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Venous thromboembolism is a complex disease with a high heritability. There are significant associations among Factor XI (FXI) levels and SNPs in the KNG1 and F11 loci. Our aim was to identify the genetic variation of KNG1 and F11 that might account for the variability of FXI levels. The KNG1 and F11 loci were sequenced completely in 110 unrelated individuals from the GAIT-2 (Genetic Analysis of Idiopathic Thrombophilia 2) Project using Next Generation Sequencing on an Illumina MiSeq. The GAIT-2 Project is a study of 935 individuals in 35 extended Spanish families selected through a proband with idiopathic thrombophilia. Among the 110 individuals, a subset of 40 individuals was chosen as a discovery sample for identifying variants. A total of 762 genetic variants were detected. Several significant associations were established among common variants and low-frequency variants sets in KNG1 and F11 with FXI levels using the PLINK and SKAT packages. Among these associations, those of rs710446 and five low-frequency variant sets in KNG1 with FXI level variation were significant after multiple testing correction and permutation. Also, two putative pathogenic mutations related to high and low FXI levels were identified by data filtering and in silico predictions. This study of KNG1 and F11 loci should help to understand the connection between genotypic variation and variation in FXI levels. The functional genetic variants should be useful as markers of thromboembolic risk.

Femoral neck geometry parameters are believed to be as good as bone mineral density as independent factors in predicting hip fracture risk. This study was conducted to analyze the roles of genetic and environmental factors in femoral properties measured in a sample of Spanish families with osteoporotic fractures and extended genealogy. The "Genetic Analysis of Osteoporosis (GAO) Project" involved 11 extended families with a total number of 376 individuals. We studied three categorical phenotypes of particular clinical interest and we used a Hip structural analysis based on DXA to analyze 17 strength and geometrical phenotypes of the hip. All the femoral properties had highly significant heritability, ranging from 0.252 to 0.586. The most significant correlations were observed at the genetic level (rho(G)). Osteoporotic fracture status (Affected 2) and, particularly, low bone mass and osteoporotic condition (Affected 3) had the highest number of significant genetic correlations with diverse femoral properties. In conclusion, our findings suggest that a relatively simple and easy to use method based on DXA studies can provide useful data on properties of the Hip in clinical practice. Furthermore, our results provide a strong motivation for further studies in order to improve the understanding of the pathophysiological mechanism underlying bone architecture and the genetics of osteoporosis.

Introducció: La malaltia venosa crònica (MVC) és una de les patologies més prevalent a la nostra societat, ocasionant importants costos socioeconòmics i sanitaris. La seva etiologia és desconeguda, i es proposa factors de risc com l’edat, el sexe, l’obesitat o els antecedents familiars. Clàssicament s’ha donat com a cert el component familiar, però la seva relació amb la malaltia tromboembòlica venosa (MTEV) és controvertida. Pretenem demostrar que hi ha prou evidència per confirmar l’origen genètic de la MVC i la relació amb la MTEV. Justificació Científica: No hi ha fins ara cap estudi realitzat en famílies extenses on es demostri origen genètic de la MVC. No hem pogut demostrar la relació genètica entre MVC i MTEV. Aquests resultats poden oferir un nou enfocament a les dues patologies i noves vies de recerca. Hipòtesi: · La MVC és una malaltia multifactorial i complexa el desenvolupament i la progressió de la qual depèn de factors ambientals i genètics, i els factors genètics són l’element preponderant. · Hi ha variants genètiques concretes associades a un augment del risc de MVC. · Hi ha una relació entre la MVC i la MTEV amb una base genètica comuna que influeix en la predisposició a patir les dues patologies. Objectius: · Estimar l’heretabilitat de la MVC a un estudi de famílies extenses. · Identificar gens que participin en la variabilitat de fenotips, associats a la predisposició a presentar MVC, mitjançant l’anàlisi del genoma complet. · Realitzar una anàlisi d’associació de tot el genoma, determinant el mecanisme d’acció de les variants genètiques identificades. · Analitzar les correlacions genètiques i ambientals