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

1. 在本研究中，我们开发了基于等位基因特异性酶切pcr扩增DNA的聚合酶链反应片段多态性（PCR-RFLP）方法，作为一种可靠、方便和实用的HLA II类DNA分型技术，取代了多序列特异性寡核苷酸探针（PCR-SSO）杂交技术。特别是含有信息酶的改良PCR-RFLP方法，该方法在扩增区域的某些等位基因上有单一的识别位点，而在其他等位基因上没有识别位点，主要通过检查扩增的dna是否被消化来确定基因型，因此更简单，更敏感。这使得生成的RFLP频带模式的读取更加容易，因此除了三对等位基因（DRB1*1103和DRB1*1104， DQB1*0602和DQB1*0603， DRB5*0201和DRB5*0202）使用29种限制性内切酶外，所有II类等位基因（DRB1、DRB3、DRB5、DQA1、DQB1、DPAL和DPBL）都可以被清晰地定义为纯合子和杂合子。我们将这种PCR-RFLP方法应用于法医鉴定、hla疾病关联和移植配型的调查。我们可以从毛发、少量全血、新鲜或旧牙髓组织中提取DNA样本来确定hla II类基因型。在hla -疾病相关性研究中，我们可以解释日本患者自身免疫性肝炎的易感基因。13号位的碱性氨基酸只存在于DR2和DR4Bl分子上（DR2上的Arg和DR4上的His），导致了对这种疾病的易感性。在骨髓移植中，我们可以解释DP差异在发生严重的急性移植物抗宿主病中发挥了重要作用。

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.