SCREENING OF THE TAP1 GENE BY DENATURING GRADIENT GEL-ELECTROPHORESISIN INSULIN-DEPENDENT DIABETES-MELLITUS - DETECTION AND COMPARISON OF NEW POLYMORPHISMS BETWEEN PATIENTS AND CONTROLS

New protective or disease-associated polymorphisms in the TAP1 gene we re sought in insulin-dependent diabetes mellitus (IDDM) patients with the use of denaturing gradient gel electrophoresis (DGGE) screening of genomic DNA. The TAP1 gene is located in the human leukocyte antigen (HLA) class II region of the genome and encodes components of a peptid e transporter essential for antigen presentation by HLA class I molecu les. Fragments of TAP1 corresponding to the 5' promoter, each of the 1 1 exons (with portions of adjacent intronic regions) and the 3' flanki ng region were amplified by the polymerase chain reaction and then sub jected to DGGE. DNA fragments of TAP1 yielded DGGE bands with patterns whose frequencies differed between IDDM patients and controls. Specif ic DGGE band patterns with fragments corresponding to the promoter, ex ons or introns 3, 6, 7, 8, 9 or 10 of TAP1 were detected exclusively i n either patients or controls. Sequencing of TAP1 fragments encompassi ng exon 7 gave rise to a DGGE band pattern exclusively observed in an IDDM patient and sequencing revealed a previously unidentified polymor phisms at codon 518 (GTC-->ATC, Val-->Ile). Another unique polymorphis m uncovered by DGGE revealed by sequencing a polymorphism in intron 2 in a diabetic patient. The genotypes of additional HLA class II matche d patients and controls were determined with regard to five exonic and one intronic TAP1 polymorphism. A 10 base pair intronic insertion in intron 9 was exclusively identified in controls and missing from patie nts (P = 0.017). Further large population-based studies may reveal whe ther these newly identified at risk or protective TAP1 variants confer markers of statistical risk in diverse population groups.