ANALYSIS OF HLA CLASS-II POLYMORPHISM USING POLYMERASE CHAIN-REACTION

A simple, rapid, and precise method of typing HLA class II polymorphis m would be valuable in the areas of disease susceptibility, tissue tra nsplantation, individual identification, and anthropological genetics. Herein, we describe a method of analyzing class II sequence polymorph ism based on polymerase chain reaction (PCR) amplification and hybridi zation with oligonucleotide probes. Many more DNA-defined alleles at t he class II loci have been identified than can be distinguished by con ventional serologic typing. Consequently, matching transplant donors a nd recipients by PCR/oligonucleotide typing should reduce graft reject ion and graft-vs-host disease. Also, the ability to identify alleles c onferring genetic predisposition to specific diseases (eg, insulin-dep endent diabetes mellitus) is significantly enhanced by distinguishing the many alleles or ''subtypes'' within a serologic type (eg, DR4). On e valuable property of sequence-based HLA typing strategies, like olig onucleotide probe hybridization, is that they reveal how and where two alleles differ, not simply that they can be operationally distinguish ed. The nature and location of HLA polymorphisms appears to be critica l in disease association studies and are important in tissue typing fo r transplantation. New alleles at the DRB1, DPB1, and DQB1 loci are li kely to be identified as this technology is applied to more and more s amples, particularly in nonwhite ethnic groups. A new allele is uncove red as an unusual pattern of probe binding and then confirmed by seque ncing. This pattern is observed because class II polymorphism is local ized to specific regions and virtually all ''new'' alleles represent ' 'shuffled'' combinations of polymorphic sequences found in previously known alleles. Since these polymorphisms are in the region of probe bi nding, these new alleles can be detected without increasing the probe panel. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available would not be revealed by oligonucleotide typing. With the PCR primers and probes described here, 7 DQalpha1 alleles, 15 DQbeta1 alleles, 18 DPB1 alleles, and 32 DRB1 alleles are distinguished. Additional primers and/or probes can, of course, increase the allelic discrimination of PCR/oligonucleotide probe typing. These horseradish peroxidase-labeled oligonucleotide pro bes are stable (>2 years when stored at 4-degrees-C) and the typing sy stem is simple and robust. Although this dot blot/oligonucleotide hybr idization procedure is a powerful and precise method of HLA class II t yping, the complexity of the procedure increases as the number of prob es required for analysis increases. The reverse dot blot method, based on an array of immobilized probes, allows the typing of individual sa mples in one single hybridization reaction. In this approach, a panel of unlabeled oligonucleotides are immobilized to a nylon membrane. The PCR product is labeled during the amplification reaction by using bio tinylated primers and hybridized to the membrane. The presence of boun d PCR product specifically hybridized to a given probe is detected usi ng a streptavidin-horseradish-peroxidase conjugate and either chromoge nic or chemoluminescent substrates. This method, which has also been a pplied to the detection of beta-thalassemia and cystic fibrosis mutati ons, offers the simplest and most rapid approach to the HLA typing of clinical samples.