Dataset Information

INPP5K and SIL1 associated pathologies with overlapping clinical phenotypes converge through dysregulation of PHGDH

ABSTRACT: In this study, we describe new patients suffering from INPP5K mutations and hereby expand the mutational and clinical spectrum of the underlying disease. Pathogenicity of a new INPP5K missense mutation has been functionally confirmed. In addition, we systematically addressed the need to identify common molecular key players to link phenotypically similar rare diseases. For this purpose, utilizing cells derived from INPP5K and SIL1 patients, we performed proteomic profiling and identified PHGDH, as a protein significantly altered in abundance in the in vitro systems of both diseases. Prompted by the striking molecular link between MSS, INPP5K- and PHGDH-phenotypes and the fact that PHGDH-patients notoriously respond to L-serine treatment, respective zebrafish models have been generated and responseto L-serine treatment has been addressed in vivo. Apart from the biochemical link between MSS, INPP5K-and PHGDH-phenotypes, affection of CDK9, a protein modulating the activity of CTDP1 biochemically links the INPP5K-phenotype to CCFDN. Thus, our study not only allowed to build molecular bridges between four phenotypically overlapping rare diseases but more importantly allowed to transfer the acquiredmolecular knowledge to the pre-clinical testing of a therapeutic intervention concept.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin, Fibroblast

DISEASE(S): Marinesco-sjogren Syndrome

SUBMITTER: Laxmikanth Kollipara

LAB HEAD: Dr. Andreas Roos

PROVIDER: PXD009297 | Pride | 2021-04-06

REPOSITORIES: Pride

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Publications

INPP5K and SIL1 associated pathologies with overlapping clinical phenotypes converge through dysregulation of PHGDH.

Hathazi Denisa D Cox Dan D D'Amico Adele A Tasca Giorgio G Charlton Richard R Carlier Robert-Yves RY Baumann Jennifer J Kollipara Laxmikanth L Zahedi René P RP Feldmann Ingo I Deleuze Jean-Francois JF Torella Annalaura A Cohn Ronald R Robinson Emily E Ricci Francesco F Jungbluth Heinz H Fattori Fabiana F Boland Anne A O'Connor Emily E Horvath Rita R Barresi Rita R Lochmüller Hanns H Urtizberea Andoni A Jacquemont Marie-Line ML Nelson Isabelle I Swan Laura L Bonne Gisèle G Roos Andreas A

Brain : a journal of neurology 20210901 8

Marinesco-Sjögren syndrome is a rare human disorder caused by biallelic mutations in SIL1 characterized by cataracts in infancy, myopathy and ataxia, symptoms which are also associated with a novel disorder caused by mutations in INPP5K. While these phenotypic similarities may suggest commonalties at a molecular level, an overlapping pathomechanism has not been established yet. In this study, we present six new INPP5K patients and expand the current mutational and phenotypical spectrum of the di ...[more]

PMID: 33792664

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Project description:Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance. This is often accompanied by abnormal postural and facial features, brachydactyly and dermal calcifications, a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. Typically, this is the result of maternal inheritance of a mutation (or epigenetic defect) in the GNAS complex locus, encoding for the GTPase subunit Gsα. However, accurate diagnosis is often difficult. In blood platelets, Gsα couples to receptors for prostaglandin E1 and iloprost, and provides a master signal for platelet inhibition via adenylyl cyclase and cAMP-dependent protein kinase A (PKA). Here, we evaluated the iloprost-induced functional changes and phosphoproteome in platelets from eight patients with confirmed or suspected PHP Ia, one of the largest cohorts examined. Our aim was to asses how current proteomics techniques can help to find phosphorylation aberrations in these patients, and hence to improve future diagnosis Platelets from six out of eight patients (4 families), with confirmed GNAS mutations, displayed impairments in Gsα-dependent functional responses, i.e. VASP phosphorylation, aggregation, and microfluidic thrombus formation. Detailed analysis of platelet phosphoproteome revealed a consistent set of 2,516 protein phosphorylation sites, of which 453 were recognised as regulated by iloprost-Gsα, and 263 were upregulated. In five patients with AHO, we detected impairments in the iloprost-induced platelet phosphoproteome that were linked to a dysregulated platelet inhibition. The changes in these patients concerned: (i) both upregulated and downregulated phosphopeptides; (ii) a high Gsα and PKA dependency of the upregulated phosphopeptides; (iii) multiple phosphoproteins implicated in key platelet functions and biological pathways. Confirmation of these changes in iloprost-induced phosphoproteome was obtained using a panel of 14 targeted phosphopeptide assays. In contrast, or one patient with GNAS mutation diagnosed as non-AHO, we noticed reversed changes in the iloprost phosphoproteome. This combination of phosphoproteomic approaches can be a powerful tool to assist in future molecular diagnosis of patients with suspected PHP Ia.

