A mutated HLA-A*0101 allele in the colorectal cell line HCA-7.
The colorectal cell line HCA-7 expresses surface human leucocyte antigen-A*0201 (HLA-A*0201), but lacks expression of HLA-A*0101 whilst the normal B-cell line (EVA-1224), derived from the same individual, expresses both surface HLA-A1 and HLA-A2. Amplification refractory mutation system-polymerase chain reaction analysis, using sequence-specific primers, suggested that HCA-7 has a mutation in a 7 base pair (bp) cytosine repeat sequence located at the beginning of Exon 4 (bp 621-627). Cloning and sequencing revealed HCA-7 to have eight cytosine residues in this repeat sequence. In contrast, EVA-1224 contained only 7 cytosines. Analysis of the mRNA for HLA-A*010 using reverse trancriptase-polymerase chain reaction (RT-PCR), with an allele-specific 5' primer in exon 2 (bp 253-271) and a series of 3' primers in exons 3, 4 and 7 and in the 3'untranslated region, revealed that HCA-7 contained a shortened message terminating in the region of the exon 3/4 boundary. The insertion of an extra cytosine in this region, which is only two bases from the exon 3/4 splice site, is presumed to lead to a splicing defect between exons 3 and 4 resulting in the lack of expression of a functional HLA-A*0101 product. HCA-7 is mismatch repair (MMR) defective due to lack of expression of hMLH1 resulting from hypermethylation of the promoter region. The consequential increase in errors in single-nucleotide repeat stretches of DNA can account for the HLA-A*0101 mutation. This has probably then been selected for in the tumour to enable escape from immune attack against an HLA-A*0101-restricted tumour-specific determinant that has also arisen as a result of the tumour being MMR defective.
Bicknell
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DC
Cancer and Immunogenetics Laboratory, Cancer Research UK, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.
Bodmer
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WF
D020413:3' Untranslated Regions; D000483:Alleles; D020137:Base Pair Mismatch; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000235:genetics*; D003596:Cytosine; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D005091:Exons; D005787:Gene Frequency; D015234:HLA-A Antigens / Q000235:genetics*; D015788:HLA-A1 Antigen; D015789:HLA-A2 Antigen; D006801:Humans; D007525:Isoelectric Focusing; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D020413:3' Untranslated Regions; D017931:DNA Primers; D015234:HLA-A Antigens; C506470:HLA-A*01:01 antigen; C435939:HLA-A*02:01 antigen; D015788:HLA-A1 Antigen; D015789:HLA-A2 Antigen; D012313:RNA; D003596:Cytosine; D004247:DNA