BRIEF COMMUNICATION
Five New TaqI DRB1 Polymorphisms D. Middleton, D. A. Savage, C. CuUen, and J. Martin
ABSTRACT: Five new restriction fragment length polymorphisms (RFLPs) using Taql and a DRfl probe have been found in Caucasian individuals. The families of these individuals have been tissue typed by serological
and RFLP methods. The new RFLPs are similar to previously established RFLPs except for the size of one fragment in each instance. Human Immunology 3l. 145-147 (1991~
ABBREVIATION KFLP restriction fragment length polymorphisms INTRODUCTION We have used conventional serology for class I typing but D N A restriction fragment length polymorphism (RFLP) for class II typing while tissue typing potential unrelated bone marrow donors. The frequency of Taql allogenotypes in this donor population has been reported [1]. During the typing of the first 2000 donors we found five new RFLPs. We report here the confirmation of these patterns in family members and the comparison with class II serology results. MATERIALS AND METHODS The five new RFLPs were found in 2000 D N A typed blood transfusion donors resident in Northern Ireland who had volunteered to be unrelated bone marrow donors. Blood was then obtained from family members. All individuals with the new RFLPs were Caucasian. Genomic D N A was prepared from peripheral blood leukocytes by the "salting-out" method [2]. Samples of genomic D N A (8/zg) were digested to completion with the restriction eodonuclease TaqI (Bethesda Research Laboratories, 5 U/tzg) and the fragments resolved by electrophoresis in agarose gels as previously described [3], with the exception that the gels were run at 40 V for 18 hr. Membranes were hybridized sequentially with the following radiolabeled e D N A probes: HLAFrom the Northern Inland Tissue Typing Sen'ice, City Hospital, Bdfaut, Northtrn Inland. Address reprint requests to O. Middleton, Northern Ireland Tissue Typing Service, City Hospital. Belfast BT9 TAD, Northern Ireland. ReceivedJMy 1 I, 1990; accepted Noveraber 16, 1990.
HumanImmunologySt, 145-147 (1991) © AmericanSocietyfor Histocompatibilityand lmmtmogenedcs,1991
DRfl pRTVI [4], HLA-DQfl plI-fl-1 [5], and HLAD Q a pDCH1 [6]. Associations between TaqI RFLVs and serologically defined HLA class H antigens and nomenclature used have previously been described [ 1, 7t. Molecular weights of hybridization signals were determined using a sem/logarithm/c standard curve [log,0 Mr (base pairs) versus electrophoretlc mobility (ram)l, calibrated with a HindIII digest of bacteriophage ~, (fragment sizes 23130, 9416, 6557, 4361, 2322, 2027, 564, and 125 bp). Conventional serological methods were performed for class I and II for all family members. Cells for class II typing were isolated using magnetic beads coated wich a class II monoclonal antibody (Dynal). RESULTS The five new RFLPs each differ from a previously reported RFLP in one fragment size and have been considered to be variants of these "parent" RFLPs. The sizes of the five new RFLPs are given in Table 1 and compared with the sizes of the "parent" RFLP. T~e haplotypes containing the new RFLPs are also giver in Table 1. Four of the new RFLPs type serologicall~ as the HLA-DR antigen associated with the "parent" ~ L P . The new RFLP PL typed as HLA-DR blank. DISCUSSION We have found a frequency of 0.25% of new HLA-DR RFLPs in a relatively homogeneous Caucasian population (there is little immigration to Northern ireland). X45 0198-8859/91/53.50
146
D. Middleton et al.
T A B L E 1 Comparison of fragment sizes (kb) of new RFLPs with previously defined RFLPs 10 IH WS (kh)" 13.81 11.48 Fragment size (kb)
Local (kb) DRBI2 DM DRa4 JR BG DR#7.1 PL TW
A32,CwI,B27,DRw12,DQw3 A1,Cw3,B62,DR4,DQw3 A2,Cw5,B44,DR4,DQw3 A23,Cw4,B44,DRBLANK,DQw3 A32,Cw2,B44.DR7,DQw2 "Tenth
14.8
13.0
7.9
5.80 5.22 4.12 4.12 4.12 7.7 6.1
5.4
+ +
4.3 + +
+ + + + +
+ +
+ + +
+ + +
4.2
4.1
2.69 2.60 2.7
2.6
+ +
+ + +
+ + +
+
InternationalHistocompatibilityWorkshop nomenclature.
