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HLA-DRB4 Gene Encoded HLA-DR53 Specificity Segregating with the HLA-DR7, -DQ9 Haplotype: Unusual Association
HLA-DRB4 Gene Encoded HLA-DR53 Specificity Segregating with the HLA-DR7, -DQ9 Haplotype: Unusual Association N. M. Lardy, A. R. van der Horst, M. J. van de Weerd, L. P. de Waal, and R. E. Bontrop ABSTRACT: HLA phenotyping of a leukemia patient of Caucasoid origin revealed the presence of the serological HLA-DR53 specificity. Comprehensive pedigree analysis demonstrated that the HLA-DR53 specificity segregated with the HLA-DR7, -DQ3 haplotype. High resolution PCR- SSP genotyping of the HLA class II genes revealed the presence of the HLA-DRB4*0101101 allele segregating together with the HLA-DRB1*0701, -DQA1*0201 and DQB1*03032 alleles. This finding is in contrast to
INTRODUCTION The HLA-DR53 specificity is a serological epitope expressed by the product of the HLA-DRB4 gene that is usually found in association with HLA-DR4, -DR7 and -DR9 positive haplotypes. The exception to this rule is provided by the HLA-DR7, -DQ9 haplotype (usually seen in association with HLA-B57) which lacks the serological HLA-DR53 specificity [1– 4]. Nucleotide sequence analyses have revealed the existence of at least eight alleles at the HLA-DRB4 locus [5]. Several of these alleles contain deleterious mutations and consequently do not encode cell surface products. In the case of the HLA-DR7, -DQ9 associated HLA-DRB4 allele, restriction fragment length polymorphism analysis have failed to detect any alterations between this gene and the expressed HLA-DRB4 sister genes [6]. However, nucleotide sequence analysis has demonstrated that an altered From the Department of HLA Diagnostics, Central Laboratory of the Dutch Red Cross Blood Transfusion Service, The Netherlands (N.M.L., A.R.v.d.H. M.J.v.d.W.), Department of Transplantation Immunology and Bloodbank, Central Laboratory of the Dutch Red Cross Blood Transfusion Service, The Netherlands (L.P.d.W.) and Department of Immunobiology, Biochemical Primate Research Centre, Rijswijk, The Netherlands (R.E.B.). Address reprint requests to: N. M. Lardy, Department of HLA-Diagnostics, Central Laboratory of the Dutch Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands; Tel: 131-20-512 3637; Fax: 131-20-512 3474. Received August 26, 1997; revised November 28, 1997; accepted December 4, 1997. Human Immunology 59, 115–118 (1998) © American Society for Histocompatibility and Immunogenetics, 1998 Published by Elsevier Science Inc.
known linkages in that thus far, the HLA-DR7, -DQ9 haplotype has only been described in association with the non-expressed HLA-DRB4*0103102N allele. The existence of this “novel” haplotype may be explained by a homologous recombinational event that occured between the HLA-DR7, -DR53, -DQ2 and the HLA-DR7, -DQ9 haplotypes. Human Immunology 59, 115–118 (1998). © American Society for Histocompatibility and Immunogenetics, 1998. Published by Elsevier Science Inc.
splice site at the 39 end of the first intron prohibits correct processing of the HLA- DRB4 transcripts [7, 8]. This non-expressed HLA-DRB4 allele has been refered to as HLA-DRB4null and is officially designated HLADRB4*0103102N [5]. In addition, recently Robbins et al demonstrated the existence of two new HLADRB4null alleles. The defects observed in these HLADRB4 alleles ranged from a deletion in the second exon of the HLA-DRB4*0201N allele to the complete lack of the second exon in the HLA-DRB4*0301N allele [9]. In general, cells positive for the HLA-DR7, -DQ9 haplotype are used to procure HLA antibodies that react solely with HLA-DRB4 gene product. Consequently, putative HLA-DR53 antibodies that react with cells bearing the HLA-DR7, -DQ9 haplotype are recarded as cross reactive with an epitope on the HLA-DRB1 gene product of HLA-DR7 positive cells [4]. In search for a suitable allogeneic bone marrow donor, routine HLA class II phenotyping of a patient suffering from leukemia revealed the presence of the serological HLA-DR53 specificity in association with the HLADR7, -DQ9 haplotype. The alleles present on this haplotype were characterized by molecular typing techniques, in order to define the HLA-DRB4 gene and investigate how such a haplotype may have been generated in an evolutionary context. 0198-8859/98/$19.00 PII S0198-8859(97)00264-4
