Serology, Restriction Fragment Length Polymorphism, and Sequence Analysis of a Unique HLA Class II Antigen, DR5x6 Harriet J. Noreen, Pere Santamaria, Maurine L. Davidson, Stephen S. Rich, and Miriam SegaU
ABSTRACT: We analyzed a new class II HLA haplotype, which we have designated DR5x6, by serology, restriction fragment length polymorphism (RFLP), and sequence analysis. As the name DRSx6 implies, the antigen is serologically closely related to both DR5 and DRw6. RFLP analysis of this haplotype suggests a close similarity with DKwll haplo~pes. The DNA sequences encoded by the second exon of its DRB1, DRB3, and DQB1 genes were also determined. Comparison of these sequences with those of alleles at
these loci in other haplotypes suggests that this haplotype could have evolved from a DKwll ances,o~ haplotype (DRwl 1-DRw52b (Dw25)-DQwT) by means of: (a) a gene conversion at the DRB1 locus involving DRw8 (Dw8.3) as the sequence donor, plus a point mutation or a gene conversion involving DR4-Dw4; and (b) a recombination event by which this haplotype would have acquired the DRwSa (Dw24) allele at the DRB3 locus. Human lmmunolog~ 30, 168-173 (199l)
ABBREVIATIONS CDC complement-dependent cytotoxicity HTC homozygous typing cells
RFLP
restriction fragment length polymorphism
INTRODUCTION The extensive polymorphism of the HLA class II molecules is almost exclusively localized to the N-terminal domain, largely encoded by the second exon of the class II genes. Molecular studies and studies of allorecognition by T-cell clones have revealed a greater polymorphism than identified by serological techniques: currently, four sequences have been described which encode the serologieaUy defined antigen D R w l l , one for DRwl2, two for DRwl 3, two for DRwl4, and three for DRw8 [1]. Recently, two new DRw6-related DRB1 genes have been described in the Japanese population [2, 3]. Some, but not all, of these individual sequences have a serological or cellular designation. We describe
From the Univenlty of Miune~ota lmmunobiology Research Center, Department of Laboratory Medicine and Pathology, Institute of Human Genetics and Division of Endocrinology ned l~aabolism, Minneapolis, Minnesota. Address all reprint requtsts to HarrietJ. Noreen, Univenity of Minntsota, lmmunobiologyResearchCenter, Box 724, Minneapolis, MN 55455. Recei*,edApril 24. 1990; acceptedSeptember8, 1990. 168 0198-8859/911S3.50
here the serological and molecular characteristics of a new class II haplotype which we have designated DR5x6, the name being a description of the original serological specificities it most closely fits.
MATERIALS A N D METHODS Serology. H L A class I1 serology was performed by complement-dependent cytorexicity (CDC). B cells were purified either ~,y nylon wool columns [4] or by immunomagnetic beads [5] and the CDC was performed by dye exclusion or fluorescence staining, respectively. Tenth Workshop, local and commercial antlsera were used to define the class II specificities. Restriction fragment length polymorphism (RFLP). Genomic DNA was extracted from peripheral blood using a non-phenol method. Restriction enzyme digests were prepared using Taql. Southern blot and hybridization were performed as previously described [6], using hi-
HumanImmunology30, 168-173(1991) © AmericanSocietyfor Histocompatibility~d ]mmunosenetics,1991
Analysis of DR5x6
TABLE 1
169
H L A class II serology analysis
A. 10thWorkshop antisera
B. Local/commercial" ~ntisera DR specificity
Reactions with DRSx6-positi~e cells DRwll 1096 1103 1113 1114 DRwI2 9050 9999 DRw8 1085
1086 1087 1089 1091 DRwl3 1126 1133 1134 DRw14 9060 DRw52 1152 1198 1199 DQw7 1176 1179 1184
-
-
-
-
±
-+
Serum Dub Smi Mil Mat 383 101~ 850 Cod Mau
Specificity 11, 12.8 3, 5, 8 11, 12 5, 8 5 5 5 I1 II
5x6
11
12
13
+
+ +
+ +
-
+
+
+
-
+ + + + -
+ + + + + +
+ ± + + -
-
-
Sel
1 [
+
-
Gaz Fie 616 7ll 432
11 3.6, 8. 11 11, 13 3,6 3,6, 12
+ *
.
