Depletion of ALMS1 affects TGF-β signalling pathway and downstream processes such as cell migration and adhesion capacity

Background: ALMS1 is a ubiquitous gene associated with Alström syndrome (ALMS). The main symptoms of ALMS affect multiple organs and tissues, generating at last, multi-organic fibrosis in the lungs, kidneys and liver. TGF- β is one of the main pathways implicated in fibrosis, controlling the cell cycle, apoptosis, cell migration, cell adhesion and epithelial-mesenchymal transition (EMT). Nevertheless, the role of ALMS1 gene in fibrosis generation and other implicated processes such as cell migration or cell adhesion via the TGF- β pathway has not been elucidated yet. Methods: Initially, we evaluated how depletion of ALMS1 affects different processes like apoptosis, cell cycle and mitochondrial activity in HeLa cells. Then, we performed proteomic profiling with TGF-β stimuli in HeLa ALMS1 −/− cells and validated the results by examining different EMT biomarkers using qPCR. The expression of these EMT biomarkers were also studied in hTERT-BJ- 5ta ALMS1 −/−. Finally, we evaluated the SMAD3 and SMAD2 phosphorylation and cell migration capacity in both models. Results: Depletion of ALMS1 generated apoptosis resistance to thapsigargin (THAP) and C2-Ceramide (C2-C), and G2/M cell cycle arrest in HeLa cells. For mitochondrial activity, results did not show significant differences between ALMS1 +/+ and ALMS1 −/−. Proteomic results showed inhibition of downstream pathways regulated by TGF-β. The protein-coding genes (PCG) were associated with processes like focal adhesion or cell-substrate adherens junction in HeLa. SNAI1 showed an opposite pattern to what would be expected when activating the EMT in HeLa and BJ-5ta. Finally, in BJ-5ta model a reduced activation of SMAD3 but not SMAD2 were also observed. In HeLa model no alterations in the canonical TGF-β pathway were observed but both cell lines showed a reduction in migration capacity. Conclusion: ALMS1 has a role in controlling the cell cycle and the apoptosis processes. Moreover, the depletion of ALMS1 affects the signal transduction through the TGF-β and other processes like the cell migration and adhesion capacity.Ministerio de Educación, Cultura y Deporte | Ref. FPU17/01567Instituto de Salud Carlos III | Ref. PI15/00049Instituto de Salud Carlos III | Ref. PI19/00332Xunta de Galicia | Ref. ED431G-2019/06Xunta de Galicia | Ref. ED431C-2018/5

Loss of the centrosomal protein ALMS1 alters lipid metabolism and the regulation of extracellular matrix-related processes

BACKGROUND: Alström syndrome (ALMS) is a rare autosomal recessive disease that is associated with mutations in ALMS1 gene. The main clinical manifestations of ALMS are retinal dystrophy, obesity, type 2 diabetes mellitus, dilated cardiomyopathy and multi-organ fibrosis, characteristic in kidneys and liver. Depletion of the protein encoded by ALMS1 has been associated with the alteration of different processes regulated via the primary cilium, such as the NOTCH or TGF-β signalling pathways. However, the cellular impact of these deregulated pathways in the absence of ALMS1 remains unknown. METHODS: In this study, we integrated RNA-seq and proteomic analysis to determine the gene expression profile of hTERT-BJ-5ta ALMS1 knockout fibroblasts after TGF-β stimulation. In addition, we studied alterations in cross-signalling between the TGF-β pathway and the AKT pathway in this cell line. RESULTS: We found that ALMS1 depletion affects the TGF-β pathway and its cross-signalling with other pathways such as PI3K/AKT, EGFR1 or p53. In addition, alterations associated with ALMS1 depletion clustered around the processes of extracellular matrix regulation and lipid metabolism in both the transcriptome and proteome. By studying the enriched pathways of common genes differentially expressed in the transcriptome and proteome, collagen fibril organisation, β-oxidation of fatty acids and eicosanoid metabolism emerged as key processes altered by the absence of ALMS1. Finally, an overactivation of the AKT pathway was determined in the absence of ALMS1 that could be explained by a decrease in PTEN gene expression. CONCLUSION: ALMS1 deficiency disrupts cross-signalling between the TGF-β pathway and other dependent pathways in hTERT-BJ-5ta cells. Furthermore, altered cross-signalling impacts the regulation of extracellular matrix-related processes and fatty acid metabolism, and leads to over-activation of the AKT pathway

