Genes, environment and evolution in eye development and malformations

Lead Research Organisation: MRC Human Genetics Unit

Abstract

We have identified three genes which, when mutated, can cause developmental malformations of the eye. These cause absence of the iris (or aniridia), and no eyes or small eyes. All three genes produce DNA-binding proteins which regulate the expression of other genes during development. We are exploring the complex regulation of these genes in cultured cells and in mouse and zebrafish models. Using existing information on what DNA sequences bind the aniridia gene PAX6, we can predict other targets for this gene and check their validity in the fish model. These targets are almost certainly conserved in mammals as well. Genes identified in this way are part of an interacting network that may include new genes causing other eye diseases. We are also exploring why no eyes and small eyes may not always manifest, even when the causative mutations are there. One protein that we have shown to play a role in this variability is called a chaperone because it helps newly formed and accidentally unfolded proteins to fold properly. The chaperone protein has a network of helpers that tells us how environmental factors influence gene function.

Technical Summary

Our aim is to identify genes implicated in developmental malformations of the eye in a large collection of severe eye anomaly cases. Following gene identification, we explore the spectrum of disease-causing mutations and deduce underlying genetic mechanisms and phenotype associations and study the role of these genes in normal development. Three major transcription factors involved in brain as well as eye development have been identified: PAX6 causing aniridia; SOX2 and OTX2 mutated in anophthalmia and microphthalmia. Heterozygous loss of gene function is associated with each phenotype. Missense mutations in PAX6 give rise to some distinct phenotypes, including microphthalmia. Familial PAX6 mutations segregate as Mendelian dominant traits. PAX6 loss of function is often mediated by chromosomal disruption more than 150 kb downstream of the transcribed gene. We have been exploring the mechanisms of such long range control of gene expression using transgenic mouse and zebrafish models and shown that regulatory changes play a key role in evolution. Recently we created a series of YAC transgenic reporter mice which have provided insight into PAX6 regulation, including autoregulation. Other transgenic studies have revealed enhancers working in adult tissues. Using Hidden Markov Modelling, we are identifying PAX6 target genes and validating these using zebrafish transgenesis and chromatin immunoprecipitation. SOX2 and PAX6 also interact at the protein level and we are exploring the effect of different missense mutations on this interaction. SOX2 mutations arise anew in each case; OTX2 mutations are incompletely penetrant so that non-Mendelian inheritance patterns are seen. We have shown that phenotypes may be modulated by the stress-response chaperone HSP90 in zebrafish and consider that perturbation of this system may account for incomplete penetrance in some cases of human malformations. We have identified a pax6b missense mutation in zebrafish, and showed increased severity of the phenotype. We can now pose the question: may stress signals during critical periods in pregnancy lead to altered disease severity in families. We are exploring, at the cell and molecular biological level the way in which the very abundant Hsp90 chaperone protein fulfils its multiple functions. Identification of novel HSP90-interacting proteins is giving us exciting new insights into how environmental factors can modulate gene function. Some of this work may have relevance to the prevention of recurrent malformations within families.

Publications

10 25 50

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Aijaz S (2004) Absence of SIX6 mutations in microphthalmia, anophthalmia, and coloboma. in Investigative ophthalmology & visual science

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Asai-Coakwell M (2007) GDF6, a novel locus for a spectrum of ocular developmental anomalies. in American journal of human genetics

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Axton R (1997) Combined SSCP/heteroduplex analysis in the screening for PAX6 mutations in Molecular and Cellular Probes

 
Description Home Office guidelines for human-animal hybrids research released February 2016, based on AMS 2011 Report, where co-author
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact This should lead to improvements in production of some therapeutic products
URL http://www.acmedsci.ac.uk/more/news/home-office-releases-new-guidelines-for-human-animal-hybrids/
 
Description EU Collaborative Grant NeuroXsys
Amount € 395,440 (EUR)
Funding ID Health-F4-2009-223262 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 06/2009 
End 12/2012
 
Description NIH RO1
Amount $288,000 (USD)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 01/2009 
End 12/2012
 
Title OTX2 mutation database 
Description Includes all reported independent mutations arising in the gene OTX2, in cases of congenital eye anomaly, predominantly microphthalmia and anophthalmia. There are also non-ocular phenotypes such as growth retardation associated with this gene. The database is regularly curated and updated 
Type Of Material Database/Collection of data 
Year Produced 2007 
Provided To Others? Yes  
Impact Insight into mechanisms of disease gene causation and effects of specific mutations in OTX2 . 
URL http://lsdb.hgu.mrc.ac.uk/home.php?select_db=OTX2
 
Title PAX6 mutation database 
Description Up-to-date regularly curated database of PAX6 gene mutations in patients with aniridia and related anomalies. Includes all published and submitted cases. Only one family member included where several affected family members know. So this is a record of all independent mutations reported in this gene. The database is publicly available and downloadable 
Type Of Material Database/Collection of data 
Year Produced 2007 
Provided To Others? Yes  
Impact Able to obtain correlations between genotype and phenotype and observe the spectrum of changes that lead to (eye) disease 
URL http://lsdb.hgu.mrc.ac.uk/home.php?select_db=PAX6
 
Title SOX2 mutation database 
Description Collection of reported mutations in the gene SOX2 which is associated with severe microphthalmia and anophthalmia. The database is regularly curated and updated 
Type Of Material Database/Collection of data 
Year Produced 2007 
Provided To Others? Yes  
Impact The database is freely available to consult and download. It provides insight into the mechanisms that cause severe developmental abnormalities of the eye 
URL http://lsdb.hgu.mrc.ac.uk/home.php?select_db=SOX2
 
Title Multiple monoclonal antibodies 
Description Multiple monoclonal antibodies licensed to multiple companies by MRCT 
IP Reference  
Protection Protection not required
Year Protection Granted
Licensed Yes
Impact Our group has been receiving about £30K of the profits annually for several years
 
Description Ophthalmic Genetics Course, Bologna 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Met a long term collaborator, an ophthalmologist, for the first time. We recently submitted a very nice paper as a joint publication in which the spetrum of mutations in severe developmental eye disease is set out.

I was asked to participate again in 2012, but could not fit the event into my diary. It is good to meet young clinicians and established ones on the faculty and also the young genetics scientists learning about the disease angle.
Year(s) Of Engagement Activity 2010,2011
 
Description Patient/Clinician/Researcher Meeting organised 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Small two-day meeting attended by representatives of the patient group Aniridia UK (some with guide dogs), clinicians and research scientists working on aniridia. Meeting organized in Edinburgh MRC Unit at request of small charity from Canada

Aniridia UK members in continuing contact with me and a London colleague who is studying the brain structure and function of volunteers
Year(s) Of Engagement Activity 2010
 
Description University of the Third Age 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact About 50-60 older people (over 50) attended this invited talk, part of their regular schedule of talks. I set out to explain how disease genes can be identified and how knowing their identity allowsus to understand about normal eye development and function as well as about the disease and the study provides insight into how genes work more generally.

Not sure, but some of my colleagues have also been invited to talk to this group
Year(s) Of Engagement Activity 2011