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Danon disease is a fatal X-linked recessive disease caused by a lack of expression of the lysosomal associated membrane protein type 2 (LAMP2), leading to severe vacuolar cardiomyopathy. Most patients with Danon progress to end-stage heart failure or death without advanced therapies. In this study, we investigated the therapeutic efficacy of systemic transplantation of ex vivo gene-modified Lamp2-/- (Lamp2 KO) hematopoietic stem and progenitor cells (HSPCs) using a lentiviral vector containing the human LAMP2B transgene, pCCL-LAMP2B, in the mouse model of Danon disease, Lamp2 KO mice. Transplanted pCCL-LAMP2B-HSPCs efficiently engrafted and differentiated into macrophages in heart. LAMP2B was found in cardiomyocytes and improved cardiac systolic as well as locomotor functions were observed in pCCL-LAMP2B-HSPCs recipient mice compared to non-treated or Lamp2 KO mice receiving Lamp2 KO HSPCs. In addition, we also demonstrated that pCCL-LAMP2B-HSPCs rescued autophagic flux and activity in the heart. In vitro, we cocultured WT macrophages with Lamp2 KO fibroblasts and observed transfer of LAMP2B and rescue of the autophagic flux in the diseased cells confirming cross-correction despite LAMP2B being a lysosomal transmembrane protein.

Patients with inherited retinal dystrophies (IRDs) were recruited from two understudied populations: Mexico and Pakistan as well as a third well-studied population of European Americans to define the genetic architecture of IRD by performing whole-genome sequencing (WGS). Whole-genome analysis was performed on 409 individuals from 108 unrelated pedigrees with IRDs. All patients underwent an ophthalmic evaluation to establish the retinal phenotype. Although the 108 pedigrees in this study had previously been examined for mutations in known IRD genes using a wide range of methodologies including targeted gene(s) or mutation(s) screening, linkage analysis and exome sequencing, the gene mutations responsible for IRD in these 108 pedigrees were not determined. WGS was performed on these pedigrees using Illumina X10 at a minimum of 30X depth. The sequence reads were mapped against hg19 followed by variant calling using GATK. The genome variants were annotated using SnpEff, PolyPhen2, and CADD score; the structural variants (SVs) were called using GenomeSTRiP and LUMPY. We identified potential causative sequence alterations in 61 pedigrees (57%), including 39 novel and 54 reported variants in IRD genes. For 57 of these pedigrees the observed genotype was consistent with the initial clinical diagnosis, the remaining 4 had the clinical diagnosis reclassified based on our findings. In seven pedigrees (12%) we observed atypical causal variants, i.e. unexpected genotype(s), including 4 pedigrees with causal variants in more than one IRD gene within all affected family members, one pedigree with intrafamilial genetic heterogeneity (different affected family members carrying causal variants in different IRD genes), one pedigree carrying a dominant causative variant present in pseudo-recessive form due to consanguinity and one pedigree with a de-novo variant in the affected family member. Combined atypical and large structural variants contributed to about 20% of cases. Among the novel mutations, 75% were detected in Mexican and 50% found in European American pedigrees and have not been reported in any other population while only 20% were detected in Pakistani pedigrees and were not previously reported. The remaining novel IRD causative variants were listed in gnomAD but were found to be very rare and population specific. Mutations in known IRD associated genes contributed to pathology in 63% Mexican, 60% Pakistani and 45% European American pedigrees analyzed. Overall, contribution of known IRD gene variants to disease pathology in these three populations was similar to that observed in other populations worldwide. This study revealed a spectrum of mutations contributing to IRD in three populations, identified a large proportion of novel potentially causative variants that are specific to the corresponding population or not reported in gnomAD and shed light on the genetic architecture of IRD in these diverse global populations., Author summary The study was performed to identify the underlying cause of inherited retinal degeneration (IRD) in 409 individuals from 108 families. Primarily, these families were recruited from three different geographic regions: Mexico, Pakistan and European Americans from the United States. Blood samples were collected from all individuals for genome analysis. This analysis detected causative variants in 61 out of the 108 pedigrees. A total of 93 gene variants were found in the 61 families. Among these, 54 were previously reported as causative variants and the remaining 39 have not been reported in IRD pedigrees. Interestingly, 54% of these novel variants were not listed in gnomAD. In addition to these findings, complex causative genotypes were observed in 20% of pedigrees. Overall, causative variants were detected in 63% Mexican, 60% Pakistani and 45% European American pedigrees. This study revealed the distribution of IRD causative variants in pedigrees with diverse ethnic and geographic backgrounds.