Evaluation of the stability of newborn hospital parenteral nutrition solutions

(1) Background: parenteral nutrition (PN) solutions are an extremely complex mixture. It is composed of a multitude of chemical elements that can give rise to a large number of interactions that condition its stability and safety. The aim of this study was to evaluate the stability of PN solutions for preterm infants. (2) Methods: eight samples were prepared according to the protocol for prescribing PN in preterm infants. Samples PN1–PN7 had the normal progression of macronutrients and standard amounts of micronutrients for a 1 kg preterm infant. The PN8 sample had a high concentration of electrolytes, with the idea of forcing stability limits. Samples were stored both at room temperature and under refrigeration. Measurements of globule size, pH, density, and viscosity were performed in both storage protocols on different days after processing. (3) Results: the changes in the composition of the samples did not affect the evolution of the stability at the different measurement times and temperatures. Viscosity was affected by the compositional changes made in the PN samples, but no alterations due to time or temperature were observed. Density and pH remained stable, without significant changes due to time, storage temperature, or different composition. (4) Conclusion: all samples remained stable during the study period and did not undergo significant alterations due to compositional changes or different experimental conditions

Allelic overload and its clinical modifier effect in Bardet-Biedl syndrome

Bardet–Biedl syndrome (BBS) is an autosomal recessive ciliopathy characterized by extensive inter- and intra-familial variability, in which oligogenic interactions have been also reported. Our main goal is to elucidate the role of mutational load in the clinical variability of BBS. A cohort of 99 patients from 77 different families with biallelic pathogenic variants in a BBS-associated gene was retrospectively recruited. Human Phenotype Ontology terms were used in the annotation of clinical symptoms. The mutational load in 39 BBS-related genes was studied in index cases using different molecular and next-generation sequencing (NGS) approaches. Candidate allele combinations were analysed using the in silico tools ORVAL and DiGePred. After clinical annotation, 76 out of the 99 cases a priori fulfilled established criteria for diagnosis of BBS or BBS-like. BBS1 alleles, found in 42% of families, were the most represented in our cohort. An increased mutational load was excluded in 41% of the index cases (22/54). Oligogenic inheritance was suspected in 52% of the screened families (23/45), being 40 tested by means of NGS data and 5 only by traditional methods. Together, ORVAL and DiGePred platforms predicted an oligogenic effect in 44% of the triallelic families (10/23). Intrafamilial variable severity could be clinically confirmed in six of the families. Our findings show that the presence of more than two alleles in BBSassociated genes correlated in six families with a more severe phenotype and associated with specific findings, highlighting the role of the mutational load in the management of BBS casesInstituto de Salud Carlos III | Ref. PI15/00049Instituto de Salud Carlos III | Ref. PI16/00425Instituto de Salud Carlos III | Ref. PI19/00321Instituto de Salud Carlos III | Ref. PI19/00332CIBERER | Ref. 07/06/0036IIS-FJD BioBank | Ref. PT13/0010/0012Comunidad de Madrid | Ref. B2017/BMD-3721Xunta de Galicia | Ref. ED431G-2019/06Xunta de Galicia | Ref. ED431C-2018/54ISCIII | Ref. FI17/00192Ministerio de Educación, Cultura y Deporte | Ref. FPU 19/00175ISCIII | Ref. CP16/0011

Descifrando as bases moleculares da síndrome de Alström: correlacións xenotipo-fenotipo e un enfoque multiómico na regulación da vía TGF-B.