entre la MVC i la MTEV utilitzant fenotips intermediaris relacionats. Disseny de l’estudi: Estudi de cohorts en famílies extenses prospectiu, observacional, analític i unicèntric. Per als estudis genètics en famílies extenses no es pot calcular la mida mostral, ja que no podem preveure els efectes ni els gens implicats en la patologia estudiada. Resultats: La mostra va consistir en 895 individus. Van ser diagnosticats de MVC 60, 39 dones (65%) i 21 homes (35%). Entre les variables poblacionals analitzades van mostrar una relació estadísticament significativa amb la MVC l’edat (p=2,3x10-06), i el sexe (p=0,008). L’heretabilitat (h2) de l’EVC va ser del 78% (p=2,75x10-8). En els fenotips de l’hemostàsia es van correlacionar genèticament l’antitrombina III (ATIIIf) (Rhog = -0,444; p = 0,0045), la creatinina (Rhog = 0,29; p = 0,04) i la vitamina B12 (Rhog = 0,16; p = 0,048). Es va trobar una significació estadística amb els SNPs relacionats amb la coagulació i el sistema ABO: rs5985 (p = 0,016), rs657152 (p=0.009), rs11925694 (p=0.016), rs10965243 (p=0), rs7901695 (p=0.03), rs2036914 (p=0.03). La presència de l’al·lel A del gen ABO es relaciona amb MVC (9.42% vs 3.48%, p=0.002), si són dos al·lels A augmenta la prevalença (12.15% vs 8.67%, p=0.012). Els portadors d’un al·lel O van tenir menys EVC que els que no en tenen cap (5.76% vs 11.8%, p=0.0032), l’efecte és additiu, la presència de dos al·lels O tenia un efecte protector més gran (3.83% vs 7.06%, p=0.00042). A l’Estudi d’Associació de tot el Genoma (GWAS) es van analitzar 10.333.120 SNPs, dels quals 16 van mostrar una significació estadística amb una p=10-7. Es va trobar una correlació genètica clara entre l’EVC i l’ETEV (ρg = 0.80, amb una p = 0.0069). Conclusions: Els resultats obtinguts confirmen una heretabilitat de la MVC del 78%, una de les més altes conegudes, demostrant la importància del component genètic a la seva etiologia. L’absència de gens directament relacionats amb l’aparició de la MVC sembla que indica la presència d’interaccions de múltiples factors genètics a petita escala. La gran correlació genètica trobada entre MVC i MTEV indica que hi ha un substrat genètic comú entre les dues patologies. Introducción: La enfermedad venosa crónica (EVC) es una de las patologías más prevalentes en nuestra sociedad, ocasionando importantes costes socioeconómicos y sanitarios. Su etiología es desconocida, proponiéndose factores de riesgo como la edad, sexo, obesidad o antecedentes familiares. Clásicamente se ha dado como cierto el componente familiar, pero su relación con la enfermedad tromboembólica venosa (ETEV) es controvertida. Pretendemos demostrar que existe suficiente evidencia para confirmar el origen genético de la EVC y su relación con la ETEV. Justificación Científica: No existe hasta la fecha ningún estudio realizado en familias extensas donde se demuestre origen genético de la EVC. No se ha podido demostrar la relación genética entre EVC y ETEV. Estos resultados pueden ofrecer un nuevo enfoque a ambas patologías y nuevas vías de investigación. Hipótesis: · La EVC es una enfermedad multifactorial y compleja cuyo desarrollo y progresión depende de factores ambientales y genéticos, siendo los factores genéticos el elemento preponderante. · Existen variantes genéticas concretas asociadas a un aumento del riesgo de EVC. · Existe una relación entre la EVC y la ETEV con una base genética común que influye en la predisposición a padecer ambas patologías. Objetivos: · Estimar la heredabilidad de la EVC en un estudio de familias extensas. · Identificar genes que participen en la variabilidad de fenotipos, asociados a la predisposición a presentar EVC, mediante el análisis del genoma completo. · Realizar un análisis de asociación de todo el genoma, determinando el mecanismo de acción de las variantes genéticas identificadas. · Analizar las correlaciones genéticas y ambientales entre la EVC y la ETEV utilizando fenotipos intermediarios relacionados. Diseño del estudio: Estudio de cohortes en familias extensas prospectivo, observacional, analítico y unicéntrico. Para los estudios genéticos en familias extensas no se puede calcular el tamaño muestral ya que no podemos prever los efectos ni los genes implicados en la patología estudiada. Resultados: La muestra consistió en 895 individuos. Fueron diagnosticados de EVC 60, 39 mujeres (65%) y 21 hombres (35%). Entre las variables poblacionales analizadas mostraron una relación estadísticamente significativa con la EVC la edad (p=2,3x10-06), y el sexo (p=0,008). La heredabilidad (h2) de la EVC fue del 78% (p=2,75x10-8). En