Patients harboring homozygous c.498_499insC mutations in MFRP demonstrate hyperopia, microphthalmia, retinitis pigmentosa, retinal pigment epithelial atrophy, variable degrees of foveal edema, and ...

Late‐onset retinal degeneration (L‐ORD) is an autosomal dominant macular degeneration characterized by the formation of sub‐retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L‐ORD results from mutations in the C1q‐tumor necrosis factor‐5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L‐ORD pathology, we used a human cDNA library yeast two‐hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM‐Ch) from wild‐type (Wt), heterozygous S163R Ctrp5 mutation knock‐in (Ctrp5S163R/wt), and homozygous knock‐in (Ctrp5S163R/S163R) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C‐terminal PDZ‐binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R‐CTRP5 protein also binds to HTRA1 but is resistant to HTRA1‐mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM‐Ch of Ctrp5S163R/S163R and Ctrp5S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L‐ORD pathology.

Danon disease (DD) is a rare X-linked autophagic vacuolar myopathy associated with multiorgan dysfunction, including the heart, skeletal muscle, and liver. There are no specific treatments, and most male patients die from advanced heart failure during the second or third decade of life. DD is caused by mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene, a key mediator of autophagy. LAMP2 has three isoforms: LAMP2A, LAMP2B, and LAMP2C. LAMP2B is the predominant isoform expressed in cardiomyocytes. This study evaluates the efficacy of human LAMP2B gene transfer using a recombinant adeno-associated virus 9 carrying human LAMP2B (AAV9.LAMP2B) in a Lamp2 knockout (KO) mouse, a DD model. AAV9.LAMP2B was intravenously injected into 2- and 6-month-old Lamp2 KO male mice to assess efficacy in adolescent and adult phenotypes. Lamp2 KO mice receiving AAV9.LAMP2B demonstrated dose-dependent restoration of human LAMP2B protein in the heart, liver, and skeletal muscle tissue. Impaired autophagic flux, evidenced by increased LC3-II, was abrogated by LAMP2B gene transfer in all tissues in both cohorts. Cardiac function was also improved, and transaminases were reduced in AAV9.LAMP2B-treated KO mice, indicating favorable effects on the heart and liver. Survival was also higher in the older cohort receiving high vector doses. No anti-LAMP2 antibodies were detected in mice that received AAV9.LAMP2B. In summary, LAMP2B gene transfer improves metabolic and physiologic function in a DD murine model, suggesting that a similar therapeutic approach may be effective for treating patients with this highly morbid disease.

Patients harboring homozygous c.498_499insC mutations in MFRP demonstrate hyperopia, microphthalmia, retinitis pigmentosa, retinal pigment epithelial atrophy, variable degrees of foveal edema, and optic disc drusen. The disease phenotype is variable, however, with some patients maintaining good central vision and cone function till late in the disease. A knock-in mouse model with the c.498_499insC mutation in Mfrp (Mfrp KI/KI) was developed to understand the effects of these mutations in the retina. The model shares many of the features of human clinical disease, including reduced axial length, hyperopia, retinal degeneration, retinal pigment epithelial atrophy, and decreased electrophysiological responses. In addition, the eyes of these mice had a significantly greater refractive error (p

Whole genome sequencing (WGS) was performed to identify the variants responsible for inherited retinal degeneration (IRD) in a Caucasian family. Segregation analysis of selected rare variants with pathogenic potential identified a set of compound heterozygous changes p.Arg266*:c.796C>T and p.Ala568Thr:c.1702G>A in the intraflagellar transport protein-88 (IFT88) gene segregating with IRD. Expression of IFT88 with the p.Arg266* and p.Ala568Thr mutations in mIMDC3 cells by transient transfection and in HeLa cells by introducing the mutations using CRISPR-cas9 system suggested that both mutations result in the formation of abnormal ciliary structures. The introduction of the IFT88 p.Arg266* variant in the homozygous state in HeLa cells by CRISPR-Cas9 genome-editing revealed that the mutant transcript undergoes nonsense-mediated decay leading to a significant depletion of IFT88 transcript. Additionally, abnormal ciliogenesis was observed in these cells. These observations suggest that the rare and unique combination of IFT88 alleles observed in this study provide insight into the physiological role of IFT88 in humans and the likely mechanism underlying retinal pathology in the pedigree with IRD.

