Epigenetic modifiers of disease phenotypes: EB as a paradigm

Patients with epidermolysis bullosa (EB) often also referred to as butterfly children suffer from a rare, heritable skin disorder. Due to distinct gene mutations, skin layers lack sufficient adherence and consequently separate already after minor trauma (e.g. friction of a T-shirt, bumping against edges), which leads to blister formation and wounding. These injuries are frequently accompanied by persisting pain and itch, and in some patients also mucous membranes (e.g. mouth) are affected. Over time, recurring wounds can lead to severe complications, like infections and the development of particularly aggressive skin tumours. Even though in most patients the cause of the disease i.e. the respective gene mutation in an EB- associated gene - is known, for some patients they could not be identified. As a proper diagnosis is crucial to making the best treatment decisions, an important part of this project is to identify disease- causing gene mutations in cases where these have not yet been resolved. To do this, we will apply up- to-date technologies such as next generation sequencing. Another phenomenon observed in EB, is that patients present with diverse symptoms, even though they harbour the same gene mutation. Even in identical twins it was reported that one sibling showed a rather mild phenotype while the other was much more severely affected. Finding out what causes these differences is another major aim of this project. In this context, we will focus on the investigation of epigenetic events. These are changes that do not directly affect the genetic sequence, but rather refer to the additional layer of regulation that exists, which in part determines which genes become expressed and which do not. Epigenetic mechanisms can be linked to external factors. Therefore, tracing the events that lead to such differences in disease severity can provide important new information that will direct patient care and support the development of novel therapies. Finally, we will also investigate epigenetic changes as a direct consequence of EB mutation that impact the ability of skin cells to self-renew. This consequently affects skin repair and therefore disease severity. We will study these especially in the context of patient cells that have been repaired. Clarifying these aspects should also help guide the improvement of gene therapeutic approaches. A notable aspect of this project is that it falls within the framework of the recently established Austrian- Israeli scientific research program, and represents the first time a project of this scale, which is conducted in collaboration with two leading research institutes in Israel, is (co-)funded by the Austrian Science Fund (FWF).

Genetics alone does not explain it: Why the severity of EB varies Patients with epidermolysis bullosa (EB)-often referred to as "butterfly children"-suffer from a rare, genetic skin disorder. Specific genetic alterations cause the skin layers to be insufficiently connected, so that even minor mechanical stress (e.g., friction from a T-shirt) can cause the skin layers to tear apart, which becomes visible as blisters or wounds. These injuries are associated with persistent pain and itching, and in some patients not only the outer skin but also the mucous membranes are affected. Over time, wounds can lead to severe complications, including infections and the development of a particularly aggressive skin cancers. Although in most patients the cause of the disease can be identified - namely specific mutations in a gene known to cause EB - in some patients who carry the same genetic alteration are affected to very different degrees. Even in twins, one may be more severely affected while the other has a milder form of EB. This shows that it is not the defective gene alone that determines how severe EB will be. This research project investigated which additional factors might influence the course of the disease. The focus was on so-called epigenetic changes - factors that do not alter the genetic material itself but regulate how genes function. The results showed that people with severe EB have a different composition of immune cells in their blood than more mildly affected patients, and that the levels of certain inflammatory mediators (signaling molecules of the immune system) differ significantly and are associated with disease severity. Two inflammatory mediators in particular - interleukin-6 and TGF-1 - were examined in more detail. When skin cells were exposed to these substances, they underwent lasting changes, which may promote the development of skin cancer. This means that chronic inflammation can worsen the disease in the long term, independent of the original genetic defect. Additional factors also play a role in junctional EB (JEB), another form of the disease. In this case, a protein called collagen-17 is affected by mutation. It is generally described that differing amounts of collagen-17 during wound healing and skin renewal lead to competition between skin stem cells, which, among other things, drives aging processes and can be disadvantageous for gene therapy. To investigate this phenomenon, we compared cells in which collagen-17 had been corrected by gene therapy with uncorrected cells. Indeed, we found a rejuvenation trend in the corrected skin cells, as well as an influence on stem cell properties. A special feature of this project is that, for the first time, cooperation with two research groups in Israel was supported, enabling the project to achieve this considerable scope.