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

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

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

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

Frecuencia reportada de enfermedad pulpo-periodontal por prestadores de servicios de salud en Colombia 2015-2022

Objective: To present the frequency of pulp and periodontal diseases based on the reports of health service providers to the territorial entities in Colombia. Materials and methods: A cross-sectional and descriptive quantitative study was carried out, based on information obtained from SISPRO, of the Colombian population attended by all types of providers registered at the national level, according to the different levels of affiliation to the system and whose main diagnosis was periodontitis or some type of pulpo-periapical pathology. Results: For the period 2015-2022, in general the reported frequency of pulpo-periodontal diseases was 57.7% for gingivitis, pulpitis 15.8%, and periodontitis 6.3%, likewise pulpitis, Necrosis and tooth loss occurred with a significantly higher frequency in the subsidized regime. Conclusions: The frequency of pulp and periodontal diseases from these reports reflects lower frequency than those reported in previous national studies, the results should be interpreted with caution since it may be due to a positive impact of the health system interventions or may indicate deficiencies in the information systems and reporting of health service providers.Objetivo: Presentar la frecuencia reportada de enfermedades pulpares y periodontales a partir del reporte de los prestadores de servicios de salud a las entidades territoriales en Colombia. Materiales y Métodos: Se realizo un estudio de tipo cuantitativo transversal y descriptivo, a partir de información obtenida de El SISPRO, de población colombiana atendidas por todos los tipos de prestadores inscritos a nivel nacional, según los diferentes niveles de afiliación al sistema y cuyo diagnóstico principal fue periodontitis o algún tipo de patología pulpo periapical. Resultados: Para el periodo 2015-2022, de forma general la frecuencia reportada de enfermedades pulpo periodontales fue de 57,7% para la gingivitis, la pulpitis de 15,8%, y la periodontitis 6,3%, así mismo la pulpitis, la necrosis y la perdida de los dientes se presentaron con una frecuencia significativamente mayor en el régimen subsidiado. Conclusiones: La frecuencia de las enfermedades pulpares y periodontales a partir de estos reportes son más bajas que las reportadas en los estudios nacionales previos, los resultados deben interpretarse con cautela ya que puede obedecer a un impacto positivo de las intervenciones del sistema de salud o puede indicar deficiencias en los sistemas de información y de reporte de los prestadores de servicios de salud

Investigating interactions between epicardial adipose tissue and cardiac myocytes: what can we learn from different approaches?

Heart disease is a major cause of morbidity and mortality throughout the world. Some cardiovascular conditions can be modulated by lifestyle factors such as increased exercise or a healthier diet, but many require surgical or pharmacological interventions for their management. More targeted and less invasive therapies would be beneficial. Recently it has become apparent that epicardial adipose tissue plays an important role in normal and pathological cardiac function, and it is now the focus of considerable research. Epicardial adipose tissue can be studied by imaging of various kinds, and these approaches have yielded much useful information. However at a molecular level it is more difficult to study as it is relatively scarce in animal models and, for practical and ethical reasons, not always available in sufficient quantities from patients. What is needed is a robust model system in which the interactions between epicardial adipocytes and cardiac myocytes can be studied, and physiologically relevant manipulations performed. There are drawbacks to conventional culture methods, not least the difficulty of culturing both cardiac myocytes and adipocytes, each of which has special requirements. We discuss the benefits of a three-dimensional co-culture model in which in vivo interactions can be replicated

Validation of Reference Genes for the Relative Quantification of Gene Expression in Human Epicardial Adipose Tissue

BACKGROUND: Relative quantification is a commonly used method for assessing gene expression, however its accuracy and reliability is dependent upon the choice of an optimal endogenous control gene, and such choice cannot be made a priori. There is limited information available on suitable reference genes to be used for studies involving human epicardial adipose tissue. The objective of the current study was to evaluate and identify optimal reference genes for use in the relative quantification of gene expression in human epicardial fat depots of lean, overweight and obese subjects. METHODOLOGY/PRINCIPAL FINDINGS: Some of the commonly used reference genes including 18S, ACTB, RPL27, HPRT, CYCA, GAPDH, RPLPO, POLR2A and B2M were quantified using real-time PCR analysis. The expression stability of these genes was evaluated using Genorm, Normfinder and Bestkeeper algorithms. In addition, the effect of sample size on the validation process was studied by randomly categorizing subjects in two cohorts of n = 2 and n = 33. CONCLUSIONS/SIGNIFICANCE: CYCA, GAPDH and RPL27 were identified as the most stable genes common to all three algorithms and both sample sizes. Their use as reference gene pairs might contribute to the enhanced robustness of relative quantification in the studies involving the human epicardial adipose tissue

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