We zugges: t:~,t in other populations, especially o f mixed race, there will be a much higher frequency of new RFLPs. Recently, new HLA-DR TaqI RFLPs have been found which are confined to South African individuals of black or mixed ancestry [8].
FIGURE 1 RFLPs showing new fragment size. I~ = new fragment. (a) S = RFLP BG (14.8, 7.7, 5.4, 2.7 kb) and/37.1 (14.8, 7.9, 4.1, 2.6 kb), C = Cox cell line DR/317 (11.1, 7.8, 4.2 kb). (b) Pl -- RFLP TW (9.5, 7.9, 4.1, 2.6 kb) and #1 (6.1, 4.4 kb), P2 = RFLP DR/~8 (9.6 kb) and/34 (14.8, 6.1, 5.4, 2.7 kb), S = RFLP TW (9.5, 7.9, 4.1, 2.6 kb) and/34 (1~1.8, 6.1, 5.4, 2.7 kb). MW (kb)
7.23 9.5
S
C
PI P2 S
~i ~
9-4 -
.... ~.....
i~ . . . .
* ~
6.6-
4,4-
•
la
Ib
.....
In general, the RFLPs detected by the methods described herein identify restriction endonuclease sites not in the second exon but in the noncoding sequences. These sites are in strong linkage with the coding sequences. Therefore RFLPs reported in this paper are most probably due to differences in noncoding sequences and therefore the coding sequences are not changed. Indeed, in four instances the epitope detected by serology has not been affected. However, it may be that the RFLP of PL is detecting variation in the exon sequence as the individuals carrying this RFLP do not type serologically for HLA-DR7. Interestingly, the RFLP of PL still retains the 7.9-kb fragment association only with HLA-DR 7.1 individuals. It appears that in three of the new RFLPs a fragment previously found in other haplotypes is found. Therefore, in these instances it appears that the RFLPs are of sequences that have been reshuffled from another region very much along the lines that give rise to the polymorphism of HLA-DP. However, in two of the new RFLPs a new fragment has been found. In RFLP B G a fragment has been found at 7.7 kb (Fig. la) and in RFLP T W a fragment has been found at 9.5 kb (Fig. lb). These may have arisen through point mutations. The haplotypes associated with four of the new RFLPs have the same D Q association, both by serology and by RFLP~ as that found with the haplotype of the "parent" RFLP. The exception is PL, which is HLA-DQw3 positive which is normally associated with HLA-DR 7.2 [7]. Individuals who are HLA-DR 7.2, DQw3 are positive with the HLA-DR7 antisera that we use, so this would not be a reason for these sera being negative with PL. Recently, there have been two reports which may correspond to two o f the RFLPs reported here. Awad et al. [9] have reported the 6.1-kb band missing in a Greek individual positive for HLA-DR4 but this individual has a band at 7.8 kb due to the HLA-DR antigen on the other haplotype and so it is not clear if he is the same as BG, whose 6.1-kb band is replaced by a 7.7-kb band.
Five New Taql DRBI Polymorphisms
Abe and colleagues [10] have sequenced two subtypes of D R w l 2 which differed at amino acid residue number 67. We are not able to tell if these subtypes correspond to those reported here. However, it is our intention to sequence the five new RFLPs. ACKNOWLEDGMENTS We thank those people mentioned in the text for supplying us with the probes and Miss K. Mills for typing the manuscript. REFERENCES 1. Cullen CG, Middleton D, Savage DA. Frequency and associations of HLA-class I1 Taql allogenotypes in the Northern Ireland population. Hum lmmunol 29:92, 1000.
