{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/390746"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/390746","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Deconvoluting genetic variants associated with aortic disease using stem cell models","abstract":"Thoracic aortic disease (TAD) is usually silent until a catastrophic, life-threatening complication occurs. Identification and treatment at an early stage of disease can be informed by utilising genetic data, which in recent years has rapidly increased with the expansion of next-generation sequencing technologies. However, a critical challenge in genomic medicine is determining which variants identified by whole genome sequencing are causal for disease. Identifying causal variants aids disease diagnosis, prognostication, and determination of cascade screening requirements. This study aimed to utilise induced pluripotent stem cell (iPSC) models of TAD to analyse genetic variants from the 100,000 Genomes Project by determining causality, disease mechanisms and identifying drug targets. We focused on three Tier One variants, protein truncating in known aortopathy-related genes. Variants in lysyl oxidase (LOX, 122070160 T>TA), myosin light chain kinase (MYLK, 123401069 ACT>A) and mothers against decapentaplegic homolog 4 (SMAD4, 51076665 C>T) were selected. CRISPR-Cas9 genetic editing introduced these mutations into wild type iPSC lines. The mutant lines were differentiated into vascular smooth muscle cells (VSMC), analysed with a multi-omic approach, and the findings were validated in vitro. This work is the first to perform deep molecular phenotyping on LOX, MYLK and SMAD4 genetic variants in the context of aortic disease. TAD variant heterozygous knock-ins were confirmed through molecular cloning and Sanger sequencing. Mutant VSMCs demonstrated TAD transcriptomic signatures, with common & distinct pathways mediating disease pathogenesis in different variants. Active or basal contraction defects were revealed in both 2D monolayer and 3D engineered vascular tissue (EVT) models in all genetic variants. Proteomics of 3D EVT indicated shared deregulated matrix proteins alongside global metabolic perturbation. Commonly enriched signalling pathways in mutants suggested shared central hubs of dysregulation. A pro-proliferative phenotype was demonstrated in LOX and SMAD4 mutants, whereas MYLK and SMAD4 mutants were pro-apoptotic, suggesting an imbalance in cell number contributing to disease. LOX and MYLK mutant VSMCs showed hallmarks of abnormal synthetic phenotypic switching, driven by increased matrix stiffness, confirming the importance of VSMC state in aortic wall homeostasis. Increased mitochondrial basal respiration revealed metabolic defects in MYLK and SMAD4 mutants. Distinct phenotypes were observed, including perturbed calcium signalling in the MYLK mutant, or an inflammatory phenotype most strongly associated with SMAD4. A high-throughput drug screening assay identified Coenzyme Q10 as improving contraction in wild type and SMAD4 VSMCs and MYLK mutant EVT active contraction was restored by targeting the Rho kinase pathway. A pipeline was established between national flagship population genomic studies and detailed ‘disease-in-a-dish’ models. iPSC-derived VSMCs demonstrated TAD signatures when combined with multi-omic analyses and, as such, offer a valuable human platform to study aortic disease genetic variants. Despite distinct genetic initiators, there is a convergence on abnormal extracellular matrix turnover and VSMC contractility, which, in combination, are likely the driving factors behind reduced aortic wall integrity. Both changes contribute to dysfunctional VSMC mechanosensing, further exacerbating the loss of extracellular matrix homeostasis through abnormal remodelling. This work points to abnormal VSMC phenotypic switching accelerating disease and adds weight to the argument that reduced TGF- signalling is causal in TAD. A hypothesis of improving VSMC contraction and reducing ECM degradation, thus retaining the VSMC mechanotransduction capabilities and reducing abnormal phenotypic switching, is put forward, which could be achieved with combination drug therapy. Overall, this thesis demonstrated an iPSC ‘disease-in-a-dish’ approach to determine variant causality, key phenotypes associated with disease and identify drug targets.","abstract_html":"Thoracic aortic disease (TAD) is usually silent until a catastrophic, life-threatening complication occurs. Identification and treatment at an early stage of disease can be informed by utilising genetic data, which in recent years has rapidly increased with the expansion of next-generation sequencing technologies. However, a critical challenge in genomic medicine is determining which variants identified by whole genome sequencing are causal for disease. Identifying causal variants aids disease diagnosis, prognostication, and determination of cascade screening