+ +
+
1192
2,
+
998 710 Rey Tou MCbI
2, 15 14, 1, 10, 16 3.5, 13 3, 6 2, 6, l I
13
. ~
8
-
+ +
+
. =.
+
+
+
+
+
+ +
+
+
+
+
.
14
+
+ + + +
-
+ +-
-
+ + + + + +
• H L A classII typingt~ys from Biorest.
trocellulose m e m b r a n e s . P r o b e s used w e r e D R B described by Bidwell and Jarruld [7], D Q B described by L a r h a m m e r et al. [8], and D Q A described by Auffray et al.
[9].
DRBI, DRB3, DQB1 sequence analysis of the DR5x6 haplotype. T o t al cellular R N A (0.5 to 1 ~ g ) obtained from peripheral b l o o d m o n o n u c l e a r cells o f two unrelased subjects typed as D R 5 x 6 was reverse transcribed with M o l o n e y routine l e u k e m i a virus reverse transcriptase using locus-specific antisense p r i m e r s ( C O D Q B 7 and C O D R B 2 0 ) , enzymatically amplified with conserved a n d / o r d e g e n e r a t e 5' p r i m e r s ( C O D Q B 1 3 , C O D R B 11, D O D R B 1 6 , D O D R B 1 7 , D O D R B 1 2 ) , and directly s e q u e n c e d with T a q polymerase by the dideoxy chain t e r m i n a tio n m e t h o d using D R B - and D Q B - s p e cific p r i m e r s ( S E D Q B S , C O D Q B 1 3 , D O D R B 1 6 , DODRBI7, S E D R B I 2 ) . T h e sequences of these p r i m e r s as well as the amplification and sequencing conditions u s e d w e r e essentially as described [ 10].
RESULTS AND DISCUSSION
Serologicalanalysis. Serologically the
D R S x 6 antigen reacts with s o m e alloantisera w h i c h define D R 5 and D R w 6 and is associated with D R w 5 2 and D Q w T . T w o D R S x 6 cells ( M D : D R 3 , 5x6 and S K : D R w 8 , 5x6) f r o m n o r m a l d o n o r s w e r e studied extensively with T e n t h W o r k s h o p , local, and commercial antisera. T h e cells appear to have a D R blank haplotype associated with D R w 5 2 and D Q ~ 7 w h e n analyzed only with the best D R S , D R w 6 , and D R w 8 T e n t h W o r k s h o p sera (Table 1A). D R w 6 W o r k s h o p antisera 1133 ( D R w 1 3 ) and 9 0 6 0 ( D R w l 4 ) react weakly with D R S x 6 cells indicating that these sera are m o r e c o m p l e x than the W o r k shop analysis demonstrates. Many duospecific and muhispecific antisera containing D R w I 1 , 12, 13, 14, a n d / o r 8 react with D R 5 x 6 cells as seen in T a b l e IB. D u e to the reactivity with these c o m p l e x antisera, it is n o t surprising that D R 5 x 6 ceils are often typed serologically as D R 5 o r D R w 6 or as subtypes thereof.
170
H . J . Noreen et al.
TABLE 2
RFLP analysis"
DR
5x6 11
Dw DRw52
? 24
11.5 kb 9.4 kb 8.5 kb 6.8 kb 5.9 kb 4.3 kb
12
13 13 13 I4
14 8
5~ DB6 18 18 19 9 I6 8' 25 25 24 25 26 25 24 - +
+
+
+ +
17 17 B8 B18 3 3 24 25
+ +
+ +
+
hybridized with a D R B probe o f D N A digested with TaqI from family SM in which the patient is D R w l l , DR5x6; the father is D R 7 , D R w 1 1 ; and the mother is D R w 1 3 , DRSx6.
