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BACKGROUNDThe 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.METHODSUsing genomic editing with CRISPR-CAS9 and phosphoproteomics we have studied the TGF-β signalling pathway in knockout (KO) models forALMS1andBBS1genes. We have developed a network diffusion-based analysis pipeline to expand the data initially obtained and to be able to determinate which processes were deregulated in TGF-β pathway. Finally, we have analysed protein-protein interactions to prioritise candidate genes in the regulation of the TGF-β pathway in Alström and Bardet-Biedl syndrome.RESULTSAnalysis of differentially phosphorylated proteins identified 10 candidate proteins in theALMS1KO model and 41 in theBBS1KO model. After network expansion using a random walk with a restart model, we were able to obtain processes related to TGF-β signalling such as endocytosis in the case ofALMS1or extracellular matrix regulation inBBS1. Protein interaction analyses demonstrated the involvement of CDC42 as a central protein in the interactome inALMS1and CDK2 in the case ofBBS1.CONCLUSIONIn conclusion, the depletion ofALMS1andBBS1affects the TGF-β signalling pathway, conditioning the phosphorylation and activation of several proteins, including CDC42 in the case ofALMS1and CDK2 in the case ofBBS1.

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 cases Instituto de Salud Carlos III | Ref. PI15/00049 Instituto de Salud Carlos III | Ref. PI16/00425 Instituto de Salud Carlos III | Ref. PI19/00321 Instituto de Salud Carlos III | Ref. PI19/00332 CIBERER | Ref. 07/06/0036 IIS-FJD BioBank | Ref. PT13/0010/0012 Comunidad de Madrid | Ref. B2017/BMD-3721 Xunta de Galicia | Ref. ED431G-2019/06 Xunta de Galicia | Ref. ED431C-2018/54 ISCIII | Ref. FI17/00192 Ministerio de Educación, Cultura y Deporte | Ref. FPU 19/00175 ISCIII | Ref. CP16/00116

14 pages, 7 figures, 1 table.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY), Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 (EDN1) is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the most common targeted by current treatments. We sequenced the EDN1 untranslated regions of a small subset of patients with PAH, predicted the effect in silico, and used a luciferase assay with the different genotypes to analyze its influence on gene expression. Finally, we used siRNAs against the major transcription factors (TFs) predicted for these regions [peroxisome proliferator-activated receptor γ (PPARγ), Krüppel-Like Factor 4 (KLF4), and vitamin D receptor (VDR)] to assess EDN1 expression in cell culture and validate the binding sites. First, we detected a single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR; rs397751713) and another in the 3'regulatory region (rs2859338) that altered luciferase activity in vitro depending on their genotype. We determined in silico that KLF4/PPARγ could bind to the rs397751713 and VDR to rs2859338. By using siRNAs and luciferase assays, we determined that PPARγ binds differentially to rs397751713. PPARγ and VDR Knock-Down (KD) increased the EDN1 mRNA levels and EDN1 production in porcine aortic endothelial cells (PAECs), while PPARγ and KLF4 KD increased the EDN1 production in HeLa. In conclusion, common variants in EDN1 regulatory regions could alter EDN1 levels. We were able to validate that PPARγ binds in rs397751713 and is a key regulator of EDN1. In addition, KLF4 and VDR regulate EDN1 production in a cell-dependent manner, but VDR does not bind directly to the regions we studied, This work was funded by the Cardiovascular Research Network of Instituto de Salud Carlos III de Madrid (RD06/0003/0012) Spanish Ministry of Science and Innovation PI18/01233 and Janssen Pharmaceuticals. CINBIO has financial support from Xunta de Galicia and the European Union (European Regional Development Fund-ERDF) (PO FEDER ED431G/02). ML-D and LM-M are supported by a Xunta de Galicia predoctoral fellowship (ED481A-2018/304; IN606A-2020/006). CS is supported by a Ministerio de Universidades FPU predoctoral fellowship (FPU19/00175)

Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 (EDN1) is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the most common targeted by current treatments. We sequenced the EDN1 untranslated regions of a small subset of patients with PAH, predicted the effect