Genomics to elucidate the molecular basis of calcific aortic valve disease

Le rétrécissement valvulaire aortique (RVA) est causé par une calcification et une fibrose progressive de la valve aortique. Le risque de développer la maladie augmente avec l’âge. À cause de l'augmentation de l'espérance de vie, le RVA est devenu un problème de santé publique. Le RVA est fatal en absence de traitement médical. Actuellement, la chirurgie est le seul traitement pour le stade sévère de la maladie, mais près de 50% des individus avec RVA n’y sont pas éligibles, principalement due à la présence de comorbidités. Plusieurs processus biologiques ont été associés à la maladie, mais les voies moléculaires spécifiques et les gènes impliqués dans le développement et la progression du RVA ne sont pas connus. Il est donc urgent de découvrir les gènes de susceptibilité pour le RVA afin d’identifier les personnes à risque ainsi que les biomarqueurs et les cibles thérapeutiques pouvant mener au développement de médicaments pour inverser ou limiter la progression de la maladie. L'objectif de cette thèse de doctorat était d'identifier la base moléculaire du RVA. Des approches modernes en génomique, incluant l’étude de gènes candidats et le criblage génomique par association (GWAS), ont été réalisées à l’aide de collections d’ADN provenant d’un grand nombre de patients bien caractérisés pour le RVA. Des études complémentaires en transciptomique ont comparé le profil d’expression global des gènes entre des valves calcifiées et non-calcifiées à l’aide de biopuces à ADN et de séquençage de l'ARN. Une première étude a identifié des variations dans le gène NOTCH1 et suggère pour la première fois la présence d'un polymorphisme commun dans ce gène conférant une susceptibilité au RVA. La deuxième étude a combiné par méta-analyse deux GWAS de patients provenant de la ville de Québec et Paris (France) aux données transcriptomiques. Cette étude de génomique intégrative a confirmé le rôle de RUNX2 dans le RVA et a permis l’identification d’un nouveau gène de susceptibilité, CACNA1C. Les troisième et quatrième études sur l’expression des gènes ont permis de mieux comprendre les bases moléculaires de la calcification des valves aortiques bicuspides et ainsi d’identifier de nouvelles cibles thérapeutiques pour le RVA. Les données générées par ce projet sont la base de futures découvertes importantes qui permettront d'améliorer les options de traitement et la qualité de vie des patients atteints du RVA.Calcific aortic valve disease (CAVD) is a common disease that causes the narrowing of the aortic valve due to fibrosis and calcification of the valve leaflets. The risk of CAVD increases with age. Due to the increase in life expectancy, CAVD is becoming a major public health problem. CAVD is fatal in the absence of medical treatment. Currently, surgery is the only treatment for severe stages of the disease, but nearly 50% of individual with CAVD are not eligible for surgery; mainly because of the presence of comorbidities. Several biological processes have been associated with the disease but the specific cell signaling pathways and genes implicated in CAVD development and progression are yet to be discovered. Thus, it is urgent to discover the susceptibility genes for CAVD, which will help identify individuals at risk as well as biomarkers and therapeutic targets for developing medication to reverse or limit disease progression. The objective of this thesis was to identify the molecular basis of CAVD. Modern genomic approaches including candidate gene and genome-wide association studies (GWAS) were performed with large DNA collections of patients well-characterized for CAVD. Whole-genome gene expression studies were also performed to compare calcified bicuspid and tricuspid valves with normal aortic valves using microarrays and RNA-Sequencing. A GWAS meta-analysis was performed using two cohorts of patients with CAVD from Quebec City and Paris. The integration of different whole-genome approaches revealed a new gene associated with CAVD called CACNA1C. This work also confirmed the potential role of NOTCH1 and RUNX2 in CAVD. In addition, this work identified new genes differentially expressed in calcified compared to normal aortic valves that are implicated in biological processes involved in the disease. These new developments are important to better understand the pathophysiological processes implicated in aortic valve calcification. Several genes differentially expressed in calcified compared to normal valves are targets for existing and emerging drugs. In general, this work has increased the knowledge about the etiology of CAVD in patients with bicuspid and tricuspid aortic valves and has identified new susceptibility genes for the development of this disease. The data generated by this project are the base of future important discoveries that will improve treatment options and the quality of life of patients with CAVD

