Development of HLA typing from forensic samples using molecular biological techniques.

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1. In this study, we have developed the polymerase chain-reaction fragment polymorphism(PCR-RFLP)method based on digestion of PCRamplified DNAs with allele specific enzymes as a reliable, convenient and practical HLA class II DNA typing technique in stead of hybridization with multiple sequence specific oligonucleotide probes(PCR-SSO). Especially, the modified PCR-RFLP method incorporating informative enzymes, which have a single recognition site in some alleles but none in other alleles in the amplified regions, is simpler and more sensitive because genotypes can be defined mainly just by checking whether the amplified DNAs are digested or not. This modified one makes reading of the generated RFLP band patterns much easier, and thus all of the class II alleles(DRB1, DRB3, DRB5, DQA1, DQB1, DPAL and DPBL)can be clearly defined both for homozygotes and heterozygotes except three pairs of the alleles(DRB1*1103 and DRB1*1104, DQB1*0602 and DQB1*0603, and DRB5*0201 and DRB5*0202)using 29 restriction enzymes.2. We have applied this PCR-RFLP method to investigation of forensic works, HLA-disease association and transplantation matching.we could define HLA-class II genotype of DNA samples extracted from hairs, a small volume of whole blood, fresh or old dental pulp tissues at autopsy.In HLA-disease association study, we could explain the susceptibility gene to autoimmune hepatitis in the Japanese patients. The basic amino acid at position 13, which is present only on the DR2 and DR4Bl molecules(Arg on DR2 and His on DR4), contributed to the susceptibility to this disease.In bone marrow transplantation, we could explain that the DP disparity played an important role developing severe acute graft-versus-host disease.