Engineering a Pro-Osteogenic Secretome through the Transient Silencing of the Gene Encoding Secreted Frizzled Related Protein 1.

The evidence sustaining the regenerative properties of mesenchymal stem cells' (MSCs) secretome has prompted a paradigm change, where MSCs have shifted from being considered direct contributors to tissue regeneration toward being seen as cell factories for producing biotech medicines. We have previously designed a method to prime MSCs towards osteogenic differentiation by silencing the Wnt/ß-Catenin inhibitor Sfpr1. This approach produces a significant increase in bone formation in osteoporotic mice. In this current work, we set to investigate the contribution of the secretome from the MSCs where Sfrp1 has been silenced, to the positive effect seen on bone regeneration in vivo. The conditioned media (CM) of the murine MSCs line C3H10T1/2, where Sfrp1 has been transiently silenced (CM-Sfrp1), was found to induce, in vitro, an increase in the osteogenic differentiation of this same cell line, as well as a decrease of the expression of the Wnt inhibitor Dkk1 in murine osteocytes ex vivo. A reduction in the RANKL/OPG ratio was also detected ex vivo, suggesting a negative effect of CM-Sfrp1 on osteoclastogenesis. Moreover, this CM significantly increases the mineralization of human primary MSCs isolated from osteoportotic patients in vitro. Proteomic analysis identified enrichment of proteins involved in osteogenesis within the soluble and vesicular fractions of this secretome. Altogether, we demonstrate the pro-osteogenic potential of the secretome of MSCs primmed in this fashion, suggesting that this is a valid approach to enhance the osteo-regenerative properties of MSCs' secretome.

Nanoparticle-mediated selective Sfrp-1 silencing enhances bone density in osteoporotic mice.

Osteoporosis (OP) is characterized by a loss in bone mass and mineral density. The stimulation of the canonical Wnt/ß-catenin pathway has been reported to promote bone formation, this pathway is controlled by several regulators as secreted frizzled-related protein-1 (Sfrp-1), antagonist of the pathway. Thus, Sfrp-1 silencing therapies could be suitable for enhancing bone growth. However, the systemic stimulation of Wnt/ß-catenin has been correlated with side effects. This work hypothesizes the administration of lipid-polymer NPs (LPNPs) functionalized with a MSC specific aptamer (Apt) and carrying a SFRP1 silencing GapmeR, could favor bone formation in OP with minimal undesired effects. Suitable SFRP1 GapmeR-loaded Apt-LPNPs (Apt-LPNPs-SFRP1) were administered in osteoporotic mice and their biodistribution, toxicity and bone induction capacity were evaluated. The aptamer functionalization of the NPs modified their biodistribution profile showing a four-fold increase in the bone accumulation and a ten-fold decrease in the hepatic accumulation compared to naked LPNPs. Moreover, the histological evaluation revealed evident changes in bone structure observing a more compact trabecular bone and a cortical bone thickness increase in the Apt-LPNPs-SFRP1 treated mice with no toxic effects. Therefore, these LPNPs showed suitable properties and biodistribution profiles leading to an enhancement on the bone density of osteoporotic mice.

Cytotoxicity analysis of oxazine 4-perchlorate fluorescence nerve potential clinical biomarker for guided surgery.

Biological tissue discrimination is relevant in guided surgery. Nerve identification is critical to avoid potentially severe collateral damage. Fluorescence imaging by oxazine 4-perchlorate (O4P) has been recently proposed. In this work, the cytotoxicity of O4P on U87 human-derived glioma cells has been investigated as a function of concentration and operating room irradiation modes. A custom-built optical irradiation device was employed for controlled optical dosimetry. DNA damage and O4P intracellular localization was also investigated by immunofluorescence and confocal microscopy. The results show that concentration below 100 µM can be considered safe. These results contribute to the assessment of the feasibility of O4P as a nerve biomarker.

Effective Osteogenic Priming of Mesenchymal Stem Cells through LNA-ASOs-Mediated Sfrp1 Gene Silencing.

