Preparatory study for the cytogenetic analysis of "Drosera" genus in Galicia

[Resumen] El objetivo de este proyecto será dar los primeros pasos que permitan el diseño y la realización de un estudio citogenético para las especies Drosera intermedia Hayne y Drosera rotundifolia L. Los principales problemas que pretendemos resolver serían: (i) asegurar el mantenimiento de las plantas en el laboratorio de forma que se tenga una fuente constante de ápices radiculares y (ii) asegurar mediante un estudio filogenético que las especies de estudio son un buen modelo para el estudio citogenético. Asimismo se realizaron las primeras pruebas para la observación de los cromosomas. El cultivo in vivo en vermiculita con agua destilada ofreció los mejores resultados, mientras que los in vitro resultaron complejos y no ofrecieron resultados satisfactorios. El análisis filogenético mostró que las especies a estudio se encuentran en la misma sección del género Drosera, siendo taxa próximos pero diferenciados. Se trata de especies adecuadas para realizar estudios citogenéticos que permitirán clarificar la taxonomía y evolución del grupo. Las primeras pruebas citogenéticas realizadas han permitido la observación de cromosomas pero con un elevado nivel de artefactación. Será necesario optimizar el protocolo utilizado para continuar con el estudio.[Abstract] The aim of this proyect is to take the inital steps in order be able to conduct a cytogenetic study focused on Drosera intermedia Hayne and Drosera rotundifolia L. The main problems that we try to solve are: (i) to ensure the maintenance of these plants in the laboratory, obtaining a constant source of root tips and (ii) to ensure, using a phylogenetic study, that these species are a suitable model for a cytogenetic study. In addition to this, we have also performed the first attempts at observing chromosomes in Drosera. The in vivo culture with distilled water yielded the best results, in stark contrast with the in vitro cultivation, which were very complex and didn’t produce any successful results. The phylogenetic analyses showed that these species belong to the Drosera section, being closely related but different taxa. These species are suitable to develop cytogenetic studies that can help us to unravel the taxonomy and evolution of the group. The first cytogenetic tests allowed us to observe highly altered chromosomes. It would be necessary to improve the used protocols in order to continue with this study.Traballo fin de grao (UDC.CIE). Bioloxía. Curso 2014/201

Isolation, characterization and analysis of human stromal cells obtained from bone marrow

Traballo fin de mestrado (UDC.CIE). Biotecnoloxía avanzada. Curso 2016/201

Caracterización genética del fenotipo de progresión rápida en la patología artrósica

