Milda Narmontė defended her dissertation at the VU Life Sciences Center. Her thesis is entitled "High-resolution detection of genomic 5-hydroxymethylcytosine and its application to epigenetic studies of human diseases" for the degree of PhD in Biochemistry. Scientific supervisor - Dr Edita Kriukienė.
High-resolution genomic 5-hydroxymethylcytosine detection method and its application to epigenetic studies of human diseases 5-hydroxymethylcytosine (5hmC) is an important DNA modification involved in the regulation of gene expression in human physiological and pathological states.
The gold standard single-base-resolution methods for 5hmC gene mapping are based on bisulfite conversion, which results in DNA loss and challenges data interpretation due to altered base composition. In addition, the high cost of whole genome sequencing makes these methods unsuitable for disease or population studies.
This work presents the development of a new cost-effective 5hmC profiling method based on covalent DNA-tagged sequencing, hmTOP-seq (5hmC-specific tethered oligonucleotide-primed sequencing). The main advantages of hmTOP-seq were demonstrated: high resolution, 5hmC specificity, good reproducibility and correlation with other methods and the provision of DNA strand-specific hydroxymethylation information. The developed hmTOP-seq method has been successfully applied in epigenetic analyses of the human diseases neuroblastoma (NB) and chromosome 21 trisomy (Down syndrome).
Using hmTOP-seq and uTOP-seq, the young researcher performed a detailed multi-omic (5hmC, unmodified CG sequences and transcriptomic) analysis of different NB cell types, which allowed her to identify changes in gene hydroxymethylation and expression under hypoxia and to comprehensively investigate the intracellular differences in NB.
We also demonstrated that the hmTOP-seq method can be applied for epigenetic non-invasive prenatal testing of the foetus by real-time PCR or sequencing of foetal chromosome 21 trisomies from free circulating non-cellular plasma DNA of pregnant women.