Chen M, Maerz W, Manfras B J, Kuehnl P, Usadel K H, Boehm B O
Center of Internal Medicine, University Hospital of Frankfurt Medical School, Germany.
Hum Immunol. 1993 Mar;36(3):199-203. doi: 10.1016/0198-8859(93)90126-l.
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-DRB30101, 0201, 0202, and 0301) were distinguished from one another by the migration of the corresponding homoduplex with the exception that DRB30201 was indistinguishable from DRB30202. The latter two alleles, however, were resolved by the artificial heteroduplexing approach. Arginine in position 74 of the DRB3 gene product (i.e., HLA-DRB30101) was significantly more frequent in Graves' patients than in controls. The relative risk conferred by the presence of the DRB30101 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.
开发了一种使用TGGE分析DRB3等位基因的HLA - DRB3分型系统。通过PCR从四个纯合分型细胞系、223名健康个体和102名格雷夫斯病患者中扩增HLA - DRB3基因的多态性第二外显子。PCR产物在35℃至70℃的温度梯度中进行电泳，所得片段通过银染可视化。通过相应同源双链体的迁移可区分出四个DRB3等位基因（HLA - DRB30101、0201、0202和0301），但DRB30201与DRB30202无法区分。然而，后两个等位基因可通过人工异源双链体方法进行分辨。DRB3基因产物（即HLA - DRB30101）第74位的精氨酸在格雷夫斯病患者中比在对照组中明显更常见。DRB30101的存在所赋予的相对风险为15.8（p≤0.001）。在我们的研究人群中，第74位精氨酸的存在导致病因分数为75%。PCR - TGGE技术是一种简单的非同位素方法，可能有助于对大量人群进行HLA疾病标志物的快速筛查。