Niveles de Expansión del Trinucleótido CGG en el Gen Fmr-1 de Pacientes con Características Fenotípicas del Síndrome del X Frágil

RESUMEN El Síndrome del X Frágil es la primera causa de retraso mental hereditario, la enfermedad es causada por el silenciamiento transcripcional inducido por la metilación del gen FMR-1 ubicado en la región q27.3 del cromosoma X, que a su vez es el resultado de la expansión de una repetición CGG en la región 5' no traducida en el primer exón del gen. Dado que las metodologías citogenéticas sólo permiten diagnosticar el 5% de las personas afectadas, en este trabajo se implementó el uso de técnicas moleculares para determinar el nivel de expansión del trinucleótido CGG en 13 individuos de la ciudad de Armenia (Colombia) que presentan características fenotípicas del síndrome y en algunas de sus respectivas madres. Para cada individuo se realizó la extracción del ADN mediante la técnica de desalamiento y se realizó la amplificación de la región portadora de la repetición CGG en el gen FMR-1 utilizando iniciadores específicos. Ocho de los 13 individuos estudiados (62% de la muestra) presentaron más de 50 repeticiones CGG, caracterizándolos como portadores de premutación mientras que los seis restantes presentaron expansión del trinucleótido en el rango normal; en cinco de nueve casos madre-hijo el número de repeticiones se mantuvo estable. Estos resultados permiten ampliar la base de datos para Colombia relacionada con la incidencia de este síndrome y adicionalmente se ha implementado una herramienta de diagnóstico molecular eficaz para quienes presentan las características fenotípicas del síndrome del X - Frágil.  Palabras claves: X-frágil, tripletas, expansión, FMR-1, retardo mental. ABSTRACT Fragile X Syndrome is the first cause of inherited mental retardation; this disease is due to the transcriptional silence of FMR-1 gene by metilation of the FMR-1 gene located in q27.3 region of X chromosome, at time is successful of expansion of CGG repeat in 5´untranslated region in the first exon of the gen. Because the cytogenetics methodologies can diagnostic only the 5% of the diseased people, in this work we use molecular techniques to determinate the level of expansion of the CGG trinucleotide in 13 citizen of Armenia (Colombia) whit the phenotypic characteristics of the syndrome and his respective mothers. For each patient we do the DNA extraction by the salting out technique and the amplification of the region with the CGG repetition with specifics primers. Eight of 13 studied patients (62%) presented more that 50 CGG repetitions, that dressed how permutation carriers, while the six rested showed expansion in the normal range; in five of nine cases the mothers-son the expansions was normal. This results let to extend the date base for Colombia related with the incidence of the syndrome and additionally is implemented an efficacy diagnostic tool for who showed phenotypic characteristics of the Fragile X Syndrome.  Key words: X-fragil, FMR-1, triplets, mental retardation

Niveles de Expansión del Trinucleótido CGG en el Gen Fmr-1 de Pacientes con Características Fenotípicas del Síndrome del X Frágil

