IMPC Testing dst1 in susceptibility to infection

Necrotising fasciitis (NF) due to group A streptococcus is a lethal infection that destroys the connective tissues beneath the skin; although rare, it kills 30% of those affected. The number of cases in the UK has risen over the last 20 years and deaths can occur despite surgery, antibiotics and supportive treatment. Many people are left permanently scarred or disabled despite life-saving surgery. Cases frequently arise following comparatively trivial skin breaks including chickenpox, or injections, presumably by allowing group A streptococci to enter and pass through the outer skin layer. However, around a quarter of patients have no prior injury or skin break at all.

There are roughly 400 cases of group A strep NF per year in England; unlike other types of NF, patients affected often have no other medical or surgical illnesses. As such, the infection is devastating when it occurs. It seems likely that there could be a genetic factor that makes some people more likely to develop group A strep NF than others. Together with the UK's NF patient support group, we undertook a small study to identify genetic factors that might be over-represented in patients who have had group A strep NF.
We found that genetic differences in one particular gene called DST were present in two children who had very severe NF; this finding was also seen in some of the adults with NF. The DST gene produces a protein that is a major part of so-called hemidesmosomes - these are sticky patches that hold the layers of skin together. Some of the patients with NF had gene defects in other proteins that are also essential in hemidesmosomes, suggesting that abnormalities of the hemidesmosome might be a general risk factor for NF.

Hemidesmosomes allow the bottom-most layer of skin cells in the epidermis to stick to a layer known as the basement membrane, below which lies the deeper tissues known as the dermis and subcutaneous connective tissue or fascia. If one gene copy of DST is faulty (as in the case of the patients we studied) or missing, it is possible that the skin layers are less strongly bound together, and bacteria such as group A strep might find it easier to penetrate into the deeper tissues to trigger infection of these layers and NF.
In order to test this experimentally, we will use mice that lack one copy of the DST gene that have already been generated; these mice are otherwise healthy. We will determine whether these mice are more prone to develop early changes of group A strep NF, compared with mice that have intact copies of the gene. Experiments will involve skin exposure to group A strep. followed by measurements of group A strep under the skin. Each experiment will be of short duration hence mice will not become sick. It will be possible to make measurements of bacteria at an early stage of infection before NF begins. Together the results will reveal whether having only one healthy copy of the DST gene results in increased risk of invasive group A strep infection and NF. This will be important because, at present, the presence of just one abnormal DST gene is not recognised to be harmful. If there is a link to group A strep NF, such patients could be alerted to the increased risk, and might, for example, be advised to receive prompt antibiotic treatment for sore throats or other infections likely to be caused by group A streptococci.

Invasive group A streptococcal (GAS) infection has a mortality of 15-20%, however the highest mortality, 30%, is accounted for by cases of necrotising fasciitis (NF), a rapidly progressive necrotic infection of the subcutaneous tissues and fascia. The condition is very rare but often occurs without any antecedent medical illness or skin injury, consistent with a genetic predisposition. In an effort to identify possible genetic determinants of GAS-associated NF, we carried out a whole exome-sequencing study with a view to identifying rare deleterious variants contributing to susceptibility. We identified six missense variations in a gene called DST, a major component of hemidesmosomes, protein structures that link the epidermis to the dermis by anchoring the lowest cells in the epidermis to the basement membrane.

In this project, we will experimentally determine the role played by DST in GAS-associated NF using mice that lack one copy of the gene. We will compare heterozygotes with wildtype littermates of the same sex to determine if heterozygous DST deletion:
1. Increases permeability of the skin to GAS following topical application of GAS in presence or absence of a small skin abrasion;
2. Facilitates spread of GAS to subcutaneous tissues following intradermal injection of GAS;
3. Enables systemic dissemination of GAS from the nasopharynx.

These data will have the potential to demonstrate the importance of DST in GAS invasion and will provide a basis for further experimental study of the hemidesmosomes function in susceptibility to lethal human bacterial infection using refined, predominantly mild models of infection.