Mesenchymal stem cell (MSC) transplantation has emerged as a promising approach for bone regeneration. Importantly, the beneficial effects of MSCs can be improved by modulating the expression levels of specific genes to stimulate MSC osteogenic differentiation. We have previously shown that Smurf1 silencing by using Locked Nucleic Acid-Antisense Oligonucleotides, in combination with a scaffold that sustainably releases low doses of BMP-2, was able to increase the osteogenic potential of MSCs in the presence of BMP-2 doses significantly smaller than those currently used in the clinic. This would potentially allow an important reduction in this protein in MSs-based treatments, and thus of the side effects linked to its administration. We have further improved this system by specifically targeting the Wnt pathway modulator Sfrp1. This approach not only increases MSC bone regeneration efficiency, but is also able to induce osteogenic differentiation in osteoporotic human MSCs, bypassing the need for BMP-2 induction, underscoring the regenerative potential of this system. Achieving successful osteogenesis with the sole use of LNA-ASOs, without the need of administering pro-osteogenic factors such as BMP-2, would not only reduce the cost of treatments, but would also open the possibility of targeting these LNA-ASOs specifically to MSCs in the bone marrow, allowing us to treat systemic bone loss such as that associated with osteoporosis.

Smurf1 Silencing Using a LNA-ASOs/Lipid Nanoparticle System to Promote Bone Regeneration.

Despite the great advance of bone tissue engineering in the last few years, repair of bone defects remains a major problem. Low cell engraftment and dose-dependent side effects linked to the concomitant administration of bone morphogenetic proteins (BMPs) are the main problems currently hindering the clinical use of mesenchymal stem cell (MSC)-based therapies in this field. We have managed to bypass these drawbacks by combining the silencing the Smurf1 ubiquitin ligase in MSCs with the use of a scaffold that sustainably releases low doses of BMP-2. In this system, Smurf1 silencing is achieved by using GapmeRs, a clinically safe method that avoids the use of viral vectors, facilitating its translation to the clinic. Here, we show that a single transient transfection with a small quantity of a Smurf1-specific GapmeR is able to induce a significant level of silencing of the target gene, enough to prime MSCs for osteogenic differentiation. Smurf1 silencing highly increases MSCs responsiveness to BMP-2, allowing a dramatic reduction of the dose needed to achieve the desired therapeutic effect. The combination of these primed cells with alginate scaffolds designed to sustainably and locally release low doses of BMP-2 to the defect microenvironment is able to induce the formation of a mature bone matrix both in an osteoporotic rat calvaria system and in a mouse ectopic model. Importantly, this approach also enhances osteogenic differentiation in MSCs from osteoporotic patients, characterized by a reduced bone-forming potential, even at low BMP doses, underscoring the regenerative potential of this system. Stem Cells Translational Medicine 2019;8:1306&1317.

ABCG5/G8 gene is associated with hypercholesterolemias without mutation in candidate genes and noncholesterol sterols.

CONTEXT: Approximately 20% to 40% of clinically defined familial hypercholesterolemia (FH) cases do not show a causative mutation in candidate genes (mutation-negative FH), and some of them may have a polygenic origin. OBJECTIVE: The aim of this work was to study the prevalence of ABCG5/G8 genetic variants in mutation-negative FH, as defects in these genes relate to intestinal hyperabsorption of cholesterol and thus ABCG5/G8 variants could explain in part the mechanism of hypercholesterolemia. DESIGN, SETTING, AND PATIENTS: We sequenced the ABCG5/G8 genes in 214 mutation-negative FH and 97 controls. Surrogate markers of cholesterol absorption (5α-cholestanol, ß-sitosterol, campesterol, stigmasterol, and sitostanol) were quantified by high-performance liquid chromatography-tandem mass spectrometry in both studied groups. RESULTS: We found 8 mutation-negative FH patients (3.73%) with a pathogenic mutation in ABCG5/G8 genes. We observed significantly higher concentration of surrogate markers of cholesterol absorption in mutation-negative FH than in controls. In addition, we found significantly higher concentrations of cholesterol absorption markers in mutation-negative FH with ABCG5/G8 defects than in mutation-negative, ABCG5/G8-negative FH. A gene score reflecting the number of common single nucleotide variants associated with hypercholesterolemia was significantly higher in cases than in controls (P = .032). Subjects with a gene score above the mean had significantly higher 5α-cholestanol and stigmasterol than those with a lower gene score. CONCLUSIONS: Mutation-negative FH subjects accumulate an excess of rare and common gene variations in ABCG5/G8 genes. This variation is associated with increased intestinal absorption of cholesterol, as determined by surrogate makers, suggesting that these loci contribute to hypercholesterolemia by enhancing intestinal cholesterol absorption.

Functional analysis of new 3' untranslated regions genetic variants in genes associated with genetic hypercholesterolemias.

BACKGROUND: Familial hypercholesterolemia (FH) is the best-described autosomal dominant genetic hypercholesterolemia (GH). Mutations in candidate genes can explain a high proportion of FH cases, but for many, no causative mutations are detected (designed non-FG-GH), suggesting the existence of additional genetic variants associated with the disease. OBJECTIVE: We aimed to identify new single-nucleotide variants (SNVs) located at the 3' untranslated regions (3'UTRs) of the low-density lipoprotein receptor, low-density lipoprotein receptor-related protein-associated protein 1, ATP-binding cassette sub-family G member 5, and sterol regulatory element-binding protein 2 genes in non-FH-GH individuals and investigated whether the association of these SNVs with non-FH-GH could be explained by changes in the affinity of regulatory microRNAs (miRNA) targeting the sequences modified by the SNVs. METHODS: The study includes probands with non-FH-GH attending 2 lipid clinics in Spain. We performed functional analyses of selected variants using a luciferase reporter system. Through in silico target-prediction tools, we identified miRNAs, which binding to the 3'UTR could be affected by the presence of specific SNVs. We used analogs and inhibitors of these miRNAs to test this possibility. RESULTS: We identified 11 new SNVs showing significant association with non-FH-GH. We show that the presence of 4 of these SNVs leads to significant changes in the transcriptional levels of the reporter gene. Through mechanistic analysis, we identified 2 miRNAs (miR-27a and miR-133-3p) targeting the 3'UTR of sterol regulatory element-binding protein 2 and an additional miRNA (miR-92a) targeting the 3'UTR of low-density lipoprotein receptor-related protein-associated protein 1. CONCLUSION: Our findings reveal novel regulatory links between certain miRNAs and key genes regulating cholesterol homeostasis. They also highlight the potential of miRNAs as therapeutic targets for the treatment of FH.

Rare genetic variants with large effect on triglycerides in subjects with a clinical diagnosis of familial vs nonfamilial hypertriglyceridemia.

BACKGROUND: Most primary severe hypertriglyceridemias (HTGs) are diagnosed in adults, but their molecular foundations have not been completely elucidated. OBJECTIVE: We aimed to identify rare dysfunctional mutations in genes encoding regulators of lipoprotein lipase (LPL) function in patients with familial and non-familial primary HTG. METHODS: We sequenced promoters, exons, and exon-intron boundaries of LPL, APOA5, LMF1, and GPIHBP1 in 118 patients with severe primary HTG (triglycerides >500 mg/dL) and 53 normolipidemic controls. Variant functionality was analyzed using predictive software and functional assays for mutations in regulatory regions. RESULTS: We identified 29 rare variants, 10 of which had not been previously described: c.(-16A>G), c.(1018+2G>A), and p.(His80Arg) in LPL; p.(Arg143Alafs*57) in APOA5; p.(Val140Ile), p.(Leu235Ile), p.(Lys520*), and p.(Leu552Arg) in LMF1; and c.(-83G>A) and c.(-192A>G) in GPIHBP1. The c.(1018+2G>A) variant led to deletion of exon 6 in LPL cDNA, whereas the c.(-16A>G) analysis showed differences in the affinity for nuclear proteins. Overall, 20 (17.0%) of the patients carried at least one allele with a rare pathogenic variant in LPL, APOA5, LMF1, or GPIHBP1. The presence of a rare pathogenic variant was not associated with lipid values, family history of HTG, clinical diagnosis, or previous pancreatitis. CONCLUSIONS: Less than one in five subjects with triglycerides >500 mg/dL and no major secondary cause for HTG may carry a rare pathogenic mutation in LPL, APOA5, LMF1, or GPIHBP1. The presence of a rare pathogenic variant is not associated with a differential phenotype.

Early growth response 1 (EGR-1) is a transcriptional regulator of mitochondrial carrier homolog 1 (MTCH 1)/presenilin 1-associated protein (PSAP).

Attempts to elucidate the cellular function of MTCH1 (mitochondrial carrier homolog 1) have not yet rendered a clear insight into the function of this outer mitochondrial membrane protein. Classical biochemical and cell biology approaches have not produced the expected outcome. In vitro experiments have indicated a likely role in the regulation of cell death by apoptosis, and its reported interaction with presenilin 1 suggests a role in the cellular pathways in which this membrane protease participates, nevertheless in vivo data are missing. In an attempt to identify cellular pathways in which this protein might participate, we have studied its promoter looking for transcriptional regulators. We have identified several putative binding sites for EGR-1 (Early growth response 1; a protein involved in growth, proliferation and differentiation), in the proximal region of the MTCH1 promoter. Chromatin immunoprecipitation showed an enrichment of these sequences in genomic DNA bound to EGR-1 and transient overexpression of EGR-1 in cultured HEK293T cells induces an increase of endogenous MTCH1 levels. We also show that MTCH1 levels increase in response to treatment of cells with doxorubicin, an apoptosis inducer through DNA damage. The endogenous levels of MTCH1 decrease when EGR-1 levels are lowered by RNA interference. Our results indicate that EGR-1 is a transcriptional regulator of MTCH1 and give some clues about the cellular processes in which MTCH1 might participate.

Ovine HSP90AA1 gene promoter: functional study and epigenetic modifications.

When environmental temperatures exceed a certain threshold, the upregulation of the ovine HSP90AA1 gene is produced to cope with cellular injuries caused by heat stress. It has been previously pointed out that several polymorphisms located at the promoter region of this gene seem to be the main responsible for the differences in the heat stress response observed among alternative genotypes in terms of gene expression rate. The present study, focused on the functional study of those candidate polymorphisms by electrophoretic mobility shift assay (EMSA) and in vitro luciferase expression assays, has revealed that the observed differences in the transcriptional activity of the HSP90AA1 gene as response to heat stress are caused by the presence of a cytosine insertion (rs397514115) and a C to G transversion (rs397514116) at the promoter region. Next, we discovered the presence of epigenetic marks at the promoter and along the gene body founding an allele-specific methylation of the rs397514116 mutation in DNA extracted from blood samples. This regulatory mechanism interacts synergistically to modulate gene expression depending on environmental circumstances. Taking into account the results obtained, it is suggested that the transcription of the HSP90AA1 ovine gene is regulated by a cooperative action of transcription factors (TFs) whose binding sites are polymorphic and where the influence of epigenetic events should be also taken into account.

Lípidos y diabetes tipo 2. Desarrollo de nuevas terapias basadas en el metabolismo lipídico / Lipid metabolism in type 2 diabetes and development of new lipid- based therapies

La diabetes tipo 2 es una de las principales causas de morbilidad a nivel global. La enfermedad se desarrolla tras una fase más o menos prolongada en la que los tejidos presentan resistencia a insulina. En el músculo, la resistencia a insulina se manifiesta como un bloqueo a la entrada de glucosa, mientras que en el hígado da lugar a un aumento de la gluconeogénesis y de la síntesis de lípidos. En ambos tejidos la propia acumulación de lípidos es la principal causa de la resistencia, que se produce por un aumento de la cantidad de diacilgliceroles intracelulares. Estos activan diversas proteín quinasas de la familia PKC que a su vez interfieren con el proceso de señalización por insulina. En el hígado esta interferencia afecta parcialmente a la señalización. La insulina produce un aumento de la síntesis de lípidos, sin ser capaz de bloquear la gluconeogénesis. El resultado es una progresiva acumulación de lípidos que agrava el fenómeno de resistencia y que, de no revertirse, progresará hasta una diabetes tipo 2. La regulación a largo plazo de la síntesis de lípidos por insulina está mediada por el factor de transcripción SREBP-1c. La síntesis de éste está regulada por numerosos receptores nucleares, y la utilización de ligandos de estos últimos está proporcionando una nueva vía para la prevención y el tratamiento de la diabetes tipo 2 (AU)

Type 2 diabetes is one of the major causes of global morbidity. The disease develops after a period of insulin resistance of variable length. In skeletal muscle, insulin resistance is characterized by an impairment of glucose transport whereas in liver there are increases in both gluconeogenesis and lipid synthesis. In both tissues lipid accumulation is responsible for insulin resistance because of increases in intracellular diacylglycerols. These activate several members of the PKC family of protein kinases, which, in turn, impair the insulin-signaling pathway. Insulin signaling is only partially affected in the resistant liver and thus insulin induces an increase in lipid synthesis without being able to block gluconeogenesis. As a consequence, lipids progressively accumulate and worsen insulin resistance, in a process, which, if not reversed, will result in an overt, type 2 diabetes. The transcription factor SREBP-1c is the major effector of long-term regulation of lipid synthesis by insulin. The synthesis of SREBP-1c is regulated by several nuclear receptors and the utilization of their ligands is opening a new therapeutic approach for the prevention and treatment of type 2 diabetes (AU)

Characterization of variants in the glucosylceramide synthase gene and their association with type 1 Gaucher disease severity.

The extreme phenotypic variability of patients with Gaucher disease (GD) is not completely explained by glucocerebrosidase gene mutations. It has been proposed that genetic modifiers might influence GD phenotype. We examined seven polymorphisms of the glucosylceramide synthase gene (UGCG) and their correlation with severity of GD. Five UGCG variants were significantly associated with disease severity, according to the DS3 scoring system: c.-295C>T, c.-232_-241ins10, c.98+50A>G, c.98+68A>T, and c.861A>G. Heterozygous [N370S]+[L444P] patients with c.[-232_-241ins10;98+50G] haplotype had a significantly lower DS3 score in relation to patients carrying only one of these polymorphisms. Electrophoretic mobility shift assay analysis showed an increased nuclear protein binding ability for the G allele at the cDNA position c.98+50, as well as an altered pattern for the c.-232_-241ins10 allele. The promoter activity of the haplotypes decreased significantly with respect to wild type activity in HepG2 and COS-7 cells (-14% and -16% for the c.[-232_-241ins10;98+50A] haplotype, -44% and -25% for c.[-222nonins;98+50G] haplotypes, and -64% and -75% for c.[-232_-241ins10;98+50G] haplotype, respectively). These data indicate that the c.-232_-241ins10 and c.98+50A>G variants are modifying factors of GD severity, which can partly explain the variability in severity of the disease.

Role of BMPs in the regulation of sclerostin as revealed by an epigenetic modifier of human bone cells.

Sclerostin, encoded by the SOST gene, is specifically expressed by osteocytes. However osteoblasts bear a heavily methylated SOST promoter and therefore do not express SOST. Thus, studying the regulation of human SOST is challenged by the absence of human osteocytic cell lines. Herein, we explore the feasibility of using the induction of SOST expression in osteoblasts by a demethylating agent to study the mechanisms underlying SOST transcription, and specifically, the influence of bone morphogenetic proteins (BMPs). Microarray analysis and quantitative PCR showed that AzadC up-regulated the expression of several BMPs, including BMP-2, BMP-4 and BMP-6, as well as several BMP downstream targets. Recombinant BMP-2 increased the transcriptional activity of the SOST promoter cloned into a reporter vector. Likewise, exposing cells transfected with the vector to AzadC also resulted in increased transcription. On the other hand, inhibition of the canonical BMP signaling blunted the effect of AzadC on SOST. These results show that the AzadC-induced demethylation of the SOST promoter in human osteoblastic cells may be a valuable tool to study the regulation of SOST expression. As a proof of concept, it allowed us to demonstrate that BMPs stimulate SOST expression by a mechanism involving BMPR1A receptors and downstream Smad-dependent pathways.

Influence of aquaporin-1 gene polymorphism on water retention in liver cirrhosis.

UNLABELLED: Water retention is a major clinical problem in patients with liver cirrhosis. The factors that predispose to water retention are poorly understood but may involve genetic factors. Recent research suggests that renal aquaporins may be a pathophysiological factor involved in this condition. Aquaporin-1 (AQP1) is expressed in the proximal tubule and aquaporin-2 (AQP2) in the renal collecting duct cells. The aim of our study was to investigate the distribution of single nucleotide polymorphisms (SNPs) of AQP1: rs1049305 (C/G) and AQP2: rs3741559 (A/G) and rs467323 (C/T) in 100 cirrhotic patients with ascites and to analyze their relationship with dilutional hyponatremia. METHODS: Genomic DNA was extracted from peripheral blood. Genotyping for the presence of different polymorphisms was performed using the Custom Taqman SNP Genotyping Assays. The possible influence of rs1049305 (C/G) in AQP1 gene expression was evaluated by luciferase assays in vitro. RESULTS: The allelic frequencies of the AQP1 gene were the following: CC = 15%; CG = 49%; GG = 36%. Patients with CC genotype had significantly lower plasma sodium concentration than those with CG or GG genotype. Luciferase assays showed that the rs1049305 (C/G) in the AQP1 gene functionally affected the expression level in vitro. In addition, we did not find any relationship between AQP2 SNPs observed and plasma sodium concentration. CONCLUSIONS: Our results suggest that the rs1049305 (C/G, UTR3) AQP1 polymorphism could be involved in the genetic susceptibility to develop water retention in patients with liver cirrhosis.

Análisis funcional de mutaciones en el promotor del LDLR y su relación con la hipercolesterolemia familiar / Functional analysis of LDLR promoter mutations and their relationship with familial hypercholesterolemia

Introducción La hipercolesterolemia familiar (HF) es una enfermedad autosómica dominante, causada principalmente por mutaciones en la región codificante del gen del receptor de las LDL (LDLR). Sin embargo, varias mutaciones situadas en el promotor de LDLR se han asociado con la HF. La búsqueda de mutaciones en sujetos clínicamente diagnosticados como HF reveló la presencia en la zona promotora de LDLR de 4 mutaciones nuevas en heterocigosidad. Objetivo Estudiar la funcionalidad de las 4 mutaciones nuevas en el promotor del LDLR (c.-36T>G, c.-136C>G, c.-140C>G y c.-208A>T) encontradas en España mediante el uso de la plataforma LIPOchip® en pacientes con sospecha clínica de HF. Métodos Se realizó el análisis funcional de las mutaciones mediante ensayos de retardo de la movilidad electroforética (EMSA) y transfección de promotores mutados con el gen reportero de la luciferasa en cultivos celulares de HepG2. Resultados Las mutaciones c.-136G y c.-140G localizadas en el elemento regulador R3 mostraron un cambio significativo en el patrón de afinidad por las proteínas nucleares en los EMSA. Además, estas mutaciones produjeron una reducción de la actividad del promotor LDLR del 88-93%. Las mutaciones c.-36G y c.-208T no provocaron cambios significativos ni en los experimentos EMSA ni con genes reporteros. Conclusiones Las mutaciones localizadas en el elemento R3 se asocian con HF, mientras que las que se encuentran fuera de los elementos reguladores del promotor de LDLR no son causa directa de hipercolesterolemia. Nuestros resultados revelan la importancia de realizar análisis de funcionalidad de las variantes encontradas en la región promotora de LDLR con objeto de conocer su implicación con el fenotipo HF (AU)

Introduction: Familial hypercholesterolemia (FH) is an autosomal dominant disorder mainly caused by mutations in the coding region of the LDLR gene. However, a variety of mutations within the LDLR promoter have been associated with FH. Genetic screening in persons clinically diagnosed with HF revealed the presence of four new heterozygous mutations within the promoter region. Objective: To study the functionality of the four new LDLR promoter mutations (c.-36T>G, c.-136C>G, c.-140C>G and c.-208A>T) found in Spain, using the LIPOchip® platform in patients with clinically suspected FH. Methods: The functional analysis of mutations was carried out by using electrophoretic mobility shift assays (EMSA) and luciferase reporter gene expression in HepG2 transfected cells with the mutated promoters. Results: Two mutations, c.-136G and c.-140, located within the R3 regulatory element, showed a significant change in the pattern of nuclear binding protein affinity. Moreover, these mutations reduced the residual activity of the LDLR promoter by 88-93%. However, mutations c.-36Gand c.-208T, located outside the response elements, produced no significant changes in EMSA experiments or reporter genes. Conclusions: Mutations within the R3 element are associated with FH, while those located outside the regulatory elements of the LDLR promoter are not a direct cause of FH. Our results reveal the importance of functional analysis of the new variants in the LDLR promoter region to identify their role in the FH phenotype (AU)

Functional analysis of LDLR promoter and 5' UTR mutations in subjects with clinical diagnosis of familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a dominant disorder due to mutations in the LDLR gene. Several mutations in the LDLR promoter are associated with FH. Screening of 3,705 Spanish FH patients identified 10 variants in the promoter and 5' UTR. Here, we analyse the functionality of six newly identified LDLR variants. Mutations located in the LDLR promoter regulatory elements R2 and R3 (c.-155_-150delACCCCinsTTCTGCAAACTCCTCCC, c.-136C>G, c.-140C>G, and c.-140C>T) resulted in 6 to 15% residual activity in reporter expression experiments and changes in nuclear protein binding affinity compared to wild type. No reduction was observed when cells were transfected with c.-208T, c.-88A, and c.-36G mutant fragments. Our results indicate that mutations localized in R2 and R3 are associated with hypercholesterolemia, whereas mutations outside the LDLR response elements are not a cause of FH. This data emphasizes the importance of functional analysis of variants in the LDLR promoter to determine their association with the FH phenotype.

El polimorfismo -1131 T>C del promotor del gen de la apolipoproteína A5 altera la unión de NRF2 (nuclear respiratory factor-2) y disminuye la actividad del promotor / -1131 T>C polymorphism of the apolipoprotein A5 gene promoter alters the binding of NRF2 (nuclear respiratory factor 2) and decreases promoter activity

Introducción. El papel que desempeñan los triglicéridos (TG) ingeridos en la dieta en la aparición de aterosclerosis es controvertido. Sin embargo, recientemente se ha descrito que los valores de TG en estado posprandial son un factor de riesgo independiente para la aparición de esta enfermedad. Recientemente, se ha identificado la apolipoproteína (Apo) AV como una proteína clave en la regulación del metabolismo de los TG. El polimorfismo ¿1131 T>C del gen de la ApoA5 se ha asociado con cambios en los valores plasmáticos de TG y de ApoAV. El objetivo de este trabajo fue determinar el papel del polimorfismo ¿1131 T>C en la expresión del gen ApoA5. Material y métodos. Con el fin de estudiar la influencia de este single nucleotide polymorphisms (SNP) en la expresión de este promotor, se han utilizado construcciones de genes reporteros de ambos alelos. Mediante experimentos de retardo en gel se demostró que este polimorfismo también altera la secuencia de unión a NRF-2. Conclusiones. Nuestros resultados indicaron que la actividad del promotor de ApoA5 que porta la variante T del SNP ¿1131 T>C es mayor que la del promotor que lleva el alelo C. Además, este cambio de nucleótido se traduce en un cambio de afinidad proteínas-ácido desoxirribonucleico. Estos resultados indican que un cambio en la actividad del promotor del gen ApoA5 podría ser la causa del incremento de los valores plasmáticos de TG asociados con el alelo C (AU)

Introduction. Postprandial triglycerides (TG) have been established as an independent risk factor for atherosclerosis, even though the role of fasting TG in the pathogenesis of this disease remains controversial. Recently, apolipoprotein AV (ApoAV) has been identified as a key player in TG metabolism. The ¿1131 T>C polymorphism in the ApoA5 gene promoter has been associated with changes in plasma triglyceride and ApoAV levels. Our objective was the functional analysis of the SNP ¿1131 T>C located on ApoA5 promoter to clarify his effect on ApoA5 expression. Material and methods. In order to test if this SNP influences promoter function, we have performed reporter gene assays. Our results indicated that the promoter carrying the T allele is stronger than its C- allele variant. Additionally electrophoretic ¿ mobility shift assays showed that the variant also produces a change in a NRF-2 binding site. Conclusions. Together, these results suggest that a change in the activity of ApoA5 promoter could be responsible for the increases in plasma TG levels associated with the C allele (AU)

Estudio funcional del promotor del gen NPC1L1 / Functional analysis of the NPC1L1 gene promoter

Introducción. En la absorción intestinal de colesterol intervienen distintos transportadores, uno de gran importancia y diana del fármaco ezetimiba, la proteína NPC1L1. Se ha demostrado una asociación entre distintas variantes genéticas de NPC1L1, la eficiencia en la absorción de esteroles y la concentración plasmática de colesterol unido a lipoproteínas de baja densidad. El objetivo de este estudio fue identificar y analizar el efecto funcional de los polimorfismos genéticos potencialmente más relevantes del gen NPC1L1sobre su actividad transcripcional. Material y métodos. Como zona de estudio se seleccionó 2 kb de la región promotora del genNPC1L1. Se realizó una búsqueda en bases de datos para localizar variantes descritas en las secuencias seleccionadas. Se diseñaron 3fragmentos, que se amplificaron mediante PCR y posteriormente se secuenciaron. La funcionalidad del polimorfismo –133A>G se determinó mediante ensayos de retardo en gel y medida de la actividadluciferasa. Resultados. El análisis de la zona promotora de102 sujetos normolipidémicos mostró 5 nuevas variantes polimórficas (–1485C>T, –1425C>G,–982G>C, –292T>C y –18C>A) no identificadas previamente. Los resultados de los ensayos de retardo en gel con el polimorfismo –133A>Grevelaron mayor afinidad de unión de las proteínas nucleares con la sonda portadora de la variante–133A. Por otra parte, la actividad del promotor deNPC1L1 con la variante –133G mostró un aumento de 2,5 veces respecto a la variante –133A.Conclusión. Nuestros resultados demuestran que hay diferencias en la afinidad de unión y actividad transcripcional de NPC1L1 en función del polimorfismo –133A>G. Esta variante génica podría contribuir a la variabilidad interindividual de la absorción intestinal de esteroles (AU)

Introduction. Cholesterol absorption is a process which involves the participation of numerous transporters. One of these transporters is NPC1L1,identified as a critical protein for intestinal cholesterol absorption and the molecular target of ezetimibe. It has been shown that NPC1L1 variants are associated with variations in sterol absorption and plasma levels of LDL-C. The aim of this work was to identify and analyse the potential functional effect on the transcriptional activity of NPC1L1gene polymorphisms. Material and methods. The previously describedNPC1L1 promoter variants were located by means of SNP database search. The NPC1L1 promoter was analysed by three PCR reactions followed by automatic capillary sequencing. Functional study of the –133A>G polymorphism was performed by EMSA and Luciferase assays. Results. NPC1L1 promoter analysis in 102normolipemic subjects showed five (..) (AU)

The g.763G>C SNP of the bovine FASN gene affects its promoter activity via Sp-mediated regulation: implications for the bovine lactating mammary gland.

Fatty acid synthase (FASN) is an enzyme that catalyzes de novo synthesis of fatty acids in cells. The bovine FASN gene maps to BTA 19, where several quantitative trait loci for fat-related traits have been described. Our group recently reported the identification of a single nucleotide polymorphism (SNP), g.763G>C, in the bovine FASN 5' flanking region that was significantly associated with milk fat content in dairy cattle. The g.763G>C SNP was part of a GC-rich region that may constitute a cis element for members of the Sp transcription factor family. Thus the SNP could alter the transcription factor binding ability of the FASN promoter and consequently affect the promoter activity of the gene. However, the functional consequences of the SNP on FASN gene expression are unknown. The present study was therefore directed at elucidating the underlying molecular mechanism that could explain the association of the SNP with milk fat content. Three cellular lines (3T3L1, HepG2, and MCF-7) were used to test the promoter and the transcription factor binding activities by luciferase reporter assays and electrophoretic mobility shift assays, respectively. Band shift assays were also carried out with nuclear extracts from lactating mammary gland (LMG) to further investigate the role of the SNP in this tissue. Our results demonstrate that the SNP alters the bovine FASN promoter activity in vitro and the Sp1/Sp3 binding ability of the sequence. In bovine LMG, the specific binding of Sp3 may account for the association with milk fat content.

Mutational screening of phospholamban gene in hypertrophic and idiopathic dilated cardiomyopathy and functional study of the PLN -42 C>G mutation.

BACKGROUND: Phospholamban is an endogenous sarcoplasmic reticulum calcium ATPase inhibitor with a regulatory effect on cardiac contraction/relaxation coupling. Mutations in the phospholamban gene (PLN) have been associated with primary cardiomyopathies. AIMS: To screen for PLN mutations in our population of patients with primary cardiomyopathies and to perform functional analysis of the mutations identified. METHODS: We performed SSCP mutational screening and DNA sequencing of the PLN gene in 186 patients with either hypertrophic or dilated cardiomyopathy. To study promoter strength we constructed reporter plasmids containing the luciferase gene and performed transient transfection analysis in C6 and C2C12 cell lines. RESULTS: The PLN -42 C>G mutation was found in one patient with late onset familial apical hypertrophic cardiomyopathy. This mutation decreased phospholamban promoter activity by 43% and 47%, in C6 and C2C12 cell lines respectively. One son had mild apical hypertrophic cardiomyopathy and carried the mutation, another son with normal ECG and echocardiogram also had the mutation. CONCLUSION: The PLN -42 C>G mutation is associated with a benign form of apical hypertrophic cardiomyopathy in this family, though the presence of a healthy adult carrier suggests that other genetic and environmental factors could be involved. Otherwise, mutations in the PLN gene are not a frequent cause of cardiomyopathies in our population.