Research projects

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Identification of cis- and trans-acting genetic modifiers of somatic expansion as novel therapeutic targets in the repeat expansion disorders

Supervisor: Darren Monckton

Project description: At least 40 human disorders, including Huntington disease and myotonic dystrophy, are caused by the expansion of a simple sequence repeat [1].

More repeats are associated with a more severe form of the disease. Expanded disease-associated alleles are highly unstable and frequently expand during intergenerational transmission accounting for the anticipation observed in these disorders. Expanded disease-associated alleles are also unstable in the soma, in a process that is age-dependent, tissue-specific, and expansion-biased [2].

Notably, large expansions accumulate in affected tissues, such as the brain in Huntington disease and muscle in myotonic dystrophy, driving the tissue specificity and progressive nature of the symptoms. We have also established that residual variation in age at onset and disease severity not accounted for by inherited repeat length is inversely associated with residual variation in somatic expansion rates not accounted for by inherited repeat length and age (i.e., individuals in who the repeat expands more rapidly, get earlier and more severe symptoms than expected)[3-6].

As such, prevention of somatic expansions presents as a novel therapeutic target in these disorders. Insights from animal and cell models have revealed that expansions are critically dependent on the DNA mismatch repair pathway. Using candidate gene and genome-wide association studies we have also revealed that common polymorphisms in the some DNA repair genes modify the rate of somatic expansion and disease severity in both myotonic dystrophy type 1 and Huntington disease [5,7-10] However, powerful as such approaches are, the application of genome wide association studies in the repeat expansion disorders is limited by the rarity of the conditions that generally precludes the assembly of the very large cohorts needed to conduct them.

However, although expansions at the disease associated loci are rare, at least one locus, ERDA1, presents with a high frequency of expanded alleles (~20%) in the general population. These alleles are not associated with a disease state, but are genetically unstable. It is our hypothesis that, as we have done at the Huntington disease [5,11] and myotonic dystrophy type 1 loci, we can use high-throughput ultra-deep sequencing to derive individual-specific measures of mutational dynamics that act as biomarkers of genetic instability and can be used as molecular phenotypes in genome wide association studies. To this end, the student will address the following aims:

1. Develop assay. Develop high-throughput ultra-deep sequencing assay for sequencing the triplet repeat at the ERDA1 locus.
2. Determine the range of ERDA1 alleles present in general population. Sequence large numbers of alleles in the general population (from the Generation Scotland collection) to derive allele length distribution and identify potential variant repeats that we have shown to have a profound effect as cis-acting modifiers of somatic mutational dynamics and disease severity in myotonic dystrophy type 1 and Huntington disease [5,12].
3. Measure somatic instability. The data generated will be used to quantify the degree of somatic mosaicism in the general population and determine the role of sequence purity, allele length and age in mediating the degree of somatic instability.
4. Identification of therapeutic targets. After correcting for sequence purity, allele length and age, residual variation in somatic mosaicism will be used as a molecular phenotype in a genome wide association study in the Generation Scotland cohort. The results will identify novel therapeutic targets for the repeat expansion disorders.

Training outcomes: The studentship will provide training in state-of-the-art DNA sequencing technologies, bioinformatics (including use of the Galaxy platform), computational modelling and core skills such as the use of mathematics and statistics in handling large datasets and experimental design.

References

1. Monckton. 2021. The contribution of somatic expansion of the CAG repeat to symptomatic development in Huntington’s disease: a historical perspective. J Huntingtons Dis 10, 7–33. https://doi.org/10.3233/JHD-200429
2. Monckton et al. 1995. Somatic mosaicism, germline expansions, germline reversions and intergenerational reductions in myotonic dystrophy males: small pool PCR analyses. Hum Mol Genet 4, 1–8. https://doi.org/10.1093/hmg/4.1.1
3. Overend et al. 2019. Allele length of the DMPK CTG repeat is a predictor of progressive myotonic dystrophy type 1 phenotypes. Hum Mol Genet 28, 2245–2254. https://doi.org/10.1093/hmg/ddz055
4. Cumming et al. 2019. Genetic determinants of disease severity in the myotonic dystrophy type 1 OPTIMISTIC cohort. Neurology 93, e995-e1009. https://doi.org/10.1212/WNL.0000000000008056
5. Ciosi et al. 2019. A genetic association study of glutamine-encoding DNA sequence structures, somatic CAG expansion, and DNA repair gene variants, with Huntington disease clinical outcomes. EBioMedicine 48, 568–580. https://doi.org/10.1016/j.ebiom.2019.09.020
6. Morales et al. 2012. Somatic instability of the expanded CTG triplet repeat in myotonic dystrophy type 1 is a heritable quantitative trait and modifier of disease severity. Hum Mol Genet 21, 3558–3567. https://doi.org/10.1093/hmg/dds185
7. Morales et al. 2016. A polymorphism in the MSH3 mismatch repair gene is associated with the levels of somatic instability of the expanded CTG repeat in the blood DNA of myotonic dystrophy type 1 patients. DNA Repair 40, 57-66. https://doi.org/10.1016/j.dnarep.2016.01.001
8. Genetic Modifiers of Huntington’s Disease Consortium et al. 2019. CAG repeat not polyglutamine length determines timing of Huntington’s disease onset. Cell 178, 887–900. https://doi.org/10.1016/j.cell.2019.06.036
9. Flower et al. 2019. MSH3 modifies somatic instability and disease severity in Huntington's and myotonic dystrophy type 1. Brain 142, 1876–1886. https://doi.org/10.1093/brain/awz115
10. Morales et al. 2016. A polymorphism in the MSH3 mismatch repair gene is associated with the levels of somatic instability of the expanded CTG repeat in the blood DNA of myotonic dystrophy type 1 patients. DNA Repair 40, 57–66. https://doi.org/10.1016/j.dnarep.2016.01.001
11. Ciosi et al. 2018. Library preparation and MiSeq sequencing for the genotyping-by-sequencing of the Huntington disease HTT exon one trinucleotide repeat and the quantification of somatic mosaicism. Protocol Exchange. https://doi.org/10.1038/protex.2018.089
12. Cumming et al. 2018. De novo gain of variant repeats in myotonic dystrophy type 1 is associated with reduced somatic instability and mild or absent clinical features. Eur J Hum Genet 26, 1635–1647. https://doi.org/10.1038/s41431-018-0156-9