RESUMEN El Síndrome del X Frágil es la primera causa de retraso mental hereditario, la enfermedad es causada por el silenciamiento transcripcional inducido por la metilación del gen FMR-1 ubicado en la región q27.3 del cromosoma X, que a su vez es el resultado de la expansión de una repetición CGG en la región 5' no traducida en el primer exón del gen. Dado que las metodologías citogenéticas sólo permiten diagnosticar el 5% de las personas afectadas, en este trabajo se implementó el uso de técnicas moleculares para determinar el nivel de expansión del trinucleótido CGG en 13 individuos de la ciudad de Armenia (Colombia) que presentan características fenotípicas del síndrome y en algunas de sus respectivas madres. Para cada individuo se realizó la extracción del ADN mediante la técnica de desalamiento y se realizó la amplificación de la región portadora de la repetición CGG en el gen FMR-1 utilizando iniciadores específicos. Ocho de los 13 individuos estudiados (62% de la muestra) presentaron más de 50 repeticiones CGG, caracterizándolos como portadores de premutación mientras que los seis restantes presentaron expansión del trinucleótido en el rango normal; en cinco de nueve casos madre-hijo el número de repeticiones se mantuvo estable. Estos resultados permiten ampliar la base de datos para Colombia relacionada con la incidencia de este síndrome y adicionalmente se ha implementado una herramienta de diagnóstico molecular eficaz para quienes presentan las características fenotípicas del síndrome del X - Frágil.  Palabras claves: X-frágil, tripletas, expansión, FMR-1, retardo mental. ABSTRACT Fragile X Syndrome is the first cause of inherited mental retardation; this disease is due to the transcriptional silence of FMR-1 gene by metilation of the FMR-1 gene located in q27.3 region of X chromosome, at time is successful of expansion of CGG repeat in 5´untranslated region in the first exon of the gen. Because the cytogenetics methodologies can diagnostic only the 5% of the diseased people, in this work we use molecular techniques to determinate the level of expansion of the CGG trinucleotide in 13 citizen of Armenia (Colombia) whit the phenotypic characteristics of the syndrome and his respective mothers. For each patient we do the DNA extraction by the salting out technique and the amplification of the region with the CGG repetition with specifics primers. Eight of 13 studied patients (62%) presented more that 50 CGG repetitions, that dressed how permutation carriers, while the six rested showed expansion in the normal range; in five of nine cases the mothers-son the expansions was normal. This results let to extend the date base for Colombia related with the incidence of the syndrome and additionally is implemented an efficacy diagnostic tool for who showed phenotypic characteristics of the Fragile X Syndrome.  Key words: X-fragil, FMR-1, triplets, mental retardation

NOTCH1 genetic variants in patients with tricuspid calcific aortic valve stenosis

BACKGROUND AND AIM OF THE STUDY: Calcific aortic valve stenosis (AS) affects 2-5% of the population aged > 65 years. Functional DNA variants at the NOTCH1 locus result in bicuspid aortic valve (BAV) and severe valve calcification. The contribution of these variants to AS in the population with tricuspid aortic valve (TAV) remains to be determined. METHODS: Fourteen genetic variants surrounding the NOTCH1 gene were genotyped, including rare mutations previously reported, and common polymorphisms. The study involved 457 French Canadian patients with severe tricuspid AS. Genotyping was carried out using the Illumina BeadXpress platform. Allele frequencies of common single nucleotide polymorphisms (SNPs) for patients with AS were compared to a shared control group of European ancestry (n = 3,294). In total, 88 ancestry-informative markers were used to correct for population stratification. RESULTS: The mutation R1107X, previously associated with AS and BAV, was identified in a relatively young patient (aged 58 years). The mutations R1279H and V2285I were detected in 18 and 14 heterozygotes, respectively. A common polymorphism (rs13290979) located in intron 2 was significantly associated with AS (p = 0.003), which remained significant after correction for multiple testing. However, this association was no longer significant after accounting for population stratification (p = 0.088). CONCLUSION: In this study, rare functional variants were found in the NOTCH1 gene in a French Canadian population of patients with severe tricuspid AS. This also suggests, for the first time, the presence of a common polymorphism in this gene conferring susceptibility to AS

Differences in Caries Status and Risk Factors among Privileged and Unprivileged Children in Colombia

