Methylated circulating free DNA (cfDNA) as biomarker of stromal remodelling

Blood based biomarkers have long been used for diagnosis and monitoring of disease. Traditionally they included circulating proteins, cells and electrolytes. More recently however circulating cell-free nucleic acids (including DNA, mRNA, microRNA and exosomes) have been successfully used to aid diagnosis in congenital abnormalities (such as Down’s syndrome) and acquired disease (such as cancer. Testing for circulating free (cf) DNA has generally been restricted to situations where there is likely to be somatic mutation or copy number change. However interrogation of modified DNA has not been extensively explored.

DNA methylation is a reversible modification of DNA structure in which a methyl group is added to cytosine residues. Methylation of DNA (for example in gene promoters) can alter gene expression through structural changes. It plays a vital role in regulating cell biology and highly cell-type specific methylation patterns can be seen. This has allowed cell-type specific methylation profiles to be used to monitor disease conditions. For example a short DNA methylation pattern specific to pancreatic β cells has been identified. Subsequently this motif was measured in cell free DNA as a marker of pancreatic β cell damage. Comparison of cfDNA isolated from the blood of individuals with and without Type-1 diabetes demonstrated that the pancreatic β cell specific methylation profile was detectable only in the blood of diabetics [1].

In the current proposal we hypothesise that methylation profiles in cfDNA, together with circulating free microRNA, can be used as a biomarkers of cell specific tissue damage and stromal remodelling in inflammatory pathologies (specifically asthma) and in colorectal cancer.

Stromal remodelling in asthma and colorectal cancer:
Asthma is a disease of the respiratory tract characterised by recurrent episodes of inflammation. The triggers for the inflammation are unknown and, in the chronic state, there is damage to the bronchiolo-alveolar epithelium, stromal fibrosis and neuromuscular hyperplasia of the airways. Circulating biomarkers may allow detection of inflammatory damage before clinical signs become apparent, monitoring of disease activity and response to therapy.

Colorectal cancer is caused by mutation in the epithelial cells of the colon and rectum. Sequential accumulation of mutations converts a normal cell into a malignant cell which has the capacity to invade into the surrounding tissue. In most cases, invasive colorectal cancers are associated with remodelling of the extracellular matrix and development of a tumour-associated stroma. Circulating biomarkers may allow detection of stromal remodelling during invasive malignancy, monitoring of disease progression and responsiveness to chemotherapy.

In both conditions, there is epithelial and stromal change. It is possible that similar mechanisms are involved and thus discoveries in each disease process may inform on changes in the other.

Experimental plan:
Ultimately, the project aims to investigate whether methylation profiles of cfDNA alone, or in combination with other circulating biomarkers such as miRNAs, can be used to predict, monitor and stratify inflammatory and neoplastic diseases. It will involve:
• generating genome wide DNA methylation data from epithelial and stromal cell types (following microdissection) which are relevant to the specific pathologies
• Analysing and validating DNA methylation data to generate highly cell type specific DNA methylation profiles characteristic of specific pathologies
• Isolating cell free DNA, miRNA and exosomes from blood samples taken from patients and healthy volunteers and optimise the methodology for testing
• Measuring the presence of the cell type specific methylation markers in circulating cell free DNA clinically / histologically defined disease conditions to determine whether the cfDNA profile of that disease condition reflects that seen in tissue samples
• Quantify changes in the cfDNA methylation profiles during disease episodes to see how these change during treatment.

The project will require the student to acquire a number of skills in molecular diagnostics and PCR based assays as well as bioinformatics.

1. Lehmann-Werman, R., et al., Identification of tissue-specific cell death using methylation patterns of circulating DNA. Proc Natl Acad Sci U S A, 2016. 113(13): p. E1826-34.