Applications accepted all year round.

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Mechanisms and applications of DNA site-specific recombinases

Supervisor: Marshall Stark

Outline & aim: Site-specific recombinases are enzymes that promote rearrangements of DNA molecules, by cutting and rejoining DNA strands at precise places within short target sequences (sites). For example, a specific piece of DNA can be cut out of a larger molecule, or its orientation can be reversed. Our research group aims to understand in detail how recombinases catalyse these reactions, and how they are controlled. To do this we use high-resolution structural data and advanced techniques for laboratory analysis. Site-specific recombinases have tremendous potential as tools for manipulating DNA in the fields of biotechnology, synthetic biology and gene therapy. We are investigating how to engineer “designer recombinases” that are suitable for these purposes, and how to use them for novel applications.

The aim of the research project will be to advance our understanding in one of the areas outlined above. For example, the project might be an investigation of the mechanism of DNA strand cutting and rejoining, using novel “single-molecule” methodologies, or to develop novel designer recombinases suitable for targeting specific genes in a living organism for deletion or modification.

Techniques:

• analysis of high-resolution structures
• protein expression, purification and biochemistry
• methods for manipulation of DNA in E. coli
• cloning, sequencing, sequence analysis
• synthetic biology
• novel methods for gene assembly
• advanced methods for analysis of protein-DNA complexes, including single-molecule methods

References

1. Olorunniji, F.J. Rosser, S.J. and Stark, W.M. (2016) Site-specific recombinases: molecular machines for the Genetic Revolution. Biochem. J. 473, 673-684.
2. Olorunniji, F.J. et al. (2017). Control of serine integrase recombination directionality by fusion with the directionality factor . Nucleic Acids Res. 45, 8635-8645.
3. Proudfoot, C., McPherson, A.L., Kolb, A.F. and Stark, W.M. (2011) Zinc Finger recombinases with adaptable DNA sequence specificity. PLoS ONE 6, e19537.

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Overview

Our staff have leading expertise and funding in human and model organism research including Drososophila melanogaster and microbes; as well as ‘omics - genomics, transcriptomics and metabolomics. They utilise a multi-disciplinary approach within the School of Molecular Biosciences with core links to Glasgow Polyomics; and have good external links with industry. Research in molecular genetics has strong alignment with the broad areas of agriculture and food security; bioscience for health; and industrial biotechnology and bioenergy.

Research topics are allied to ongoing research within the School. Projects can be related to either fundamental or translational science including clinical science and sustainable agriculture. Multi-disciplinary research approaches applied within the molecular genetics research programme include molecular genetics (human, Drosophila and microbial genetics), biochemistry, molecular biology, systems biology, ‘omics (genomics, transcriptomics, proteomics, metabolomics), bioinformatics and synthetic biology, as well as cellular imaging of biological functions using advanced technology. Specific areas of interest include:

• modelling kidney disease in Drosophila
• insect neuropeptides and GPCRs
• ‘omics and network modelling in Drosophila
• designer recombinases for genome engineering
• DNA rearrangements for Synthetic Biology
• topoisomerases and DNA topology
• mutational dynamics of unstable DNA in human disease
• new approaches to transcriptomic data analysis
• computational frameworks for omics data integration

Our PhD programme provides excellent training in cutting edge technologies that will be applicable to career prospects in both academia and industry. Many of our PhD graduates go on to research positions in academia, as well as to industry, and to policy and government organisations.

We have strong research connections with international collaborators. Funds are available through the College of Medical, Veterinary and Life Sciences to allow visits to international laboratories where part of your project can be carried out. This provides an excellent opportunity for networking and increasing the scientific knowledge and skill set of our PhD students.

Study options

PhD

• Duration: 3/4 years full-time; 5 years part-time

Individual research projects are tailored around the expertise of principal investigators.

Integrated PhD programmes (5 years)

Our Integrated PhD allows you to combine masters level teaching with your chosen research direction in a 1+3+1 format. 

International students with MSc and PhD scholarships/funding do not have to apply for 2 visas or exit and re-enter the country between programmes. International and UK/EU students may apply.

Year 1

Taught masters level modules are taken alongside students on our masters programmes. Our research-led teaching supports you to fine tune your research ideas and discuss these with potential PhD supervisors. You will gain a valuable introduction to academic topics, research methods, laboratory skills and the critical evaluation of research data. Your grades must meet our requirements in order to gain entry on to your pre-selected PhD research project. If not, you will have the options to pay outstanding MSc fees and complete with masters degree only.

Years 2, 3 and 4

PhD programme with research/lab work, completing an examinable piece of independent research in year 4.

Year 5

Thesis write up.

MSc (Research)

• Duration: 1 year full-time; 2 years part-time