Retinitis pigmentosa (RP) causes progressive photoreceptor loss resulting from mutations in over 80 genes. This study identified the genetic cause of RP in three members of a non-consanguineous pedigree. Detailed ophthalmic evaluation was performed in the three affected family members. Whole exome sequencing (WES) and whole genome sequencing (WGS) were performed in the three affected and the two unaffected family members and variants were filtered to detect rare, potentially deleterious variants segregating with disease. WES and WGS did not identify potentially pathogenic variants shared by all three affected members. However, WES identified a previously reported homozygous nonsense mutation in KIZ (c.226C>T, p.Arg76*) in two affected sisters, but not in their affected second cousin. WGS revealed a novel 1.135 kb homozygous deletion in a retina transcript of C21orf2 and a novel 30.651 kb heterozygous deletion in CACNA2D4 in the affected second cousin. The sisters with the KIZ mutation carried no copies of the C21orf2 or CACNA2D4 deletions, while the second cousin with the C21orf2 and CACNA2D4 deletions carried no copies of the KIZ mutation. This study identified two independent, homozygous mutations in genes previously reported in autosomal recessive RP in a non-consanguineous family, and demonstrated the value of WGS when WES fails to identify likely disease-causing mutations.

Not all patients with osteoarthritis (OA) show the same disease progression, as some of them remain relatively stable over time, while others progress to severe structural deterioration of the joint. In this sense, the main goal of both genetic and protein biomarkers in OA is to predict not only the risk of OA at an earlier stage of the disease but also which OA patients are more likely to progress to severe disease. Taking into account the incidence of the mitochondria and the mtDNA haplogroups in the pathogenesis of OA, the main objective of this work was to evaluate the incidence of the mtDNA haplogroups in the radiographic progression of the OA disease in a well-characterized follow-up cohort of Spanish patients. DNA from 281 OA patients from Hospital Universitario A Coruña was isolated to determine the European mtDNA haplogroups. Knee or hip radiographs from all affected joints were obtained at two time points with at least 36 months apart. Radiographs were evaluated using the Kellgren/Lawrence (K/L) scale; radiographic OA progression was defined as any radiographic worsening of the K/L joint score. Statistical analyses included Kaplan-Meier survival curves and Cox regression models. Patients belonging to the cluster TJ showed a slower radiographic OA progression than patients in the cluster KU (p = 0.036). Moreover, patients carrying the most common mtDNA haplogroup H are more apt to require total joint replacement surgery than non-H patients (p = 0.049). The inherited mitochondrial variants influence the radiographic progression of OA and could be considered among the genetic variants taken into account when the radiographic progression of OA is analyzed.

Objective To compare the frequency distribution of the mtDNA haplogroups in OA patients and healthy controls between the United Kingdom (UK) and Spain. Methods We used the single base extension (SBE) assay to obtain the European mtDNA haplogroups in 1471 OA patients and 406 healthy controls from Spain, and 453 OA patients and 280 healthy controls from the UK. Some differential haplogroup J-related single nucleotide polymorphisms (SNPs) between both populations were analyzed. The whole data was analyzed with SPSS software (v.18) following appropriate approaches that included chi-square contingency tables and logistic regression models adjusting by gender and age. Results The haplogroup J appeared underrepresented in OA patients from Spain when compared with healthy controls (OR = 0.636; 95% CI: 0.444–0.911; p = 0.013). Individuals from the UK carrying the haplogroup T showed a decreased risk of OA (OR = 0.574; 95% CI: 0.350–0.939; p = 0.027). The comparison of the frequency distribution of the haplogroup J between the UK and Spain showed a decreased presence of this haplogroup in healthy controls from the UK when compared with healthy controls from Spain that is in borderline of the statistical significance (p = 0.06). The analysis of some haplogroup J-related SNPs in OA patients and healthy controls from Spain and the UK showed that the SNP m.3394t>c appeared underrepresented in the UK cohort (p = 0.038). Conclusions The proposed mitochondrial uncoupling mechanism derived from the mtDNA haplogroups J and T could be behind their protective role against OA. The different association found in Spain and the UK could reflect the adaptation of the mtDNA haplogroups to different climatic patterns. The genetic composition of the haplogroup J between the UK and Spain seems to be slightly different, being the m.3394t>c SNP one of the differentially expressed haplogroup J-related polymorphisms.

