- Email: [email protected]
Southern blotting analysis of the IDD-associated DR2 and related haplotypes
Abstracts
193
HLA-DQ SUBREGION POLYMORPHISMS DETECTED BY SEROLOGIC, CELLULAR,AND MOLECULAR GENETIC TECHNIQUES. Edward J. Ball, Lori J. Dombrausky, Gabriel Nufiez, Linda K. Myers, and Peter Stasmy; Department of Internal Medicine, The University of Texas Health Science Center at Dallas, Southwestern Medical School. Dallas, T X The complexity of antigenic determinants of the HLA-DQ subregion-encoded molecules has only recently been appreciated. In addition to the broad specificities D Q w l , DQw2, and DQw3, more restricted specificities have been identified. In an attempt to begin to define this polymorphic system, we have used selected alloantisera, cloned T cell lines, and restriction fragment length polymorphisms (RFLP), using a D Q alpha cDNA clone in panel studies. Each approach identified previously unrecognized polymorphisms of the DQ molecule(s) or the D Q genetic subregion. DQ-reactive alloantisera and T cell lines were identified by blocking with monoclonal antibodies. DQ-reactive antisera were found which recognized determinants in linkage disequilibrium with DR2 (r = 0.44), DRwl 3 (r = 0.63), and D R w l l (r = 0.61-0.82). DQ-reactive T cell lines recognizing determinants associated with DRw11 (r = 0.65) and DRw13 (r = 0.43) were also identified. Using a D Q alpha cDNA probe and the enzyme Pst I, RFLP bands were found that correlated with D Qw l (12 kb, 15 of 15 DQwl-positive cells), DRw52 (4.5 kb, 21 of 29 DRw52 cells), and DRw53 (2.3 kb, 26 of 26 DRw53 cells). An additional 8.8 kb band was found in 14 of 46 individuals tested that was not correlated with any D-region antigens. Thus far, most of the new antigenic specificities of the DQ molecules and the RFLP patterns detected with the DQ alpha probe by ourselves and others have shown striking associations with antigens of the DR-subregion. These results fit the notion of strong linkage disequilibrium between the DR and DQ subregions. The observation that D Q molecules carry antigenic epitopes that are largely indistinguishable by panel studies from DR-subregion-encoded alloantigens, points out the need to reexamine and possibly reinterpret the associations of DR antigens with functional attributes and with disease predisposition.
SOUTHERN BLOTTING ANALYSIS OF THE IDD-ASSOCIATED DR2 AND RELATED HAPLOTYPES. M. Segall, H. Noreen, L. Schluender, and F.H. Bach; University of Minnesota, Minneapolis, MN Substantial levels of D N A restriction fragment length polymorphism (RFLP) have been demonstrated between cells expressing different serologically defined DR specificities. The specificity DR2 is associated with several different Dw subtypes defined with homozygous typing cells (HTCs). Using both DR 3 and D Q /3 probes, we have demonstrated RFLPs characteristic for the Dw2, Dw12, and LD5a subtypes of DR2. The frequency of DR2 is significantly decreased in patients with insulin-dependent diabetes (IDD), and DR2 + IDD patients have a decreased frequency of Dw2 and an increased frequency of the primed lymphocyte typing (PLT) specificity LD-MN2. LD-MN2 appears by PLT testing to be related to the DB9 group of HTCs of the Eighth and Ninth Histocompatibility Workshops (LD-5a, FJO, WJR, AZH). We have studied the RFLP of LD-MN2 and the DB9-related HTCs. The LD-MN2 RFLP pattern is similar but not identical to that of LD-5a. It is identical to RFLP patterns of the HTCs FJO, AZH, and WJR, although these three HTCs do not have identical Dw specificities and are not identical to LD-MN2. This situation is similar to that which we have seen with DR4 where clearly different Dw subtypes did not have characteristic differences in RFLP patterns. The similarity of the DQ/3-probe RFLP pattern of DR2-LD-MN2 to that of DR1-Dwl, as well as the sharing of some fragments between DR2-LD-MN2 and DR4, is being further explored and will be discussed.
