An integrated multiplex-PCR and PCR-RFLP typing system for markers associated with seronegative arthritides

The system was designed with emphasis on the identification HLA-B alleles and genotypes associated or potentially associated with seronegative arthritides. By using a combination of multiplex SSP and PCR-RFLPs, the assays can be economically performed on a large range of sample sizes in diagnosis and epidemiology. 24 HLA-B alleles and subtypes can be discriminated, including options for PCR-RFLP or sequence specific amplification of the allele groups B27 and B60 (B*4001 and B*4007). In addition, the internal control carries central MHC polymorphisms, which can help to identify HLA extended halplotypes. False negatives, caused by preferential amplification of the internal control under suboptimal PCR conditions, were prevented by employing new, optimized PCR buffer. Four of the HLA-B primers were pooled into a multiplex reaction whose products were subtyped by digestion with seven restriction endonucleases. Specificity and sensitivity were verified in a panel of 68 homozygous cell lines and 200 heterozygous samples. An HLA-B*27-B*40 hybrid allele was observed in 3 out of 95 B*27-positive individuals from Berlin, Germany. Such an allele could be mistyped by some published assays as a B*27/B*40 heterozygote, a genotype reported to confer an increased risk for ankylosing spondylitis.