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MATERIALS AND METHODS HLA Typing HLA phenotyping for the World Health Organisation (WHO) defined HLA class II specificities i.e. DR1DR16, DR51-DR53, and DQ1-DQ9 was performed in the microlymphocytotoxicity assay using the two-color fluorescence technique (TCF) [10]. HLA class II typing sera were obtained either locally or by means of sera exchange. Specific characterization of the serological HLA-DR53 specificity was achieved by means of the lymphocytotoxic allo-antiserum MSD51. This HLADR53 allo-antiserum has previously been extensively characterized and reacts with all HLA-DR53 positive cells with the exception of cells expressing the HLADR7, -DQ3 phenotype [11]. To distinguish T and B cells, FITC-labeled goat-anti-human Ig was used. The test was performed with 0.5 ml serum and 0.5 ml PBLs at a concentration of 10 3 106 incubated for 1 h at 22°C. Rabbit complement was added for a further incubation of 2 h. Cell lysis was assessed by fluorescence microscopy using ethidium bromide. PCR-SSP Analysis HLA class II genotyping was performed by means of the commercially available DynalR PCR-SSP typing kits (Dynal A.S., Oslo, Norway). Briefly, 100 ng genomic DNA was prepared with the aid of the Qiagen DNA isolation kit (Diagen, Hilden, Germany) and amplified in a 10 ml PCR mixture containing 0.25 mM allele- and group specific primers, 500 mM KCl, 15 mM MgCl2, 100 mM Tris-HCl pH8.3, 0.01% w/v gelatin, 10 mM of each of the four deoxyribo- nucleotide phosphates and 0.375 units of Taq polymerase (Promega Co., Leiden, the Netherlands). The DNA amplification consisted of 1 cycle for 2 min at 94°C followed by 10 cycles each for 10 s at 94°C, 60 s at 65°C and 20 cycles each for 10 s at 94°C, 50 s at 61°C and 30 s at 72°C. The resolution of the DynalR PCR-SSP kits used are: DR “low resolution” kit identifies allHLA-DRB1, -DRB3, -DRB4 and -DRB5 alleles, DRB4 SSP kit identifies the DRB4*0101101 -DRB4*0201N alleles, DQA1 SSP kit identifies the DQA1*0101-DQA1*0105, DQA1*0201, DQA1*0301-DQA1*0303, DQA1*0401, DQA1*0501-DQA1*0503, DQA1*0601 alleles and the DQ “low resolution” kit identifies the DQB1*0501DQB1*0504, DQB1*06011-DQB1*0612, DQB1*0201DQB1*0203, DQB1*0301-DQB1*0306 and the DQB1* 0401-DQB1*0402 alleles. RESULTS HLA Typing Conventional HLA class II typing revealed the presence of the serological HLA-DR53 specificity on cells of a
N. M. Lardy et al.
FIGURE 1 Serological typing of HLA antigens segregating in family ELE. The individual (II-3), in which the “novel” haplotype was originally detected, is indicated by shading. HLA class I and class II antigens are given according to their official nomenclature. The “novel” haplotype is composed of the HLA-A2, -B41, -DR7, -DR53, -DQ9 phenotype.
patient suffering from leukemia. The individual was HLA phenotyped as HLA-A2, -A3, -B41, -B62, -Cw7, -DR7, -DR15, -DR51, -DR53, -DQ9, -DQ6 positive. Family investigation was performed to define the haplotype. Figure 1 demonstrates that the detected HLADR53 specificity is maternally derived and segregates with the HLA-A2, -B41, -DR7, -DQ9 haplotype. This “novel” haplotype is present in individuals I-1, II-2 and II-3 (patient). Furthermore, pedigree analysis revealed that individual II-2 is positive for two distinct HLADR7 haplotypes i.e. the “novel” HLA-DR7, -DR53, -DQ9 haplotype and the well documented HLA-B57, -DR7, -DQ9 haplotype which is associated with the non-expressed HLA-DRB4*0103102N allele. The detection of the HLA-DR53 specificity raised the question whether the serological reactivity was indeed the result of an expressed HLA-DRB4 gene product or due to a cross reactive epitope present on another HLA-DR molecule. The presence of the serological HLA-DR53 specificity was assessed by means of the well characterized lymphocytotoxic allo- anti HLA-DR53 serum MSD51. Table 1 demonstrates that the allo-antiserum MSD51 reacted with cells derived from individuals I-1, II-2, II-3 (patient) and also with the control cells 29-102 and 29-315. These control cells exhibit the HLA-DR7, -DQ2 phenotype which is associated with the HLA-DRB4*0101101 allele encoded HLA-DR53 specificity. However, no reactivity was found with cells derived from individual I-2, II-1 or the control cells 29-244 and 29-355. The control cells 29-244 and 29-355 are positive for the HLADR7, -DQ9 phenotype associated with the HLA-