[Resumen] La artrosis es la enfermedad reumatológica más común y se caracteriza por una degeneración del cartilago articular junto con el resto de estructuras que forman parte de la articulación. Su prevalencia es elevada y se espera que se incremente notablemente en las próximas décadas debido al envejecimiento de la población. Es la principal causa de incapacidad laboral y una de las principales razones de consulta en medicina general. La artrosis conlleva unos elevados costes económicos tanto desde el punto de vista laboral, como médicos y de calidad de vida. Es una enfermedad compleja y multifactorial en la que interactúan tanto factores ambientales como genéticos. En los últimos años se ha puesto de manifiesto la implicación de la mitocondria en la artrosis a través de diferentes estudios. A pesar de que el metabolismo energético del condrocito es fundamentalmente glucolítico, la mitocondria tiene un papel relevante en la artrosis no solo por la aportación energética que hace a través del proceso de fosforilación oxidativa en la producción de Arp, sino que además la mitocondria constituye la principal fuente de generación de especies reactivas de oxígeno, además de regular los mecanismos de apoptosis en el condrocito, único tipo celular presente en el cartilago, entre otras funciones celulares. La mitocondria posee su propio material genético que, debido a la adaptación a las condiciones climáticas y ambientales sufridas a lo largo de las diferentes migraciones de la especie humana desde su origen africano, ha adquirido una serie de mutaciones estables que permiten caracterizar a las poblaciones humanas según su origen geográfico. Los diferentes conjuntos de polimorfismos característicos de cada una de estas poblaciones han dado lugar a los haplogrupos mitocondriales. Debido a su origen adaptativo a las condiciones ambientales, cada haplogrupo presenta unos mecanismos y funciones metabólicas diferentes. Estas diferencias se han relacionado con la predisposición a padecer diferentes enfermedades, entre las que se encuentra la artrosis. El objetivo de esta tesis es replicar los resultados obtenidos por RegoPérez et al. sobre la influencia de los haplogrupos mitocondriales en la prevalencia de la artrosis en diferentes cohortes mundiales, así como evaluar la participación de los haplogrupos en la progresión de la enfermedad. El primer trabajo de esta tesis analiza la implicación de los haplogrupos en la artrosis comparando los porcentajes de prevalencia en una cohorte del norte de España con los de una cohorte del Reino Unido. Los resultados obtenidos mostraron que existía una asociación diferencial entre las frecuencias de los haplogrupos de ambas cohortes con la artrosis. De manera que, en la cohorte española es el haplogrupo J el que actúa como factor protector frente a la artrosis, mientras que en la cohorte británica es el haplogrupo T el que muestra una asociación estadísticamente significativa como factor protector de artrosis. En el segundo trabajo de esta tesis se analizó la relación de los haplogrupos con la progresión en artrosis. Para ello se utilizaron los datos radiográficos de una cohorte de pacientes del hospital Universitario de A Coruña considerando progresión radiográfica como el incremento en el grado KL durante un periodo de seguimiento de al menos 36 meses. Los resultados obtenidos pusieron de manifiesto la influencia de los haplogrupos sobre la progresión en artrosis, siendo los pacientes pertenecientes al clúster TJ los que presentaban una progresión más lenta de la patología mientras que los portadores del haplogrupo H mostraron una mayor predisposición a alcanzar la prótesis articular .. El tercer trabajo consistió en replicar los resultados obtenidos en el segundo trabajo en una amplia y bien caracterizada cohorte Norteamericana de pacientes artrósicos de rodilla pertenecientes a la Osteoarthritis Initiative. En este estudio se analizó la influencia de los haplogrupos en la progresión de la enfermedad a través de las magnitudes (grado KL, presencia de osteofitos, esclerosis subcondral y la reducción de espacio articular) obtenidas de las imágenes radiográficas de las rodillas de los pacientes incluidos en el estudio. Asimismo, también se estudió la degradación estructural de la articulación mediante las medidas del grosor y el volumen de cartílago en las diferentes zonas que componen la rodilla, obtenidas a partir de imágenes