2. MiUar SA, Dykes DD, Polesky HF: A simple salting-out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215, 1988. 3. Middleton D, Savage DA, BidwellJL Cullen C, Martin J: Association of HLA-DRBr with HLA-DQw3. Tissue Antigens 31:79, 1988. 4. Bidwell JL, Jarrold EA: HLA-DR allogenotyping using exon-specific cDNA probes and application of rapid minigel methods. Moi immuno123:1111, 1986. 5. Larhammar D, Schenning I., Gustafsson K, Wiman K, Claesson L, Rask L, Peterson PA: Complete amino acid
147
sequence of an HLA-DR antigen-like chain as pred/cted from the nucleotide seque~e: Similarities with immunoglobulins and HLA-A,B and C antigens. Proc Natl Acad Sci USA 79:3687, 1982. 6. Auffray C, LiUieJW, Arnot D, Grossberger D, Kappes D, $trominger JL: lsoqrpic and allotypic variation of human class II histocompadbility antigen-chain genes. Nature 308:327, 1984. 7. Bidwell JL, Bidwell EA, Savage DA, Midd~eton D, Klouda FF, Bradley BA: A DNA-RFLP typing system that positively identifies serolog/cally well-defined and illdefined HLA-DR and DQ alleles, including DRwl0. Transplantation 45:640, 1988. 8. Martell RW, Oudshoorn M, Arendse B, DuToit ED: Polymorphism of DRw52 and its association wi~h DRwl 1 and DRwl2 in South African blacks (negroes~ and individuals of mixed ancestry (Cape coloredsL Hum Immunol 28:32, 1990. 9. Awad J, Oilier w: Cutbush S, Papasteriadis C, Gupta A, Carthy D, McCio,key D, Brown CJ, Boki K, Fostizopoulos G, Festenstein H: Heterogeneity of HLA-DR,~ in Greeks including a unique DR,~-DQw2 association. Tissue Antigens 35:40, lC~0. t0. Abe A, ho I, Ohkubo M, Kaneko 2", lto K, Kato H, Kashiwagi N, Obata F: Two distinct subtypes of the HLA-DRw12 haplotypes in the Japanese population detected by nucleotide ~"queoce analysis and oligonudeotide genotyping, lmmunogenetics 30:422, 1989.
Five New TaqI DRB1 Polymorphisms D. Middleton, D. A. Savage, C. CuUen, and J. Martin
ABSTRACT: Five new restriction fragment length polymorphisms (RFLPs) using Taql and a DRfl probe have been found in Caucasian individuals. The families of these individuals have been tissue typed by serological
and RFLP methods. The new RFLPs are similar to previously established RFLPs except for the size of one fragment in each instance. Human Immunology 3l. 145-147 (1991~
ABBREVIATION KFLP restriction fragment length polymorphisms INTRODUCTION We have used conventional serology for class I typing but D N A restriction fragment length polymorphism (RFLP) for class II typing while tissue typing potential unrelated bone marrow donors. The frequency of Taql allogenotypes in this donor population has been reported [1]. During the typing of the first 2000 donors we found five new RFLPs. We report here the confirmation of these patterns in family members and the comparison with class II serology results. MATERIALS AND METHODS The five new RFLPs were found in 2000 D N A typed blood transfusion donors resident in Northern Ireland who had volunteered to be unrelated bone marrow donors. Blood was then obtained from family members. All individuals with the new RFLPs were Caucasian. Genomic D N A was prepared from peripheral blood leukocytes by the "salting-out" method [2]. Samples of genomic D N A (8/zg) were digested to completion with the restriction eodonuclease TaqI (Bethesda Research Laboratories, 5 U/tzg) and the fragments resolved by electrophoresis in agarose gels as previously described [3], with the exception that the gels were run at 40 V for 18 hr. Membranes were hybridized sequentially with the following radiolabeled e D N A probes: HLAFrom the Northern Inland Tissue Typing Sen'ice, City Hospital, Bdfaut, Northtrn Inland. Address reprint requests to O. Middleton, Northern Ireland Tissue Typing Service, City Hospital. Belfast BT9 TAD, Northern Ireland. ReceivedJMy 1 I, 1990; accepted Noveraber 16, 1990.
