Rapid and simple subtyping of the HLA-DRB3 gene in Graves' disease by using temperature-gradient gel electrophoresis

A HLA-DRB3-subtyping system that uses TGGE for analyzing DRB3 alleles was developed. The polymorphic second exon of the HLA-DRB3 gene was amplified from four homozygous typing cell lines, 223 healthy individuals, and 102 patients with Graves' disease by using the PCR. The PCR products were electrophoresed in a temperature gradient from 35 degrees C to 70 degrees C, and the resulting fragments were visualized by silver staining. Four DRB3 alleles (HLA-DRB3*0101, *0201, *0202, and *0301) were distinguished from one another by the migration of the corresponding homoduplex with the exception that DRB3*0201 was indistinguishable from DRB3*0202. The latter two alleles, however, were resolved by the artificial heteroduplexing approach. Arginine in position 74 of the DRB3 gene product (i.e., HLA-DRB3*0101) was significantly more frequent in Graves' patients than in controls. The relative risk conferred by the presence of the DRB3*0101 was 15.8 (p < or = 0.001). The presence of arginine in position 74 contributed to an etiologic 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.