This thesis project will deal with the ALMS1 gene and its implication in the TGF-B; pathway. Recent studies in our group determined that this protein could be interacting with the TGF-B; pathway, one of the main signal transduction pathways, and that it has already been extensively studied in other diseases, such as cancer, but little described in this disease. Deregulation in the TGF-B; pathway is related to the development of fibrosis, which can occur in two independent ways: the canonical pathway (SMAD 2/3) or non-canonical pathway (p38 / MAPK). The preliminary results of our group indicate that the reduction of the levels of ALMS1 decreased the magnitude of the signaling, in addition to producing a reduction of the rapid canonical response capacity to the stimulation of the TGF-B; and BMP pathways, as well as in the case of signaling mediated by pERK1 / 2 associated with the TGF-B; pathway (while stimulating the BMP pathway, a non-canonical response of lesser magnitude was observed but without affecting the duration thereof). This project will focus on clarifying whether fibrosis production in Alström patients occurs through the canonical Smad-dependent pathway or a non-canonical pathway such as the P38 / MAPK pathway.Este proyecto de tesis tratará sobre el gen ALMS1 y su implicación en la ruta TGF-B;. Estudios recientes en nuestro grupo determinaron que esta proteína podría estar interactuando con la ruta de TGF-B;, una de las principales vías de transducción de señales, y que ya ha sido estudiado extensamente en otras enfermedades, como el cáncer, pero poco descrita en esta enfermedad. La desregulación en la ruta de TGF-B; se relacionan con el desarrollo de fibrosis, que puede ocurrir de dos maneras independientes: la vía canónica (SMAD 2/3) o vía no canónica (p38 / MAPK). Los resultados preliminares de nuestro grupo indican que la reducción de los niveles de ALMS1 disminuyó la magnitud de la señalización, además de producir una reducción de la capacidad de respuesta canónica rápida a la estimulación de las vías TGF-B; y BMP, así como en el caso de la señalización mediada por pERK1/2 asociada a la vía TGF-B; (mientras que al estimular la vía BMP, se observó una respuesta no canónica de menor magnitud pero sin afectar a la duración de la misma). Este proyecto se centrará en aclarar si la producción de fibrosis en pacientes Alström se produce a través de la vía canónica Smad-dependiente o una vía no canónica como la ruta de la P38 / MAPK.Este proxecto de tesis versará sobre o xene ALMS1 e a sua implicación na ruta TGF-B;. Recentes estudos no noso grupo determinaron que dita proteína podería estar interactuando coa ruta TFG-B;, unha das principais rutas de transdución de sinais, e a cal xa foi amplamente estudada noutras patoloxía coma o cáncer, pero pouco descrita nesta enfermidade. Desregulacións na ruta TGF-B; están relacionadas co desenvolvemento de fibrose, a cal pode producirse por dúas vías independentes: a vía canónica (SMAD 2/3) ou a vía non canónica (p38/MAPK). Os resultados preliminais do noso grupo indican que a redución dos niveis de ALMS1 diminuían a magnitude da sinalización, ademais de producir unha redución da capacidade de resposta canónica rápida á estimulación das vías TGF-B; e BMP, así como no caso da sinalización mediada por pERK1/2 asociada a vía TGF-B; (mentres que o estimular a vía BMP, observouse unha resposta non canónica de menor magnitude, pero sen afectar á duración da mesma). Este proxecto centraráse en dilucidar se a producción de fibrose en pacientes de Alström producese pola vía canónica dependente de SMAD ou por unha vía non canónica como pode ser a vía da P38/MAPK.Instituto de Salud Carlos III | Ref. PI19/00332Ministerio de Economía y Competitividad | Ref. PI15/0004

Loss of the centrosomal protein ALMS1 alters lipid metabolism and the regulation of extracellular matrix-related processes

Abstract Background Alström syndrome (ALMS) is a rare autosomal recessive disease that is associated with mutations in ALMS1 gene. The main clinical manifestations of ALMS are retinal dystrophy, obesity, type 2 diabetes mellitus, dilated cardiomyopathy and multi-organ fibrosis, characteristic in kidneys and liver. Depletion of the protein encoded by ALMS1 has been associated with the alteration of different processes regulated via the primary cilium, such as the NOTCH or TGF-β signalling pathways. However, the cellular impact of these deregulated pathways in the absence of ALMS1 remains unknown. Methods In this study, we integrated RNA-seq and proteomic analysis to determine the gene expression profile of hTERT-BJ-5ta ALMS1 knockout fibroblasts after TGF-β stimulation. In addition, we studied alterations in cross-signalling between the TGF-β pathway and the AKT pathway in this cell line. Results We found that ALMS1 depletion affects the TGF-β pathway and its cross-signalling with other pathways such as PI3K/AKT, EGFR1 or p53. In addition, alterations associated with ALMS1 depletion clustered around the processes of extracellular matrix regulation and lipid metabolism in both the transcriptome and proteome. By studying the enriched pathways of common genes differentially expressed in the transcriptome and proteome, collagen fibril organisation, β-oxidation of fatty acids and eicosanoid metabolism emerged as key processes altered by the absence of ALMS1. Finally, an overactivation of the AKT pathway was determined in the absence of ALMS1 that could be explained by a decrease in PTEN gene expression. Conclusion ALMS1 deficiency disrupts cross-signalling between the TGF-β pathway and other dependent pathways in hTERT-BJ-5ta cells. Furthermore, altered cross-signalling impacts the regulation of extracellular matrix-related processes and fatty acid metabolism, and leads to over-activation of the AKT pathway

ALMS1 regulates TGF-β signaling and morphology of primary cilia

In this study, we aimed to evaluate the role of ALMS1 in the morphology of primary cilia and regulation of cellular signaling using a knockdown model of the hTERT-RPE1 cell line. ALMS1 depletion resulted in the formation of longer cilia, which often displayed altered morphology as evidenced by extensive twisting and bending of the axoneme. Transforming growth factor beta/bone morphogenetic protein (TGF-β/BMP) signaling, which is regulated by primary cilia, was similarly affected by ALMS1 depletion as judged by reduced levels of TGFβ-1-mediated activation of SMAD2/3. These results provide novel information on the role of ALMS1 in the function of primary cilia and processing of cellular signaling, which when aberrantly regulated may underlie Alström syndrome.Ministerio de Educación, Cultura y Deporte | Ref. FPU12/01442Ministerio de Educación, Cultura y Deporte | Ref. FPU13/01835Ministerio de Educación, Cultura y Deporte | Ref. FPU17/01567Ministerio de Educación, Cultura y Deporte | Ref. FPU19/00175Ministerio de Economía y Competitividad | Ref. PI15/00049Ministerio de Ciencia, Innovación y Universidades | Ref. PI19/00332Xunta de Galicia | Ref. ED481A-2018/304Xunta de Galicia | Ref. ED431G2019-0