Novel HLA-DR7, -DR53, -DQ9 association
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TABLE 1 Reactivity pattern of the allo-anti HLA-DR53 serum ‘‘MSD51’’
MSD51
I-1
I-2
II-1
II-2
II-3
29-102
29-315
29-244
29-355
1
2
2
1
1
1
1
2
2
I-1 mother (A1, A2, B8, B41, DR17, DR7, DR52, DR53, DQ2, DQ9), I-2 father (A2, A31, B57, B62, Cw7, Cw7, DR7, DR15, DQ3, DQ1), II-1 sibling 1 (A1, A31, B8, B62, Cw7, DR17, DR15, DR52, DR51, DQ2, DQ6), II-2 sibling 2 (A2, A2, B41, B57, Cw6, DR7, DR7, DR53, DQ9, DQ3), II-3 patient (A2, A31, B41, B62, Cw7, DR7, DR15, DR53, DR51, DQ9, DQ6). 29-102, 29-315, 29-244 and 29-355 are well characterized HLA-DR7 positive control cells with the following HLA class II phenotypes: 29-102 (DR7, DR10, DR53, DQ2, DQ5), 29-315 (DR7, DR13, DR52, DR53, DQ6, DQ2), 29-244 (DR7, DR13, DR52, DQ7, DQ9) and 29-355 (DR14, DR7, DR52, DQ5, DQ9)
DRB4*0103102N allele that does not encode the serological HLA-DR53 specificity. PCR-SSP Analysis Unfortunately it was not possible to obtain further material from family members I-2 and II-2. Thus, DNA from individuals I-1 and II-3 were subjected to PCR-SSP analysis to establish the HLA class II alleles comprising this unreported HLA-DR association. In addition, DNA of the genotypically well characterized homozygous cellines PITOUT (HLA-DRB*0701, -DRB4*0101101, -DQA1*0201, -DQB1*0202) and DBB (HLA-DRB1*0701, -DRB4*0103102N, -DQA1*0201, -DQB1*03032) expressing respectively the HLA-DR7, -DR53, -DQ2 and the HLA-DR7, -DQ9 phenotypes were included in the analysis to exclude genotyping errors or misinterpretations. Table 2 demonstrates that the “novel” detected HLA-DR haplotype segregating in individual I-1 and II-3 is composed of the HLA-DRB1*0701, -DQA1*0201, -DQB1*03032 alleles in association with the HLADRB4*0101101 allele. This finding is in contrast to known linkages in that thus far the HLA-DRB1*0701, -DQA1*0201, -DQB1*03032 haplotype has been found only in association with the non-expressed HLADRB4*0103102N allele. The complete HLA class II genotypes of individual I-1, II-1, II-3 and the reference cell lines PITOUT and DBB are given in Table 2.
DISCUSSION The serological HLA-DR53 specificity, encoded by the HLA-DRB4 gene, is normally present on the HLA-DR4, -DR7 and -DR9 positive haplotypes. However, it has previously been demonstrated that the HLA-DR7, -DQ9 haplotype is associated with an HLA-DRB4null allele that does not encode the serological HLA-DR53 specificity [2, 3]. The HLA-DR7, -DQ9 associated HLADRB4 gene has a single base substitution in the acceptor splice site at the 39 end of the first intron that abrogates functional expression of this gene [8]. This nonexpressed HLA-DRB4 gene is designated HLADRB4*0103102N [5]. In the present communication we describe the existence of an HLA-DR7, -DQ9 haplotype which possesses an expressed HLA-DRB4 gene product. Family investigation demonstrated that the HLA-DR53 specificity segregated unequivocally with the HLA-A2, -B41, -DR7, -DQ9 haplotype. Repeated HLA typing and extensive serological analysis using the well characterized anti HLA-DR53 serum MSD51 excluded the possibility that the detected HLA-DR53 reactivity was due to serological misinterpretation. High resolution PCR-SSP analysis of DNA obtained from individuals I-1 and II-3 revealed that the “novel” HLA-DR association consisted of the HLA-DRB1*0701, -DQA1*0201, -DQB1*03032 alleles in association with the expressed HLA-
TABLE 2 PCR-SSP genotyping of family ELE Id. I-1 II-1 II-3 PITOUT DBB
Phenotype DR17, DR7, DR52, DR53, DQ2, DQ9 DR17, DR15, DR52, DR53, DQ2, DQ6 DR15, DR7, DR51, DR53, DQ6, DQ9 DR7, DR53, DQ2 DR7, DQ3
DRB1*
DRB3*
0301/0701
0101
0301/15
0101
DRB4* 0101101 —
15/0701
—
0101101
0701 0701
— —
0101101 0103102N
DRB5*
DQA1*
DQB1*
—
0501/0201
0201/03032
0101
0501/0102
0201/06
0101
0102/0201
06/03032
0201 0201
0202 03032
— —
I-1: mother (HLA-DR17, 2DR7, 2DR52, 2DR53, 2DQ2, 2DQ9), II-1: sibling (HLA-DR17, 2DR15, 2DR52, 2DR51, 2DQ2, 2DQ6), II-3: patient (HLA-DR15, 2DR7, 2DR51, 2DR53, 2DQ6, 2DQ9). PITOUT and DBB are well characterized HLA-DR7 positive control cells. PITOUT is phenotypically typed as HLA-DR7, 2DR53, 2DQ2 and is associated with the expressed HLA-DRB4*0101101 allele, DBB has the HLA-DR7, 2DQ3 phenotype and is associated with the non-expressed HLA-DRB4*0103102N allele. The specificities given in bold denotes the ‘‘novel’’ HLA class II association.
