Methylation found in normal but not in the paired tumor..WHY - (Jun/06/2005 )

Hi,

Can anybody explain why, is some cases, methylation was detected in normal but not in the paired tumour tissue? Anybody experience this before? Thank you.

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Congratulations! You may have found a gene that is hypomethylated in tumor (relative to normal tissue). Does the gene express higher in tumor than in normal cells? If you search pubmed with hypomethylation AND cancer you will find quite a few papers on this.

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HI pcrman,

Thanks for your replied. The genes that I studied are tumour suppressor gene which involve in tumour invasion. I expect methylation in tumour but not in normal tissue. However the reverse case was happened.

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That's interesting. What method do you use for methylation analysis and what region have you analzed for your gene?

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mrpcr,

you could well have uncovered a new or already described oncogene or proto-oncogene.

sounds very exciting indeed. hypomethylation is linked to the CIN phenotype, or chromosome instability which is a known phenotype in tumors.

Nick

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pcrman, methylnick,

I study DAPK and E-cadherin using MSP. 4 out of 12 CRC patients shown methylation in normal but not in tumour tissues, and 2 out of 12 shown methylation in tumour but not in normal tissues, in DAPK. For E-cad, only one patient shown methylation in normal but not in tumour tissue and this patient also shown DAP methylation positve for normal but not tumour tissue.

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Hi mrpcr,

I think the number of samples (12) is too samll to get a conclusion. On the other hand, if you want to make a surprising discovery, the MSP results have to be confirmed by bisulfite genomic sequencing. If you do go ahead with sequencing, don't use MSP product for sequencing.

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Hi pcrman,

Thank you very much. I will tried on more samples. Why do you said that MSP product is not appropriate for sequencing? What other things that you can tell from bisulfite genomic sequecing, apart from the methylation status? If I would want to detect novel methylated genes, what methods are available and which one would you recommend? Thank you.

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MSP product cannot be used for sequencing because the primers are biased to either methylated or unmethylated DNA. What else you can get from bisulfite sequencing? First it is a high resolution methylation mapping mthod; second, if you do cloning before sequencing, the result may tell you how methylation is spread along the DNA. You have to use BSP primers or universial primers which contain no CpG 'C's to amplified the modified DNA.

Regarding finding novel methylated genes, there are several methods available such as RLGS, CpG island microarray or oligo microarray. RLGS is too hard to carry out and I have not seen any meaningful things coming out of it. The same is ture of CpG island microarray. I favor an expresion-to-methylation strategy in which you can treat cells with 5aza followed by a cDNA microarray and then screen those upregulated gene for methylation. For tissue samples, do a microarray comparing normal and tumor to see what genes are silenced in tumor and then map those genes for methylation.

Good luck.

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Hi pcrman,

My fren just thrown me a question that I can't really answer. Maybe you can help.
We know that clinical tissue (said normal colon tissue) contain mixture of cells like mucosa, muscle cells, stroma cells, T-cell, B-cell and so on, and all these cells contain nucleus. Let say I want to check the methylation status of E-cadherin in normal tissue (using MSP), I would expect no methylation being detected because E-cad is a anti-metastasis gene. However, when the normal tissue was stained with E-cad antibody, only mucosa showed positive signal. This is to say cells other than mucosa are not expressing E-cad. So, cells that are not expressing E-cad could be dued to methylation or others mechanism. If it is because of methylation, could we detect methylation in normal tissue?

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