de resonancia magnética. Los resultados mostraron que el haplogrupo T era el que presentaba una progresión radiográfica más lenta que el resto de haplogrupos así como una menor pérdida de grosor y volumen de cartílago en el tiempo en diferentes zonas del compartimento medial de la rodilla. Para finalizar se realizó también un estudio sobre la capacidad de los haplogrupos para distinguir entre diferentes fenotipos de pacientes artrósicos analizando los niveles en suero de algunos de los principales marcadores moleculares de artrosis. Los resultados obtenidos permitieron distinguir claramente dos fenotipos de artrosis; de este modo, los pacientes con mayores niveles séricos de marcadores catabólicos fueron los pacientes portadores del haplogrupo H frente a los pacientes portadores del haplogrupo J. Los resultados de este trabajo mostraron que los haplogrupos mitocondriales son posibles biomarcadores complementarios de artrosis con aplicación en la práctica clínica. En resumen, los resultados de los 4 trabajos que forman parte de esta tesis ponen de manifiesto la influencia de los haplogrupos mitocondriales en la patología artrósica. Las diferentes asociaciones encontradas entre las distintas cohortes analizadas podrían reflejar la adpatación de los haplogrupos a los diferentes patrones climáticos., [Resumo] A artrose é a enfermidade reumatolóxica máis común e caracterízase por unha dexeneración da cartilaxe articular xunto co resto de estruturas que forman parte da articulación. A súa prevalencia é elevada e esperase que se incremente notablemente nas próximas décadas debido o envellecemento da poboación. É a principal causa de incapacidade laboral e unha das principais razóns de consulta na medicina xeral. A artrose leva consigo uns elevados custos económicos tanto dende o punto de vista laboral como médico e de calidade de vida. É unha enfermidade complexa e multifactorial na que interactuan tanto factores ambientais como xenéticos. Nos últimos anos púxose de manifesto a implicación da mitocondria na artrose a través de diferentes estudos. A pesar de que o metabolismo enerxético do condrocito é fundamentalmente glucolítico, a mitocondria ten un papel relevante na amose non só pola achega enerxética que fai a través do proceso de fosforilación oxidativa na produción de ATP, senón que ademais a mitocondria constitúe a principal fonte de xeración de especies reactivas de oslxeno, ademais de regular os mecanismos de apoptose no condrocito, único tipo celular presente na cartilaxe, entre outras funcións celulares. A mitocondria posúe ademais o seu propio material xenético que, debido á adaptación ás condicións climáticas e ambientais sufridas ao longo das diferentes migracións da especie humana dende a súa orixe africana, adquiriu unha serie de mutación s estables que permitiron caracterizar as poboacións humanas segundo a súa orixe xeográfica. Os diferentes conxuntos de polimorfismos característicos de cada unha de stas poboaci6ns de ron lugar aos haplogrupos mitocondriais. Debido á súa orixe adaptativa ás condicións ambientais, cada haplogrupo presenta uns mecanismos e funcións metabólicas diferentes. Estas diferenzas relacionáronse coa predisposición a padecer diferentes enfermidades, entre as que se en contra a artrose. O obxectivo desta tese é replicar os resultados obtidos por Rego-Pérez et al. sobre a influencia dos haplogrupos mitocondriais na prevalencia da artrose en diferentes cohortes mundiais, así como avaliar a participación dos haplogrupos na progresión da enfermidade. O primeiro traballo desta tese analiza a implicación dos haplogrupos na artrose comparando as porcentaxes de prevalencia nunha cohorte do norte de España cos dunha cohorte do Reino Unido. Os resultados obtidos mostraron que existía unha asociación diferencial entre as frecuencias dos haplogrupos de ambas as dúas cohortes coa artrose. De maneira que, na cohorte española é o haplogrupo laque actúa como factor protector fronte á artrose, mentres que na cohorte británica é o haplogrupo T o que mostra unha asociación estatísticamente significativa como factor protector de artrose. No segundo trabaIlo desta tese analizouse a relación dos haplogrupos coa progresión en artrose. Para iso utilizáronse os datos clínicos e radiográficos dunha cohorte de pacientes do hospital Universitario da Coruña avaliando a progresión radiográfica a través do incremento no grao KL durante un período de