Sequence analysis. Sequence analysis was performed in two unrelated subjects ( M D and SK) which had been typed as D R S x 6 by serology and RFLP. T h e nucleotide
+ + +
+ +
+
+
+
+
+
+
+
+
+
~
+
+
+
+
+
• A schematicrepresentationof RFLPpa~rernsmsodated withDR.Dwspedfidties. DNA fromTenth InternationalWorkshopHTC and hetemzygousnormal individualswas digestedwithTaql and hybridizedwith a DRB probe. HTC Sweig.JVM.Tisi.SPO,FPA.and he[emzygo~ DRwl I ceils,as wellas ' HTC Mad~a. OLGA,TAB089,and heter~ygous DRw8cellsdemonstrate the samegFLPpattern despiteD regioaheterogeneityas definedby sequence analysis [1 10] and T
ABSTRACT: We analyzed a new class II HLA haplotype, which we have designated DR5x6, by serology, restriction fragment length polymorphism (RFLP), and sequence analysis. As the name DRSx6 implies, the antigen is serologically closely related to both DR5 and DRw6. RFLP analysis of this haplotype suggests a close similarity with DKwll haplo~pes. The DNA sequences encoded by the second exon of its DRB1, DRB3, and DQB1 genes were also determined. Comparison of these sequences with those of alleles at
these loci in other haplotypes suggests that this haplotype could have evolved from a DKwll ances,o~ haplotype (DRwl 1-DRw52b (Dw25)-DQwT) by means of: (a) a gene conversion at the DRB1 locus involving DRw8 (Dw8.3) as the sequence donor, plus a point mutation or a gene conversion involving DR4-Dw4; and (b) a recombination event by which this haplotype would have acquired the DRwSa (Dw24) allele at the DRB3 locus. Human lmmunolog~ 30, 168-173 (199l)
ABBREVIATIONS CDC complement-dependent cytotoxicity HTC homozygous typing cells
RFLP
restriction fragment length polymorphism
INTRODUCTION The extensive polymorphism of the HLA class II molecules is almost exclusively localized to the N-terminal domain, largely encoded by the second exon of the class II genes. Molecular studies and studies of allorecognition by T-cell clones have revealed a greater polymorphism than identified by serological techniques: currently, four sequences have been described which encode the serologieaUy defined antigen D R w l l , one for DRwl2, two for DRwl 3, two for DRwl4, and three for DRw8 [1]. Recently, two new DRw6-related DRB1 genes have been described in the Japanese population [2, 3]. Some, but not all, of these individual sequences have a serological or cellular designation. We describe
From the Univenlty of Miune~ota lmmunobiology Research Center, Department of Laboratory Medicine and Pathology, Institute of Human Genetics and Division of Endocrinology ned l~aabolism, Minneapolis, Minnesota. Address all reprint requtsts to HarrietJ. Noreen, Univenity of Minntsota, lmmunobiologyResearchCenter, Box 724, Minneapolis, MN 55455. Recei*,edApril 24. 1990; acceptedSeptember8, 1990. 168 0198-8859/911S3.50
here the serological and molecular characteristics of a new class II haplotype which we have designated DR5x6, the name being a description of the original serological specificities it most closely fits.
MATERIALS A N D METHODS Serology. H L A class I1 serology was performed by complement-dependent cytorexicity (CDC). B cells were purified either ~,y nylon wool columns [4] or by immunomagnetic beads [5] and the CDC was performed by dye exclusion or fluorescence staining, respectively. Tenth Workshop, local and commercial antlsera were used to define the class II specificities. Restriction fragment length polymorphism (RFLP). Genomic DNA was extracted from peripheral blood using a non-phenol method. Restriction enzyme digests were prepared using Taql. Southern blot and hybridization were performed as previously described [6], using hi-
HumanImmunology30, 168-173(1991) © AmericanSocietyfor Histocompatibility~d ]mmunosenetics,1991
Analysis of DR5x6
TABLE 1
169
H L A class II serology analysis
A. 10thWorkshop antisera
B. Local/commercial" ~ntisera DR specificity
Reactions with DRSx6-positi~e cells DRwll 1096 1103 1113 1114 DRwI2 9050 9999 DRw8 1085
1086 1087 1089 1091 DRwl3 1126 1133 1134 DRw14 9060 DRw52 1152 1198 1199 DQw7 1176 1179 1184
-
-
-
-
±
-+
Serum Dub Smi Mil Mat 383 101~ 850 Cod Mau
Specificity 11, 12.8 3, 5, 8 11, 12 5, 8 5 5 5 I1 II
5x6
11
12
13
+
+ +
+ +
-
+
+
+
-
+ + + + -
+ + + + + +
+ ± + + -
-
-
Sel
1 [
+
-
Gaz Fie 616 7ll 432
11 3.6, 8. 11 11, 13 3,6 3,6, 12
+ *
.
+ +
+
1192
2,
+
998 710 Rey Tou MCbI
2, 15 14, 1, 10, 16 3.5, 13 3, 6 2, 6, l I
13
. ~
8
-
+ +
+
. =.
