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#89
DIFFERENTIATION OF HLA-B*44 ALLELES BY PCR-SSP.
Emyr L. Harries, BSc, Jonathan Downing, BSc DipRCPath and Christopher Darke, PhD. Pontyclun Wales, United Kingdom, Welsh Blood Service, CF72 9WB, Welsh Transplantation and Immunogenetics Laboratory.

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HLA-B*44 is the most frequent HLA-B allele family in Caucasoids and disparity for its alleles in haematopoietic stem cell (HSC) transplantation can induce allogeneic responses leading to acute graft-versus-host disease and graft failure.
We designed a PCR-SSP system for B*44 allele typing using 51 primers in 48 mixtures to differentiate 36 B*44 alleles. Successful validation for specificity used 31 ‘reference’ DNA samples possessing: B*440201, *440301, *4404, *4405, *4408, *4409, *4411, *4412, *4413, *4414, *4417, *4419N, *4423N, *4427, *4431 and 166 random ‘reference’ subjects typed for 11 HLA loci.
B*44 allele typing and analysis of 503 HLA typed random ‘Welsh’ blood donors showed that all HLA-A, -B, -C, DRB1, -DQB1 genotypes gave a good fit to Hardy-Weinberg expectations (all p>0.8) and that B*44 consisted of B*440201 and B*440301 with carriage and gene frequencies of 22.3%, 0.11928 and 10.3%, 0.05268, respectively. The highest frequency 3-locus B*4402-bearing haplotypes were: A*02, Cw*05; Cw*05, DRB1*04; DRB1*04, DQB1*07 and B*4403-bearing were: A*29, Cw*16; Cw*16, DRB1*07; DRB1*07, DQB1*02.
Tests on 89 subjects with rare B*44/C haplotypes (B*44 with Cw*01, *02, *03, *06, *08, *12, *14, *15) identified the haplotype B*4405, Cw*02 and showed that A*24, B*4405, Cw*02, DRB1*01 was a common B*4405-bearing haplotype. A significant association was seen between B*440201 and Cw*0303 and examples of B*4404, B*440302 and B*4422 were identified.
This B*44 allele typing system will be updated as new B*44 alleles emerge and requires continuing review as further examples of rare B*44 alleles become available. However, it currently provides us with a well-validated PCR-SSP system to differentiate B*44 alleles during HSC transplant patient/donor selection.