Programa Oficial de Doutoramento en Ciencias da Saúde. 5007V01[Resumo] Esta tese de doutoramento trata de identificar variantes xenéticas capaces de alterar o risco de desenvolver o fenotipo de artrose de xeonllo con progresión rápida. Para iso utilizáronse técnicas de secuenciación para atopar polimorfismos presentes no ADN nuclear e mitocondrial que poidan favorecer o desenvolvemento do fenotipo de interese. Detectouse unha variante mitocondrial, presente na posición m.16519C, que estivo significativamente asociada coa rápida progresión. Despois de realizar un pequeno estudo funcional, detectáronse niveis máis altos de interleucina 6 e cicloxixenasa 2 nos cíbridos portadores da variante, indicando que, o aumento da resposta inflamatoria podería ser un mecanismo desencadeante para unha progresión rápida. Por outra banda, detectouse por primeira vez unha interacción entre o ADN nuclear e o mitocondrial no campo da artrose. A presenza do haplogrupo mitocondrial UK e do polimorfismo nuclear rs12107036 tamén aumenta o risco de progresión rápida na artrose do xeonllo. Por último, xerouse un modelo preditivo de gran relevancia clínica e farmacolóxica, baseado en variables xenéticas e clínicas capaces de predecir o risco dun individuo de desenvolver o fenotipo de artrose de xeonllo con progresión rápida.[Resumen] Esta tesis doctoral trata de identificar variantes genéticas capaces de alterar el riesgo de desarrollar el fenotipo de artrosis de rodilla con progresión rápida. Para ello, se han empleado técnicas de secuenciación para buscar polimorfismos presentes en el ADN nuclear y en el mitocondrial que puedan favorecer el desarrollo del fenotipo de interés. Se detectó una variante mitocondrial, presente en la posición m.16519C, que se asoció significativamente con la progresión rápida. Tras realizar un pequeño estudio funcional, se detectaron mayores niveles de interleuquina 6 y ciclooxigenasa 2 en los cíbridos portadores de la variante, pudiendo ser el aumento de la respuesta inflamatoria un mecanismo desencadenante en la progresión rápida. Por otro lado, se ha detectado por primera vez en el campo de la artrosis una interacción entre el ADN nuclear y el mitocondrial. La presencia del haplogrupo mitocondrial UK y el polimorfismo nuclear rs12107036 también aumenta el riesgo de progresión rápida en la artrosis de rodilla. Finalmente, se generó un modelo predictivo de gran relevancia clínica y farmacológica, basado en variables genéticas y clínicas capaz de predecir el riesgo que tiene un individuo de desarrollar el fenotipo de artrosis de rodilla con progresión rápida.[Abstract] This thesis project tries to identify genetic variants capable of altering the risk of developing the rapidly progressive phenotype in knee osteoarthritis. To achieve this, sequencing techniques have been used to search for polymorphisms present in nuclear and mitochondrial DNA that may favor the development of the phenotype of interest. A mitochondrial variant, present at m.16519C, was detected and was significantly associated with rapid progression. After carrying out a small functional study, higher levels of interleukin 6 and cyclooxygenase 2 were detected in cybrids carrying this variant, and the increased inflammatory response could be a triggering mechanism for rapid progression. On the other hand, an interaction between nuclear and mitochondrial DNA has been detected for the first time in the field of osteoarthritis. The presence of the mitochondrial haplogroup UK and the rs12107036 nuclear polymorphism also increases the risk of rapid progression in knee osteoarthritis. Finally, a predictive model of great clinical and pharmacological relevance was generated, based on genetic and clinical variables, which is capable of predicting the risk of developing the rapidly progressive phenotype of knee osteoarthritis

Mitochondrial genetics and epigenetics in osteoarthritis

Review[Abstract] During recent years, the significant influence of mitochondria on osteoarthritis (OA), the most common joint disease, has been consistently demonstrated. Not only mitochondrial dysfunction but also mitochondrial genetic polymorphisms, specifically the mitochondrial DNA haplogroups, have been shown to have an important influence on different OA-related features, including the prevalence, severity, incidence, and progression of the disease. This influence could probably be mediated by the role of mitochondria in the regulation of different processes involved in the pathogenesis of OA, such as energy production, the generation of reactive oxygen and nitrogen species, apoptosis, and inflammation. The regulation of these processes is at least partially controlled by the bi-directional communication between the nucleus and mitochondria, which permits the regulation of adaptation to a wide range of stressors and the maintenance of cellular homeostasis. This bi-directional communication consists of an “anterograde regulation” by which the nucleus regulates mitochondrial biogenesis and activity and a “retrograde regulation” by which both mitochondria and mitochondrial genetic variation exert a regulatory signaling control over the nuclear epigenome, which leads to the modulation of nuclear genes. Throughout this mini review, we will describe the evidence that demonstrates the profound influence of the mitochondrial genetic background in the pathogenesis of OA, as well as its influence on the nuclear DNA methylome of the only cell type present in the articular cartilage, the chondrocyte. This evidence leads to serious consideration of the mitochondrion as an important therapeutic target in OA.Instituto de Salud Carlos III; CIBERCB06/01/0040Instituto de Salud Carlos III; RETIC-RIER-RD16/0012/0002Instituto de Salud Carlos III; PRB2-ISCIII-PT17/0019/0014Instituto de Salud Carlos III; PI14/01254Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PI17/00210Instituto de Salud Carlos III; (CPII17/00026

