Tomas Ekstrom
Department of Clinical Neuroscience; Karolinska Institutet
Despite the prospect of employing global DNA-methylation as a potentially useful marker of malignancy, no method has been available to determine methylation changes in a high throughput manner. We have developed a novel method for assaying the level of global DNA methylation. The LUminometric Methylation Assay (LUMA) method is based on DNA cleavage by methylation sensitive restriction enzymes combined with a polymerase extension assay in a Pyrosequencing� format. The method also employs EcoRI, which serves as an internal control for DNA input.
The method is quantitative, highly reproducible and easy to scale up. The total assay time is only six hours, requires 200 — 500 ng of genomic DNA and is less labour intensive than previously reported methods. We asked whether LUMA could 1) grade chronic lymphocytic leukemia (CLL) cells at the level of global methylation changes, 2) whether Hairy cell leukaemia (HCL) and acute lymphocytic leukaemia (ALL) could be distinguished using LUMA, and 3) whether systemic inflammation in patients with end stage renal disease (ESRD) affects global methylation in peripheral blood leukocytes.
Hypomethylation of the ZAP70 promoter has previously been correlated with worse outcome for CLL patients compared to hypermethylation. 1) The results of the LUMA analysis revealed a homogenous group of normally methylated cells which also showed ZAP70 methylation. 2). Application of LUMA to normal peripheral leukocytes, HCL and ALL, revealed significant differences in genomic DNA methylation between these patients. 3). The results from the ESRD patients showed a significant correlation between inflammatory markers and hypermethylation of PBL. Furthermore, a correlation between PBL hypermethylation and death caused by cardiac failure was evident. In addition to these data, we found increasing peripheral blood cell DNA-methylation with age in apparently healthy individuals.
The LUMA assay may be a useful method to assess genome wide global DNA methylation for a variety of purposes including etiology, diagnosis and prognosis of some cancers as well as other pathological states. It may also be employed to analyze general epigenetic alterations related directly and indirectly to cancer, environmental influences like malnutrition, infection or other impact.