193
HLA-DQ SUBREGION POLYMORPHISMS DETECTED BY SEROLOGIC, CELLULAR,AND MOLECULAR GENETIC TECHNIQUES. Edward J. Ball, Lori J. Dombrausky, Gabriel Nufiez, Linda K. Myers, and Peter Stasmy; Department of Internal Medicine, The University of Texas Health Science Center at Dallas, Southwestern Medical School. Dallas, T X The complexity of antigenic determinants of the HLA-DQ subregion-encoded molecules has only recently been appreciated. In addition to the broad specificities D Q w l , DQw2, and DQw3, more restricted specificities have been identified. In an attempt to begin to define this polymorphic system, we have used selected alloantisera, cloned T cell lines, and restriction fragment length polymorphisms (RFLP), using a D Q alpha cDNA clone in panel studies. Each approach identified previously unrecognized polymorphisms of the DQ molecule(s) or the D Q genetic subregion. DQ-reactive alloantisera and T cell lines were identified by blocking with monoclonal antibodies. DQ-reactive antisera were found which recognized determinants in linkage disequilibrium with DR2 (r = 0.44), DRwl 3 (r = 0.63), and D R w l l (r = 0.61-0.82). DQ-reactive T cell lines recognizing determinants associated with DRw11 (r = 0.65) and DRw13 (r = 0.43) were also identified. Using a D Q alpha cDNA probe and the enzyme Pst I, RFLP bands were found that correlated with D Qw l (12 kb, 15 of 15 DQwl-positive cells), DRw52 (4.5 kb, 21 of 29 DRw52 cells), and DRw53 (2.3 kb, 26 of 26 DRw53 cells). An additional 8.8 kb band was found in 14 of 46 individuals tested that was not correlated with any D-region antigens. Thus far, most of the new antigenic specificities of the DQ molecules and the RFLP patterns detected with the DQ alpha probe by ourselves and others have shown striking associations with antigens of the DR-subregion. These results fit the notion of strong linkage disequilibrium between the DR and DQ subregions. The observation that D Q molecules carry antigenic epitopes that are largely indistinguishable by panel studies from DR-subregion-encoded alloantigens, points out the need to reexamine and possibly reinterpret the associations of DR antigens with functional attributes and with disease predisposition.
SOUTHERN BLOTTING ANALYSIS OF THE IDD-ASSOCIATED DR2 AND RELATED HAPLOTYPES. M. Segall, H. Noreen, L. Schluender, and F.H. Bach; University of Minnesota, Minneapolis, MN Substantial levels of D N A restriction fragment length polymorphism (RFLP) have been demonstrated between cells expressing different serologically defined DR specificities. The specificity DR2 is associated with several different Dw subtypes defined with homozygous typing cells (HTCs). Using both DR 3 and D Q /3 probes, we have demonstrated RFLPs characteristic for the Dw2, Dw12, and LD5a subtypes of DR2. The frequency of DR2 is significantly decreased in patients with insulin-dependent diabetes (IDD), and DR2 + IDD patients have a decreased frequency of Dw2 and an increased frequency of the primed lymphocyte typing (PLT) specificity LD-MN2. LD-MN2 appears by PLT testing to be related to the DB9 group of HTCs of the Eighth and Ninth Histocompatibility Workshops (LD-5a, FJO, WJR, AZH). We have studied the RFLP of LD-MN2 and the DB9-related HTCs. The LD-MN2 RFLP pattern is similar but not identical to that of LD-5a. It is identical to RFLP patterns of the HTCs FJO, AZH, and WJR, although these three HTCs do not have identical Dw specificities and are not identical to LD-MN2. This situation is similar to that which we have seen with DR4 where clearly different Dw subtypes did not have characteristic differences in RFLP patterns. The similarity of the DQ/3-probe RFLP pattern of DR2-LD-MN2 to that of DR1-Dwl, as well as the sharing of some fragments between DR2-LD-MN2 and DR4, is being further explored and will be discussed.