Publication
Unravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis : Association with disease activity and progression
| Summary: | Multiple Sclerosis (MS) is a chronic neuroinflammatory and neurodegenerative disease with a multifactorial undetermined etiology that causes demyelination and neuroaxonal loss in the central nervous system (CNS), leading to disability, particularly in women. Mitochondria are pivotal for MS disease activity and progression: (I) in the CNS, mitochondrial dysfunction is the main trigger for neuroaxonal loss and brain atrophy; and (II) in the periphery, immunometabolism is critical for CNS autoimmunity, while patients with MS (PwMS) show inappropriate mitochondrial phenotypes and/or oxidative phosphorylation (OXPHOS) insufficiency, particularly in CD4+ T cells. Nonetheless, no studies have explored if mitochondrial genotype could be causing the mitochondrial dysfunction observed in CD4+ T cells from PwMS. In contrast, most studies have opted for whole blood analysis, despite the significant tissue-specificity of mitochondrial DNA (mtDNA) variants, including T cells. Overall, next-generation sequencing (NGS) approaches to mtDNA have been hindered by fewer resources and less uniformity, in comparison with nuclear DNA (nDNA). A notable example is the Applied Biosystems™ Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific, Waltham, MA, USA), mostly used in forensic research, which achieves mtDNA whole genome sequencing (WGS) with very low DNA amounts. Despite this, its bioinformatic processing occurs in the enterprise Ion Torrent Suite™ Software (TSS), yielding BAM files aligned to an unorthodox version of the revised Cambridge Reference Sequence (rCRS), thus impeding further bioinformatic processing with third-party tools, with a relatively high heteroplasmy threshold level of 10%. In order to better understand the role of mitochondrial genotype in MS activity and progression, I aimed to: (I) devise a novel approach for the Precision ID mtDNA Whole Genome Panel, which yielded an output in the correct rCRS and a lower heteroplasmy threshold; (II) explore differences in mtDNA in CD4+ T cells between newly diagnosed PwMS and healthy controls (HC); and (III) analyze baseline and longitudinal mtDNA changes regarding MS clinical phenotypes, enabling a correlation with disease activity and progression. Accordingly, an alternative customizable and open-source pipeline, the PrecisionCallerPipeline (PCP), was developed for processing samples with the correct rCRS output after Ion Torrent sequencing with the Precision ID library kit. Using 18 samples (3 original samples and 15 mixtures) derived from the 1000 Genomes Project, PCP achieved overall improved performance metrics in comparison with the proprietary TSS, with optimal performance at a 2.5% heteroplasmy threshold. These findings were further validated with 50 samples from an independent cohort of stroke patients, with PCP finding 98.31% of TSS’s variants (TSS found 57.92% of PCP’s variants), with a significant correlation between the variant levels (VLs) of variants found with both pipelines. Regarding the MS/Clinically Isolated Syndrome (CIS) cohort, it was possible to fully sequence the mtDNA of 61 PwMS at two clinical visits, six months (V1) and 36 months (V2) after disease onset, as well as their age- and sex-matched HC, following the successful magnetic enrichment in CD4+ T cells — mean fold change in paired samples of 2.59, 95% confidence interval (CI) [2.31–2.87]. Ultimately, however, there were no statistically significant differences in mitochondrial genotype between PwMS and HC, as well as between clinical visits V1 and V2 within PwMS. Thus, mitochondrial genotype does not seem to determine a higher risk of developing MS, nor a higher disease disability in PwMS. Furthermore, mitochondrial dysfunction in CD4+ T cells is unlikely to derive from mitochondrial genotype. |
|---|---|
| Main Authors: | Cortes-Figueiredo, Filipe |
| Subject: | ADN mitocondrial Esclerose Múltipla linfócitos T CD4+ Precision ID sequenciação de próxima geração CD4+ T cells mitochondrial DNA Multiple Sclerosis next-generation sequencing Precision ID |
| Year: | 2022 |
| Country: | Portugal |
| Document type: | doctoral thesis |
| Access type: | open access |
| Associated institution: | Universidade de Lisboa |
| Language: | English |
| Origin: | Repositório da Universidade de Lisboa |
| _version_ | 1866810329737986048 |
|---|---|
| author | Cortes-Figueiredo, Filipe |
| author_facet | Cortes-Figueiredo, Filipe |
| author_role | author |
| contributor_name_str_mv | Paul, Friedemann Morais, Vanessa Alexandra Repositório Científico de Acesso Aberto da ULisboa |
| country_str | PT |
