Study proposal: HNPCC: SNP-LOH Study
Zdena Bartosova, PhD. Laboratory of Cancer Genetics Cancer Research Institute of Slovak Academy of Sciences Vlarska 7 833 91 Bratislava Slovak Republic e-mail: exonbar@savba.sk
Background
The majority of patients with HNPCC carry germline mutations in one allele of the hMSH2 or hMLH1 gene. In the tumours, the normal allele of the respective gene is silenced by LOH. This finding could be utilized in the search for HNPCC germline mutations. Thus, assuming that LOH affects loci harbouring mutated genes, identification of a LOH event in e.g. the hMLH1 locus suggests that the patient is a carrier of a germline hMLH1 mutation.
So far, the detection of LOH at the hMLH1 and hMSH2 loci was carried out using microsatellite markers. However, the use of MS markers in MMR deficient tumours that display MSI gives rise to several bands, the significance of which is often disputable. We have developed a rapid LOH detection method at the hMLH1 and hMSH2 loci, which is based on the use of the most frequent intragenic single nucleotide polymorphisms (SNP). Our assay is based on the Multiplex SNaPshot system, in which 12 SNPs are evaluated simultaneously in a single reaction. So far, we have analyzed 37 tumor samples of patients with suspected HNPCC diagnosis and found that the SNP markers were substantially more informative and detected LOH more accurately than the MS markers D3S1611 and D2S123. The results were presented by the poster P14 (Bujalkova M., Krivulcik T., Wolf B., de Wind N., Jiricny J., and Bartosova Z.: Detection of LOH at hMLH1 and hMSH2 loci by SNPs: a potential diagnostic tool for directed mutation screening of HNPCC), and I can send more details to those who will be interested.
Aim of the proposed study:
The aim is to evaluate the potential of SNP-based LOH analysis in the determination of mutated gene using the HNPCC patients samples with confirmed germline mutation. The study may also shed more light on the question how often LOH actually occurs at hMLH1 or hMSH2. We search for collaborators (potential co-authors of the paper) who have DNA from tumour and normal tissue of HNPCC patients carrying already characterized germline mutations in hMLH1 or hMSH2 (any kind of mutation, including large deletions/duplications).
Samples to be sent in our laboratory:
1. Paired samples of DNA isolated from peripheral blood leukocytes (or normal mucosa) and fresh frozen tumor tissue (or paraffin-embedded) of the same patient. DNA from fresh frozen tumours is preferrable, if the choice can be made.
Amount of DNA:
in the case of DNA isolated from fresh frozen tissue or peripheral blood:
depending on concentration, 20 – 100 ml of 50 – 100 ng/ml of DNA We will run the test gel for integrity of DNA and measure again the DNA concentration.
In the case of DNA isolated from paraffin block: As we know how problematic these samples may be, please send us any amount you can offer (even very little) with any kind of information you have about the performance, quality, and concentration. We will see what can be done. Maybe some samples will need whole genome amplification.
2. If tumour DNA has been spent, but you have a block, you can send us 50 mg of sliced section of paraffin block with tumor tissue and we will isolate DNA in our laboratory; in a „worse“ case, a whole block can be sent also.
Information to be sent along with the samples:
1. The description of germline mutation: the gene, change and its position (the reference, in the case of published mutation).
2. Not necessarily, however, if available: MSI status, IHC results, LOH data - by MSI markers.
Please, do not hesitate to send me an e-mail if you have any kind of question.
Looking forward for the collaboration.
Zdena Bartosova