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DRB4*0101101 allele. Thus, the detected serological HLA-DR53 specificity is indeed the product of an HLADRB4 gene, namely the HLA-DRB4*0101101 allele. Thus far the existence of two different HLA-DR7 haplotypes has been demonstrated, namely the HLA-DR7, -DQ2 haplotype which is associated with the serological HLA-DR53 specificity and the HLA-DR7, - DQ9 haplotype which does not express the HLA-DR53 specificity. The finding presented in this study is in contrast to known HLA-DR linkage disequilibrium and demonstrates the existence of a third HLA-DR7 haplotype. Surprisingly, during the preparation of this manuscript the “novel” described HLA-DRB1*0701, -DRB4*0101101 -DQA1*0201, -DQB1*03032 haplotype was again detected in an unrelated healthy blood donor with the following HLA phenotype: A2/A68, -B41/B57, -Cw7, -DR7/DR13, -DR53/DR52, -DQ9/ DQ6. The detection of the expressed HLA-DRB4*0101101 allele segregating with the HLA-DRB1*0701, -DQA1*0201, -DQB1*03032 haplotype in unrelated individuals raises the question whether it is a new haplotype and how it originated in the population. Although not a single case of crossing-over events in the HLA-DR region has been documented [12], it is tempting to speculate that the HLA-DRB1*0701, -DRB4*0101101 -DQA1*0201, -DQB1*03032 haplotype arose due to a homologous exchange of HLA-DRB4 genes between the HLA-DRB1*0701, -DRB4*0101101 -DQA1*0201, -DQB1*0201 and the HLA-DRB1*0701, -DRB4* 0103102N, -DQA1*0201, -DQB1*03032 haplotypes. Evidence for this supposition will be the existence of an HLA-DR7, -DQ2 haplotype that possesses an HLADRB4 null allele that does not encode the HLA-DR53 specificity. REFERENCES 1. Mukai R, Suzuki M, Yabe T, Hamaguchi H, Maeda H: Identification of the MT3 molecule from HLA-DR4, 7, w9 homozygous cell lines. J Immunol 33:321, 1984. 2. Knowles RW, Flomenberg N, Horibe K, Winchester R, Radka SF, Dupont B: Complexity of the supertypic HLADRw53 specificity: Two distinct epitopes differentially expressed on one or all of the DR b-chains depending on the HLA-DR allotype. J Immunol 137:2618, 1986.
