A DNA-based detection and screening system for identifying HLA class I expression variants by sequence-specific primers

Molecular methods are now commonplace for HLA typing and they have replaced traditional serological methods in many histocompatibility laboratories. A consequence of reliance on molecular methods using primers or probes based on existing sequence information is that unsequenced or partially-sequence d null, or low expressed variants are not discriminated from expressed alle les. Failure to identify null alleles might have deleterious implications f or allogeneic transplants. Expression variants may be classified into two c ategories: unique mutations and repeat mutations. For example, the alleles A*0303N, A*2409N, and B*1526N have apparently unique mutations In contrast, repeat mutations may occur frequently at points where unusual nucleotide s equences make accurate DNA replication by DNA polymerases difficult. One ex ample is between nucleotide positions 621-627, where HLA class I alleles ma y exhibit between three and seven consecutive cytosine residues. Incorrect insertion of an extra cytosine in this region is the cause of expression fa ilure in A*2411N and A*0104N alleles. We hypothesise that insertion of an e xtra cytosine into cytosine island between nucleotide positions 621-627 is likely to recur not only in other HLA-B alleles but also in HLA-B and even HLA-C alleles. We describe here a polymerase chain reaction using sequence- specific primers (PCR-SSP) system that can not only detect all previously-s equenced HLA class I expression variants but ran also screen for mutations between positions 621-627 in HLA-A, B or C alleles which may give rise to p otentially null alleles. Overall, in this study HLA class I expression vari ants were identified in 5 of 931 tested samples (0.53%).