Periodic Reporting for period 1 - TTNPred (Development of novel computational biology pipeline for the efficient classification of titin SNPs for clinical use)
Reporting period: 2018-01-01 to 2019-12-31
Mutations in the giant muscle protein titin are a major cause of heart disorders in human populations. Routine DNA screening of patient cohorts is now becoming feasible, with a staggering number of titin truncations and missense single nucleotide polymorphisms (mSNPs) rapidly accumulating in genomics databases (>17,000 mSNPs). While the link between titin truncation and disease is now becoming clarified, detecting the pathogenic potential of mSNPs remains a substantial challenge. In mSNPs classification, bioinformatics evaluation is a necessary first filtering step, but existing predictors are poorly reliable. To address this problem, we aim to develop a new titin-centric scoring function that predicts the mechanistic effect of an mSNP exchange in the titin protein by considering the specific characteristics of its poly-domain chain. For this work, we built a medium-throughput molecular diagnostic pipeline that harvests existent structural models of titin components in estimating mSNPs-induced changes in free energy and conformational dynamics in the protein. To develop this methodology, we used mSNPs from a dataset of healthy people over 60 whose SNPs can be assumed to not cause disease. Identified positions which are tolerant to change will be extrapolated to the rest of the titin chain by exploiting the repetition of structural and functional loci within the chain. This allowed us to reinterpret SNP data from other public databases, as well as expand the structural knowledge from one area of titin to another. A titin map of vulnerability was then calculated and will be distributed to the research community. Ultimately, we aim to produce a tool that can aid clinicians to identify patients at risk of developing a titin-based heart condition at early disease stages, where intervention is still possible.
Titin has a repetitive beads-on-a-string arrangement of small domains. Many of these domains are structurally similar. Therefore, information from one domain can be used to predict how another related domain would behave should it have a SNP. In this work I have firstly identified that this assumption – that information form one can be used to predict another’s behaviour- is correct and then used it to expand the knowledge that we have from well-characterised areas to those areas that have not been studied directly. Training in mSNP annotation was received at Prof Richard’s lab (Généthon, Évry France), During the training exchange I was made aware of several publicly available databases and how to correctly interpret the information with in them. In particular, a data set of healthy individuals over 60 from Brazil (of mixed ethnic backgrounds) was used to identify tolerated SNVs.
During this work a total 10 new 3D domain structures across four areas of titin have been solved. increasing the structural coverage of titin domains, allowing for the better assessment of uniqueness and repetition across the titin chain. These will be deposited in the PDB - a public database of structures- when their accompanying papers detailing their biological significance are also published. The preparation of samples for crystal structure experiments resulted in the development of a new phosphorylation purification method (Adams, M et. al., 2019, Protein Journal. 38(2):181-189). In addition, due to the expertise gained from titin domain structural analysis, I also contributed towards a biomimetics project resulting in two publications (Hill, CJ et. al. Advanced Materials. 31(17):e1807521, 2019; Nesterenko, Y et. al., 2018, International Journal of Molecular Sciences. 20(17), 4299).
Several scripts have been developed to extract, organise and assimilate the data into diagnostic scores. Upon publication of the final tool, these will be made publicly available for those wishing to use or alter them. During this work to determine which methods would be suitable for incorporation into the molecular diagnostics pipeline, I undertook a series of detailed analysis to serve as case studies for structural mSNP assessment in titin. Firstly, I expanded on previous work using molecular dynamics to assess the effect of mSNPs on interdomain conformations (Fleming, JR et. al., 2020, Journal of Biomolecular Structure and Dynamics). Then, I investigated a cardiomyopathy-linked mSNP involved in a stretch induced molecular phenotype (manuscript submitted for review), as well as the effects of SNPs on a unique area of titin involved in binding stress response factors (manuscript in preparation). Details of additional methods and supporting data, confirming the applicability the chosen approaches integrated into the final tool and creation of the overall pipeline, are currently being prepared into several manuscripts. The final tool will be released and disseminated to the clinical community upon publication of these supporting manuscripts.
