ABERRANT SPLICING OF INTRON 1 CREATES A NOVEL NULL HLA-AB*1501 ALLELE.
D Middleton, MD Curran, F Williams and BK Rima, Histocompatibilty and Immunogenetics Laboratory, City Hospital, Belfast, Northern Ireland.


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A comparative analysis of our HLA serological and DNA typing data for our bone marrow panel (5000 members) revealed an inconsistent typing result at the B locus in one panel member. While the serological data indicated a homozygous B8 allele, DNA typing using our SSOP HLA-B typing scheme revealed the presence of a B15 allele along with B8. Genomic clones for the entire HLA-B locus were first prepared using long range PCR and the B15 specific plasmid clones sequenced. An examination of the sequences of exon 2, 3 and 4 revealed complete identity with B*1501. In view of this result, cDNA clones specific for the entire coding region of the HLA-B gene were prepared from the RNA extracted from the PBMLs of this patient using RT-PCR. cDNA plasmid clones specific for the B15 allele were also isolated, indicating normal transcription for this gene, and subjected to nucleotide sequence analysis. While the coding region of the clones again displayed complete identity with B*1501, all 7 clones examined retained the intron 1 sequence. Examination of the intron 1 sequence revealed a 10 bp deletion just 17bp upstream of the 3' end of intron 1. This deletion was also confirmed to be present in the genomic clones. RT-PCR specific for the HLA-B region spanning exon 1 to exon 3 in conjunction with Southern blot analysis with probes specific for B15 and B8 clearly demonstrated that the larger PCR product containing intron 1 was exclusively the B15 allele. The location of this deletion in intron 1 will interfere with lariat formation and lead to inefficient splicing of intron 1 fron the RNA. The null phenotype is therefore more than likely due to the retention of the deleted intron 1 in the B15 mRNA which creates a frameshift resulting in an in frame stop codon at nucleotide position 13-15 of exon 2.