Molecular and cellular characterisation of an important new human DNA repair disorder

There are a group of rare inherited genetic disorders that are caused by the mutation of genes that normally act to protect our genes from damage. The chemical that comprises our genetic information, DNA, is susceptible to damage from a wide variety of sources, including environmental exposure or cellular metabolism. Individuals that inherit these DNA damage sensitivity disorders have distinctive combinations of symptoms, resulting from the abnormal behaviour of the mutated genes. These symptoms can include premature ageing, growth defects, neurodegeneration, sunlight or X-ray sensitivity and abnormal skin pigmentation.

We recently identified a new syndrome in the Ohio Amish community that is distinct from, but appears related to, known DNA damage sensitivity conditions such as the disorders known as XP (xeroderma pigmentosum), CS (Cockayne's syndrome) or AT (ataxia telangiectasia). The affected individuals have a unique combination of symptoms that led us to hypothesize that a previously unreported mutation must be causing the syndrome. Using state of the art genetic mapping techniques and genome sequencing we have identified the gene and the mutation responsible for causing this disorder.

We now need to understand what this particular genetic change does in order to cause the symptoms of the affected individuals. This is important for three reasons. Firstly, if we understand better the problems faced by the cells of affected individuals we should be able to manage their condition more effectively. Secondly, understanding how cells behave when this gene is altered will give us important information about how the unmutated gene normally functions. This is crucial because all our cells are constantly facing DNA damage, and failure to deal with this damage properly can lead to mutations that can result in cancer, ageing and neurodegeneration. A knowledge of the processes that cells normally use to detect, repair or tolerate DNA damage can therefore be hugely valuable in understanding how cancer and neurodegeneration are normally prevented, and what has to go wrong in order for them to occur. Finally, in the clinical setting we use chemicals and treatments that induce DNA damage as chemo- and radio-therapies for many different type of cancer. A better understanding of how our cells tolerate DNA damage, and how specific cells can be made more susceptible to these treatments, can help research aimed at improving therapies and developing novel treatments for malignant disease.

Through a longstanding community collaborative program we have unique access to cellular material derived from skin biopsies and blood samples from the affected individuals and unaffected family members. Our proposal is to test these cells in the laboratory to find out how their behaviour differs from normal cells, for example when treated with DNA damaging agents. We will use state-of the-art molecular biology, microscopy and biochemical approaches to uncover why the mutated gene causes these altered cell behaviours.

We will throughout the project keep in close communication with the local clinicians to ensure that our findings are used to optimise the treatment of the affected individuals, and we will also make sure that other researchers are informed about our results (via publication, networking and presentation at conferences), so that our research can be translated to generate maximum benefit for human health.

We have recently identified a novel inherited syndrome in the Ohio Amish community. Affected individuals have features which overlap with known DNA repair disorders including Cockayne's syndrome (CS), ataxia telangiectasia (AT) and xeroderma pigmentosum (XP). The eldest affected individuals also show signs suggestive of premalignant changes in their skin. Because the features of this syndrome overlap with those of multiple DNA repair disorders, we hypothesize that the underlying molecular defect probably affects more than one DNA repair process.

We have identified the causative mutation in the Amish community and find it to be a missense alteration in an essential gene.

We have access to unique patient material (comprising fibroblast and lymphoblastoid cell lines from affected individuals and unaffected family members) and plan to use these and other established resources to develop a clearer understanding of the role of this protein in DNA damage responses and other cellular pathways, with the aim of understanding how the molecular defect results in the cellular phenotypes and eventually the symptoms of the affected individuals. To do this we will use cell-based functional assays, and molecular biology approaches to investigate protein interactions.

We have already developed a substantial body of unpublished work and technical resources for this project and so are perfectly positioned to make rapid and effective progress. Successful completion of the project will have implications for our understanding of cellular pathways that regulate genome stability. This is vital as the correct operation of these processes is crucial to limit mutagenesis and prevent tumorigenesis and neurodegeneration in normal individuals. Our findings will also have direct patient benefit because they will provide a better understanding of the nature this condition, facilitating the clinical management of affected individuals.

The research here will directly impact upon strategies for treatment and management of individuals affected with this novel syndrome, and the dissemination of our findings may lead to molecular diagnosis of others with the same disorder, improving the chances that they also receive appropriate treatment.

DNA damage occurs in all cells at all times, due to the effects of cellular metabolism (such as DNA replication processes) as well as external chemicals (e.g. in tobacco smoke or pollutants) or agents (e.g. the UV component of sunlight, or cosmic radiation). Our understanding of the cellular response to DNA damage has increased dramatically in the last 20 years, and this has had enormous impact on clinical medicine (in particular for oncology) and also in other areas (for example the beauty industry).
Understanding of DNA damage responses has directly led to the development of drugs and targets for chemotherapy, for altered management of radiotherapy regimes and for better combinations of therapies to increase patient welfare by reduction of side effects as well as by enhancing efficacy of treatments and by personalised medicine approaches. Thus the research proposed here, that will lead to a better understanding of DNA damage responses, has the potential in the long term to benefit patients undergoing chemotherapy in the future by improving treatment regimes.

Because we here propose experiments that will explain why the affected individuals symptoms result from this molecular defect, we will be furthering understanding of DNA repair and replication in general, in a previously unexplored direction. This may well in the future lead to potential benefits in terms of treating and preventing cancers and neurodegeneration. Thus it may be a new line of investigation for bioscience companies wishing to develop strategies for diagnosis, prognosis and treatment of such disorders.

During the proposal we will also develop experimental techniques that can subsequenlty be applied to the analysis of other biological questions involving altered protein-protein interactions. We will publish our methods in full in open access journals so that other researchers, even in unrelated scientific fields can make use of our research.

The proposal will also enable training of a postdoctoral fellow and of Dr Baple, a clinical research fellow, not only in the technical skills required for the project, but also in project management, scientific writing and presentation skills that will be of use throughout their future careers.