Project description:Marinesco-Sjogren syndrome (MSS) is a rare multisystem pediatric disorder, caused by loss of function of the endoplasmic reticulum cochaperone SIL1 in about 60% of the patients. SIL1 acts as a nucleotide exchange factor for BiP, which plays a central role in secretory protein folding. SIL1-deficient cells have reduced BiP-assisted protein folding, cannot fulfil their protein needs and experience chronic activation of the unfolded protein response (UPR). Maladaptive UPR may explain the cerebellar and skeletal muscle degeneration re-sponsible for the ataxia and muscle weakness typical of MSS. However, the cause of other, more variable, clinical manifestations, such as mild to severe mental retardation, hypogonadism, short stature and skeletal deformities, are less clear. To gain insights into the pathogenic mechanisms of MSS, we carried out cell bio-logical and proteomic investigations in skin fibroblasts derived from a young patient carrying the SIL1 ... mutation. Despite fibroblasts are not overtly affected in MSS we found morphological and biochemical changes consistent with UPR and metabolic alterations. All the cell machineries involved in RNA splicing and translation were strongly downregulated, while protein degradation via lysosome-based structures was boosted, consistent with an attempt of the cell to reduce the workload of the endoplasmic reticulum and dis-pose misfolded proteins. Cell metabolism was extensively affected as we observed a reduction in lipid syn-thesis, an increase in beta oxidation and an enhancement of the tricarboxylic acid cycle, with upregulation of eight of its enzymes. Finally, the catabolic pathways of various amino acids, including valine, leucine, isoleu-cine, tryptophane, lysine, aspartate and phenylalanine, were enhanced, while the biosynthetic pathways of arginine, serine, glycine and cysteine were reduced. These results indicate profound metabolic alterations in MSS cells in addition to UPR activation and increased protein degradation, which may contribute to make them resistant to SIL1 loss.

Project description:D-3-Phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95) is a necessary enzyme for de novo L-serine biosynthesis via the phosphorylated pathway. We demonstrated previously that Phgdh is expressed exclusively by neuroepithelium and radial glia in developing mouse brain and later mainly by astrocytes. Mutations in the human PHGDH gene cause serine deficiency disorders (SDD) associated with severe neurological symptoms such as congenital microcephaly, psychomotor retardation, and intractable seizures. We recently demonstrated that genetically engineered mice, in which the gene for Phgdh has been disrupted, have significantly decreased levels of serine and glycine, and exhibit malformation of brain such as microcephaly. The Phgdh null (KO) embryos exhibit lethal phenotype after gestational day 14, indicating that the phosphorylated pathway is essential for embryogenesis, especially for brain development. It is worth noting that the Phgdh knockout (KO) embryos primarily displayed microcephaly, which is the most conspicuous phenotype of patients with SDD. Thus, Phgdh KO mice are a useful animal model for studying the effect of diminished L-serine levels on development of the central nervous system and other organs. To better understand the mechanism underlying the molecular pathogenesis of SDD, we sought to examine whether gene expression is altered in the Phgdh KO mouse model. We identify genes that have altered expression in the head of the Phgdh KO embryos using the GeneChip array. Some of the genes identified by this method belong in functional categories that are relevant to the biochemical and morphological aberrations of the Phgdh deletion. Experiment Overall Design: Total RNA samples were prepared from head tissues from 2 embryos of Phgdh knockout and littermate wild-type controls. Experiment Overall Design: RNA of 4 biological replicates was hybridized to Affymetrix Mouse Genome 430 2.0 arrays. Five microgram total RNA was labelled according to the ENZO-protocol, fragmented and hybridized according to Affymetrix's protocols.

Dataset Information

INPP5K and SIL1 associated pathologies with overlapping clinical phenotypes converge through dysregulation of PHGDH

Publications

INPP5K and SIL1 associated pathologies with overlapping clinical phenotypes converge through dysregulation of PHGDH.

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