+
+
+
+
+
+ +
+
+
+
+
.
14
+
+ + + +
-
+ +-
-
+ + + + + +
• H L A classII typingt~ys from Biorest.
trocellulose m e m b r a n e s . P r o b e s used w e r e D R B described by Bidwell and Jarruld [7], D Q B described by L a r h a m m e r et al. [8], and D Q A described by Auffray et al.
[9].
DRBI, DRB3, DQB1 sequence analysis of the DR5x6 haplotype. T o t al cellular R N A (0.5 to 1 ~ g ) obtained from peripheral b l o o d m o n o n u c l e a r cells o f two unrelased subjects typed as D R 5 x 6 was reverse transcribed with M o l o n e y routine l e u k e m i a virus reverse transcriptase using locus-specific antisense p r i m e r s ( C O D Q B 7 and C O D R B 2 0 ) , enzymatically amplified with conserved a n d / o r d e g e n e r a t e 5' p r i m e r s ( C O D Q B 1 3 , C O D R B 11, D O D R B 1 6 , D O D R B 1 7 , D O D R B 1 2 ) , and directly s e q u e n c e d with T a q polymerase by the dideoxy chain t e r m i n a tio n m e t h o d using D R B - and D Q B - s p e cific p r i m e r s ( S E D Q B S , C O D Q B 1 3 , D O D R B 1 6 , DODRBI7, S E D R B I 2 ) . T h e sequences of these p r i m e r s as well as the amplification and sequencing conditions u s e d w e r e essentially as described [ 10].
RESULTS AND DISCUSSION
Serologicalanalysis. Serologically the
D R S x 6 antigen reacts with s o m e alloantisera w h i c h define D R 5 and D R w 6 and is associated with D R w 5 2 and D Q w T . T w o D R S x 6 cells ( M D : D R 3 , 5x6 and S K : D R w 8 , 5x6) f r o m n o r m a l d o n o r s w e r e studied extensively with T e n t h W o r k s h o p , local, and commercial antisera. T h e cells appear to have a D R blank haplotype associated with D R w 5 2 and D Q ~ 7 w h e n analyzed only with the best D R S , D R w 6 , and D R w 8 T e n t h W o r k s h o p sera (Table 1A). D R w 6 W o r k s h o p antisera 1133 ( D R w 1 3 ) and 9 0 6 0 ( D R w l 4 ) react weakly with D R S x 6 cells indicating that these sera are m o r e c o m p l e x than the W o r k shop analysis demonstrates. Many duospecific and muhispecific antisera containing D R w I 1 , 12, 13, 14, a n d / o r 8 react with D R 5 x 6 cells as seen in T a b l e IB. D u e to the reactivity with these c o m p l e x antisera, it is n o t surprising that D R 5 x 6 ceils are often typed serologically as D R 5 o r D R w 6 or as subtypes thereof.
170
H . J . Noreen et al.
TABLE 2
RFLP analysis"
DR
5x6 11
Dw DRw52
? 24
11.5 kb 9.4 kb 8.5 kb 6.8 kb 5.9 kb 4.3 kb
12
13 13 13 I4
14 8
5~ DB6 18 18 19 9 I6 8' 25 25 24 25 26 25 24 - +
+
+
+ +
17 17 B8 B18 3 3 24 25
+ +
+ +
+
hybridized with a D R B probe o f D N A digested with TaqI from family SM in which the patient is D R w l l , DR5x6; the father is D R 7 , D R w 1 1 ; and the mother is D R w 1 3 , DRSx6.
Sequence analysis. Sequence analysis was performed in two unrelated subjects ( M D and SK) which had been typed as D R S x 6 by serology and RFLP. T h e nucleotide
+ + +
+ +
+
+
+
+
+
+
+
+
+
~
+
+
+
+
+
• A schematicrepresentationof RFLPpa~rernsmsodated withDR.Dwspedfidties. DNA fromTenth InternationalWorkshopHTC and hetemzygousnormal individualswas digestedwithTaql and hybridizedwith a DRB probe. HTC Sweig.JVM.Tisi.SPO,FPA.and he[emzygo~ DRwl I ceils,as wellas ' HTC Mad~a. OLGA,TAB089,and heter~ygous DRw8cellsdemonstrate the samegFLPpattern despiteD regioaheterogeneityas definedby sequence analysis [1 10] and T
