RAPID DNA TYPING FOR CLASS-II HLA ANTIGENS - SUBTYPING OF DRW52-ASSOCIATED DRB1 ALLELES

Serologic typing for MHC class II antigens is incapable of identifying important subtypes for certain DRB1 alleles and occasionally leads to errors of assignment, particularly with the DR antigens associated with DRw52. To simplify DNA typing of DRw52-associated DRB1 alleles, we have developed a new rapid method using PCR-RFLP. The PCR-RFLP method is based on allele-specific amplification followed by digestion of PCR-amplified DNA with restriction enzymes. Group-specific amplification of the second exon of DR3, DR5, DR6 and DR8 was achieved using a 5' primer specific for the first hypervariable region sequence common to all alleles in this group and generic 3' primers. Human genomic DNA was amplified in a Perkin-Elmer Thermocycler. The presence of a 265 bp fragment was confirmed by agarose gel electrophoresis. Restriction enzyme digestion using Rsa I followed by polyacrylamide gel electrophoresis gave a pattern unique for some alleles and placed the remainder in subgroups. Digestion of the PCR product with one or two of the following enzymes (Asp 700, Hae II, Mnl I, Mbo II, Ksp I and Hph I) permitted the identification of 21 of the 22 alleles. DRB1*1103 and DRB1*1104 are not distinguished by this method and can be distinguished by SSOP or by using a specific 3' primer. For some heterozygous combinations, additional primers are used to provide full subtyping. This method provides a rapid and less costly alternative to PCR-SSOP for DRw52 subtyping in the smaller laboratory as only one amplification is required (two primers) for the majority of samples. The patterns produced by restriction digest are easy to interpret and complete subtyping using three additional primers can be accomplished in less than 2 days. This method should significantly improve the selection of unrelated donors for bone marrow or solid organ transplantation and facilitate immunogenetic studies in autoimmune diseases.