| creators_json_txt | [{\"Person.name\":\"Cortes-Figueiredo, Filipe\",\"Person.identifier.orcid\":\"0000-0002-3946-3868\"}] |
| datacite.contributors.contributor.contributorName.fl_str_mv | Paul, Friedemann Morais, Vanessa Alexandra Repositório Científico de Acesso Aberto da ULisboa |
| datacite.creators.creator.creatorName.fl_str_mv | Cortes-Figueiredo, Filipe |
| datacite.date.Accepted.fl_str_mv | 2022-09-01T00:00:00Z |
| datacite.date.available.fl_str_mv | 2025-03-01T01:30:16Z |
| datacite.date.embargoed.fl_str_mv | 2025-03-01T01:30:16Z |
| datacite.rights.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| datacite.subjects.subject.fl_str_mv | ADN mitocondrial Esclerose Múltipla linfócitos T CD4+ Precision ID sequenciação de próxima geração CD4+ T cells mitochondrial DNA Multiple Sclerosis next-generation sequencing Precision ID |
| datacite.titles.title.fl_str_mv | Unravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis : Association with disease activity and progression |
| dc.contributor.none.fl_str_mv | Paul, Friedemann Morais, Vanessa Alexandra Repositório Científico de Acesso Aberto da ULisboa |
| dc.creator.none.fl_str_mv | Cortes-Figueiredo, Filipe |
| dc.date.Accepted.fl_str_mv | 2022-09-01T00:00:00Z |
| dc.date.available.fl_str_mv | 2025-03-01T01:30:16Z |
| dc.date.embargoed.fl_str_mv | 2025-03-01T01:30:16Z |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | http://hdl.handle.net/10451/63575 |
| dc.language.none.fl_str_mv | eng |
| dc.rights.none.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| dc.subject.none.fl_str_mv | ADN mitocondrial Esclerose Múltipla linfócitos T CD4+ Precision ID sequenciação de próxima geração CD4+ T cells mitochondrial DNA Multiple Sclerosis next-generation sequencing Precision ID |
| dc.title.fl_str_mv | Unravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis : Association with disease activity and progression |
| dc.type.none.fl_str_mv | http://purl.org/coar/resource_type/c_db06 |
| description | Multiple Sclerosis (MS) is a chronic neuroinflammatory and neurodegenerative disease with a multifactorial undetermined etiology that causes demyelination and neuroaxonal loss in the central nervous system (CNS), leading to disability, particularly in women. Mitochondria are pivotal for MS disease activity and progression: (I) in the CNS, mitochondrial dysfunction is the main trigger for neuroaxonal loss and brain atrophy; and (II) in the periphery, immunometabolism is critical for CNS autoimmunity, while patients with MS (PwMS) show inappropriate mitochondrial phenotypes and/or oxidative phosphorylation (OXPHOS) insufficiency, particularly in CD4+ T cells. Nonetheless, no studies have explored if mitochondrial genotype could be causing the mitochondrial dysfunction observed in CD4+ T cells from PwMS. In contrast, most studies have opted for whole blood analysis, despite the significant tissue-specificity of mitochondrial DNA (mtDNA) variants, including T cells. Overall, next-generation sequencing (NGS) approaches to mtDNA have been hindered by fewer resources and less uniformity, in comparison with nuclear DNA (nDNA). A notable example is the Applied Biosystems™ Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific, Waltham, MA, USA), mostly used in forensic research, which achieves mtDNA whole genome sequencing (WGS) with very low DNA amounts. Despite this, its bioinformatic processing occurs in the enterprise Ion Torrent Suite™ Software (TSS), yielding BAM files aligned to an unorthodox version of the revised Cambridge Reference Sequence (rCRS), thus impeding further bioinformatic processing with third-party tools, with a relatively high heteroplasmy threshold level of 10%. In order to better understand the role of mitochondrial genotype in MS activity and progression, I aimed to: (I) devise a novel approach for the Precision ID mtDNA Whole Genome Panel, which yielded an output in the correct rCRS and a lower heteroplasmy threshold; (II) explore differences in mtDNA in CD4+ T cells between newly diagnosed PwMS and healthy controls (HC); and (III) analyze baseline and longitudinal mtDNA changes regarding MS clinical phenotypes, enabling a correlation with disease activity and progression. Accordingly, an alternative customizable and open-source pipeline, the PrecisionCallerPipeline (PCP), was developed for processing samples with the correct rCRS output after Ion Torrent sequencing with the Precision ID library kit. Using 18 samples (3 original samples and 15 mixtures) derived from the 1000 Genomes Project, PCP achieved overall improved performance metrics in comparison with the proprietary TSS, with optimal performance at a 2.5% heteroplasmy threshold. These findings were further