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3. Matsuyama T, Schwenzer J, Silver J, Winchester R: Structural Relationships between the DRb2 subunits in DR4, 7, and w9 haplotypes and the DRw53 (MT3) specificity. J Immunol 137:934, 1986. 4. Marsh SGE, Du Toit ED, Bunce M, Moses JH, Martel R, Morris P, De Waal L, Johnson A, Fotino M: Antigen Society no. 208/209: HLA-DR52, -DR53. In Tsuji K, Aizawa M, Sasazuki T (eds): HLA 1991, vol I. Proceedings of the Eleventh International Histocompatibility Workshop and Conference. Oxford, Oxford University Press, 1992, p 376. 5. Bodmer JG, Marsh SGE, Albert ED, Bodmer WF, Bontrop RE, Charron D, Dupont B, Erlich HA, Fauchet R, Mach B, Mayr WR, Parham P, Sasazuki T, Schreuder GMTh, Strominger JL, Svegaard A, Terasaki PI: Nomenclature for the factors of the HLA system, 1996. Tissue Antigens 9:297, 1997. 6. Sutton VR, Knowles RW: Mapping of a restriction fragment length polymorphism associated with defective DRb4 chain expression to the HLA-DRB1 gene. Hum Immunol 22:123, 1988. 7. Sutton VR, Kienzle BK, Knowles RW: An altered splice site is found in the DRB4 gene that is not expressed in HLA- DR7,Dw11 individuals. Immunogenetics 29:317, 1989. 8. Sutton VR, Knowles RW: An abberant DRB4 null gene transcript is found that could encode a novel HLA-DRb chain. Immunogenetics 31:112, 1990. 9. Robbins F, Hurley CK, Tang T, Yao H, Lin YS, Wade J, Goeken N, Hartzman RJ: Diversity associated with the second expressed HLA-DRB locus in the human population. Immunogenetics 46:104, 1997. 10. van Rood JJ, van Leeuwen A, Ploem JS: Simultaneous detection of two cell populations by two-colour fluorescence and application to the recognition of B-cell determinants. Nature 262:795, 1976. 11. Lardy NM, van der Horst AR, van den Berg-Loonen EM, Bontrop RE, de Waal LP: Fine Specificity of the Alloantiserum MSD-51: Epitope Mapping of HLA-DRw53 Determinants. Hum immunol 32:65, 1991. 12. Gregersen PK, Kao H, Nunez-Roldan A, Hurley CK, Karr RW, Silver J: Recombination sites in the HLA class II region are haplotype-dependent. J Immunol 141:1365, 1988.
INTRODUCTION The HLA-DR53 specificity is a serological epitope expressed by the product of the HLA-DRB4 gene that is usually found in association with HLA-DR4, -DR7 and -DR9 positive haplotypes. The exception to this rule is provided by the HLA-DR7, -DQ9 haplotype (usually seen in association with HLA-B57) which lacks the serological HLA-DR53 specificity [1– 4]. Nucleotide sequence analyses have revealed the existence of at least eight alleles at the HLA-DRB4 locus [5]. Several of these alleles contain deleterious mutations and consequently do not encode cell surface products. In the case of the HLA-DR7, -DQ9 associated HLA-DRB4 allele, restriction fragment length polymorphism analysis have failed to detect any alterations between this gene and the expressed HLA-DRB4 sister genes [6]. However, nucleotide sequence analysis has demonstrated that an altered From the Department of HLA Diagnostics, Central Laboratory of the Dutch Red Cross Blood Transfusion Service, The Netherlands (N.M.L., A.R.v.d.H. M.J.v.d.W.), Department of Transplantation Immunology and Bloodbank, Central Laboratory of the Dutch Red Cross Blood Transfusion Service, The Netherlands (L.P.d.W.) and Department of Immunobiology, Biochemical Primate Research Centre, Rijswijk, The Netherlands (R.E.B.). Address reprint requests to: N. M. Lardy, Department of HLA-Diagnostics, Central Laboratory of the Dutch Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands; Tel: 131-20-512 3637; Fax: 131-20-512 3474. Received August 26, 1997; revised November 28, 1997; accepted December 4, 1997. Human Immunology 59, 115–118 (1998) © American Society for Histocompatibility and Immunogenetics, 1998 Published by Elsevier Science Inc.
known linkages in that thus far, the HLA-DR7, -DQ9 haplotype has only been described in association with the non-expressed HLA-DRB4*0103102N allele. The existence of this “novel” haplotype may be explained by a homologous recombinational event that occured between the HLA-DR7, -DR53, -DQ2 and the HLA-DR7, -DQ9 haplotypes. Human Immunology 59, 115–118 (1998). © American Society for Histocompatibility and Immunogenetics, 1998. Published by Elsevier Science Inc.