Cilj: Željela se usporediti prevalencija karijesa prema klasifikaciji ICDAS-a II i čimbenici povezani s karijesom između djece iz seoskih i gradskih škola u Pastou u Kolumbiji. Materijali i metode: Istraživanje je obuhvatilo 120 djece (od 4 do 6 godina) iz seoskih i gradskih škola. Postojanje karijesnih lezija ocijenjeno je prema kriterijima ICDAS-a II. Primijenjena je i anketa o čimbenicima povezanima s karijesom. Hi-kvadrat i Fisherovi testovi upotrijebljeni su za procjenu razlika u svakoj varijabli između dviju skupina. Za usporedbu broja zuba između skupina prema kategoriji ICDAS-a II odabran je Mann-Whitneyjev U test. Negativna binomna regresija korištena je za procjenu postotne promjene srednjeg broja zuba prema kategoriji ICDAS-a II među seoskim i gradskim učenicima. Rezultati: Utvrđene su statistički značajne razlike između učenika iz seoskih i gradskih škola za ICDAS-II 0 i 3. do 6. kategorije (p < 0,001). Srednji broj zuba s umjerenim do teškim stupnjem karijesa povećao se za 233 % kod djece iz seoskih škola u usporedbi s onima koji su pohađali škole u gradu (p = 0,0). Učestalost četkanja zuba (p = 0,006), kariogena prehrana, vrijeme proteklo od posljednjeg posjeta stomatologu, socijalno-ekonomski status i vrsta zdravstvenog osiguranja (p < 0,001) bili su među značajnim čimbenicima prema kojima su se razlikovale škole u ruralnom i urbanom području. Zaključak: Ovo je prvo istraživanje koje je uspoređivalo dentalni status prema klasifikaciji ICDAS-a II između učenika seoskih i gradskih škola u Kolumbiji. Kod učenika iz seoskih škola ustanovljen je lošiji oralni status. Ovo istraživanje identificiralo je socijalno-ekonomske i kliničke čimbenike koji mogu poslužiti kao smjernice za specifične intervencije kod seoske djece primjenom programa promicanja oralnoga zdravlja i prevencije bolesti.Objective: The objective of this study was to compare the ICDAS-II caries status and caries-related factors among children from rural and urban schools in Pasto, Colombia. Materials and Methods: The study included 120 children (4 - 6 year- old children) from rural (privileged) and urban (unprivileged) schools. Caries was evaluated using the ICDAS-II criteria. A survey about the factors related to the presence of caries was applied. Chi-square and Fisher’s tests were used to assess the differences in each study variable between the two groups. A Mann–Whitney U test was used to compare the number of teeth, per ICDAS-II category, between the groups. Negative binomial regression was used to estimate the percentage change in the mean number of teeth, per ICDAS-II category, among the rural and urban students. Results: Significant differences were found between the rural and urban students for the ICDAS-II 0 and 3-6 categories (p<0.001). The mean number of teeth with moderate-to-severe caries status increased 233% in children from the rural school compared to those attending the urban school (p=0.0). Toothbrushing frequency (p=0.006), cariogenic diet, time elapsed from last dental visit, socioeconomic status, and type of health regime (p<0.001) were among the significant factors related to the rural and urban schools. Conclusions: This was the first study to compare ICDAS-II caries status between rural and urban students in Colombia. A worse caries status was found in rural students. This study identified the socioeconomic and clinical factors to guide specific interventions for rural children by modifying the available oral health promotion and disease prevention programs

Quantitative profiling of the UGT transcriptome in human drug metabolizing tissues

Alternative splicing as a mean to control gene expression and diversify function is suspected to considerably influence drug response and clearance. We report the quantitative expression profiles of the human UGT genes including alternatively spliced variants not previously annotated established by deep RNA-sequencing in tissues of pharmacological importance. We reveal a comprehensive quantification of the alternative UGT transcriptome that differ across tissues and among individuals. Alternative transcripts that comprise novel in-frame sequences associated or not with truncations of the 5’ and/or 3’ termini, significantly contribute to the total expression levels of each UGT1 and UGT2 gene averaging 21% in normal tissues, with expression of UGT2 variants surpassing those of UGT1. Quantitative data expose preferential tissue expression patterns and remodelling in favour of alternative variants upon tumorigenesis. These complex alternative splicing programs have the strong potential to contribute to interindividual variability in drug metabolism in addition to diversify the UGT proteome

The Transcriptome of Human Epicardial, Mediastinal and Subcutaneous Adipose Tissues in Men with Coronary Artery Disease

