17 April 2018 from 10:30 to 11:30

PhD Defence: Resolving allele-specific genetic and architectural variation: Proximity ligations from benchtop to bedside

In this thesis I describe the results of two main projects that on first glance have very little in common. One project, Monogenic Prenatal Diagnostic (MG-NIPD), shows how we can diagnose an unborn fetus at risk of inheriting a monogenic disease using maternal blood. In another project we designed a novel method, Multi-Contact Chromatin Conformation Capture (MC-4C), to investigate the 3D folding of DNA stored within the cell nucleus. To find the common factor is this work, one has to look at the methods rather than the subjects. Our MG-NIPD method relies in a large part on Targeted Locus Amplification (TLA). TLA and MC-4C, are both derived from the Chromatin Conformation Capture (3C) method, where DNA is fixated, digested and ligated within the cell nucleus. As a result, DNA elements in close physical proximity are joined in what is known as a proximity ligation. Such ligation events are indicative of interactions between functional DNA elements such as promotors and enhancers.

Previously, analysis of proximity ligations was limited to pairwise observations, resulting in population wide averages. To further elucidate the organisation of the genome, we developed MC-4C, which relies on a novel sequencing method (nanopore sequencing). For the first time we can now interrogate multi-way proximity ligations. Using MC-4C we were able to detect the formation of an active chromatin hub, where multiple genes and multiple enhancers interact simultaneously. Additionally, we applied MC-4C in cells where the cohesin displacing factor WAPL was depleted. As a result, novel CTCF-CTCF interactions are formed over longer distances. Our analysis showed that this is at least partially the result of multi-way CTCF clustering. We propose that the underlying mechanism may be excessive loop collision, resulting in a “cohesin traffic jam”.

Proximity ligations are however not limited to functional interactions, and also contain random ligation events between elements of the same chromosome. This aspect of proximity ligations is used in TLA to map the genetic polymorphisms that are present on the same chromosome and are therefore co-inherited. We applied this knowledge in the setting of risk families expecting a child. First we determined the neutral genetic polymorphisms that are present on the disease and wild-type chromosomes in both parents. Subsequently we isolated and sequenced cell-free DNA from maternal blood during pregnancy, which contains a small percentage of fetal DNA.  Quantification of the previously mapped polymorphisms in the cell-free DNA allowed us to then determine which parental genes were inherited by the fetus, and to thereby deduce the fetal disease status. We provided proof-of-principle for the genes responsible for cystic fibrosis and for Beta-thalassemia.

Start date and time
17 April 2018 10:30
End date and time
17 April 2018 11:30
PhD candidate
Carlo Vermeulen
Dissertation
Resolving allele-specific genetic and architectural variation: Proximity ligations from benchtop to bedside
PhD supervisor(s)
Wouter de Laat
Entrance fee
Free