EYS TYR PHE PHE ASH GLY IHR GLU ARG VAL ARG GGG GAC ACC AGA £CA CGT TTC TIG GAG TAC TCT ACG GGI GAG TGT TAT TTC IIC AAT GGG ACG GAG CGG GTG CGG ......... C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................................................. ARG
DRBI*0804 ORBI*0801 DRBI*0802 DRBI*0803
DRBI*0804 DRBI*0801
30 40 50 PHE LEU ASP ARG TYR PHE TYR ASH GLN GLU GLU TYR VAL ARG PHE ASP SER ASP VAL GLY GLU TYR ARG ALA VAL TTC CTG GAC AGA TAC TTC TAT AAC CAA GAG GAG TAC GIG CGC TTC GAC AGC GAC GTG GGG GAG TAC CGG GCG GTG
60 70 THR GLU LEU GLY ARG PRO ASP ALA GLU TYR TRP ASH SER GLH LYS ASP PHE LEU GLU ASP ARG ARG ALA LEU VAL ACG GAG CTG GGG CGG CCT GAT GCC GAG TAC IGG AAC AGC CAG AAG GAC IIC CTG GAA GAC AGG CGG GC¢ CTG GTG AGC
DRBI*0802 DRBI*0803
AGC
SER
;LE
80 90 ASP THR TYR CYS ARG HIS ASN TYR GLY VAL VAL GLU SER PHE THR VAL GLH ARG ARG
DRBI*0804 DRBI*0801 DRBI*0802 DRBI*0803
GAC ACC IAC IG{ AGA CAC AAC IAC GGGGII GIG GAG AGC IlC ACA GTG CAG CGG CGA . . . . . . . . . . GT -gT -GT GLY
FIGURE 1 First domain cDNA sequences and predicted amino acid sequences of DRBl*0804 (1066,1127 (DRw8,DQwT,D-)), DRBI*0801 (MADURA (DRw8, DQw4,DwS.1) [10]), DRBI*0802 (SPL (DRwS, DQw4,Dw8.2) [9]; OLGA (DRw8,DQw4,Dw8.2) [5]), and DRB1*0803 (TAB (DRw8,DQw6,Dw8.3) [5]). A dash indicate's identity with DRBI*0804.
region of the DR # genes. In the three-dimensional model of the class II molecule, residue 86 lies at the end of the/3-chain c~ helix and may point into the peptide bindingsite [16]. Since the size of the valine and glycine side chains differ substantially, this substitution may change the topography of the binding site [17] and, thus, may have some relevance in the positioning of antigenic peptide in the groove of the molecule. Although the relative importance of each class II molecule and each amino acid sequence difference in generating a mixed lymphocyte reaction is not known, the difference in HLA-D typing between DRw8 haplotypes appears to reside, at least in part, in differences in DRB1 allelic products. This is most clearly seen in a comparison of DRw8,DQw4,Dw8.1 and DRw8, DQw4,Dw8.2 haplotypes which appear to differ only in the expressed DRw8 molecules [5, 10]. However, since the first domain of the DRB1 allele encoded by the DRwS,DQw7,D- haplotype in American blacks differs by only a single amino acid at position 86 from the DRw8,DQw4,Dw8.2, differences in the DQ allelic products may also contribute to the relatively robust
stimulation noted in an MLC between cells bearing these haplotypes. The position of the DRBI*0804 allele in a hypothetical evolutionary pathway giving rise to DRw8 is shown in Fig. 2. It has been postulated that DRw8 and DRwl 1 arose from the same precursor [5]. This is supported by the observation that DRB 1"0804 is found in association with DQw7 as is DRwl 1. Comparison of all DRw8associated alleles also places DRBI*0804 at the initial step in the diversification of DRw8. Since all other DRw8 microvariants exhibit GGT (Gly) at residue 86, the simplest explanation is that DRB 1"0804 may be the original allele in the DRw8 pathway which was then altered by a gene conversion event at position 86. In addition, all other members of the DRw52 family share the AGA c0don at position 4 with DRBl*0804 (and DRBl*0802) suggesting that DRBI*0804 and DRBl*0802 may be older than DRBI*0801, which carties a CGA codon at this position. Finally, DRBl*0803 appears to be the most recently derived DRw8 allele since it appears to differ from all other DRw8 alleles at positions 36 and 67. Like other DRw8 haplotypes, the haplotype encoding DRBI®0804 lacks a DRB3 gene when examined by restriction fragment length polymorphism analysis using a DR ~ probe [18]. The position of DRBI*0804 in the evolutionary pathway is consistent with the theory that American blacks carry genes of the oldest human population, African blacks [19, 20]. It is interestingthat the DRw8 microvariants differ in DQ association (Fig. 2). Supporting its origin from a DRw8/DRwl 1 precursor haplotype, DRBIO0804 is as-
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C.K. Hurley et al.
! DRw 11
DRw8
4, A
GAG GAT TTO GTG
AGA GAG GAT
"rTC GGT ~
GAG AGO TTC
4, DQW4 ~.|OaO~ . ~ . .
GGT
GAA AGC ATC GGT
~DQw§ ~ ~060:1~ I
FIGURE 2 Hypothetical evolutionary scheme for the origin of the DRw8 microvariants. It has been hypothesized that the DRw8 haplotype may have arisen from a gene deletion event which fused portions of the DRB 1 and DRB3 loci [5]. An X indicates a postulated reciprocal recombination event which altered the DR/DQ association.