To support the dissemination of my work to a general audience I partook in several outreach activities. In 2019 I participated in two events associated with the European Researchers’ Night. The first was giving a public engagement talk on the project “Wrestling with the muscular giant, Titin” in Ely, UK. The second I participated in the Cambridge Life Lab in Peterborough, which was publicised in the local press. This was a series of hands on experiments for children set up in a shopping centre. This event was very popular (with over 300 visits in the day) and provided the opportunity not only engage children and also their parents. I have also created a profile as part of the women in science (WISE) campaign role model initiative (https://www.myskillsmylife.org.uk/my-skills-role-model/1002671(opens in new window)) to help inspire young girls into STEM careers. At a local level, I have become a member of the nascent University of Konstanz 500 women scientist ‘pod’ (https://500womenscientists.org/(opens in new window)). We aim to increase local awareness of issue affecting female researchers and contribute to global schemes to advocate for science in society and women in science (such as organising wiki-thons where female academics profiles are written and translated).
During this work a total 10 new 3D domain structures across four areas of titin have been solved. increasing the structural coverage of titin domains, allowing for the better assessment of uniqueness and repetition across the titin chain. These will be deposited in the PDB - a public database of structures- when their accompanying papers detailing their biological significance are also published. The preparation of samples for crystal structure experiments resulted in the development of a new phosphorylation purification method (Adams, M et. al., 2019, Protein Journal. 38(2):181-189). In addition, due to the expertise gained from titin domain structural analysis, I also contributed towards a biomimetics project resulting in two publications (Hill, CJ et. al. Advanced Materials. 31(17):e1807521, 2019; Nesterenko, Y et. al., 2018, International Journal of Molecular Sciences. 20(17), 4299).
Several scripts have been developed to extract, organise and assimilate the data into diagnostic scores. Upon publication of the final tool, these will be made publicly available for those wishing to use or alter them. During this work to determine which methods would be suitable for incorporation into the molecular diagnostics pipeline, I undertook a series of detailed analysis to serve as case studies for structural mSNP assessment in titin. Firstly, I expanded on previous work using molecular dynamics to assess the effect of mSNPs on interdomain conformations (Fleming, JR et. al., 2020, Journal of Biomolecular Structure and Dynamics). Then, I investigated a cardiomyopathy-linked mSNP involved in a stretch induced molecular phenotype (manuscript submitted for review), as well as the effects of SNPs on a unique area of titin involved in binding stress response factors (manuscript in preparation). Details of additional methods and supporting data, confirming the applicability the chosen approaches integrated into the final tool and creation of the overall pipeline, are currently being prepared into several manuscripts. The final tool will be released and disseminated to the clinical community upon publication of these supporting manuscripts.
To support the dissemination of my work to a general audience I partook in several outreach activities. In 2019 I participated in two events associated with the European Researchers’ Night. The first was giving a public engagement talk on the project “Wrestling with the muscular giant, Titin” in Ely, UK. The second I participated in the Cambridge Life Lab in Peterborough, which was publicised in the local press. This was a series of hands on experiments for children set up in a shopping centre. This event was very popular (with over 300 visits in the day) and provided the opportunity not only engage children and also their parents. I have also created a profile as part of the women in science (WISE) campaign role model initiative (https://www.myskillsmylife.org.uk/my-skills-role-model/1002671(opens in new window)) to help inspire young girls into STEM careers. At a local level, I have become a member of the nascent University of Konstanz 500 women scientist ‘pod’ (https://500womenscientists.org/(opens in new window)). We aim to increase local awareness of issue affecting female researchers and contribute to global schemes to advocate for science in society and women in science (such as organising wiki-thons where female academics profiles are written and translated).
Muscle disease, particularly heart disease, has over the last few decades increased in prevalence in the aging, sedentary societies of EU. These disorders are an enormous burden to the health and welfare systems of European economies. This research successfully evaluated the extent to which computational treatment of mSNPs could be used as reliable avenue for the accelerated analysis of patient genomes in the clinic. I have now evaluated different approaches and developed an analysis pipeline for titin mSNP interpretation, which I aim to soon release to the titin community to provide clinicians with a tool to identify patients at risk of developing a titin-based heart condition at early disease stages, where pharmacological or surgical interventions are still possible. Additionally, I contributed to the understanding of two titin mechanosensing hubs which will have a wider impact the understanding of muscle disease and function as the foundation for further research into new therapies and medical interventions.