HumanImmunologySt, 145-147 (1991) © AmericanSocietyfor Histocompatibilityand lmmtmogenedcs,1991
DRfl pRTVI [4], HLA-DQfl plI-fl-1 [5], and HLAD Q a pDCH1 [6]. Associations between TaqI RFLVs and serologically defined HLA class H antigens and nomenclature used have previously been described [ 1, 7t. Molecular weights of hybridization signals were determined using a sem/logarithm/c standard curve [log,0 Mr (base pairs) versus electrophoretlc mobility (ram)l, calibrated with a HindIII digest of bacteriophage ~, (fragment sizes 23130, 9416, 6557, 4361, 2322, 2027, 564, and 125 bp). Conventional serological methods were performed for class I and II for all family members. Cells for class II typing were isolated using magnetic beads coated wich a class II monoclonal antibody (Dynal). RESULTS The five new RFLPs each differ from a previously reported RFLP in one fragment size and have been considered to be variants of these "parent" RFLPs. The sizes of the five new RFLPs are given in Table 1 and compared with the sizes of the "parent" RFLP. T~e haplotypes containing the new RFLPs are also giver in Table 1. Four of the new RFLPs type serologicall~ as the HLA-DR antigen associated with the "parent" ~ L P . The new RFLP PL typed as HLA-DR blank. DISCUSSION We have found a frequency of 0.25% of new HLA-DR RFLPs in a relatively homogeneous Caucasian population (there is little immigration to Northern ireland). X45 0198-8859/91/53.50
146
D. Middleton et al.
T A B L E 1 Comparison of fragment sizes (kb) of new RFLPs with previously defined RFLPs 10 IH WS (kh)" 13.81 11.48 Fragment size (kb)
Local (kb) DRBI2 DM DRa4 JR BG DR#7.1 PL TW
A32,CwI,B27,DRw12,DQw3 A1,Cw3,B62,DR4,DQw3 A2,Cw5,B44,DR4,DQw3 A23,Cw4,B44,DRBLANK,DQw3 A32,Cw2,B44.DR7,DQw2 "Tenth
14.8
13.0
7.9
5.80 5.22 4.12 4.12 4.12 7.7 6.1
5.4
+ +
4.3 + +
+ + + + +
+ +
+ + +
+ + +
4.2
4.1
2.69 2.60 2.7
2.6
+ +
+ + +
+ + +
+
InternationalHistocompatibilityWorkshop nomenclature.
We zugges: t:~,t in other populations, especially o f mixed race, there will be a much higher frequency of new RFLPs. Recently, new HLA-DR TaqI RFLPs have been found which are confined to South African individuals of black or mixed ancestry [8].
FIGURE 1 RFLPs showing new fragment size. I~ = new fragment. (a) S = RFLP BG (14.8, 7.7, 5.4, 2.7 kb) and/37.1 (14.8, 7.9, 4.1, 2.6 kb), C = Cox cell line DR/317 (11.1, 7.8, 4.2 kb). (b) Pl -- RFLP TW (9.5, 7.9, 4.1, 2.6 kb) and #1 (6.1, 4.4 kb), P2 = RFLP DR/~8 (9.6 kb) and/34 (14.8, 6.1, 5.4, 2.7 kb), S = RFLP TW (9.5, 7.9, 4.1, 2.6 kb) and/34 (1~1.8, 6.1, 5.4, 2.7 kb). MW (kb)
7.23 9.5
S
C
PI P2 S
~i ~
9-4 -
.... ~.....
i~ . . . .
* ~
6.6-
4,4-
•
la
Ib
.....