Genetic biomarkers in osteoarthritis: a quick overview

[Abstract] Osteoarthritis (OA) is a chronic musculoskeletal disease with a polygenic and heterogeneous nature. In addition, when clinical manifestations appear, the evolution of the disease is usually already irreversible. Therefore, the efforts on OA research are focused mainly on the discovery of therapeutic targets and reliable biomarkers that permit the early identification of different OA-related parameters such as diagnosis, prognosis, or phenotype identification. To date, potential candidate protein biomarkers have been associated with different aspects of the disease; however, there is currently no gold standard. In this sense, genomic data could act as complementary biomarkers of diagnosis and prognosis or even help to identify therapeutic targets of the disease. In this review, we will describe the most recent advances in genetic biomarkers in OA over the past three years.Instituto de Salud Carlos III; PI17/00210Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PI20/00614Instituto de salud Carlos III; RETIC-RIER-RD16/0012/0002Instituto de Salud Carlos III; PRB3-ISCIII-PT17/0019/0014Xunta de Galicia; IN607A2017/1

Oleate prevents palmitate-induced mitochondrial dysfunction in chondrocytes

[Abstract] The association between obesity and osteoarthritis (OA) in joints not subjected to mechanical overload, together with the relationship between OA and metabolic syndrome, suggests that there are systemic factors related to metabolic disorders that are involved in the metabolic phenotype of OA. The aim of this work is study the effects of palmitate and oleate on cellular metabolism in an “in vitro” model of human chondrocytes. The TC28a2 chondrocyte cell line was used to analyze the effect of palmitate and oleate on mitochondrial and glycolytic function, Adenosine triphosphate (ATP) production and lipid droplets accumulation. Palmitate, but not oleate, produces mitochondrial dysfunction observed with a lower coupling efficiency, maximal respiration and spare respiratory capacity. Glycolytic function showed lower rates both glycolytic capacity and glycolytic reserve when cells were incubated with fatty acids (FAs). The production rate of total and mitochondrial ATP showed lower values in chondrocytes incubated with palmitic acid (PA). The formation of lipid droplets increased in FA conditions, being significantly higher when the cells were incubated with oleic acid (OL). These results may help explain, at least in part, the close relationship of metabolic pathologies with OA, as well as help to elucidate some of the factors that can define a metabolic phenotype in OA.Instituto de Salud Carlos III; PI14/01254Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; RETIC-RIER-RD16/0012/0002Instituto de Salud Carlos III; PRB3-ISCIII-PT17/0019/0014Xunta de Galicia; IN607A2017/1

Single nucleotide polymorphism genes and mitochondrial DNA haplogroups as biomarkers for early prediction of knee osteoarthritis structural progressors: use of supervised machine learning classifiers