3.
4. sociated with a DQw7 allele as is DRwl 1. The recombination that generated the DQw4 association then paralleled the divergence of DRBI*0802 and DRBI*0801. Finally, the recombination with a DQw6 allele paralleled the events which resulted in DRBI*0803. It is interesting that DR divergence coincides with changes in D Q association suggesting that class II alleles encoded by a haplotype may be evolving in concert. Thus, the repertoire of class II molecules expressed by an individual may be derived by selective pressure which generates optimal allele combinations on a haplotype. ACKNOWLEDGMENTS This work is supported by the National Institutes of Health Grant no. AI23371. The authors would like to thank Ms. Barbara Ziff for her technical assistance. REFERENCES 1. Mickelson EM, Nisperos B, Layrisse Z, Kim SJ, Thomas ED, Hansen JA: Analysis of the HLA-DRw8 haplotype: Recognition by HTC typing of three distinct antigen complexes in Caucasians, Native Americans, and Orientals, lmmunngenetics 17:399, 1983. 2. Herrera F, Layrisse Z, Carreno B, Qunitero M, Honeyman M, Mickelson EM, Tsuji K: Definition of Dw8.2 by
5.
6.
7.
primary and secondary mixed lymphocyte cultures. Hum Immunol 12:9, 1985. Layrisse Z, Carreno B, Svejgaard A, Platz P, Ryder LP, Kristensen T: First level testing ofDw8 HTC. In Albert ED, Baur MP, Mayr WR (eds): Histocompatability Testing 1984. New York, Springer-Verlag, 1984. Flomenberg N: Assignment of T-cell-defined (TCD) HLA class II specificides for the reference panel of Blymphoblastoid cell lines. In Dupont B (ed): lmmunobiology of HLA, vol I. Histocompatibility Testing 1987. New York, Springer-Verlag, 1989. Gorski J: The HLA-DRw8 lineage was generated by a deletion in the DR B region followed by first domain diversification. J Immunol 142:4041, 1989. Gubler U, Hoffman B: A simple and very effective method for generating eDNA libraries. Gene 25:263, 1983. Saiki R, Gelfand D, Stoffel S, Scharf S, Higuchi R, Horn G, Mullis K, Erlich H: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487, 1988.
8. Sanger F, Nicklen S, Coulson A: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463, 1977. 9. Jonsson AK,. Andersson L, Rask L: A cellular and functional split in the DRw8 haplotype is due to a single amino acid replacement (DR~r~7"~P~7). Immunogenetics 29:308, 1989. 10. BeUJ, Denney D, Foster L, Belt T, Todd J, McDevitt H: Allelic variation in the DR subregion of the human major histocompatibility complex. Proc Natl Acad Sci USA 84:6234, 1987. 11. Lanchbury J, Hall M, Welsh K, Panayi G: Sequence analysis of HLA-DR4B1 subtypes: Additional first dbmain
DRw8 Microvariation
variability is detected by oligonucleotide hydridization and nucleotide sequencing. Hum lmmuno127:136,1990. 12. Gregersen PK, Kao H, Nunez-Roldan A, Hurley CK, Kerr R, Silver J: Recombination sites in the HLA class II region are haplotype dependent. J lmmunol 141:1365, 1988. 13. Gorski J, Eckels DD, Tiercy JM, Ucla C, Mach B: Sequence analysis of the DRw13/3 chain genes: The Dw19 specificity may be encoded by the DRB3 locus. In Dupont B (ed): Immunobiology of HLA, vol 2. New York, Springer-Verlag, 1989. 14. Lee B, Rust N, McMichael A, McDevitt HO: HLA-DR2 subtypes form an additional supertypic family of DR/3 alleles. Proc Natl Acad Sci USA 84:4591, 1987. 15. Didier D, Schiffenbauer J, Shuman S, Abruzzini L, GorskiJ, Watling D, Tieber V, Schwartz B: Characterization of two distinct DR beta chain alleles at the betam locus of the DR5 haplotype: betam alleles are highly conserved. J Immunol 137:2627, 1986.
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16. Brown JH, Jardetsky T, Saper MA, Smmraoui B, Bjorkman P, Wiley DC: A hypothetical model of the foreign antigen binding site of class II tfistocompadbi|ity molecules. Nature 332:845, 1988. 17. Garrett TPJ, Saper MA, Biorkman PJ. Stromir~gcr J L Wiley DC: Specificity pockets for the side chains of peptide antigens in HLA-Aw68. Nature 342:692, 1989. 18. Hurley CK, Gregersen PK, GorskiJ, Steiner N, Robbins FM, Hartzman R, Johnson AH, Silver J: The DR3 (wl8),DQw4 haplotype differs from DR3(wlT),DQw2 haplotypes at multiple class II loci, Hum lmmuno125:37, 1989. 19. Cavalli-Sforza LL, Piazza A, Menozzi P, Mountain J: Reconstruction of human evolution: Bringing together genetic, archaeological, and linguistic data. Proc Natl Acad Sci USA 85:6002, 1988. 20. Simons E: Human origins. Science 245:1543, 1989.