requirements. This study aimed to utilise induced pluripotent stem cell (iPSC) models of TAD to analyse genetic variants from the 100,000 Genomes Project by determining causality, disease mechanisms and identifying drug targets. We focused on three Tier One variants, protein truncating in known aortopathy-related genes. Variants in lysyl oxidase (LOX, 122070160 T>TA), myosin light chain kinase (MYLK, 123401069 ACT>A) and mothers against decapentaplegic homolog 4 (SMAD4, 51076665 C>T) were selected. CRISPR-Cas9 genetic editing introduced these mutations into wild type iPSC lines. The mutant lines were differentiated into vascular smooth muscle cells (VSMC), analysed with a multi-omic approach, and the findings were validated in vitro. This work is the first to perform deep molecular phenotyping on LOX, MYLK and SMAD4 genetic variants in the context of aortic disease. TAD variant heterozygous knock-ins were confirmed through molecular cloning and Sanger sequencing. Mutant VSMCs demonstrated TAD transcriptomic signatures, with common & distinct pathways mediating disease pathogenesis in different variants. Active or basal contraction defects were revealed in both 2D monolayer and 3D engineered vascular tissue (EVT) models in all genetic variants. Proteomics of 3D EVT indicated shared deregulated matrix proteins alongside global metabolic perturbation. Commonly enriched signalling pathways in mutants suggested shared central hubs of dysregulation. A pro-proliferative phenotype was demonstrated in LOX and SMAD4 mutants, whereas MYLK and SMAD4 mutants were pro-apoptotic, suggesting an imbalance in cell number contributing to disease. LOX and MYLK mutant VSMCs showed hallmarks of abnormal synthetic phenotypic switching, driven by increased matrix stiffness, confirming the importance of VSMC state in aortic wall homeostasis. Increased mitochondrial basal respiration revealed metabolic defects in MYLK and SMAD4 mutants. Distinct phenotypes were observed, including perturbed calcium signalling in the MYLK mutant, or an inflammatory phenotype most strongly associated with SMAD4. A high-throughput drug screening assay identified Coenzyme Q10 as improving contraction in wild type and SMAD4 VSMCs and MYLK mutant EVT active contraction was restored by targeting the Rho kinase pathway. A pipeline was established between national flagship population genomic studies and detailed ‘disease-in-a-dish’ models. iPSC-derived VSMCs demonstrated TAD signatures when combined with multi-omic analyses and, as such, offer a valuable human platform to study aortic disease genetic variants. Despite distinct genetic initiators, there is a convergence on abnormal extracellular matrix turnover and VSMC contractility, which, in combination, are likely the driving factors behind reduced aortic wall integrity. Both changes contribute to dysfunctional VSMC mechanosensing, further exacerbating the loss of extracellular matrix homeostasis through abnormal remodelling. This work points to abnormal VSMC phenotypic switching accelerating disease and adds weight to the argument that reduced TGF- signalling is causal in TAD. A hypothesis of improving VSMC contraction and reducing ECM degradation, thus retaining the VSMC mechanotransduction capabilities and reducing abnormal phenotypic switching, is put forward, which could be achieved with combination drug therapy. Overall, this thesis demonstrated an iPSC ‘disease-in-a-dish’ approach to determine variant causality, key phenotypes associated with disease and identify drug targets.","abstract_has_math":false,"creators":["Singh, Aminder"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Sinha, Sanjay"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-04-24","date_published":"2025-04-24","updated_at":"2026-07-22T22:23:56Z","subjects":["aorta","aortic disease","stem cell","induced pluripotent stem cells","genetic variants","genomic medicine"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/d847f06f-b31c-4644-a045-d7d03cf158bc/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000290994162"],"render_values":[{"text":"0000-0002-9099-4162","href":"https://orcid.org/0000-0002-9099-4162","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.122202","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Sinha, Sanjay"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["This work was supported by the Wellcome Trust [227508/Z/23/Z] and a Saven Research & Development Grant from the Royal College of Surgeons of England."]