[Abstract] Background. Knee osteoarthritis is the most prevalent chronic musculoskeletal debilitating disease. Current treatments are only symptomatic, and to improve this, we need a robust prediction model to stratify patients at an early stage according to the risk of joint structure disease progression. Some genetic factors, including single nucleotide polymorphism (SNP) genes and mitochondrial (mt)DNA haplogroups/clusters, have been linked to this disease. For the first time, we aim to determine, by using machine learning, whether some SNP genes and mtDNA haplogroups/clusters alone or combined could predict early knee osteoarthritis structural progressors. Methods. Participants (901) were first classified for the probability of being structural progressors. Genotyping included SNP genes TP63, FTO, GNL3, DUS4L, GDF5, SUPT3H, MCF2L, and TGFA; mtDNA haplogroups H, J, T, Uk, and others; and clusters HV, TJ, KU, and C-others. They were considered for prediction with major risk factors of osteoarthritis, namely, age and body mass index (BMI). Seven supervised machine learning methodologies were evaluated. The support vector machine was used to generate gender-based models. The best input combination was assessed using sensitivity and synergy analyses. Validation was performed using tenfold cross-validation and an external cohort (TASOAC). Results. From 277 models, two were defined. Both used age and BMI in addition for the first one of the SNP genes TP63, DUS4L, GDF5, and FTO with an accuracy of 85.0%; the second profits from the association of mtDNA haplogroups and SNP genes FTO and SUPT3H with 82.5% accuracy. The highest impact was associated with the haplogroup H, the presence of CT alleles for rs8044769 at FTO, and the absence of AA for rs10948172 at SUPT3H. Validation accuracy with the cross-validation (about 95%) and the external cohort (90.5%, 85.7%, respectively) was excellent for both models. Conclusions. This study introduces a novel source of decision support in precision medicine in which, for the first time, two models were developed consisting of (i) age, BMI, TP63, DUS4L, GDF5, and FTO and (ii) the optimum one as it has one less variable: age, BMI, mtDNA haplogroup, FTO, and SUPT3H. Such a framework is translational and would benefit patients at risk of structural progressive knee osteoarthritis

Mitochondrial DNA (mtDNA) haplogroups J and H are differentially associated with the methylation status of articular cartilage: potential role in apoptosis and metabolic and developmental processes

[Abstract] Objective. To analyze the influence of mitochondrial genome variation on the DNA methylome of articular cartilage. Methods. DNA methylation profiling was performed using data deposited in the NCBI Gene Expression Omnibus database (accession no. GSE 43269). Data were obtained for 14 cartilage samples from subjects with haplogroup J and 20 cartilage samples from subjects with haplogroup H. Subsequent validation was performed in an independent subset of 7 subjects with haplogroup J and 9 with haplogroup H by RNA ‐seq. Correlated genes were validated by real‐time polymerase chain reaction in an independent cohort of 12 subjects with haplogroup J and 12 with haplogroup H. Appropriate analyses were performed using R Bioconductor and qB asePlus software, and gene ontology analysis was conducted using DAVID version 6.8. Results. DNA methylation profiling revealed 538 differentially methylated loci, while whole‐transcriptome profiling identified 2,384 differentially expressed genes, between cartilage samples from subjects with haplogroup H and those with haplogroup J. Seventeen genes showed an inverse correlation between methylation and expression. In terms of gene ontology, differences in correlations between methylation and expression were also detected between cartilage from subjects with haplogroup H and those with haplogroup J, highlighting a significantly enhanced apoptotic process in cartilage from subjects with haplogroup H (P = 0.007 for methylation and P = 0.019 for expression) and repressed apoptotic process in cartilage from subjects with haplogroup J (P = 0.021 for methylation), as well as a significant enrichment of genes related to metabolic processes (P = 1.93 × 10−4 for methylation and P = 6.79 x 10−4 for expression) and regulation of gene expression (P = 0.012 for methylation) in cartilage from subjects with haplogroup H, and to developmental processes (P = 0.015 for methylation and P = 8.25 x 10−12 for expression) in cartilage from subjects with haplogroup J. Conclusion. Mitochondrial DNA variation differentially associates with the methylation status of articular cartilage by acting on key mechanisms involved in osteoarthritis, such as apoptosis and metabolic and developmental processes.Instituto de Salud Carlos III; CIBERCB06/01/0040‐SpainInstituto de Salud Carlos III; CPII17/00026Instituto de Salud Carlos III; PI14/01254Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PI17/0021

A meta-analysis and a functional study support the influence of mtDNA variant m.16519C on the risk of rapid progression of knee osteoarthritis

