Abstract
Using FISH and LOH in ALL cases with microscopically visible deletions of the long arm of chromosome 6q, we previously defined a region of minimal deletion (RMD) in a spectrum of lymphoid malignancies and with other investigators have suggested that the region may harbour several tumour suppressor genes. (Sinclair et al 2004; Cancer Research, 64: 4089). 15 recognised genes were positioned in the RMD and three were found expressed in bone marrow: GRIK2, C6orf111 and Cyclin-C, the investigation of which is reported in this study.
GRIK2 analysis. Combining published RMD in ALL with our own data, we extensively analysed GRIK2 in ALL patients carrying heterozygous 6q deletion. Although a paternally inherited non-silent mutation of GRIK2 was identified in one of 14 patients, we concluded that loss of one or more other genes in the 6q15-q21 region was likely to contribute to ALL pathogenesis.
Cyclin-C (CCNC) analysis. Lymphoid cell lines without and with del (6q)(Peer, Molt-4 and NKL) were assessed for expression levels of C6orf111 and Cyclin-C and other candidate tumour suppressor genes within the region (FLASH, Q9NR58, Cx43, BACH2, BLIMP1, APG5L, TAK1 and AIM1) using real-time quantitative RT-PCR (RQ-PCR). While easily detectable in all other cell lines, no expression of CCNC was demonstrated in NKL (by RT-PCR and Western blotting), this in spite of the presence of at least one copy of the gene (as demonstrated by FISH using PAC P13743 which contains CCNC) suggesting that expression of the second allele was ablated through either micro-deletion, point mutation or epigenetic silencing. Expression levels of other candidate genes were similar, albeit with minor variations, in all cell lines. We further analysed expression of CCNC in whole PB and BM, a variety of haemopoietic cells isolated from PB and in 66 de novo ALL patients (9 T ALL; 48 B cell ALL (predominantly common ALL) and 9 other ALL). Expression was relatively high in all cell lines, intermediate in normal T-lymphocytes and low in other normal cells. Leukemic cells collected from diagnostic material from adult acute lymphoid malignancies showed a wide variation in levels of CCNC expression. To exclude the possibility that low expression in some cases may be due to the presence of a high proportion of normal cells CCNC levels were normalised against Ki67 expression (as an indicator of mitotic index) by RQ-PCR. At least 27 patients showed a CCNC level below the median value. Twenty-five cases showed value above the median value. Levels of expression of CCNC and Ki67 were correlated with survival, immunophenotype or other clinical parameters, but no statistically significant correlation was demonstrated.
Genome-wide methylation analysis identifies epigenetically inactivated candidate tumour suppressor genes in renal cell carcinoma