},{"key":"dc:creator","label":"Author","values":["Singh, Aminder"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000290994162"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2025-04-24"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/390746"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["aorta","aortic disease","stem cell","induced pluripotent stem cells","genetic variants","genomic medicine"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/d847f06f-b31c-4644-a045-d7d03cf158bc/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2026-10-13"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.122202"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/6b11ad4d-ec6a-44d3-b8fc-35357f394454/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Thoracic aortic disease (TAD) is usually silent until a catastrophic, life-threatening complication occurs. Identification and treatment at an early stage of disease can be informed by utilising genetic data, which in recent years has rapidly increased with the expansion of next-generation sequencing technologies. However, a critical challenge in genomic medicine is determining which variants identified by whole genome sequencing are causal for disease. Identifying causal variants aids disease diagnosis, prognostication, and determination of cascade screening requirements. This study aimed to utilise induced pluripotent stem cell (iPSC) models of TAD to analyse genetic variants from the 100,000 Genomes Project by determining causality, disease mechanisms and identifying drug targets. We focused on three Tier One variants, protein truncating in known aortopathy-related genes. Variants in lysyl oxidase (LOX, 122070160 T>TA), myosin light chain kinase (MYLK, 123401069 ACT>A) and mothers against decapentaplegic homolog 4 (SMAD4, 51076665 C>T) were selected. CRISPR-Cas9 genetic editing introduced these mutations into wild type iPSC lines. The mutant lines were differentiated into vascular smooth muscle cells (VSMC), analysed with a multi-omic approach, and the findings were validated in vitro. This work is the first to perform deep molecular phenotyping on LOX, MYLK and SMAD4 genetic variants in the context of aortic disease. TAD variant heterozygous knock-ins were confirmed through molecular cloning and Sanger sequencing. Mutant VSMCs demonstrated TAD transcriptomic signatures, with common & distinct pathways mediating disease pathogenesis in different variants. Active or basal contraction defects were revealed in both 2D monolayer and 3D engineered vascular tissue (EVT) models in all genetic variants. Proteomics of 3D EVT indicated shared deregulated matrix proteins alongside global metabolic perturbation. Commonly enriched signalling pathways in mutants suggested shared central hubs of dysregulation. A pro-proliferative phenotype was demonstrated in LOX and SMAD4 mutants, whereas MYLK and SMAD4 mutants were pro-apoptotic, suggesting an imbalance in cell number contributing to disease. LOX and MYLK mutant VSMCs showed hallmarks of abnormal synthetic phenotypic switching, driven by increased matrix stiffness, confirming the importance of VSMC state in aortic wall homeostasis. Increased mitochondrial basal respiration revealed metabolic defects in MYLK and SMAD4 mutants. Distinct phenotypes were observed, including perturbed calcium signalling in the MYLK mutant, or an inflammatory phenotype most strongly associated with SMAD4. A high-throughput drug screening assay identified Coenzyme Q10 as improving contraction in wild type and SMAD4 VSMCs and MYLK mutant EVT active contraction was restored by targeting the Rho kinase pathway. A pipeline was established between national flagship population genomic studies and detailed ‘disease-in-a-dish’ models. iPSC-derived VSMCs demonstrated TAD signatures when combined with multi-omic analyses and, as such, offer a valuable human platform to study aortic disease genetic variants. Despite distinct genetic initiators, there is a convergence on abnormal extracellular matrix turnover and VSMC contractility, which, in combination, are likely the driving factors behind reduced aortic wall integrity. Both changes contribute to dysfunctional VSMC mechanosensing, further exacerbating the loss of extracellular matrix homeostasis through abnormal remodelling. This work points to abnormal VSMC phenotypic switching accelerating disease and adds weight to the argument that reduced TGF- signalling is causal in TAD. A hypothesis of improving VSMC contraction and reducing ECM degradation, thus retaining the VSMC mechanotransduction capabilities and reducing abnormal phenotypic switching, is put forward, which could be achieved with combination drug therapy. Overall, this thesis demonstrated an iPSC ‘disease-in-a-dish’ approach to determine variant causality, key phenotypes associated with disease and identify drug targets."