Impact of Loss-of-function Mutations of Genes Encoding Cytosolic Aminoacyl-tRNA Synthetases on Protein Translation and Responses to Cellular Stress
试验速览
- 阶段
- 不适用
- 状态
- 撤回
- 入组人数
- 22
- 试验地点
- 2
- 主要终点
- Ribosome profiling
研究概览
简要总结
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.
详细描述
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)
研究设计
- 研究类型
- Interventional
- 分配方式
- Na
- 干预模型
- Single Group
- 主要目的
- Basic Science
- 盲法
- None
入排标准
- 性别
- All
- 接受健康志愿者
- 否
入选标准
- •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
排除标准
- •- 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
研究组 & 干预措施
Patients
Patients with mutations in genes encoding cytosolic aminoacyl-tRNA synthetases and cared at Necker Hospital.
干预措施: Skin biopsy (Other)
结局指标
主要结局
Ribosome profiling
时间窗: Day 0
Ribosome profiling by high throughput sequencing.
Incorporation of d-methionine and d-phenylalanine into proteins
时间窗: Day 0
Incorporation of methionine and phenylalanine by labelled amino-acid fluorescent assays using ready-to-use kits.
Study of polysomes profiling
时间窗: Day 0
Study of polysome profils by differential sedimentation on sucrose gradients.
Production of reactive oxygen species (ROS)
时间窗: Day 0
Production of reactive oxygen species (ROS) by fluorescent measurement after cells' incubation with 2',7'- dichlorodihydrofluorescein diacetate (H2DCFDA).
Study of the assembly of the ribosomal 43S pre-initiation complex
时间窗: 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
时间窗: Day 0
Phosphorylation of eIF2α and 4EBP and the expression of ATF4 by western blot.
Transfer RNA (tRNA) sequencing
时间窗: Day 0
Transfer RNA (tRNA) sequencing by high throughput sequencing.
Determination of total protein content
时间窗: Day 0
Determination of total protein content by Bicinchoninic acid assay.
次要结局
未报告次要终点