[Abstract] Objectives: To identify mitochondrial DNA (mtDNA) genetic variants associated with the risk of rapid progression of knee osteoarthritis (OA) and to characterise their functional significance using a cellular model of transmitochondrial cybrids. Methods: Three prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 1095 subjects, the Cohort Hip and Cohort Knee included 373 and 326 came from the PROspective Cohort of Osteoarthritis from A Coruña. mtDNA variants were screened in an initial subset of 450 subjects from the OAI by in-depth sequencing of mtDNA. A meta-analysis of the three cohorts was performed. A model of cybrids was constructed to study the functional consequences of harbouring the risk mtDNA variant by assessing: mtDNA copy number, mitochondrial biosynthesis, mitochondrial fission and fusion, mitochondrial reactive oxygen species (ROS), oxidative stress, autophagy and a whole transcriptome analysis by RNA-sequencing. Results: mtDNA variant m.16519C is over-represented in rapid progressors (combined OR 1.546; 95% CI 1.163 to 2.054; p=0.0027). Cybrids with this variant show increased mtDNA copy number and decreased mitochondrial biosynthesis; they produce higher amounts of mitochondrial ROS, are less resistant to oxidative stress, show a lower expression of the mitochondrial fission-related gene fission mitochondrial 1 and an impairment of autophagic flux. In addition, its presence modulates the transcriptome of cybrids, especially in terms of inflammation, where interleukin 6 emerges as one of the most differentially expressed genes. Conclusions: The presence of the mtDNA variant m.16519C increases the risk of rapid progression of knee OA. Among the most modulated biological processes associated with this variant, inflammation and negative regulation of cellular process stand out. The design of therapies based on the maintenance of mitochondrial function is recommended

mtDNA Haplogroup A Enhances the Effect of Obesity on the Risk of Knee OA in a Mexican Population

[Abstract] To evaluate the influence of mitochondrial DNA haplogroups on the risk of knee OA in terms of their interaction with obesity, in a population from Mexico. Samples were obtained from (n = 353) knee OA patients (KL grade ≥ I) and (n = 364) healthy controls (KL grade = 0) from Mexico city and Torreon (Mexico). Both Caucasian and Amerindian mtDNA haplogroups were assigned by single base extension assay. A set of clinical and demographic variables, including obesity status, were considered to perform appropriate statistical approaches, including chi-square contingency tables, regression models and interaction analyses. To ensure the robustness of the predictive model, a statistical cross-validation strategy of B = 1000 iterations was used. All the analyses were performed using boot, GmAMisc and epiR package from R software v4.0.2 and SPSS software v24. The frequency distribution of the mtDNA haplogroups between OA patients and healthy controls for obese and non-obese groups showed the haplogroup A as significantly over-represented in knee OA patients within the obese group (OR 2.23; 95% CI 1.22-4.05; p-value = 0.008). The subsequent logistic regression analysis, including as covariate the interaction between obesity and mtDNA haplogroup A, supported the significant association of this interaction (OR 2.57; 95% CI 1.24-5.32; p-value = 0.011). The statistical cross-validation strategy confirmed the robustness of the regression model. The data presented here indicate a link between obesity in knee OA patients and mtDNA haplogroup A.This work is supported by Grants from Fondo de Investigación Sanitaria (PI17/00210, PI16/02124, PI20/00614, RETIC-RIER-RD16/0012/0002 and PRB3-ISCIII-PT17/0019/0014) integrated in the National Plan for Scientific Program, Development and Technological Innovation 2013–2016 and funded by the ISCIII-General Subdirection of Assessment and Promotion of Research-European Regional Development Fund (FEDER) “A way of making Europe” and Grant IN607A2017/11 from Xunta de Galicia. The authors further acknowledge AE CICA-INIBIC (ED431E 2018/03) for financial support. IRP is supported by Contrato Miguel Servet-II Fondo de Investigación Sanitaria (CPII17/00026) and AD-S is supported by Grant IN606A-2018/023 from Xunta de Galicia, Spain. The Biomedical Research Networking Center (CIBER) is an initiative from Instituto de Salud Carlos III (ISCIII)Xunta de Galicia; IN607A2017/11Xunta de Galicia; ED431E 2018/03Xunta de Galicia; IN606A-2018/02