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["1aabeef47c93919887dc5c4019b3e82c","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["Deconvoluting genetic variants associated with aortic disease using stem cell models"]}]}],"canonical_facts":{"dc:contributor.advisor":["Sinha, Sanjay"],"dc:contributor.sponsor":["This work was supported by the Wellcome Trust [227508/Z/23/Z] and a Saven Research & Development Grant from the Royal College of Surgeons of England."],"dc:creator":["Singh, Aminder"],"dc:creator.authoridentifier":["0000000290994162"],"dc:date.issued":["2025-04-24"],"dc:description.abstract":["Thoracic aortic disease (TAD) is usually silent until a catastrophic, life-threatening complication occurs. Identification and treatment at an early stage of disease can be informed by utilising genetic data, which in recent years has rapidly increased with the expansion of next-generation sequencing technologies. However, a critical challenge in genomic medicine is determining which variants identified by whole genome sequencing are causal for disease. Identifying causal variants aids disease diagnosis, prognostication, and determination of cascade screening requirements. This study aimed to utilise induced pluripotent stem cell (iPSC) models of TAD to analyse genetic variants from the 100,000 Genomes Project by determining causality, disease mechanisms and identifying drug targets. We focused on three Tier One variants, protein truncating in known aortopathy-related genes. Variants in lysyl oxidase (LOX, 122070160 T>TA), myosin light chain kinase (MYLK, 123401069 ACT>A) and mothers against decapentaplegic homolog 4 (SMAD4, 51076665 C>T) were selected. CRISPR-Cas9 genetic editing introduced these mutations into wild type iPSC lines. The mutant lines were differentiated into vascular smooth muscle cells (VSMC), analysed with a multi-omic approach, and the findings were validated in vitro. This work is the first to perform deep molecular phenotyping on LOX, MYLK and SMAD4 genetic variants in the context of aortic disease. TAD variant heterozygous knock-ins were confirmed through molecular cloning and Sanger sequencing. Mutant VSMCs demonstrated TAD transcriptomic signatures, with common & distinct pathways mediating disease pathogenesis in different variants. Active or basal contraction defects were revealed in both 2D monolayer and 3D engineered vascular tissue (EVT) models in all genetic variants. Proteomics of 3D EVT indicated shared deregulated matrix proteins alongside global metabolic perturbation. Commonly enriched signalling pathways in mutants suggested shared central hubs of dysregulation. A pro-proliferative phenotype was demonstrated in LOX and SMAD4 mutants, whereas MYLK and SMAD4 mutants were pro-apoptotic, suggesting an imbalance in cell number contributing to disease. LOX and MYLK mutant VSMCs showed hallmarks of abnormal synthetic phenotypic switching, driven by increased matrix stiffness, confirming the importance of VSMC state in aortic wall homeostasis. Increased mitochondrial basal respiration revealed metabolic defects in MYLK and SMAD4 mutants. Distinct phenotypes were observed, including perturbed calcium signalling in the MYLK mutant, or an inflammatory phenotype most strongly associated with SMAD4. A high-throughput drug screening assay identified Coenzyme Q10 as improving contraction in wild type and SMAD4 VSMCs and MYLK mutant EVT active contraction was restored by targeting the Rho kinase pathway. A pipeline was established between national flagship population genomic studies and detailed ‘disease-in-a-dish’ models. iPSC-derived VSMCs demonstrated TAD signatures when combined with multi-omic analyses and, as such, offer a valuable human platform to study aortic disease genetic variants. Despite distinct genetic initiators, there is a convergence on abnormal extracellular matrix turnover and VSMC contractility, which, in combination, are likely the driving factors behind reduced aortic wall integrity. Both changes contribute to dysfunctional VSMC mechanosensing, further exacerbating the loss of extracellular matrix homeostasis through abnormal remodelling. This work points to abnormal VSMC phenotypic switching accelerating disease and adds weight to the argument that reduced TGF- signalling is causal in TAD. A hypothesis of improving VSMC contraction and reducing ECM degradation, thus retaining the VSMC mechanotransduction capabilities and reducing abnormal phenotypic switching, is put forward, which could be achieved with combination drug therapy. Overall, this thesis demonstrated an iPSC ‘disease-in-a-dish’ approach to determine variant causality, key phenotypes associated with disease and identify drug targets."],"dc:format.checksum.md5":["1aabeef47c93919887dc5c4019b3e82c","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.122202"],"dc:identifier.uri":["https://www.repository.cam.ac.uk/bitstreams/6b11ad4d-ec6a-44d3-b8fc-35357f394454/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/390746"],"dc:rights":["https://www.repository.cam.ac.uk/bitstreams/d847f06f-b31c-4644-a045-d7d03cf158bc/download","http://purl.org/NET/rdflicense/allrightsreserved"],"dc:rights.embargodate":["2026-10-13"],"dc:rights.embargotype":["embargo"],"dc:subject":["aorta","aortic disease","stem cell","induced pluripotent stem cells","genetic variants","genomic medicine"],"dc:title":["Deconvoluting genetic variants associated with aortic disease using stem cell models"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:23:56Z"}