seguimento de polo menos 36 meses. Os resultados obtidos puxeron de manifesto a influencia dos haplogrupos sobre a progresión en artrose, senda os pacientes pertencentes ao c1úster TI os que presentaban unha progreslOn máis lenta da patoloJÓa, mentres que os portadores do haplogrupo H amos aro n unha maior predisposición a alcanzar a prótese articular. O terceiro traballo desta tese consistiu en replicar os resultados obtidos no segundo traballo nunha ampla e ben caracterizada cohorte norteamericana de pacientes artrósicos de xeonllo pertencentes á Osteoarthritis lnitiative. Neste estudo analizouse a influencia dos haplogrupos na progresión da enfermidade a través das magnitudes (grao KL, presenza de osteofitos, esclerose subcondral e a redución de espazo articular) obtidas das imaxes radiográficas dos xeonllos dos pacientes incluidos no estudo. Asimesmo tamén se estudou a degradación estrutural da articulación mediante as medidas do grosor e o volume de cartilaxe nas diferentes zonas que compoñen o xeonllo, obtidas a partires de imaxes de resonancia magnética. Os resultados mostraron que o haplogrupo T era o que presentaba unha progresión radiográfica máis lenta que o resto de haplogrupos así como unha menor perda de grosor e volume de cartilaxe no tempo en diferentes zonas do compartimento medial do xeonllo. Para finalizar realizo use tamén un estudo sobre a capacidad e dos haplogrupos para distinguir entre diferentes fenotipos de pacientes artrósicos analizando os niveis en soro dalgúns dos principais marcadores moleculares de artrose. Os resultados obtidos permitiron distinguir claramente dous fenotipos de artrose, deste modo, os pacientes con maiores niveis sérico s de marcadores catabólicos foron os pacientes portadores do haplogrupo H fronte aos pacientes portadores do haplogrupo j. Os resultados deste traballo mostraron que os haplogrupos mitocondriais son posibles biomarcadores complementarios de artrose con aplicación na práctica clínica. En resumo, os resultados dos 4 traballos que forman parte desta tese poñen de manifesto a influencia dos haplogrupos mitocondriais na patoloxía artrósica. As diferentes asociacións atopadas entre as distintas poboacións analizadas poderían estar a reflexar a adaptación dos haplogrupos aos diferentes patróns climáticos., [Abstract]Osteoartritis (DA) is the most common rheumatic disease related to ageing and is characterized by the degeneration of articular cartilage together witb the rest of joint structures. Since DA is an age-related disease, its prevalence and burden are expected to increase substantially in the near future due to demographic changes and ageing population. DA is the leading cause ofwork incapacitation and one ofthe most common reasons for visiting primary care physicians. The economic cost of DA is also particularly high, resulting in a decreased quality oflife, hospitalizations and loss ofproductivity. DA is a multifactorial disease in which both enviromental and genetic factors interact. In recent years a growing body of evidence suggests the implication of the mitochondria in the pathogenesis of DA, even though the glycolitic nature of articular chondrocytes. In addition to the energetic contribution, mitocondria is the main source of reactive oxygen species which lead to cause sigRificant oxidative damage to the cell, and one of the major celular pathways of apoptosis in DA chondrocytes, among other cellular functions. Mitochondria also have their own DNA and, as the result of an adaptative process to the new climatic and environmental conditions suffered throughout the different migrations humans from their African origin, it acquired a series of stable mutations that allow nowadays the characterization of human populations according to their geographical origino So, a particular set of single nucleotide polymorphisms defines each of the mtDNA haplogroups, and these haplogroups represent populations of different ancestral origins. As a result of their enviromental adaptation, each haplogroup shows different mechanisms and metabolic functions. Recent studies provided evidence that mtDNA haplogroups inlluence the penetrance and c1inical severity of modern diseases, including DA. The aim ofthis thesis is to reply the results obtained by Rego-Pérez et al. on the inlluence of the mitochondrial haplogroups in the prevalence of DA in different world cohorts, as well as to evaluate the participation