RAPID AND SIMPLE SUBTYPING OF THE HLA-DRB3 GENE IN GRAVES-DISEASE BY USING TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS

A HLA-DPB3-subtyping system that uses TGGE for analyzing DRB3 alleles was developed. The polymorphic second exon of the HLA-DRB3 gene was am plified from four homozygous typing cell lines, 223 healthy individual s, and 102 patients with Graves' disease by using the PCR. The PCR pro ducts were electrophoresed in a temperature gradient from 35-degrees-C to 70-degrees-C, and the resulting fragments were visualized by silve r staining. Four DRB3 alleles (HLA-DRB30101, *0201, *0202, and *0301) were distinguished from one another by the migration of the correspon ding homoduplex with the exception that DRB30201 was indistinguishabl e from DRB30202. The latter two alleles, however, were resolved by th e artificial heteroduplexing approach. Arginine in position 74 of the DRB3 gene product (i.e., HLA-DRB3 0101) was significantly more freque nt in Graves' patients than in controls. The relative risk conferred b y the presence of the DPB30101 was 15.8 (p less-than-or-equal-to 0.00 1). The presence of arginine in position 74 contributed to an etiologi c fraction of 75% in our study population. The PCR-TGGE technique is a simple, nonisotopic method, which may be useful in rapid screening of large populations for HLA disease markers.