The biological functions of epicardial adipose tissue (EAT) remain largely unknown. However, the proximity of EAT to the coronary arteries suggests a role in the pathogenesis of coronary artery disease (CAD). The objectives of this study were to identify genes differentially regulated among three adipose tissues, namely EAT, mediastinal (MAT) and subcutaneous (SAT) and to study their possible relationships with the development of cardiovascular diseases.Samples were collected from subjects undergoing coronary artery bypass grafting surgeries. Gene expression was evaluated in the three adipose depots of six men using the Illumina® HumanWG-6 v3.0 expression BeadChips. Twenty-three and 73 genes were differentially up-regulated in EAT compared to MAT and SAT, respectively. Ninety-four genes were down-regulated in EAT compared to SAT. However, none were significantly down-regulated in EAT compared to MAT. More specifically, the expression of the adenosine A1 receptor (ADORA1), involved in myocardial ischemia, was significantly up-regulated in EAT. Levels of the prostaglandin D2 synthase (PTGDS) gene, recently associated with the progression of atherosclerosis, were significantly different in the three pairwise comparisons (EAT>MAT>SAT). The results of ADORA1 and PTGDS were confirmed by quantitative real-time PCR in 25 independent subjects.Overall, the transcriptional profiles of EAT and MAT were similar compared to the SAT. Despite this similarity, two genes involved in cardiovascular diseases, ADORA1 and PTGDS, were differentially up-regulated in EAT. These results provide insights about the biology of EAT and its potential implication in CAD

The transcriptome of human epicardial, mediastinal and subcutaneous adipose tissues in men with coronary artery disease

Le tissu adipeux épicardique (TAE) est localisé à la surface du cœur en contact avec les artères coronaires, ce qui suggère un rôle dans la pathogénèse de la maladie coronarienne. Les objectifs de cette étude étaient d’identifier les gènes différentiellemment régulés entre les tissus graisseux épicardique, médiastinal et sous-cutané à l’aide des biopuces d’ADN et d’étudier leurs rôles dans le développement des maladies cardiovasculaires. Les résultats ont montré une grande similarité d’expression entre les tissus adipeux épicardique et médiastinal. Toutefois, certains gènes impliqués dans les maladies cardiovasculaires étaient régulés différemment entre ces deux tissus. L’expression des gènes codant pour le récepteur A1 de l’adénosine (ADORA1) et la prostaglandine D2 synthase (PTGDS), impliqué dans les ischémies myocardiques et la progression de l’athérosclérose, respectivement, était significativement élevée dans le TAE. Cette étude est une première étape pour comprendre le rôle biologique du TAE et ses implications dans les maladies cardiovasculaires.Increased visceral adipose tissue has been associated with the development of cardiovascular diseases (CVD). Epicardial adipose tissue (EAT) is the visceral fat depot located on the surface of the heart especially around the epica rdial coronary vessels with extension into the myocardium. The proximity of EAT to the coronary arteries suggests a role in the pathogenesis of coronary artery disease (CAD). EAT thickness was significantly correlated with the severity of CAD. However, the biological functions of EAT and its relationship with the development of CVD remain largely elusive. The objectives of this study were to identify genes that were up- or down-regulated among three distinct adipose tissues, namely EAT, mediastinal and subcutaneous using whole-genome gene expression microarrays and to study the possible relationships of these genes with the development of CVD. Overall, the transcriptional profiles of EAT and mediastinal adipose tissue were similar compared to subcutaneous adipose tissue. Despite this similarity, a number of genes involved in cardiovascular diseases were up-regulated in EAT. The expression of the adenosine A1 receptor (ADORA1), involved in myocardial ischemia, was significantly up-regulated in EAT. Levels of the prostaglandin D2 synthase (PTGDS) gene, recently associated with the progression of atherosclerosis, were significantly different in the three pairwise comparisons (epicardial &gt; mediastinal &gt; subcutaneous). Overexpression of ADORA1 and PTGDS in EAT may confer cardioprotection against myocardial ischemia and CAD. This study is an important first step to understand the biological function of EAT and its potential implications in CVD

Diversidad y estructura genética de tres poblaciones afrodescendientes del suroccidente colombiano a partir de 8 STRs

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Diversidad y estructura genética de tres poblaciones afrodescendientes del suroccidente colombiano a partir de 8 STRs

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