Rowntree, Clare; (2000) Identification and characterisation of candidate tumour suppressor genes from chromosome 13q14.3, an area of frequent deletion in patients with B-cell chronic lymphocytic leukaemia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

B-cell chronic lymphocytic leukaemia (B-cell CLL) is a malignancy of circulating B lymphocytes characterised by a clonal expansion of CD5+ B cells. The aetiology of this disease is unknown. The commonest structural cytogenetic abnormality seen in B-cell CLL is deletion of chromosome 13q13.4 and it is likely that a tumour suppressor gene lies within this deleted region. The aim of the work described in this thesis was to define the region of minimal deletion at chromosome 13q14.3 in our patients with B-cell CLL and to then isolate and characterise candidate tumour suppressor gene cDNAs from this genomic region. Using known markers from the area, a physical map was constructed of the deleted region. 52% of our patients were shown to have deletion of 13q14.3 when tested by Southern blotting techniques for loss of markers from this region.. The region of minimal deletion in these patients was shown to be a maximum of 450kb. A putative exon, TA 6.35, was isolated by exon trapping techniques from this minimally deleted area. By screening a peripheral leucocyte cDNA library with this putative exon a candidate tumour suppressor gene cDNA was isolated. Using further cDNA library screening techniques and RACE PCR to characterise this cDNA, a second candidate tumour suppressor gene cDNA was also isolated from the region of deletion. The first cDNA, clone 1, consisted of 9 exons including the original TA 6.35 exon. The exons of clone 1 span a genomic distance of over 450kb with exons 2, 3 and 4 lying within our minimal region of deletion. The second cDNA, clone 2:2 consisted of 3 exons. The third exon of this clone also lies within our minimal region of deletion. The first two exons of clone 1 were shared by clone 2:2. Both clones were demonstrated to be expressed in normal and CLL lymphocytes by RT PCR analysis. cDNA clone 2:2 had a postulated open reading frame encoding for 78 amino acids. cDNA clone 1 was shown to exist in many alternatively spliced forms, non of which have a long open reading frame. It was postulated that clone 1 may not encode for a peptide but may act as an RNA regulating expression of cDNA clone 2:2. When patients with heterozygous deletion of 13q14.3 were analysed for mutation of either clone 1 or 2:2, the majority did not have mutations demonstrated within the retained allele. However, the promoter regions of these transcripts are as yet unidentified and we suggest that, therefore, they remain candidate tumour suppressor genes from 13q14.3.

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