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Cancer cells with the Replication Repair defect (RER) are subject to frequent errors, during DNA replication, particularly in DNA sequences with either single or double base pair repeats. Such a single base pair repeat occurs in the HLA*0101 gene, at the beginning of Exon 4 (bp 621-627), where a run of 7 cytosine residues are located. The colorectal cancer cell line HCA-7 has been shown to express surface HLA*0201, but to lack expression of HLA*0101 whilst its corresponding normal B cell line (EVA-1224), derived from the same individual, expresses both surface A1 and A2. HCA-7 has also been reported to have methylation of the promoter region of hMLH1, causing the loss of expression of hMLH1, and to exhibit the RER positive status as demonstrated by microsatillite instability. When analysed for HLA with sequence specific primers for HLA*A0101 in an ARMS PCR, the tumour cell line, HCA-7, appeared to have a mutation in the 7bp repeat C sequence at the beginning of Exon 4. Following cloning and sequencing HCA-7 was shown to have an inserted cytosine the run of 7 C's increasing this series of C's to 8. In contrast, the normal B cell, EVA-1224, contained the 7 bp repeat sequence expected for HLA*A0101. Analysis of the mRNA of HLA*0101 was performed using RT PCR with an ailele specific 5' primer in Exon 2 (bp 253-271) and a series of 3' primers in Exon 3 (bp 559-574), Exon 4 (bp 866-882), Exon 7 (bp 1082-1097) and in the 3' untranslated region. This revealed the presence of a shorter message in the HCA-7 cell line terminating in the region of the Exon 3/4 boundary. The insertion of an extra C in the C7 repeat region, which is only 2 bases from the Exon 3/4 splice site, may lead to a splicing defect between these two Exons resulting in a shortened message and the lack of expression of a functional A*0101 product. © 2001 Blackwell Science Ltd,.

Type

Journal article

Journal

European journal of immunogenetics

Publication Date

01/12/2001

Volume

28