Infantile Liver Failure Syndrome 1, Interstitial Lung and Liver Disease, Neurologic, Endocrine and Pancreatic Disease, Multisystem, Infantile-Onset 2, Rajab Interstitial Lung Disease With Brain Calcifications 2
Conditions
Keywords
Cytosolic aminoacyl-tRNA synthetase, Mutations in the MARS1 gene, Mutations in the LARS1 gene, Mutations in the YARS1 gene, Mutations in the FARSA gene
Brief summary
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.
Detailed description
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 control cells: * 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 the corresponding amino acid.
Interventions
OTHERSkin biopsy
A skin biopsy performed on the forearm or thigh depending on the patient's age and wishes, with a biopsy punch with a diameter of 3 to 4 mm depending on the child's age (3 for children under 3 years, 4 beyond). Culture of fibroblasts and immortalization.
Sponsors
URC-CIC Paris Descartes Necker Cochin
Assistance Publique - Hôpitaux de Paris
Study design
Allocation
NA
Intervention model
SINGLE_GROUP
Primary purpose
BASIC_SCIENCE
Masking
NONE
Eligibility
Sex/Gender
ALL
Healthy volunteers
No
Inclusion criteria
* Patients carrying mutations in genes encoding cytosolic aminoacyl-tRNA synthetases responsible for a multi-systemic phenotype * Information and consent of the patient if an adult and of the holders of parental authority if a minor patient and of the minor patient
Exclusion criteria
\- Non-consent of one of the holders of parental authority or of the minor patient or of adult patient Contrôl patients : * Fibroblasts from control patients without mutation in genes encoding cytosolic aminoacyl-tRNA synthetases, from an existing biological collection. The control patients will be selected according to the age at which the skin biopsy was performed in order to have an age match between the patients and the controls. * Information and consent of the patient if an adult and of the holders of parental authority if a minor patient and of the minor patient
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Determination of total protein content | Day 0 | Determination of total protein content by Bicinchoninic acid assay. |
| Incorporation of d-methionine and d-phenylalanine into proteins | Day 0 | Incorporation of methionine and phenylalanine by labelled amino-acid fluorescent assays using ready-to-use kits. |
| Study of polysomes profiling | Day 0 | Study of polysome profils by differential sedimentation on sucrose gradients. |
| Study of the assembly of the ribosomal 43S pre-initiation complex | Day 0 | Study of the assembly of the ribosomal 43S pre-initiation complex by co-immunoprecipitation experiments. |
| Phosphorylation of eIF2α and 4EBP and the expression of ATF4 | Day 0 | Phosphorylation of eIF2α and 4EBP and the expression of ATF4 by western blot. |
| Ribosome profiling | Day 0 | Ribosome profiling by high throughput sequencing. |
| Transfer RNA (tRNA) sequencing | Day 0 | Transfer RNA (tRNA) sequencing by high throughput sequencing. |
| Production of reactive oxygen species (ROS) | Day 0 | Production of reactive oxygen species (ROS) by fluorescent measurement after cells' incubation with 2',7'- dichlorodihydrofluorescein diacetate (H2DCFDA). |
Countries
France
Outcome results
None listed