N05029: PCR and folic acid status

Study Duration: January 2001 to January 2004

Contractor: Laboratory of the Government Chemist

In recent years a link between lack of folate, hypomethylation and cancer susceptibility has begun to emerge. It is thought that low levels lead to a reduction in methylation density of genomic DNA promoting aberrant gene expression and somatic mutation. Specific monitoring of hypomethylation could therefore be used as a suitable functional marker for a lack of dietary folate. Traditional methods for hypomethylation analysis rely on laborious, radiolabelled assays which monitor global methylation of genomic DNA. This project, therefore, seeks to overcome this problem by using specific monitoring of hypomethylation as a suitable functional biomarker for a lack of dietary folate, linking dietary folate status directly to DNA methylation. Identifying specific methylation sites in specific genes, in DNA from individuals of known folate status, which become hypomethylated under low folate conditions, would enable a direct correlation between blood folate levels and changes at the genetic level to be made. This will also be co-ordinated with the analysis of samples from colorectal cancer patients to investigate whether methylation sites identified as folate bioavailability biomarkers in healthy individuals also have a role to play as biomarkers in colorectal cancer. By identifying key 'indicator' sites present in genomic DNA obtained from blood or buccal swabs it should be possible to utilise such markers in a fluorescence or PCR-based assay to provide a rapid, high throughput assay for determination of dietary folate status. This will provide a rapid diagnostic test for effective monitoring of folate bioavailability.

Objectives:

• Obtain genomic DNA from suitable biological samples with a known folate history.
• Screening of DNA samples to identify potential biomarker sites.
• Characterisation of potential biomarker sites.
• Selection of suitable methylation sites.
• Assay development.
• Evaluation of assay.
• Report and dissemination activites.

The aim of this study is to link dietary availability of folate to hypomethylation at specific sites in genomic DNA to develop and validate functional markers of dietary folate status. Genomic DNA samples will be obtained form blood and buccal swab samples generated in N05028 and other non-FSA funded folate supplementation studies. Initially a differential DNA digestion screening protocol will be used to try and locate specific 'indicator' sites that show differences in their methylation status dependent on an individuals dietary folate status. This will be coupled with interrogation of the genomic DNA of these individuals for the methylation status of CpG sites genes that have been highlighted in previous studies as having variable methylation patterns. To identify novel hypomethylation sites a differential digestion screening protocol that allows the identification and isolation of CpG containing fragments that differ in their methylation status will be employed. Fragments containing novel methylation sites will be sequences to allow the generation of site-specific probes. An assay to distinguish hypomethylated target sites will then be developed and evaluated.

Approach
Two approaches were incorporated in order to identify sites of interest. The first was based upon prior knowledge, identifying genes which may be of interest from the literature and then characterising their methylation status in individuals with a high and low folate status. The DNA is characterised with respect to methylation status by a process referred to as bisulphite sequencing. Initially the DNA has to be treated with a chemical called sodium bisulphite, which causes the alteration of unmethylated cytosine bases, whilst preserving methylated cytosines. A process called polymerase chain reaction (PCR) then amplified the target sequence, which was generally about 600-700 bases long. PCR is a technique which allows you to isolate a specific fragment of DNA by increasing the number of copies of that sequence from a few copies to several million. The sequence of the bases in the DNA can then be determined, and those sites that were altered or maintained can be identified by comparing the sequence to a reference sequence.

The second approach which was implemented, in order to identify potential sites of interest, was a whole genome screening method. This allows identification of novel sites of interest which exhibit different methylation states between individuals with a high and low folate intake. Unlike the previous approach, this screening method requires no previous information about the DNA.

A number of different attempts were made to develop an assay to differentiate between methylation differences identified between the high and low folate groups. Differentiation of the two sequences was attempted on the basis of sequence alterations which result from bisulphite treatment. Approaches included PCR, quantitative PCR and by looking at the temperature at which the two DNA strands dissociate, which is a function of the sequence differences. A further method incorporated a short strand of DNA which would selectively bind either the altered or unaltered DNA, to allow differentiation.

Outcome/key results obtained
It was not possible to identify specific sites which displayed absolute differences, however a number of sites were identified which illustrated differing degrees of change between individuals with a high and low folate intake. This was because the changes do not occur in all copies of an individual's DNA, but only in a percentage of the DNA. Therefore both the original sequence and the altered sequence are present in all individuals, just in different proportions. It was interesting that these sites were present in one of the genes thought to be responsible for the development of cancer. Despite this, it was not possible to develop a high throughput assay to identify this site in samples of unknown folate intake for a number of reasons. The presence of both the altered and unaltered sequence required that the assay had to quantitative, furthermore the treatment of DNA with bisulphite causes the majority of the DNA to break down.

What it means and why it's important.
This work has illustrated that no absolute methylation differences could be identified at specific sites as a result of differential dietary folate status. Although, the overall ratio of altered to unaltered sites, however, can be modified in certain genes. This made it difficult to develop an assay of the type intended as the differences observed in normal healthy individuals are not of a level suitable for optimal assay design. Although it was possible to show proof of principle for the assays to differentiate different methylation states it was not possible to transfer these across to 'real' genomic DNA samples.

Final report is available from the FSA Library and Information centre.
To obtain a copy, please contact the Enquiry Desk, Dr Elsie Widdowson Library and Information Services, Food Standards Agency, tel: 020 7276 8181/8182 or email: library&info@foodstandards.gsi.gov.uk).

Contact: For any enquiries concerning this research project, please contact the relevant Programme contact or e-mail science@foodstandards.gsi.gov.uk