of the haplogrupos in the progression ofthe disease. In the first chapter of this thesis we analyze the frecuency distribution of the mtDNA haplogroups in two DA cohorts of populations from different geograpbic locations. The results obtained showed a differential association among the frequencies of the haplogroups of both cohorts with DA. The haplogroup J appeared underrepresented in DA patients from 5pain when compared with the healthy controls whereas the haplogroup T showed a decreased risk of DA in the UK cohort. In the second work of this thesis we analyzed the inlluence of the mtDNA haplogroups on the radiographic progression of DA. We evaluated the radiographic progression in a well-characterized follow-up cohort of 5panish patients, from Hospital Universitario de A Coruña, defining DA progression as any radiographic worsening of the KL score in a follow up period of not less than 36 months between radiographs. The results showed that patients belonging to cluster TJ had a slower DA progression and patients with mtDNA haplogroup H were more apt to require joint replacement surgery than non-H carriers. In the third work we evaluated the occurrence of the mtDNA haplogroups in the progression of knee DA using the longitudinal data obtained from radiographs and magentic resonance images of knee DA patients belonging to the progression subcohort of the Dsteoarthritis Initiative. In this study the progression criteria was defined as an increase of at least one KL grade in either knee; additionally, we also analyzed the progression ofjoint space narrowing, osteophytes and subchondral sclerosis in the medial compartment where the progression criteria was an increase of at least one DARSI atlas grade in either knee. We also analyzed the cartilage integrity by measuring cartilage thickness and volume through magnetic resonance images from knee DA patients. The results revealed that patients carrying mtDNA haplogroup T show a lower cumulative probability of radiographic progression as well as a significant reduced loss of knee cartilage thickness and volume over tiem in the medial compartment of the knee. Finally, the fourth study aims to assess a mitochondrion-related phenotype in DA patients. We analyzed the serum levels of DA-related biomarlers between DA patients and healthy controls in carriers of haplogroups H and ¡. The results showed two different mitochondrion-related DA phenotypes clearly defined, thereby, patients that carry haplogroup H showed higher levels of catabolic markers of DA than DA patients carrying the mtDNA haplogroup ¡. Briefly, the results of the 4 works that form part of this thesis show the influence of the mitochondrial haplogroups in the DA disease. The different association found among cohorts could reflect the adaptation of the mtDNA haplogroups to different climatic patterns.

OBJECTIVE: Several lines of evidence suggest that estrogens influence the development of osteoarthritis (OA). The aim of this study was to explore the association of two common polymorphisms within the aromatase (CYP19A1) and estrogen receptor (ER) alpha (ESR1) genes with severe OA of the lower limbs. METHODS: The rs1062033 (CYP19A1) and rs2234693 (ESR1) single nucleotide polymorphisms were genotyped in 5528 individuals (3147 patients with severe hip or knee OA, and 2381 controls) from four centres in Spain and the United Kingdom. Gene expression was measured in femoral bone samples from a group of patients. RESULTS: In the global analysis, both polymorphisms were associated with OA, but there was a significant sex interaction. The GG genotype at rs1062033 was associated with an increased risk of knee OA in women [odds ratio (OR) 1.23; P=0.04]. The CC genotype at rs2234693 tended to be associated with reduced OA risk in women (OR 0.76, P=0.028, for knee OA; OR=0.84, P=0.076 for hip OA), but with increased risk of hip OA in men (OR 1.28; P=0.029). Women with unfavourable genotypes at both loci had an OR of 1.61 for knee OA (P=0.006). The rs1062033 genotype associated with higher OA risk was also associated with reduced expression of the aromatase gene in bone. CONCLUSIONS: Common genetic variations of the aromatase and ER genes are associated with the risk of severe OA of the large joints of the lower limb in a sex-specific manner. These results are consistent with the hypothesis that estrogen activity may influence the development of large-joint OA.

