Mutation and Methylation Analysis of WWOX and CYLD on 16q; Potential Tumor Suppressor Genes in Myeloma.

We have shown that loss of heterozygosity (LOH) at 16q is an adverse prognostic marker when it occurs in combination with either t(4;14) translocation or del(17p). Using 500K Affymetrix mapping arrays we found that 16q was involved in translocations with the IgH locus, deleted, or had uniparental disomy (UPD). The deletion patterns led us to 2 regions at 16q12.1 and 16q21-q24.1, which contains 5 genes and is the location of the t(14;16) translocation. By integrating mapping and expression profiling data from presenting myeloma cases we were able to identify WWOX and CYLD as key genes deregulated in these regions. Both WWOX and CYLD are known tumor suppressor genes. WWOX is known to have a pro-apoptotic effect by participating in the TNF apoptotic pathway and via the direct physical interaction with p53 and p73. CYLD functions as a negative regulator of the NF-κB pathway as well as blocking the activation of cyclin D. WWOX is found to be methylated in other cancer tissues, whereas CYLD is frequently mutated. To determine mode of action of these genes in samples with LOH we performed mutation and methylation analysis on myeloma cell lines and pre-treatment patient samples. Variant transcripts of WWOX, which may act as dominant negatives to block the function of WWOX have been reported so we also determined the nature of these in the samples. Exons from WWOX and CYLD were PCR amplified from samples with LOH, as well as from samples with retention of heterozygosity, and sequenced directly. All differences to the consensus were checked against databases for known SNPs and mutations. Mutations in the tumor sample were confirmed by a repeat PCR, and mutations were also checked against peripheral blood DNA from the same patient to determine if these were acquired in the tumor. LOH of 16q was found in 2 out of 9 myeloma cell lines and additionally homozygous deletion of WWOX exons 5-8 in KMS11 cells was observed. cDNA analysis from these cell lines showed that KMS11 cells expressed variant 3, as could be expected, whereas the dominant transcript in other cell lines is variant 1. However, 2 cell lines also expressed an additional transcript (variant 4), which has previously been shown to act as a dominant-negative transcript. All pretreatment samples expressed the variant 1 transcript. 16 pretreatment samples with LOH were screened for mutations revealing 2 mutations in exon 9, one of which was non-synonymous. Methylation analysis of the WWOX locus is underway. Analysis of CYLD showed frequent mutation in cell lines with many synonymous and non-synonymous mutations, including several frame-shifts resulting in truncated products. Screening of 14 pretreatment samples with LOH at the CYLD locus revealed 2 mutations including a C>TT mutation in exon 11, which results in a frameshift and premature termination of translation. Alternative transcripts of CYLD are formed through splicing of exons 3 and 7. Both exon 3 variants were present but only the variant lacking exon 7 was present in myeloma cell lines. These data suggest that dysregulation of both WWOX and CYLD may be important in the pathogenesis of myeloma and contribute to the effect of del(16q) on patient survival.