los fenotipos de la hemostasia se correlacionaron genéticamente la antitrombina III (ATIIIf) (Rhog = -0,444; p = 0,0045), la creatinina (Rhog = 0,29; p = 0,04) y la vitamina B12 (Rhog = 0,16; p = 0,048). Se encontró una significación estadística con los SNPs relacionados con la coagulación y el sistema ABO: rs5985 (p = 0,016), rs657152 (p=0.009), rs11925694 (p=0.016), rs10965243 (p=0.016), rs932206 (p=0.03), rs7901695 (p=0.03), rs2036914 (p=0.03). La presencia del alelo A del gen ABO se relaciona con EVC (9.42% vs 3.48%, p=0.002), si son dos alelos A aumenta la prevalencia (12.15% vs 8.67%, p=0.012). Los portadores de un alelo O tuvieron menos EVC que los que no tienen ninguno (5.76% vs 11.8%, p=0.0032), el efecto es aditivo, la presencia de dos alelos O tenía un efecto protector mayor (3.83% vs 7.06%, p=0.00042). En el Estudio de Asociación de todo el Genoma (GWAS) se analizaron 10.333.120 SNPs, de ellos 16 mostraron una significación estadística con una p=10-7. Se encontró una clara correlación genética entre la EVC y la ETEV (ρg = 0.80, con una p = 0.0069). Conclusiones: Los resultados obtenidos confirman una heredabilidad de la EVC del 78%, una de las más altas conocidas, demostrando la importancia del componente genético en su etiología. La ausencia de genes directamente relacionados con la aparición de la EVC parece indicar la presencia de interacciones de múltiples factores genéticos a pequeña escala. La gran correlación genética hallada entre EVC y ETEV indica la existencia de un sustrato genético común entre ambas patologías. Introduction: Chronic venous disease (CVD) is one of the most prevalent pathologies in our society, causing significant socioeconomic and health costs. Its etiology is unknown, proposing risk factors such as age, sex, obesity or family history. Classically, the familial component has been considered true, but its relationship with venous thromboembolic disease (VTED) is controversial. We intend to demonstrate that there is sufficient evidence to confirm the genetic origin of CVD and its relationship with VTED. Scientific Justification: To date, there is no study carried out in extended families that demonstrate the genetic origin of CVD. The genetic relationship between CVD and VTED has not been demonstrated. These results may offer a new approach to both pathologies and new avenues of research. Hypothesis: · CVD is a multifactorial and complex disease whose development and progression depend on environmental and genetic factors, with genetic factors being the predominant element. · There are specific genetic variants associated with an increased risk of CVD. · There is a relationship between CVD and VTED with a common genetic basis that influences the predisposition to suffer from both pathologies. Goals: · Estimate the heritability of CVD in a study of extended families. · Identify genes that participate in the variability of phenotypes, associated with the predisposition to present CVD, through the analysis of the complete genome. · Perform an association analysis of the entire genome, determining the mechanism of action of the identified genetic variants. · Analyze the genetic and environmental correlations between CVD and VTED using related intermediate phenotypes. Study design: Cohort study in extended families prospective, observational, analytical and single center. For genetic studies in extended families, the sample size cannot be calculated, since we cannot predict the effects or the genes involved in the pathology studied. Results: The sample consisted of 895 individuals. 60 were diagnosed with CVD, 39 women (65%) and 21 men (35%). Among the population variables analyzed, age (p=2.3x10-06), and sex (p=0.008) showed a statistically significant relationship with CVD. The heritability (h2) of CVD was 78% (p=2.75x10-8). Antithrombin III (fATIII) (Rhog = -0.444; p = 0.0045), creatinine (Rhog = 0.29; p = 0.04) and vitamin B12 (Rhog = 0.16, p = 0.048). Statistical significance was found with the SNPs related to coagulation and the ABO system: rs5985 (p = 0.016), rs657152 (p = 0.009), rs11925694 (p = 0.016), rs10965243 (p = 0.016), rs932206 (p = 0.03), rs7901695 (p=0.03), rs2036914 (p=0.03). The presence of the A allele of the ABO gene is related to CVD (9.42% vs 3.48%, p=0.002), if there are two A alleles the prevalence increases (12.15% vs 8.67%, p=0.012). Carriers of one O allele had fewer CVDs than those without (5.76% vs 11.8%, p=0.0032), the effect is additive, the presence of two O alleles had a greater protective effect (3.83% vs 7.06%, p=0.00042). In the Genome-Wide