Objective. To conduct a replication study and meta-analysis involving the study of mtDNA variants in the radiographic progression of OA in different cohorts worldwide, including Cohort Hip and Cohort Knee (CHECK), the OA Initiative and a cohort from Spain. Methods. The influence of the haplogroups in the rate of radiographic progression at 96 months in 431 subjects from CHECK was assessed in terms of Kellgren and Lawrence (KL) grade. Progression was defined as a change from KL ⩾ 1 at baseline to any higher grade during the follow-up. Extended Cox proportional hazard models were used to analyse the influence of mtDNA variants in the rate of radiographic knee OA progression. A subsequent meta-analysis of 1603 subjects following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines was conducted to combine the data of individual studies. A sensitivity analysis was performed to validate the stability of the results. Results. CHECK subjects carrying the haplogroup T showed the lowest rate of radiographic knee OA progression [hazard ratio (HR) 0.645 (95% CI 0.419, 0.978); P < 0.05]. When pooled, subjects within the superhaplogroup JT showed the same trend [HR 0.707 (95% CI 0.501, 0.965); P < 0.05]. BMI [HR 1.046 (95% CI 1.018, 1.073); P < 0.05] and bilateral OA [HR 2.266 (95% CI 1.733, 2.954); P < 0.05] at baseline are risk factors for radiographic knee OA progression as well. In the meta-analysis there was a reduced rate of radiographic progression in subjects with haplogroup T [HR 0.612 (95% CI 0.454, 0.824); P = 0.001] or in the superhaplogroup JT [HR 0.765 (95% CI 0.624, 0.938); P = 0.009]. Sensitivity analysis revealed that the results were robust. Conclusion. The mtDNA variants in the superhaplogroup JT associate with a reduced rate of radiographic OA progression. The mtDNA polymorphisms in the superhaplogroup JT emerge as potential complementary genetic biomarkers for disease progression.

Retinitis pigmentosa (RP) causes progressive photoreceptor loss resulting from mutations in over 80 genes. This study identified the genetic cause of RP in three members of a non-consanguineous pedigree. Detailed ophthalmic evaluation was performed in the three affected family members. Whole exome sequencing (WES) and whole genome sequencing (WGS) were performed in the three affected and the two unaffected family members and variants were filtered to detect rare, potentially deleterious variants segregating with disease. WES and WGS did not identify potentially pathogenic variants shared by all three affected members. However, WES identified a previously reported homozygous nonsense mutation in KIZ (c.226C>T, p.Arg76*) in two affected sisters, but not in their affected second cousin. WGS revealed a novel 1.135 kb homozygous deletion in a retina transcript of C21orf2 and a novel 30.651 kb heterozygous deletion in CACNA2D4 in the affected second cousin. The sisters with the KIZ mutation carried no copies of the C21orf2 or CACNA2D4 deletions, while the second cousin with the C21orf2 and CACNA2D4 deletions carried no copies of the KIZ mutation. This study identified two independent, homozygous mutations in genes previously reported in autosomal recessive RP in a non-consanguineous family, and demonstrated the value of WGS when WES fails to identify likely disease-causing mutations.

Objective: To evaluate the influence of the mtDNA haplogroups on knee osteoarthritis progression in Osteoarthritis Initiative (OAI) participants through longitudinal data from radiographs and magnetic resonance imaging (MRI). Methods: Four-year knee osteoarthritis progression was analyzed as increase in Kellgren and Lawrence (KL) grade, in addition to increase in OARSI atlas grade for joint space narrowing (JSN), osteophytes and subchondral sclerosis in the tibia medial compartment of 891 Caucasian individuals from the progression subcohort. The influence of the haplogroups on the rate of structural progression was also assessed as the four-year change in minimum joint space width (mJSW in millimetres) in both knees of (n = 216) patients with baseline unilateral medial-tibiofemoral JSN. Quantitative cartilage measures from longitudinal MRI data were those related to cartilage thickness and volume with a 24 month follow-up period (n = 381). Results: During the four-year follow-up period, knee OA patients with the haplogroup T showed the lowest increase in KL grade (Hazard Risk [HR] = 0.499; 95% Confidence Interval [CI]: 0.261-0.819; p