Project title
Towards personalised medicine for rare inherited brain diseases by identifying and manipulating variant RNA transcripts
Supervisors
Background
The greatest abundance of variation in transcription occurs in the brain1, the site of significant inherited disease pathology and likely functional consequences2. We have used the most common Batten disease3 gene, CLN3 (MRC award MR/V033956), to develop a bioinformatic and experimental targeted long-read RNA sequencing pipeline to describe transcript complexity for any gene4. We have since collected many fresh blood samples for other genetic types of Batten disease and other diseases, and brain bank samples, ready for analysis. We select disease genes to investigate by analysing public (healthy control) long-read RNAseq databases5 to estimate transcript diversity, which varies from 21% (i.e. no dominant transcript, so interesting) to >90% (less interesting), and transcript distribution across organs to confirm blood is a representative tissue source for that gene.
Aims/objectives
The project will: (1) apply state-of-the-art targeted deep RNA sequencing technology to comprehensively identify disease transcript isoforms, including those missing exons or gaining new sequence, and reveal rare transcripts not accessible by traditional sequencing methods; (2) investigate interesting templates at a cell level to understand their effect on protein function or non-mediated decay predictions. Questions to be asked include: how much disease pathogenesis associated with a particular mutation is due to partial loss of activity or the acquisition of new characteristics; whether disease severity correlates with the prevalence of particular variant transcripts; and whether the proportion of variant transcripts can be manipulated using oligonucleotides to reduce or exacerbate the disease (in collaboration with Prof Haiyan Zhou).
Methods
- Purify RNA from tissue samples (already collected) and long read sequence using PacBio Sequel system with targeted capture of selected disease gene cDNA to provide true transcript diversity and proportionality.
- Study the functionality of peptides encoded by individual variant transcripts using human model cell systems - for conserved genes, the fission yeast S. pombe can also be used.
- Develop more accurate cell models to study transcripts arising from particular gene mutations to better understand their cellular consequences. e.g. brain organoid models can be developed through collaboration.
- Enhance disease pathology and pathogenesis of disease progression from mechanistic cascades.
- Design new therapeutic strategies based on enhancing the concentration of templates that are beneficial and reducing the concentration of templates that are deleterious.
Timeline
- Months 1-12 – Identify variation in transcripts produced in inherited diseases of current interest
- Months 6-30 – Functional study of selected key transcripts in cell models
- Months 18-33 – Therapeutic design and development
- Months 33-36 – Thesis writing and submission
References
- Zhang D, et al., Incomplete annotation has a disproportionate impact on our understanding of Mendelian and complex neurogenetic disorders. Sci Adv, 2020. 6: eaay8299. DOI: 10.1126/sciadv.aay8299.
- Clark MB, et al., Long-read sequencing reveals the complex splicing profile of the psychiatric risk gene CACNA1C in human brain. Mol Psychiatry, 2020. 25: 37-47. DOI: 10.1038/s41380-019-0583-1.
- Mole SE, et al., Clinical challenges and future therapeutic approaches for neuronal ceroid lipofuscinosis. Lancet Neurol, 2019. 18: 107-16. DOI: 10.1016/S1474-4422(18)30368-5.
- Zhang H-Y, et al., CLN3 transcript complexity revealed by long-read RNA sequencing analysis. BMC Med Genomics, 2024. 17. DOI: 10.1186/s12920-024-02017-z.
- Encode Project Consortium, et al., Expanded encyclopaedias of DNA elements in the human and mouse genomes. Nature, 2020. 583: 699-710. DOI: 10.1038/s41586-020-2493-4.
Who should students contact?
Professor Sara Mole (s.mole@ucl.ac.uk)
Research topic
Genetics, Epilepsy