validated with 50 samples from an independent cohort of stroke patients, with PCP finding 98.31% of TSS’s variants (TSS found 57.92% of PCP’s variants), with a significant correlation between the variant levels (VLs) of variants found with both pipelines. Regarding the MS/Clinically Isolated Syndrome (CIS) cohort, it was possible to fully sequence the mtDNA of 61 PwMS at two clinical visits, six months (V1) and 36 months (V2) after disease onset, as well as their age- and sex-matched HC, following the successful magnetic enrichment in CD4+ T cells — mean fold change in paired samples of 2.59, 95% confidence interval (CI) [2.31–2.87]. Ultimately, however, there were no statistically significant differences in mitochondrial genotype between PwMS and HC, as well as between clinical visits V1 and V2 within PwMS. Thus, mitochondrial genotype does not seem to determine a higher risk of developing MS, nor a higher disease disability in PwMS. Furthermore, mitochondrial dysfunction in CD4+ T cells is unlikely to derive from mitochondrial genotype. |
| dirty | 0 |
| eu_rights_str_mv | openAccess |
| format | doctoralThesis |
| fulltext.url.fl_str_mv | https://repositorio.ulisboa.pt/bitstreams/dc614bae-bebf-48fc-b4ab-c2a2475c0a6f/download |
| funding.funder.alternateName_str_mv | FCT |
| funding.funder.identifier_str_mv | http://doi.org/10.13039/501100001871 |
| funding.funder.name_str_mv | Fundação para a Ciência e a Tecnologia |
| funding.name_str_mv | OE |
| id | ul_5a3e0e22b0d8cbe3ac8aab4e5d3e16a4 |
| identifier.url.fl_str_mv | http://hdl.handle.net/10451/63575 |
| instacron_str | ul |
| institution | Universidade de Lisboa |
| instname_str | Universidade de Lisboa |
| language | eng |
| network_acronym_str | ul |
| network_name_str | Repositório da Universidade de Lisboa |
| oai_identifier_str | oai:repositorio.ulisboa.pt:10451/63575 |
| organization_str_mv | urn:organizationAcronym:ul |
| person_str_mv | Cortes-Figueiredo, Filipe Cortes-Figueiredo, Filipe https://www.ciencia-id.pt/D010-B998-18AC D010-B998-18AC http://orcid.org/0000-0002-3946-3868 0000-0002-3946-3868 |
| publishDate | 2022 |
| reponame_str | Repositório da Universidade de Lisboa |
| repository_id_str | urn:repositoryAcronym:ul |
| service_str_mv | urn:repositoryAcronym:ul |
| spelling | engpt_PTMultiple Sclerosis (MS) is a chronic neuroinflammatory and neurodegenerative disease with a multifactorial undetermined etiology that causes demyelination and neuroaxonal loss in the central nervous system (CNS), leading to disability, particularly in women. Mitochondria are pivotal for MS disease activity and progression: (I) in the CNS, mitochondrial dysfunction is the main trigger for neuroaxonal loss and brain atrophy; and (II) in the periphery, immunometabolism is critical for CNS autoimmunity, while patients with MS (PwMS) show inappropriate mitochondrial phenotypes and/or oxidative phosphorylation (OXPHOS) insufficiency, particularly in CD4+ T cells. Nonetheless, no studies have explored if mitochondrial genotype could be causing the mitochondrial dysfunction observed in CD4+ T cells from PwMS. In contrast, most studies have opted for whole blood analysis, despite the significant tissue-specificity of mitochondrial DNA (mtDNA) variants, including T cells. Overall, next-generation sequencing (NGS) approaches to mtDNA have been hindered by fewer resources and less uniformity, in comparison with nuclear DNA (nDNA). A notable example is the Applied Biosystems™ Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific, Waltham, MA, USA), mostly used in forensic research, which achieves mtDNA whole genome sequencing (WGS) with very low DNA amounts. Despite this, its bioinformatic processing occurs in the enterprise Ion Torrent Suite™ Software (TSS), yielding BAM files aligned to an unorthodox version of the revised Cambridge Reference Sequence (rCRS), thus impeding further bioinformatic processing with third-party tools, with a relatively high heteroplasmy threshold level of 10%. In order to better understand the role of mitochondrial genotype in MS activity and progression, I aimed to: (I) devise a novel approach for the Precision ID mtDNA Whole Genome Panel, which yielded an output in the correct rCRS and a lower heteroplasmy threshold; (II) explore differences in mtDNA in CD4+ T cells between newly diagnosed PwMS and healthy controls (HC); and (III) analyze baseline and longitudinal mtDNA changes regarding MS clinical phenotypes, enabling a correlation with disease activity and progression. Accordingly, an alternative customizable and open-source pipeline, the PrecisionCallerPipeline (PCP), was developed for processing samples with the correct rCRS output after Ion Torrent sequencing with the