Brais Bea-Mascato

The ALMS1 gene plays a crucial role in maintaining cellular homeostasis through its involvement in primary cilium assembly, cytoskeletal regulation, and signalling pathways such as NOTCH and TGF-β. Pathogenic variants in ALMS1 are associated with Alström Syndrome (ALMS), a multi-systemic ciliopathy characterised by neurosensory deficits, metabolic disorders, and multi-organ fibrosis. To better understand the tissue-dependent role of ALMS1 , we utilised CRISPR/Cas9 technology to develop a zebrafish model with alms1 depletion. Multi-tissue transcriptomic profiling revealed that alms1 depletion has pleiotropic effects on gene expression, with the brain and eyes displaying the most pronounced transcriptomic alterations, including disrupted ciliary function and immune dysregulation. Inflammatory and innate immune pathways along with glutamatergic synapse-related processes were significantly affected in the brain and eyes but with different gene expression signatures. The analysis further highlights tissue-specific processes, primarily associated with organ dysfunction. Additionally, our findings underscore the role of alms1 in regulating age-associated gene expression profiles in the brain, suggesting a link between ciliary dysfunction and accelerated brain ageing. Comparative analyses with Bardet-Biedl Syndrome iPSC models revealed shared pathways, reinforcing the potential of ciliopathies as models for ageing-related disorders. This study provides novel insights into the tissue-specific functions of alms1 and the molecular mechanisms underlying ALMS, paving the way for the development of targeted therapeutic strategies.

Background: Alström syndrome (ALMS; #203800) is an ultrarare monogenic recessive disease. This syndrome is associated with variants in the ALMS1 gene, which encodes a centrosome-associated protein involved in the regulation of several ciliary and extraciliary processes, such as centrosome cohesion, apoptosis, cell cycle control and receptor trafficking. The type of variant associated with ALMS is mostly complete loss-of-function variants (97%) and they are mainly located in exons 8, 10 and 16 of the gene. Other studies in the literature have tried to establish a genotype–phenotype correlation in this syndrome with limited success. The difficulty in recruiting a large cohort in rare diseases is the main barrier to conducting this type of study. Methods: In this study we collected all cases of ALMS published to date. We created a database of patients who had a genetic diagnosis and an individualised clinical history. Lastly, we attempted to establish a genotype–phenotype correlation using the truncation site of the patient’s longest allele as a grouping criteria. Results: We collected a total of 357 patients, of whom 227 had complete clinical information, complete genetic diagnosis and meta-information on sex and age. We have seen that there are five variants with high frequency, with p.(Arg2722Ter) being the most common variant, with 28 alleles. No gender differences in disease progression were detected. Finally, truncating variants in exon 10 seem to be correlated with a higher prevalence of liver disorders in patients with ALMS. Conclusion: Pathogenic variants in exon 10 of the ALMS1 gene were associated with a higher prevalence of liver disease. However, the location of the variant in the ALMS1 gene does not have a major impact on the phenotype developed by the patient.

BACKGROUND: The primary cilium is a sensory organelle that extends from the plasma membrane. It plays a vital role in physiological and developmental processes by controlling different signalling pathways such as WNT, Sonic hedgehog (SHh), and transforming growth factor β (TGF-β). Ciliary dysfunction has been related to different pathologies such as Alström (ALMS) or Bardet-Biedl (BBS) syndrome. The leading cause of death in adults with these syndromes is chronic kidney disease (CKD), which is characterised by fibrotic and inflammatory processes often involving the TGF-β pathway. METHODS: Using genomic editing with CRISPR-CAS9 and phosphoproteomics we have studied the TGF-β signalling pathway in knockout (KO) models for ALMS1 and BBS1 genes. We have developed a network diffusion-based analysis pipeline to expand the data initially obtained and to be able to determine which processes were deregulated in TGF-β pathway. Finally, we have analysed protein-protein and kinase-substrate interactions to prioritise candidate genes in the regulation of the TGF-β pathway in ALMS and BBS. RESULTS: Analysis of differentially phosphorylated proteins identified 10 candidate proteins in the ALMS1 KO model and 41 in the BBS1 KO model. After network expansion using a random walk with a restart algorithm, we were able to identify the TGF-β signalling pathway together with other related processes such as endocytosis in the case of ALMS1 or the regulation of the extracellular matrix in BBS1 . Protein interaction analyses demonstrated the involvement of CDC42 as a central protein in the interactome in ALMS1 and CDK2 in the case of BBS1 . CONCLUSION: In conclusion, the depletion of ALMS1 and BBS1 affects the TGF-β signalling pathway, conditioning the phosphorylation and activation of several proteins, including CDC42 in the case of ALMS1 and CDK2 in the case of BBS1 .

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.

Introduction: Alström syndrome (ALMS, #203800) is an ultra-rare monogenic recessive disease. This the syndrome is associated with mutations in the ALMS1 gene, which codes for a centrosome structural protein responsible for centrosome cohesion. The type of mutation associated with ALMS is mostly cLOF (97%) and they are mainly located in exons 8, 10 and 16 of the gene. Other studies in the literature have tried to establish a genotype-phenotype correlation in this syndrome with little success. The difficulty in recruiting a large cohort in rare diseases is the main barrier to conducting this type of study. Methods: In this study we have collected all cases published to date for ALMS. We have created a database with those patients who had a genetic diagnosis and an individualised clinical history. Finally, we have attempted to establish a genotype-phenotype correlation using the truncation site of the patient’s longest allele as a grouping criterion. Results: We collected a total of 357 patients of which 227 had complete clinical information, complete genetic diagnosis and meta information regarding sex and age. We have seen that there are 5 variants in high frequency with p.(Arg2722Ter) being the most common variant with 28 alleles. There are no gender differences in disease progression. Finally, truncating mutations in exon 10 seem to be correlated with a higher prevalence of liver disorders in patients with ALMS. Conclusion: The location of the mutation in the ALMS1 gene does not have a major impact on the phenotype developed by the patient. Only mutations in exon 10 of the ALMS1 gene were associated with a higher prevalence of liver disease.

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 also 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 models 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.

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.

Background:Alström syndrome (ALMS) is a rare disease with an estimated prevalence lower than 1 in 1,000,000. It is associated with mutations in the Alström syndrome 1 (ALMS1) gene, which codifies for a structural protein of the basal body and centrosomes. Symptomatology involves nystagmus, type 2 diabetes mellitus (DM2), obesity, dilated cardiomyopathy (DMC), neurodegenerative disorders and multi-organ fibrosis.Methods:We included the clinical data of 13 patients from 12 families, all of them from Spain. We studied the allelic frequency for the different mutations present in this cohort and we perform a haplotype analysis for the most prevalent allele.Results:Two alleles were detected in high frequency: p.(Tyr1714Ter) (0,25) and p.(Ser3872TyrfsTer19) (0,167). The segregation analysis of the mutation p.(Tyr1714Ter) in 3 families shows that it is linked to a rare missense polymorphism. Also, we detect an ancestral haplotype for ALMS1 in three families.Conclusion:Mutation p.(Tyr1714Ter) co-segregates with a rare single nucleotide polymorphism (SNP) that could be arise by a founder effect in the Iberian Peninsula.