Mutations in the genes encoding cytosolic aminoacyl-tRNA synthetases are responsible for early-onset multisystemic diseases including to varying degrees interstitial lung disease, liver damage, neurological and digestive disorders, and systemic inflammation. These are rare and severe diseases whose pathophysiology is poorly understood.~The investigative team hypothesizes that mutations within these genes are responsible for a decrease in protein translation and lead to a cellular stress response similar to that induced by amino acid deprivation. The investigative team also hypothesizes that these alterations could be corrected by high-dose supplementation in the culture medium of the corresponding amino acid.~The main objective of the study is to precisely determine the consequences of cytosolic aminoacyl-tRNA synthetase mutations at the cell level on protein translation.~The parameters below will be studied in vitro in cell culture from skin biopsies of patients and commercially available human dermal fibroblasts from newborns and adults:~Determination of total protein content~The incorporation of d-methionine, leucine, tyrosine or phenylalanine into proteins~The study of polysomes profiling~The study of the assembly of the ribosomal 43S pre-initiation complex~The phosphorylation of eIF2α and 4EBP and the expression of ATF4~Ribosome profiling~Transfer RNA (tRNA) sequencing~The production of reactive oxygen species (ROS)~The results of these studies will be compared:~Between patient cells and control cells~Between genetically corrected patient cells, by stable transfection of the wild-type cDNA of the concerned genes and uncorrected cells~Between patient cells cultured in medium enriched with methionine, phenylalanine, tyrosine or leucine and cells cultured in standard medium.
Mutations in the genes encoding cytosolic aminoacyl-tRNA synthetases are responsible for early-onset multisystemic diseases including to varying degrees interstitial lung disease, liver damage, neurological and digestive disorders, and systemic inflammation. These are rare and severe diseases whose pathophysiology is poorly understood.~The investigative team hypothesizes that mutations within these genes are responsible for a decrease in protein translation and lead to a cellular stress response similar to that induced by amino acid deprivation. The investigative team also hypothesizes that these alterations could be corrected by high-dose supplementation in the culture medium of the corresponding amino acid.~The main objective of the study is to precisely determine the consequences of cytosolic aminoacyl-tRNA synthetase mutations at the cell level on protein translation.