splice site at the 39 end of the first intron prohibits correct processing of the HLA- DRB4 transcripts [7, 8]. This non-expressed HLA-DRB4 allele has been refered to as HLA-DRB4null and is officially designated HLADRB4*0103102N [5]. In addition, recently Robbins et al demonstrated the existence of two new HLADRB4null alleles. The defects observed in these HLADRB4 alleles ranged from a deletion in the second exon of the HLA-DRB4*0201N allele to the complete lack of the second exon in the HLA-DRB4*0301N allele [9]. In general, cells positive for the HLA-DR7, -DQ9 haplotype are used to procure HLA antibodies that react solely with HLA-DRB4 gene product. Consequently, putative HLA-DR53 antibodies that react with cells bearing the HLA-DR7, -DQ9 haplotype are recarded as cross reactive with an epitope on the HLA-DRB1 gene product of HLA-DR7 positive cells [4]. In search for a suitable allogeneic bone marrow donor, routine HLA class II phenotyping of a patient suffering from leukemia revealed the presence of the serological HLA-DR53 specificity in association with the HLADR7, -DQ9 haplotype. The alleles present on this haplotype were characterized by molecular typing techniques, in order to define the HLA-DRB4 gene and investigate how such a haplotype may have been generated in an evolutionary context. 0198-8859/98/$19.00 PII S0198-8859(97)00264-4
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MATERIALS AND METHODS HLA Typing HLA phenotyping for the World Health Organisation (WHO) defined HLA class II specificities i.e. DR1DR16, DR51-DR53, and DQ1-DQ9 was performed in the microlymphocytotoxicity assay using the two-color fluorescence technique (TCF) [10]. HLA class II typing sera were obtained either locally or by means of sera exchange. Specific characterization of the serological HLA-DR53 specificity was achieved by means of the lymphocytotoxic allo-antiserum MSD51. This HLADR53 allo-antiserum has previously been extensively characterized and reacts with all HLA-DR53 positive cells with the exception of cells expressing the HLADR7, -DQ3 phenotype [11]. To distinguish T and B cells, FITC-labeled goat-anti-human Ig was used. The test was performed with 0.5 ml serum and 0.5 ml PBLs at a concentration of 10 3 106 incubated for 1 h at 22°C. Rabbit complement was added for a further incubation of 2 h. Cell lysis was assessed by fluorescence microscopy using ethidium bromide. PCR-SSP Analysis HLA class II genotyping was performed by means of the commercially available DynalR PCR-SSP typing kits (Dynal A.S., Oslo, Norway). Briefly, 100 ng genomic DNA was prepared with the aid of the Qiagen DNA isolation kit (Diagen, Hilden, Germany) and amplified in a 10 ml PCR mixture containing 0.25 mM allele- and group specific primers, 500 mM KCl, 15 mM MgCl2, 100 mM Tris-HCl pH8.3, 0.01% w/v gelatin, 10 mM of each of the four deoxyribo- nucleotide phosphates and 0.375 units of Taq polymerase (Promega Co., Leiden, the Netherlands). The DNA amplification consisted of 1 cycle for 2 min at 94°C followed by 10 cycles each for 10 s at 94°C, 60 s at 65°C and 20 cycles each for 10 s at 94°C, 50 s at 61°C and 30 s at 72°C. The resolution of the DynalR PCR-SSP kits used are: DR “low resolution” kit identifies allHLA-DRB1, -DRB3, -DRB4 and -DRB5 alleles, DRB4 SSP kit identifies the DRB4*0101101 -DRB4*0201N alleles, DQA1 SSP kit identifies the DQA1*0101-DQA1*0105, DQA1*0201, DQA1*0301-DQA1*0303, DQA1*0401, DQA1*0501-DQA1*0503, DQA1*0601 alleles and the DQ “low resolution” kit identifies the DQB1*0501DQB1*0504, DQB1*06011-DQB1*0612, DQB1*0201DQB1*0203, DQB1*0301-DQB1*0306 and the DQB1* 0401-DQB1*0402 alleles. RESULTS HLA Typing Conventional HLA class II typing revealed the presence of the serological HLA-DR53 specificity on cells of a
N. M. Lardy et al.
FIGURE 1 Serological typing of HLA antigens segregating in family ELE. The individual (II-3), in which the “novel” haplotype was originally detected, is indicated by shading. HLA class I and class II antigens are given according to their official nomenclature. The “novel” haplotype is composed of the HLA-A2, -B41, -DR7, -DR53, -DQ9 phenotype.
