The primary goal of this project is to resolve, at the DNA level, HLA-DR and -DQ haplotypes associated with susceptibility to vitiligo in African Americans. Previous studies from our laboratory have shown: 1) an association of HLA-HLA-DR4 with susceptibility to vitiligo; 2) a corresponding increase in HLA-HLA-DQw3 that may be due to linkage disequilibrium of HLA-DR4 and HLA-DQw3, 3) a HLA-DR4 association with positive family history of autoimmune diseases and early age of disease onset, and 4) an increase in HLA-DRw6 among patients with no family history of autoimmune disease and late age of disease onset. In this project, molecular typing methods will be used to refine serologically defined HLA phenotypes associated with disease susceptibility in the target population. Lymphocytes from peripheral blood samples of the patients' family members will be HLA-phenotyped in standard microcytotoxicity assays to determine haplotypes. Genomic DNA from HLA- HLA-DR4 and HLA-DRw6 patients and ethnically matched controls will be amplified using the polymerase chain reaction technique and probed with sequence specific oligonucleotide probes (SSOP) in dot-blot hybridization assays to determine DRB1 and B3 or B4, and DQA1 and DQB1 alleles. Reference protocols employing HLA-DR and DQ alleles of HLA-DR5 and HLA- DRw6 (i.e., w13 and w14) haplotypes. The results will yield important frequency data on HLA and disease-association at the DNA level, the definitive measure of polymorphism. Moreover, extrapolation from the three-dimensional structure of the HLA-Class I molecule to Class II molecules suggest that allelic hyper-variable regions responsible for HLA-DR and DQ antigen polymorphisms also encode functional epitopes involved in peptide binding and T4-cell recognition, the initial step in the induction of an immune response. Thus, the molecular characterization of HLA-DR and -DQ haplotypes at the DNA level will not only refine the definition of disease-associated alleles and/or sequences, but also be of great importance in better understanding the significance of immunogenetic factors in the etiology and pathogenesis of vitiligo in patients with an autoimmune-type disorder.
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