Neural tube defects, affecting 1-2 per 1000 pregnancies, are severe congenital malformations that arise during early embryonic development due to failure of neurulation. Neural tube closure is influenced by a complex multifactorial etiology including both genetic and environmental factors. Although more than 250 mice models are known for NTDs and many candidate genes have been investigated in patient cohorts, the molecular basis underlying most human NTDs has still to be unravelled. The methylation hypothesis suggests that folate prevents NTDs by stimulating cellular methylation reactions and that a disturbed one-carbon metabolism may underlie NTDs.
The aim of this doctoral thesis was to determine which DNA regions and signaling pathways are more sensitive to DNA methylation changes during embryogenesis and lead to NTDs. Therefore, in addition to the core study of genome-wide DNA methylation, we designed locus-specific and functional validation studies to identify the most interesting candidate genes and in order to unravel the impact of DNA methylation in neural tube development and disease.
In Chapter 1 we go back to the embryological background of NTDs and we give an overview of the genetic and environmental factors that are known to be involved in NTDs. As we suppose that epigenetics and DNA methylation might fill the gap of knowledge in the search for the pathogenesis of NTDs, Chapter 2 gives an overview of studies that have been investigating the link between folate, DNA methylation and NTDs. In Chapter 3 we first investigated DNA methylation of the Homeobox genes in patients with a MMC. We identified HOXB7 hypomethylation as a potential risk factor for NTDs. In addition, HOXB7 hypomethylation was also present in unaffected siblings of patients with a MMC. These findings are suggestive for additional (epi)genetic factors underlying NTDs. Therefore, we further analysed the genome-wide DNA methylation data without prioritizing for candidate genes in Chapter 4. Forty-five genes were significantly differentially methylated, and functional enrichment analysis revealed multiple neurodevelopmentally important gene ontology classes. Validation of the top 6 genes (ABAT, CNTNAP1, SLC1A6, SNED1, SOX18 and TEPP) showed that SOX18 hypomethylation is a potential risk factor for NTDs. DNA methylation analyses in families of patients with MMC revealed that parents had a significantly higher methylation than both patients with MMC and their unaffected siblings. These findings might suggest a maternal contribution for a disturbed one-carbon metabolism. DNA methylation analysis in a family of a patient with a paternally inherited BMP4 deletion showed that genetic and epigenetic factors might interplay in the development of NTDs.
In Chapter 5 we present the study of genome-wide DNA methylation in patients with pseudohypoparathyroidism, that was performed to obtain expertise in data analysis and filtering, subsequently used for this thesis.
In conclusion, this doctoral thesis investigated the relation between DNA methylation and NTDs. We believe that our data support the multifactorial polygenic inheritance pattern of NTDs and the importance of epigenetic changes in neural tube development and disease. Further combined (epi)genetic studies are essential to unravel the complex etiology of NTDs and to alleviate the clinical, economical and psychological burdens for patients with NTDs.
Table of Contents:
Chapter 1: Introduction to neural tube defects.
Chapter 2: Folate, DNA methylation and neural tube defects.
THESIS OBJECTIVES & OVERVIEW
Chapter 3: DNA methylation of Homeobox genes in Spina Bifida.
Chapter 4: Genome-wide DNA methylation in Spina Bifida.
Chapter 5: Genome-wide DNA methylation in Pseudohypoparathyroidism.