Association Study (GWAS), 10,333,120 SNPs were analyzed, of which 16 showed statistical significance with p=10-7. A clear genetic correlation was found between CVD and VTED (ρg = 0.80, p = 0.0069). Conclusions: The results obtained confirm a heritability of CVD of 78%, one of the highest known, demonstrating the importance of the genetic component in its etiology. The absence of genes directly related to the appearance of CVD seems to indicate the presence of interactions of multiple genetic factors on a small scale. The great genetic correlation found between CVD and VTED indicates the existence of a common genetic substrate between both pathologies. Universitat Autònoma de Barcelona. Programa de Doctorat en Cirurgia i Ciències Morfològiques

[eng] Venous Thrombosis (VT) is a frequent complex disease that involves a disruption of the balance of the hemostatic system. microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Promising findings in other complex diseases encourage their use as clinical biomarkers. However, up to now, the implication of miRNAs in VT has not been studied in-depth. The main objective of this Thesis was to study the regulatory mechanisms of the gene expression through miRNAs in VT. The specific objectives were (1) to identify a plasma miRNA profile associated with VT and to analyze its suitability as biomarker, (2) to dissect the interactions between biological layers in the biological context of VT and (3) to identify factors affecting either expression or quantification of miRNAs in plasma. In the first article, the differential expression of miRNAs in plasma was analyzed in the ‘Genetic Analysis of Idiopathic Thrombophilia’ 2 (GAIT-2) Project, which involves 935 individuals belonging to 35 extended Spanish families with idiopathic thrombophilia. First, we conducted a discovery phase, in which 752 miRNAs were measured in plasma by quantitative Polymerase Chain Reaction in 104 individuals of GAIT-2 (52 VT cases and 52 controls). Sixteen miRNAs were selected, which were measured in the entire GAIT-2 (n=935). Four of the miRNAs were significantly associated with VT (false discovery rate 0.7. The first model (VT ~ GATA2 + von Willebrand Factor) showed that the expression of GATA2 in blood was inversely correlated with the blood levels of von Willebrand Factor, and that the disruption of this relationship represents a prothrombotic phenotype. The second model (VT ~ Factor IX, ANXA2 + ENTPD1 + ILK + PDPK1 + PRKAR1A + STXBP3 +hsa-miR- 885-5p + hsa-miR-192-5p) represented an interaction between the fibrinolytic system and platelet activation through the αIIbβ3 signaling pathway. The third model (VT ~CSRP1+ LYN + hsa-miR-192-5p+ hsa-miR-885-5p) revealed the interaction between two group of genes involved in platelet activation, correlated with Protein S, and two miRNAs, in the biological context of VT. In the third article, we used the miRNA expression data of the discovery phase (103 miRNAs in 104 subjects) to analyze the effect of biological and technical factors in their expression. First, we found that the hemolysis marker represented ~10% of the shared variability of miRNA expression. Therefore, using this value as a continuous covariate, beyond as a categorical control, could help to increase consistency across miRNA studies. Second, we found that the expression of miRNAs in plasma was not biased by any blood cell count. Then, we identified 1,323 genetic variants associated with the expression of 16 miRNA genes, that represent 158 independent loci. Finally, we found that these loci were enriched in promoter regions from several tissues, though not in blood tissue. This finding is in agreement with the results regarding blood cell counts, and encourages the role of circulating miRNAs as biomarkers of tissue specific conditions., [spa] El objetivo general de esta Tesis fue estudiar los mecanismos de regulación de la expresión génica mediante microRNAs (miRNAs) en la trombosis venosa (VT). En el primer artículo, se explora la expresión diferencial de miRNAs en VT utilizando la población del proyecto ‘Genetic Analysis of Idiopathic Thrombophilia’ 2 (GAIT-2), compuesta por 935 individuos de 35 familias con trombosis idiopática. El diseño experimental implicó una fase de descubrimiento en la que 752 miRNAs plasmáticos se cuantificaron en 104 individuos del GAIT2 (52 casos y 52 controles) y se seleccionaron 16 miRNAs para la fase de validación, en la que se cuantificaron en toda la población GAIT-2 (n=935). Cuatro de los miRNAs se asociaron significativamente con la enfermedad (false discovery rate