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

Lead Research Organisation: University of Oxford
Department Name: Wellcome Trust Centre for Human Genetics

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description 2016 BSCB Honor Fell travel award to attend abcam: Chromatin, Replication and Chromosomal Stability conference, Copenhagen
Amount £360 (GBP)
Organisation British Society for Cell Biology 
Sector Learned Society
Country United Kingdom
Start 10/2016 
End 10/2016
 
Description 2016 UKEMS Travel Bursary to attend Responses to DNA damage, Egmond aan Zee, Netherlands
Amount £1,000 (GBP)
Organisation United Kingdom Environmental Mutagen Society 
Sector Learned Society
Country United Kingdom
Start 04/2016 
End 04/2016
 
Description NDM-CSC prize studentship
Amount £100,000 (GBP)
Organisation University of Oxford 
Department Nuffield Department of Medicine
Sector Academic/University
Country United Kingdom
Start 09/2015 
End 08/2019
 
Description Analysis of uracil in the genome 
Organisation Hungarian Academy of Sciences (MTA)
Department Institute of Enzymology
Country Hungary 
Sector Academic/University 
PI Contribution We provided samples
Collaborator Contribution They analysed samples and provided data and figures to be included in the next publication
Impact This is still ongoing
Start Year 2016
 
Description COMET assays 
Organisation University of Oxford
Department Division of Structural Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided cells and materials
Collaborator Contribution They trained us in methods, and assisted with data analysis
Impact We have a nearly complete dataset, which will go into the next paper
Start Year 2016
 
Description DNA combing on home made equipment 
Organisation Lancaster University
Department Faculty of Health and Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution I have hosted students from Nikki Copeland's group for training in DNA combing and associated procedures.
Collaborator Contribution They will provide materials relating to cell cycle regulation that will assist with the project.
Impact none to date
Start Year 2015
 
Description Structural and biochemical studies of the pathogenic PCNA mutation 
Organisation University of Oxford
Department Mathematical Institute Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided materials
Collaborator Contribution They assisted with crystal production, beam time and analysis.
Impact We solved the crystal structure of the mutant PCNA protein. This data was included in a published paper.
Start Year 2015
 
Description generation of pombe strains 
Organisation University of Sussex
Country United Kingdom 
Sector Academic/University 
PI Contribution We generated the intellectual property
Collaborator Contribution Felicity Watts made pombe strains and performed experiments
Impact We are generating data for use in a publication
Start Year 2015
 
Description DNA day workshop for schools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact My team assisted in a DNA workshop at the natural history museum, for a school party
Year(s) Of Engagement Activity 2016
 
Description Dissemination of findings to clinical practitioners 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Andrew and Emma had an MRC Alexander Fleming dissemination award last year that led to a very successful conference for health care workers in Wisconsin we cited the PCNA grant and the disorder was mentioned at the conference.
Year(s) Of Engagement Activity 2016
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 26 pupils attended a seminar that I gave to the schools STEM club on the topic of the effect of sunlight on skin cells.This led to a involved discussion with the children, and I have been asked to return next year.
Year(s) Of Engagement Activity 2016
 
Description Science uncovered at the Natural History Museum 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact We participated in an exhibition at the natural history museum in London. We presented the story of DNA and mutation, showing chromosomes and DNA damage and repair pathways. we spoke to at least 100 adult participants, who reported an increased understanding of DNA and mutation at the end of the evening.
Year(s) Of Engagement Activity 2016
 
Description podcast - level up human 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was part of the panel on a science-comedy podcast, level up human. We talked about topical subjects in science (I covered human genetics) and took questions from the audience.
Year(s) Of Engagement Activity 2016
 
Description podcast - oxford sparks 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I presented a podcast about the effect of UV light on human cells
Year(s) Of Engagement Activity 2016