Biallelic PI4KA variants cause a novel neurodevelopmental syndrome with hypomyelinating leukodystrophy

Phosphoinositides are lipids that play a critical role in processes such as cellular signalling, ion channel activity and membrane trafficking. When mutated, several genes that encode proteins that participate in the metabolism of these lipids give rise to neurological or developmental phenotypes. PI4KA is a phosphoinositide kinase that is highly expressed in the brain and is essential for life. Here we used whole exome or genome sequencing to identify 10 unrelated patients harbouring biallelic variants in PI4KA that caused a spectrum of conditions ranging from severe global neurodevelopmental delay with hypomyelination and developmental brain abnormalities to pure spastic paraplegia. Some patients presented immunological deficits or genito-urinary abnormalities. Functional analyses by western blotting and immunofluorescence showed decreased PI4KA levels in the patients' fibroblasts. Immunofluorescence and targeted lipidomics indicated that PI4KA activity was diminished in fibroblasts and peripheral blood mononuclear cells. In conclusion, we report a novel severe metabolic disorder caused by PI4KA malfunction, highlighting the importance of phosphoinositide signalling in human brain development and the myelin sheath

Influence d'une carence en acide folique sur la biosynthèse de la mélatonine chez le rat

LYON1-BU Santé (693882101) / SudocSudocFranceF

La carence en acide folique chez le rat (effets sur les interactions acides aminés soufrés - mélatonine et sur l'expression des gènes de l'horloge circadienne)

Les folates participent à la synthèse de la mélatonine (MLT) via le métabolisme de la méthionine. De plus, ils sont les cofacteurs des cryptochromes, protéines impliquées dans le mécanisme moléculaire de l horloge circadienne. Nous avons étudié chez le rat l'influence d'une carence nutritionnelle en folates sur le métabolisme des acides aminés soufrés et de la MLT ainsi que sur l expression des gènes-horloges au sein d oscillateurs secondaires. Après 4 semaines de régime, la balance oxydant (augmentation de l homocystéine)/anti-oxydant (diminution de la MLT et du glutathion) est altérée. De plus, les modifications de l organisation des boucles transcriptionnelles de l horloge concomitantes à la diminution de MLT suggèrent un rôle important des folates dans la régulation des rythmes circadiens. Ainsi, la carence en folates pourrait contribuer au développement de pathologies impliquant des troubles des rythmes biologiques, en particulier au cours du vieillissement, état prédisposé à cette carenceFolate acts in melatonin (MLT) synthesis by way of methionin metabolism and is a co-factor of cryptochromes, proteins involved in molecular mechanisms of the circadian clock. We investigated the impact of a nutritional folate deficiency on sulphur-containing amino acids and melatonin metabolism as well as on circadian clock gene expression in peripheral tissues of male rats. After a 4 week diet, the oxidant (increase in homocystéine)/anti-oxidant (decrease in MLT and in glutathione) balance was altered. Moreover, changes in the organization of the clock transcriptional loop associated to a decrease in MLT suggest that folate plays an important role in circadian rhythm regulation. Therefore, folate deficiency may contribute to the development of pathologies involving rhythm disturbances, in particular in aging where such a deficiency can frequently occurLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Combined Use of In Silico and In Vitro Splicing Assays for Interpretation of Genomic Variants of Unknown Significance in Cardiomyopathies and Channelopathies

The identification of molecular anomalies in patients with inherited arrhythmias or cardiomyopathies is a multi challenge due to: i) the number of genes involved; ii) the number of polymorphisms and the fact that most mutations are private; and iii) the variable degree of penetrance which complicates family segregation study. Consequently, a number of unclassified variants (UV) are found in patients’ DNA and some (outside the canonical GT/AG) may affect splicing. Mutational screening on the most prevalent genes involved in arrythmias syndromes or in cardiomyopathies was performed on a cohort made up of 740 unrelated French index probands. To identify splice variants among the identified UVs, a combination of available in silico and in vitro tools were used since transcript is not available. Using this approach, 10 previously identified UVs were reclassified as disease-causing mutations and, more precisely, as haploinsufficiency mutations rather than dominant-negative mutations. Most of them (7 of 10) were observed in MYBPC3. Our study highlighted the importance of the combined use of in silico and in vitro splicing assays to improve the prediction of the functional impact of identified genetic variants. The primary challenge now, with new sequencing technologies, is to distinguish between background polymorphisms and pathogenic mutations. Since splice site mutations can account for almost 50% of disease-causing mutations, recognizing them from among other variations is essential

Epigenetic Silencing of Lysyl Oxidase-Like-1 through DNA Hypermethylation in an Autosomal Recessive Cutis Laxa Case

International audienceWe have recently reported a case of cutis laxa caused by a fibulin-5 missense mutation (p.C217R). Skin fibroblasts from this individual showed an abnormal pattern of expression of several genes coding for elastic fiber-related proteins, including lysyl oxidase-like-1 (LOXL1). In this study we intended to elucidate the mechanism responsible for LOXL1 downregulation in these fibulin-5-mutant cells. We identified a proximal region (-442/-342) of the human LOXL1 promoter in which two binding sites for the transcription factor specific protein 1 (Sp-1) are required for gene activity in normal fibroblasts. Binding of Sp-1 to these sequences was dramatically reduced within cutis laxa cells, although Sp-1 expression was normal. Further analysis of the promoter sequence found increased methylation levels in cutis laxa cells compared with cells from unaffected individuals. When DNA methyltransferase activity was transiently inhibited in cutis laxa cells using the 5-aza-2'-deoxycytidine, we found a significant increase in LOXL1 expression. In conclusion, besides changes caused by the fibulin-5 mutation, LOXL1 gene regulation is affected by an epigenetic mechanism that can be reversed by an inhibitor of DNA methyltransferase activity. It is not yet known whether LOXL1 gene expression is affected in all cases of cutis laxa arising from fibulin-5 mutation

Modeling neuronal defects associated with a lysosomal disorder using patient-derived induced pluripotent stem cells

International audienceBy providing access to affected neurons, human induced pluripotent stem cells (iPSc) offer a unique opportunity to model human neurodegenerative diseases. We generated human iPSc from the skin fibroblasts of children with mucopolysaccharidosis type IIIB. In this fatal lysosomal storage disease, defective α-N-acetylglucosaminidase interrupts the degradation of heparan sulfate (HS) proteoglycans and induces cell disorders predominating in the central nervous system, causing relentless progression toward severe mental retardation. Partially digested proteoglycans, which affect fibroblast growth factor signaling, accumulated in patient cells. They impaired isolation of emerging iPSc unless exogenous supply of the missing enzyme cleared storage and restored cell proliferation. After several passages, patient iPSc starved of an exogenous enzyme continued to proliferate in the presence of fibroblast growth factor despite HS accumulation. Survival and neural differentiation of patient iPSc were comparable with unaffected controls. Whereas cell pathology was modest in floating neurosphere cultures, undifferentiated patient iPSc and their neuronal progeny expressed cell disorders consisting of storage vesicles and severe disorganization of Golgi ribbons associated with modified expression of the Golgi matrix protein GM130. Gene expression profiling in neural stem cells pointed to alterations of extracellular matrix constituents and cell-matrix interactions, whereas genes associated with lysosome or Golgi apparatus functions were downregulated. Taken together, these results suggest defective responses of patient undifferentiated stem cells and neurons to environmental cues, which possibly affect Golgi organization, cell migration and neuritogenesis. This could have potential consequences on post-natal neurological development, once HS proteoglycan accumulation becomes prominent in the affected child brain