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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.

Original publication

DOI

10.1111/j.1399-0039.2005.00456.x

Type

Journal article

Journal

Tissue Antigens

Publication Date

09/2005

Volume

66

Pages

231 - 237

Keywords

3' Untranslated Regions, Alleles, Base Pair Mismatch, Cell Line, Tumor, Cloning, Molecular, Colorectal Neoplasms, Cytosine, DNA, DNA Primers, DNA Repair, Exons, Gene Frequency, HLA-A Antigens, HLA-A1 Antigen, HLA-A2 Antigen, Humans, Isoelectric Focusing, Mutation, Polymerase Chain Reaction, Promoter Regions, Genetic, RNA, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA