Role of Wnt pathway genes in complex and monogenic phenotypes of low and high bone mass

[eng] Osteoporosis is a complex disease characterized by low bone mass, microarchitectural deterioration, and increased fracture risk. It is the most common bone disease that affects more than 200 million people worldwide and is responsible for more than 8.9 million fractures every year, especially affecting the elderly. This makes it one of the major causes of morbidity and mortality and generates a high social-health cost, which is expected to grow as life expectancy has been increasing considerably over the last decades. Although nowadays there are different treatments available that reduce bone loss, few of them target bone formation enhancement. Therefore, it is essential to discover new anabolic therapeutic targets that restore bone mass and skeletal architecture. In this doctoral thesis, I have used two tools to identify new therapeutic targets by elucidating the genetic bases of the disease. On the one hand, I have performed a search for variants in three Wnt pathway genes, that might explain the GWAS significant association with different bone parameters, and subsequently I characterized them functionally. Specifically, I have studied DKK1, SOST, and WNT16, identifying common and rare variants that may explain the susceptibility to the complex disease of osteoporosis or the extreme high bone mass (HBM) phenotype. Regarding DKK1, I have shown the loss of inhibitory function associated with missense mutations found in patients with HBM and/or in the general population. I have also detected for the first time a physical contact between the region enriched in BMD-GWAS signals and the DKK1 promoter and also with, LNCAROD, a lncRNA known to regulate DKK1 expression. For WNT16, I have identified a promoter region in intron 2 that interacts with different regulatory elements located in introns of the neighboring gene CPED1. Finally, in SOST, I have shown a decrease in gene expression associated with the minor allele of a variant that modifies an extended TATA box motif, a reduction in protein expression of a missense variant in the signal peptide found in an HBM women, and, for the first time, physical interactions between the SOST proximal promoter and ECR5 and several enhancer elements upstream of it. These functional studies reinforce the importance of the Wnt pathway in bone homeostasis and demonstrate the need to study in-depth the GWAS signals that often do not correspond to a functional variant. On the other hand, using whole-exome sequencing (WES), I have also investigated a family case of HBM in the search for a causal gene. Manual filtering of the low- frequency variants found in this case suggests that mutated VAV3 - along with other variants in SIK3- could determine the HBM phenotype in the family due to an inhibitory effect on osteoclastogenesis. Importantly, the results have pointed at novel genes and regions which may constitute a new therapeutic target for osteoporosis

Nickel removal from exhausted electroplatting baths by using vegetable wastes

During the last years our research group has been studying the use of industrial vegetable wastes as grape stalks and exhausted coffee to remove metals ions such as Ni(II), Cu(II), Pb(II), Zn(II), Cd(II) or Cr(VI) and Cr(III) in aqueous solution from the point of view to use these wastes as biosorbents in a low cost alternative to activated carbon for wastewater treatment. The optimal experimental conditions for the removal of each of these metal ions in synthetic solutions by using both biosorbents were determined in previous studies . In this work, the performance of grape stalks and exhausted coffee for the removal of nickel ions from an exhausted electroplating bath of a metal finishing industry from Barcelona (Spain) has been investigated. Batch and column experiments were carried out at room temperature by using grape stalk wastes (particle size 0.8-1.0 mm), meanwhile in the case of exhausted coffee, two different particle size ranges were used, 0.25-0.50 mm and 0.50-1.00 mm for batch and column experiments, respectively. Kinetics and equilibrium studies were carried out in batch mode to determine the equilibrium contact time and to obtain the sorption capacity of sorbents. The data in both studies have been treated by using different models. Column experiments were designed to establish the optimal condition for the treatment of the industrial wastewater. The experiments were performed in packed bed up flow columns of different internal diameter and bed depth in order to predict the transport and sorption parameters. In all column experiments the flow rate was around 11 mL h-1. From perspective of process modelling, the dynamic behavior was described in terms of breakthrough curves. The bed depth service time (BDST), Thomas and Yoon Nelson models were used to analyze the experimental data and to determine model parameters. Batch results show that about 1hour was the time needed to reach equilibrium when using grape stalks and around 15 h when using exhausted coffee. In the case of exhausted coffee, the pH solution decreased during the sorption process from initial pH 5,5 to lower pH than the corresponding pHpzc (point zero charge). To avoid this, pH solution was controlled to a constant pH 5,5 and the equilibrium were achieved in 1h. Kinetic data of both sorption processes fit pseudo-second order model, indicating that chemisorption could be rate limiting in the sorption step. Equilibrium data of nickel sorption onto grape stalks and exhausted coffee fit adequately Langmuir model, indicating monolayer coverage. Results showed that maximum sorption capacity of grape stalks (4,8 10-2 mmol/g; 2,84 mg/g) is slightly higher than exhausted coffee (2,9 10-2 mmol/g; 1,70 mg/g). The maximum nickel sorption capacity of both sorbents was reduced to 50% compared to maximum sorption capacity determined using synthetic Ni(II) solutions. Thus, grape stalks and exhausted coffee performance for the removal of Ni(II) from the studied industrial wastewater are negatively affected by the presence of other compounds in the industrial wastewater. In column experiments, the best results were obtained by using 2.8 cm internal diameter columns and bed depth 6 cm and 8 cm for grape stalks and exhausted coffee, respectively. Breakthrough curves were successfully modelled by the proposed columns models. The results obtained demonstrated that grape stalks sorption capacity was higher than exhausted coffee but this one presented a higher sorption rate.Postprint (published version

Valorització de residus vegetals procedents de processos industrials com a biosorbents per a l'eliminació d'ions metàl•lics d'afluents aquosos

L’objectiu d’aquest projecte és la valorització de residus vegetals procedents de processos industrials, com són el pinyol d’oliva, rapa del raïm, suro, marro de cafè, etc.,com a biosorbents per a l’eliminació de diferents ions metàl·lics, crom, arsènic, plom, cadmi, coure, níquel, etc., d’afluents aquosos.Peer Reviewe

IV Jornada d'Intercanvi d'Experiències de la Família de Serveis Socioculturals i a la Comunitat

Encara que aquesta és la primera vegada que publiquem les experiències presentades, aquestes IV Jornades, tenen un recorregut anterior i són el reflex del saber fer professional en el que es troba gran part del col·lectiu d’aquesta Família professional. En el temps transcorregut des de la última jornada s’han implantat la majoria dels cicles LOE, i encara que alguns centres fa més temps que d’altres que segueixen aquest nou camí, és un bon moment per reflexionar sobre el que s’ha fet, compartir noves experiències entre el professorat, imaginar nous escenaris per desenvolupar i seguir construint la professió docent. Els cicles LOE han suposat l’orientació definitiva de la formació per competències professionals, perquè els alumnes esdevinguin professionals competents, capaços d’aplicar en contextos de treball els coneixements adquirits i les habilitats desenvolupades. Organitzar les classes d’altres maneres que no siguin la classe magistral ha suposat esforç, creativitat i coordinació amb l’equip docent de dins i de fora dels centres per acostar la realitat laboral als nostres estudiants. Els diferents centres que imparteixen els cicles d’aquesta família professional recullen un bon grapat d’experiències en aquest sentit. Posar- les en comú, saber què fem uns i altres, obre un món de possibilitats al professorat i per això les volem fer arribar a altres docents. Les presents jornades s’han organitzat a partir d’un grup de treball de l’Institut de Ciències de la Universitat de Barcelona que han participat conjuntament en altres ocasions en temes relacionats amb aquesta família professional. Ha estat una gran oportunitat per desenvolupar un treball cooperatiu, engrescador i enriquidor que posarem al servei d’altres tasques de formació

Lipoprotein hydrophobic core lipids are partially extruded to surface in smaller HDL : "Herniated" HDL, a common feature in diabetes

Recent studies have shown that pharmacological increases in HDL cholesterol concentrations do not necessarily translate into clinical benefits for patients, raising concerns about its predictive value for cardiovascular events. Here we hypothesize that the size-modulated lipid distribution within HDL particles is compromised in metabolic disorders that have abnormal HDL particle sizes, such as type 2 diabetes mellitus (DM2). By using NMR spectroscopy combined with a biochemical volumetric model we determined the size and spatial lipid distribution of HDL subclasses in a cohort of 26 controls and 29 DM2 patients before and after two drug treatments, one with niacin plus laropiprant and another with fenofibrate as an add-on to simvastatin. We further characterized the HDL surface properties using atomic force microscopy and fluorescent probes to show an abnormal lipid distribution within smaller HDL particles, a subclass particularly enriched in the DM2 patients. The reduction in the size, force cholesterol esters and triglycerides to emerge from the HDL core to the surface, making the outer surface of HDL more hydrophobic. Interestingly, pharmacological interventions had no effect on this undesired configuration, which may explain the lack of clinical benefits in DM2 subjects

Genetics and Genomics of SOST: functional analysis of variants and genomic regulation in osteoblasts

SOST encodes the sclerostin protein, which acts as a key extracellular inhibitor of the canonical Wnt pathway in bone, playing a crucial role in skeletal development and bone homeostasis. The objective of this work was to assess the functionality of two variants previously identified (the rare variant rs570754792 and the missense variant p.Val10Ile) and to investigate the physical interactors of the SOST proximal promoter region in bone cells. Through a promoter luciferase reporter assay we show that the minor allele of rs570754792, a variant located in the extended TATA box motif, displays a significant decrease in promoter activity. Likewise, through western blot studies of extracellular and intracellular sclerostin, we observe a reduced expression of the p.Val10Ile mutant protein. Finally, using a circular chromosome conformation capture assay (4C-seq) in 3 bone cell types (MSC, hFOB, Saos-2), we have detected physical interactions between the SOST proximal promoter and the ECR5 enhancer, several additional enhancers located between EVT4 and MEOX1 and a distant region containing exon 18 of DHX8. In conclusion, SOST presents functional regulatory and missense variants that affect its expression and displays physical contacts with far reaching genomic sequences, which may play a role in its regulation within bone cells

Genetic analysis in a familial case with high bone mineral density suggests additive effects at two loci

Osteoporosis is the most common bone disease, characterized by a low bone mineral density (BMD) and increased risk of fracture. At the other end of the BMD spectrum, some individuals present strong, fracture-resistant, bones. Both osteoporosis and high BMD are heritable and their genetic architecture encompasses polygenic inheritance of common variants and some cases of monogenic highly penetrant variants in causal genes. We have investigated the genetics of high BMD in a family segregating this trait in an apparently Mendelian dominant pattern. We searched for rare causal variants by whole-exome sequencing in three affected and three nonaffected family members. Using this approach, we have identified 38 rare coding variants present in the proband and absent in the three individuals with normal BMD. Although we have found four variants shared by the three affected members of the family, we have not been able to relate any of these to the high-BMD phenotype. In contrast, we have identified missense variants in two genes, VAV3 and ADGRE5, each shared by two of out of three affected members, whose loss of function fits with the phenotype of the family. In particular, the proband, a woman displaying the highest BMD (sum Z-score = 7), carries both variants, whereas the other two affected members carry one each. VAV3 encodes a guanine-nucleotide-exchange factor with an important role in osteoclast activation and function. Although no previous cases of VAV3 mutations have been reported in humans, Vav3 knockout (KO) mice display dense bones, similarly to the high-BMD phenotype present in our family. The ADGRE5 gene encodes an adhesion G protein-coupled receptor expressed in osteoclasts whose KO mouse displays increased trabecular bone volume. Combined, these mouse and human data highlight VAV3 and ADGRE5 as novel putative high-BMD genes with additive effects, and potential therapeutic targets for osteoporosis. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research

Gene Network of Susceptibility to Atypical Femoral Fractures Related to Bisphosphonate Treatment

Atypical femoral fractures (AFF) are rare fragility fractures in the subtrocantheric or diaphysis femoral region associated with long-term bisphosphonate (BP) treatment. The etiology of AFF is still unclear even though a genetic basis is suggested. We performed whole exome sequencing (WES) analysis of 12 patients receiving BPs for at least 5 years who sustained AFFs and 4 controls, also long-term treated with BPs but without any fracture. After filtration and prioritization of rare variants predicted to be damaging and present in genes shared among at least two patients, a total of 272 variants in 132 genes were identified. Twelve of these genes were known to be involved in bone metabolism and/or AFF, highlighting DAAM2 and LRP5, both involved in the Wnt pathway, as the most representative. Afterwards, we intersected all mutated genes with a list of 34 genes obtained from a previous study of three sisters with BP-related AFF, identifying nine genes. One of these (MEX3D) harbored damaging variants in two AFF patients from the present study and one shared among the three sisters. Gene interaction analysis using the AFFNET web suggested a complex network among bone-related genes as well as with other mutated genes. BinGO biological function analysis highlighted cytoskeleton and cilium organization. In conclusion, several genes and their interactions could provide genetic susceptibility to AFF, that along with BPs treatment and in some cases with glucocorticoids may trigger this so feared complication

Predictive and Prognostic Brain Metastases Assessment in Luminal Breast Cancer Patients: FN14 and GRP94 from Diagnosis to Prophylaxis

FN14 has been implicated in many intracellular signaling pathways, and GRP94 is a well-known endoplasmic reticulum protein regulated by glucose. Recently, both have been associated with metastasis progression in breast cancer patients. We studied the usefulness of FN14 and GRP94 expression to stratify breast cancer patients according their risk of brain metastasis (BrM) progression. We analyzed FN14 and GRP94 by immunohistochemistry in a retrospective multicenter study using tissue microarrays from 208 patients with breast carcinomas, of whom 52 had developed BrM. Clinical and pathological characteristics and biomarkers expression in Luminal and non-Luminal patients were analyzed using a multivariate logistic regression model adjusted for covariates, and brain metastasis-free survival (BrMFS) was estimated using the Kaplan–Meier method and the Cox proportional hazards model. FN14 expression was associated with BrM progression mainly in Luminal breast cancer patients with a sensitivity (53.85%) and specificity (89.60%) similar to Her2 expression (46.15 and 89.84%, respectively). Moreover, the likelihood to develop BrM in FN14-positive Luminal carcinomas increased 36.70-fold (3.65–368.25, p = 0.002). Furthermore, the worst prognostic factor for BrMFS in patients with Luminal carcinomas was FN14 overexpression (HR = 8.25; 95% CI: 2.77–24.61; p = 0.00015). In these patients, GRP94 overexpression also increased the risk of BrM (HR = 3.58; 95% CI: 0.98–13.11; p = 0.054—Wald test). Therefore, FN14 expression in Luminal breast carcinomas is a predictive/prognostic biomarker of BrM, which combined with GRP94 predicts BrM progression in non-Luminal tumors 4.04-fold (1.19–8.22, p = 0.025), suggesting that both biomarkers are useful to stratify BrM risk at early diagnosis. We propose a new follow-up protocol for the early prevention of clinical BrM of breast cancer patients with BrM risk