In general, the RFLPs detected by the methods described herein identify restriction endonuclease sites not in the second exon but in the noncoding sequences. These sites are in strong linkage with the coding sequences. Therefore RFLPs reported in this paper are most probably due to differences in noncoding sequences and therefore the coding sequences are not changed. Indeed, in four instances the epitope detected by serology has not been affected. However, it may be that the RFLP of PL is detecting variation in the exon sequence as the individuals carrying this RFLP do not type serologically for HLA-DR7. Interestingly, the RFLP of PL still retains the 7.9-kb fragment association only with HLA-DR 7.1 individuals. It appears that in three of the new RFLPs a fragment previously found in other haplotypes is found. Therefore, in these instances it appears that the RFLPs are of sequences that have been reshuffled from another region very much along the lines that give rise to the polymorphism of HLA-DP. However, in two of the new RFLPs a new fragment has been found. In RFLP B G a fragment has been found at 7.7 kb (Fig. la) and in RFLP T W a fragment has been found at 9.5 kb (Fig. lb). These may have arisen through point mutations. The haplotypes associated with four of the new RFLPs have the same D Q association, both by serology and by RFLP~ as that found with the haplotype of the "parent" RFLP. The exception is PL, which is HLA-DQw3 positive which is normally associated with HLA-DR 7.2 [7]. Individuals who are HLA-DR 7.2, DQw3 are positive with the HLA-DR7 antisera that we use, so this would not be a reason for these sera being negative with PL. Recently, there have been two reports which may correspond to two o f the RFLPs reported here. Awad et al. [9] have reported the 6.1-kb band missing in a Greek individual positive for HLA-DR4 but this individual has a band at 7.8 kb due to the HLA-DR antigen on the other haplotype and so it is not clear if he is the same as BG, whose 6.1-kb band is replaced by a 7.7-kb band.
Five New Taql DRBI Polymorphisms
Abe and colleagues [10] have sequenced two subtypes of D R w l 2 which differed at amino acid residue number 67. We are not able to tell if these subtypes correspond to those reported here. However, it is our intention to sequence the five new RFLPs. ACKNOWLEDGMENTS We thank those people mentioned in the text for supplying us with the probes and Miss K. Mills for typing the manuscript. REFERENCES 1. Cullen CG, Middleton D, Savage DA. Frequency and associations of HLA-class I1 Taql allogenotypes in the Northern Ireland population. Hum lmmunol 29:92, 1000.
2. MiUar SA, Dykes DD, Polesky HF: A simple salting-out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215, 1988. 3. Middleton D, Savage DA, BidwellJL Cullen C, Martin J: Association of HLA-DRBr with HLA-DQw3. Tissue Antigens 31:79, 1988. 4. Bidwell JL, Jarrold EA: HLA-DR allogenotyping using exon-specific cDNA probes and application of rapid minigel methods. Moi immuno123:1111, 1986. 5. Larhammar D, Schenning I., Gustafsson K, Wiman K, Claesson L, Rask L, Peterson PA: Complete amino acid
147
sequence of an HLA-DR antigen-like chain as pred/cted from the nucleotide seque~e: Similarities with immunoglobulins and HLA-A,B and C antigens. Proc Natl Acad Sci USA 79:3687, 1982. 6. Auffray C, LiUieJW, Arnot D, Grossberger D, Kappes D, $trominger JL: lsoqrpic and allotypic variation of human class II histocompadbility antigen-chain genes. Nature 308:327, 1984. 7. Bidwell JL, Bidwell EA, Savage DA, Midd~eton D, Klouda FF, Bradley BA: A DNA-RFLP typing system that positively identifies serolog/cally well-defined and illdefined HLA-DR and DQ alleles, including DRwl0. Transplantation 45:640, 1988. 8. Martell RW, Oudshoorn M, Arendse B, DuToit ED: Polymorphism of DRw52 and its association wi~h DRwl 1 and DRwl2 in South African blacks (negroes~ and individuals of mixed ancestry (Cape coloredsL Hum Immunol 28:32, 1990. 9. Awad J, Oilier w: Cutbush S, Papasteriadis C, Gupta A, Carthy D, McCio,key D, Brown CJ, Boki K, Fostizopoulos G, Festenstein H: Heterogeneity of HLA-DR,~ in Greeks including a unique DR,~-DQw2 association. Tissue Antigens 35:40, lC~0. t0. Abe A, ho I, Ohkubo M, Kaneko 2", lto K, Kato H, Kashiwagi N, Obata F: Two distinct subtypes of the HLA-DRw12 haplotypes in the Japanese population detected by nucleotide ~"queoce analysis and oligonudeotide genotyping, lmmunogenetics 30:422, 1989.