Prevalent ALMS1 pathogenic variants in Spanish Alström patients

Alström syndrome (ALMS) is an ultrarare disease with an estimated prevalence lower than 1 in 1,000,000. It is associated with disease-causing mutations in the Alström syndrome 1 (ALMS1) gene, which codifies for a structural protein of the basal body and centrosomes. The symptomatology involves nystagmus, type 2 diabetes mellitus (T2D), obesity, dilated cardiomyopathy (DCM), neurodegenerative disorders and multiorgan fibrosis. We refined the clinical and genetic diagnosis data of 12 patients from 11 families, all of them from Spain. We also studied the allelic frequency of the different variants present in this cohort and performed a haplotype analysis for the most prevalent allele. The genetic analysis revealed 2 novel homozygous variants located in the exon 8, p.(Glu929Ter) and p.(His1808GlufsTer20) in 2 unrelated patients. These 2 novel variants were classified as pathogenic after an in silico experiment (computer analysis). On the other hand, 2 alleles were detected at a high frequency in our cohort: p.(Tyr1714Ter) (25%) and p.(Ser3872TyrfsTer19) (16.7%). The segregation analysis showed that the pathogenic variant p.(Tyr1714Ter) in 3 families is linked to a rare missense polymorphism, p.(Asn1787Asp). In conclusion, 2 novel pathological mutations have been discovered in homozygosis, as well as a probable founder effect in 3 unrelated families.Xunta de Galicia | Ref. ED431G-2019/06Xunta de Galicia | Ref. ED431C-2018/54Instituto de Salud Carlos III | Ref. PI15/00049Instituto de Salud Carlos III | Ref. PI19/00332Ministerio de Educación, Cultura y Deporte | Ref. FPU17/01567Ministerio de Educación, Cultura y Deporte | Ref. FPU19/0017

Physicochemical Stability of Hospital Parenteral Nutrition Solutions: Effect of Changes in Composition and Storage Protocol

(1) Background: Parenteral nutrition (PN) is a technique used for the administration of nutrients to patients for whom traditional routes cannot be used. It is performed using solutions with extremely complex compositions, which can give rise to a large number of interactions. These interactions can impact their stability and put the patient’s life at risk. The aim of this study is to determine how changes in composition and storage protocol affect the stability of NP solutions. (2) Methods: Twenty-three samples were prepared according to routine clinical practice, with modifications to the concentration of some components. The samples were stored at room temperature (RT) and refrigerated (4 °C). Measurements of the droplet diameter, pH, density and viscosity were performed for both storage protocols on days 1, 3, 10 and 14. (3) Results: The samples with the lowest concentration of lipids (PN13-17) and proteins (PN18-22) showed a larger droplet diameter than the rest of the samples throughout the experiments. The USP limits were exceeded for some of the measurements of these sample groups. The pH density and viscosity remained relatively constant under the conditions studied. (4) Conclusions: The PN samples were considered stable and safe for administration under real-world conditions, but the samples with the lowest concentrations of lipids and proteins showed a tendency towards emulsion instability

Allelic overload and its clinical modifier effect in Bardet-Biedl syndrome

Abstract Bardet–Biedl syndrome (BBS) is an autosomal recessive ciliopathy characterized by extensive inter- and intra-familial variability, in which oligogenic interactions have been also reported. Our main goal is to elucidate the role of mutational load in the clinical variability of BBS. A cohort of 99 patients from 77 different families with biallelic pathogenic variants in a BBS-associated gene was retrospectively recruited. Human Phenotype Ontology terms were used in the annotation of clinical symptoms. The mutational load in 39 BBS-related genes was studied in index cases using different molecular and next-generation sequencing (NGS) approaches. Candidate allele combinations were analysed using the in silico tools ORVAL and DiGePred. After clinical annotation, 76 out of the 99 cases a priori fulfilled established criteria for diagnosis of BBS or BBS-like. BBS1 alleles, found in 42% of families, were the most represented in our cohort. An increased mutational load was excluded in 41% of the index cases (22/54). Oligogenic inheritance was suspected in 52% of the screened families (23/45), being 40 tested by means of NGS data and 5 only by traditional methods. Together, ORVAL and DiGePred platforms predicted an oligogenic effect in 44% of the triallelic families (10/23). Intrafamilial variable severity could be clinically confirmed in six of the families. Our findings show that the presence of more than two alleles in BBS-associated genes correlated in six families with a more severe phenotype and associated with specific findings, highlighting the role of the mutational load in the management of BBS cases