patient suffering from leukemia. The individual was HLA phenotyped as HLA-A2, -A3, -B41, -B62, -Cw7, -DR7, -DR15, -DR51, -DR53, -DQ9, -DQ6 positive. Family investigation was performed to define the haplotype. Figure 1 demonstrates that the detected HLADR53 specificity is maternally derived and segregates with the HLA-A2, -B41, -DR7, -DQ9 haplotype. This “novel” haplotype is present in individuals I-1, II-2 and II-3 (patient). Furthermore, pedigree analysis revealed that individual II-2 is positive for two distinct HLADR7 haplotypes i.e. the “novel” HLA-DR7, -DR53, -DQ9 haplotype and the well documented HLA-B57, -DR7, -DQ9 haplotype which is associated with the non-expressed HLA-DRB4*0103102N allele. The detection of the HLA-DR53 specificity raised the question whether the serological reactivity was indeed the result of an expressed HLA-DRB4 gene product or due to a cross reactive epitope present on another HLA-DR molecule. The presence of the serological HLA-DR53 specificity was assessed by means of the well characterized lymphocytotoxic allo- anti HLA-DR53 serum MSD51. Table 1 demonstrates that the allo-antiserum MSD51 reacted with cells derived from individuals I-1, II-2, II-3 (patient) and also with the control cells 29-102 and 29-315. These control cells exhibit the HLA-DR7, -DQ2 phenotype which is associated with the HLA-DRB4*0101101 allele encoded HLA-DR53 specificity. However, no reactivity was found with cells derived from individual I-2, II-1 or the control cells 29-244 and 29-355. The control cells 29-244 and 29-355 are positive for the HLADR7, -DQ9 phenotype associated with the HLA-
Novel HLA-DR7, -DR53, -DQ9 association
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TABLE 1 Reactivity pattern of the allo-anti HLA-DR53 serum ‘‘MSD51’’
MSD51
I-1
I-2
II-1
II-2
II-3
29-102
29-315
29-244
29-355
1
2
2
1
1
1
1
2
2
I-1 mother (A1, A2, B8, B41, DR17, DR7, DR52, DR53, DQ2, DQ9), I-2 father (A2, A31, B57, B62, Cw7, Cw7, DR7, DR15, DQ3, DQ1), II-1 sibling 1 (A1, A31, B8, B62, Cw7, DR17, DR15, DR52, DR51, DQ2, DQ6), II-2 sibling 2 (A2, A2, B41, B57, Cw6, DR7, DR7, DR53, DQ9, DQ3), II-3 patient (A2, A31, B41, B62, Cw7, DR7, DR15, DR53, DR51, DQ9, DQ6). 29-102, 29-315, 29-244 and 29-355 are well characterized HLA-DR7 positive control cells with the following HLA class II phenotypes: 29-102 (DR7, DR10, DR53, DQ2, DQ5), 29-315 (DR7, DR13, DR52, DR53, DQ6, DQ2), 29-244 (DR7, DR13, DR52, DQ7, DQ9) and 29-355 (DR14, DR7, DR52, DQ5, DQ9)
DRB4*0103102N allele that does not encode the serological HLA-DR53 specificity. PCR-SSP Analysis Unfortunately it was not possible to obtain further material from family members I-2 and II-2. Thus, DNA from individuals I-1 and II-3 were subjected to PCR-SSP analysis to establish the HLA class II alleles comprising this unreported HLA-DR association. In addition, DNA of the genotypically well characterized homozygous cellines PITOUT (HLA-DRB*0701, -DRB4*0101101, -DQA1*0201, -DQB1*0202) and DBB (HLA-DRB1*0701, -DRB4*0103102N, -DQA1*0201, -DQB1*03032) expressing respectively the HLA-DR7, -DR53, -DQ2 and the HLA-DR7, -DQ9 phenotypes were included in the analysis to exclude genotyping errors or misinterpretations. Table 2 demonstrates that the “novel” detected HLA-DR haplotype segregating in individual I-1 and II-3 is composed of the HLA-DRB1*0701, -DQA1*0201, -DQB1*03032 alleles in association with the HLADRB4*0101101 allele. This finding is in contrast to known linkages in that thus far the HLA-DRB1*0701, -DQA1*0201, -DQB1*03032 haplotype has been found only in association with the non-expressed HLADRB4*0103102N allele. The complete HLA class II genotypes of individual I-1, II-1, II-3 and the reference cell lines PITOUT and DBB are given in Table 2.
