Study population and ethics
We performed a transversal study including one group of 13 FX and a group of 14 healthy controls, all males matched for age. Participants were recruited through the Fragile X Clinic, at the CIUSSS de l’Estrie-CHUS (Sherbrooke, Québec, Canada). Informed written consent was obtained directly from healthy controls or through a caregiver for FXS participants in accordance with requirements of the Ethics Review Board of the CIUSSS de l’Estrie-CHUS. All participants had a blood draw in the morning. FXS individuals had cognitive evaluations as well, performed by a certified neuropsychologist.
Blood samples were collected by venipuncture into ACD tubes (acid citric dextrose, BD Vacutainer®). All subsequent steps were performed at room temperature. PBMCs were subsequently extracted by density gradient using Ficoll-Paque (Ge Healthcare®) following the manufacturer protocol with minor modifications. Briefly, blood samples were centrifuged at 300 g for 10 minutes to allow separation and collection of plasma rich platelets (PRP). A volume of PBS, equal to the volume of plasma collected, was then added to each tube. The blood samples were carefully place onto a layer of Ficoll-Paque (blood:Ficoll-Paque ratio of 4:3) and centrifuged at 500 g for 30 minutes. PBMCs were thereafter isolated form the interface and collected by centrifugation (500 g, 10 minutes). Cell pellets were wash twice PBS and counted on a DXH-9000 hematology analyzer (Beckman Coulter®).
Platelets were extracted from blood sample collected in 8 mL ACD tubes as previously described . Briefly, PRP was separated from whole blood by centrifugation (300 g, 10 minutes). Afterwards, platelets where sedimented from the PRP (2400 g, 15 minutes) and wash twice with PBS containing 5 mM EDTA and counted on a DXH-9000 hematology analyzer (Beckman Coulter®).
Rate of proteins synthesis rate assay
Protein synthesis rate assay is schematised in Fig. 1. Freshly extracted PBMCs were suspended into RPMI 1640 Met−/Cys− (supplemented with 2 mM L-Glutamine, Sigma) and incubated for 30 minutes at 37 °C under 5% CO2 with gentle agitation. Following methionine and cysteine depletion, PBMCs were diluted to 2 million cells/mL and 0,5 mL of this culture were incubated with 50 µCi/mL of [35S] radiolabeled methionine and cysteine (Perkin Elmer®) at 37 °C (5% CO2) under gentle agitation for either 20, 40 or 60 minutes. The latter procedure was performed in triplicate. PBMCs were then pelleted by centrifugation (500 g, 10 minutes) and lysed in PBS containing 2% SDS. Proteins were precipitated overnight at 4 °C in 20% trichloroacetic acid and pelleted by centrifugation (14 000 g, 15 minutes). After two washes with acetone, proteins were suspended in 0.1N NaOH and their radiolabelled content was measured by liquid scintillation. Protein synthesis rate was normalised according to cell count and expressed in cpm/min/106 PBMCs. Radiolabeled amino acids incorporation was limited to 60 minutes to ensure unbiased normalisation by cell count (See Additional file 1). Experimental conditions including the number of cells, the timeframe and the concentration of radiolabeled amino acids used were rigorously optimised to ensure that the incorporation was linear and provides enough signal strength.
The same assay was used to measure protein synthesis rate in quiescent blood platelets with some minor modifications. After methionine and cysteine depletion, platelets were diluted to 200 million cells/mL and incubated with 250 µCi/mL of [35S] radiolabeled methionine and cysteine. Platelets were then incubated under gentle agitation at 37 °C under 5% CO2 for periods of 30, 60 and 120 minutes (triplicates) and subsequently collected by centrifugation (2400 g, 15 minutes). The same protocol as described for PBMCs was used for protein precipitation and liquid scintillation counting. Protein synthesis rate was also normalised to the concentration of cells and expressed in CPM/min/1006 platelets.
FXS participants had a cognitive evaluation including three different scales validated for the French-Canadian population. First, the Wechsler Adult Intelligence Scale third edition (WAIS-III) was used to evaluate intelligence quotient. Second, the Aberrant Behavior Checklist-Community Edition refactored for FXS (ABC-CFX) and the Adaptive Behavior Assessment System (ABAS-II) were administered to assesses aberrant and adaptive behavior respectively. Third, the Behavior Rating Inventory of Executive Function-Adult Version (BRIEF-A) was used to evaluate the executive functions
All results are expressed as means ± standard error of the mean (SEM). The non-parametric Mann-Whitney test was used to compare differences in protein synthesis rate between the two groups. Protein synthesis rate correlation with clinical parameters were accessed by both Pearson and Spearman correlation. All statistical tests were two-tailed, and a p < 0.05 was considered significant. All statistical analysis was conducted in GraphPad Prism (8.4.0).