Precision ID library kit. Using 18 samples (3 original samples and 15 mixtures) derived from the 1000 Genomes Project, PCP achieved overall improved performance metrics in comparison with the proprietary TSS, with optimal performance at a 2.5% heteroplasmy threshold. These findings were further validated with 50 samples from an independent cohort of stroke patients, with PCP finding 98.31% of TSS’s variants (TSS found 57.92% of PCP’s variants), with a significant correlation between the variant levels (VLs) of variants found with both pipelines. Regarding the MS/Clinically Isolated Syndrome (CIS) cohort, it was possible to fully sequence the mtDNA of 61 PwMS at two clinical visits, six months (V1) and 36 months (V2) after disease onset, as well as their age- and sex-matched HC, following the successful magnetic enrichment in CD4+ T cells — mean fold change in paired samples of 2.59, 95% confidence interval (CI) [2.31–2.87]. Ultimately, however, there were no statistically significant differences in mitochondrial genotype between PwMS and HC, as well as between clinical visits V1 and V2 within PwMS. Thus, mitochondrial genotype does not seem to determine a higher risk of developing MS, nor a higher disease disability in PwMS. Furthermore, mitochondrial dysfunction in CD4+ T cells is unlikely to derive from mitochondrial genotype.application/pdfpt_PTUnravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis : Association with disease activity and progressionPersonalCortes-Figueiredo, FilipeDSpacehttp://dspace.org/items/66b7090a-f5b3-452d-960e-e8c84eb0950aDSpacehttp://dspace.org/items/66b7090a-f5b3-452d-960e-e8c84eb0950aPereira dos Reis Cortes FigueiredoFilipe CarlosCiência IDhttps://www.ciencia-id.ptD010-B998-18ACORCIDhttp://orcid.org0000-0002-3946-3868Paul, FriedemannMorais, Vanessa AlexandraHostingInstitutionOrganizationalRepositório Científico de Acesso Aberto da ULisboae-mailmailto:repositorio@reitoria.ulisboa.ptrepositorio@reitoria.ulisboa.ptURNurn:tid:1015470482025-03-01T01:30:16Z2022-092022-052022-09-01T00:00:00ZHandlehttp://hdl.handle.net/10451/63575http://purl.org/coar/access_right/c_abf2open accessADN mitocondrialEsclerose Múltiplalinfócitos T CD4+Precision IDsequenciação de próxima geraçãoCD4+ T cellsmitochondrial DNAMultiple Sclerosisnext-generation sequencingPrecision ID23922381 bytesFundação para a Ciência e a TecnologiaUnraveling the nutritional determinants in Multiple Sclerosis Multiple Sclerosis patient samples Alterado para: Unravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis: association with disease activity and progressionOECrossref Funder IDhttp://doi.org/10.13039/501100001871literaturehttp://purl.org/coar/resource_type/c_db06doctoral thesishttp://purl.org/coar/access_right/c_abf2application/pdffulltexthttps://repositorio.ulisboa.pt/bitstreams/dc614bae-bebf-48fc-b4ab-c2a2475c0a6f/download |
| spellingShingle | Unravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis : Association with disease activity and progression Cortes-Figueiredo, Filipe ADN mitocondrial Esclerose Múltipla linfócitos T CD4+ Precision ID sequenciação de próxima geração CD4+ T cells mitochondrial DNA Multiple Sclerosis next-generation sequencing Precision ID |
| status | SINGLETON |
| subject.fl_str_mv | ADN mitocondrial Esclerose Múltipla linfócitos T CD4+ Precision ID sequenciação de próxima geração CD4+ T cells mitochondrial DNA Multiple Sclerosis next-generation sequencing Precision ID |
| title | Unravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis : Association with disease activity and progression |
| title_full | Unravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis : Association with disease activity and progression |
| title_fullStr | Unravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis : Association with disease activity and progression |
| title_full_unstemmed | Unravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis : Association with disease activity and progression |
| title_short | Unravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis : Association with disease activity and progression |
| title_sort | Unravelling longitudinal mitochondrial DNA mutations in Multiple Sclerosis : Association with disease activity and progression |
| topic | ADN mitocondrial Esclerose Múltipla linfócitos T CD4+ Precision ID sequenciação de próxima geração CD4+ T cells mitochondrial DNA Multiple Sclerosis next-generation sequencing Precision ID |
| topic_facet | ADN mitocondrial Esclerose Múltipla linfócitos T CD4+ Precision ID sequenciação de próxima geração CD4+ T cells mitochondrial DNA Multiple Sclerosis next-generation sequencing Precision ID |
| url | http://hdl.handle.net/10451/63575 |
| visible | 1 |