DISCUSSION The serological HLA-DR53 specificity, encoded by the HLA-DRB4 gene, is normally present on the HLA-DR4, -DR7 and -DR9 positive haplotypes. However, it has previously been demonstrated that the HLA-DR7, -DQ9 haplotype is associated with an HLA-DRB4null allele that does not encode the serological HLA-DR53 specificity [2, 3]. The HLA-DR7, -DQ9 associated HLADRB4 gene has a single base substitution in the acceptor splice site at the 39 end of the first intron that abrogates functional expression of this gene [8]. This nonexpressed HLA-DRB4 gene is designated HLADRB4*0103102N [5]. In the present communication we describe the existence of an HLA-DR7, -DQ9 haplotype which possesses an expressed HLA-DRB4 gene product. Family investigation demonstrated that the HLA-DR53 specificity segregated unequivocally with the HLA-A2, -B41, -DR7, -DQ9 haplotype. Repeated HLA typing and extensive serological analysis using the well characterized anti HLA-DR53 serum MSD51 excluded the possibility that the detected HLA-DR53 reactivity was due to serological misinterpretation. High resolution PCR-SSP analysis of DNA obtained from individuals I-1 and II-3 revealed that the “novel” HLA-DR association consisted of the HLA-DRB1*0701, -DQA1*0201, -DQB1*03032 alleles in association with the expressed HLA-
TABLE 2 PCR-SSP genotyping of family ELE Id. I-1 II-1 II-3 PITOUT DBB
Phenotype DR17, DR7, DR52, DR53, DQ2, DQ9 DR17, DR15, DR52, DR53, DQ2, DQ6 DR15, DR7, DR51, DR53, DQ6, DQ9 DR7, DR53, DQ2 DR7, DQ3
DRB1*
DRB3*
0301/0701
0101
0301/15
0101
DRB4* 0101101 —
15/0701
—
0101101
0701 0701
— —
0101101 0103102N
DRB5*
DQA1*
DQB1*
—
0501/0201
0201/03032
0101
0501/0102
0201/06
0101
0102/0201
06/03032
0201 0201
0202 03032
— —
I-1: mother (HLA-DR17, 2DR7, 2DR52, 2DR53, 2DQ2, 2DQ9), II-1: sibling (HLA-DR17, 2DR15, 2DR52, 2DR51, 2DQ2, 2DQ6), II-3: patient (HLA-DR15, 2DR7, 2DR51, 2DR53, 2DQ6, 2DQ9). PITOUT and DBB are well characterized HLA-DR7 positive control cells. PITOUT is phenotypically typed as HLA-DR7, 2DR53, 2DQ2 and is associated with the expressed HLA-DRB4*0101101 allele, DBB has the HLA-DR7, 2DQ3 phenotype and is associated with the non-expressed HLA-DRB4*0103102N allele. The specificities given in bold denotes the ‘‘novel’’ HLA class II association.
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DRB4*0101101 allele. Thus, the detected serological HLA-DR53 specificity is indeed the product of an HLADRB4 gene, namely the HLA-DRB4*0101101 allele. Thus far the existence of two different HLA-DR7 haplotypes has been demonstrated, namely the HLA-DR7, -DQ2 haplotype which is associated with the serological HLA-DR53 specificity and the HLA-DR7, - DQ9 haplotype which does not express the HLA-DR53 specificity. The finding presented in this study is in contrast to known HLA-DR linkage disequilibrium and demonstrates the existence of a third HLA-DR7 haplotype. Surprisingly, during the preparation of this manuscript the “novel” described HLA-DRB1*0701, -DRB4*0101101 -DQA1*0201, -DQB1*03032 haplotype was again detected in an unrelated healthy blood donor with the following HLA phenotype: A2/A68, -B41/B57, -Cw7, -DR7/DR13, -DR53/DR52, -DQ9/ DQ6. The detection of the expressed HLA-DRB4*0101101 allele segregating with the HLA-DRB1*0701, -DQA1*0201, -DQB1*03032 haplotype in unrelated individuals raises the question whether it is a new haplotype and how it originated in the population. Although not a single case of crossing-over events in the HLA-DR region has been documented [12], it is tempting to speculate that the HLA-DRB1*0701, -DRB4*0101101 -DQA1*0201, -DQB1*03032 haplotype arose due to a homologous exchange of HLA-DRB4 genes between the HLA-DRB1*0701, -DRB4*0101101 -DQA1*0201, -DQB1*0201 and the HLA-DRB1*0701, -DRB4* 0103102N, -DQA1*0201, -DQB1*03032 haplotypes. Evidence for this supposition will be the existence of an HLA-DR7, -DQ2 haplotype that possesses an HLADRB4 null allele that does not encode the HLA-DR53 specificity. REFERENCES 1. Mukai R, Suzuki M, Yabe T, Hamaguchi H, Maeda H: Identification of the MT3 molecule from HLA-DR4, 7, w9 homozygous cell lines. J Immunol 33:321, 1984. 2. Knowles RW, Flomenberg N, Horibe K, Winchester R, Radka SF, Dupont B: Complexity of the supertypic HLADRw53 specificity: Two distinct epitopes differentially expressed on one or all of the DR b-chains depending on the HLA-DR allotype. J Immunol 137:2618, 1986.
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