Functional characterization and transcriptional profiling of fibroblasts from patients with mutations in SLC16A2 gene

Background/Objectives: Genetics variants in SLC16A2 gene encoding for the monocarboxylate transporter 8 (MCT8) cause a severe X-linked intellectual deficit known as Allan-Herndon-Dudley syndrome (AHDS). MCT8 promotes cellular uptake and efflux of thyroid hormones. Active T3 and retinoid X receptors (RXR) can form heterodimer complexes which bind to hormone response elements (HREs) that leads to activate or repress transcription. Our aim is to investigate the impact of MCT8 mutations on the pathogenetic mechanisms of AHDS. Methods: Fibroblasts were obtained from skin biopsies of 2 AHDS mutated patients and matched controls. RNA was extracted with TRIzoLTM. Total RNA sequencing was performed with the CORALL Total RNA-Seq Library Prep Kit using Illumina NextSeq 500 Sequencing. Protein expression was valuated via western blot and immunofluorescence. MTT assay was used to compare cell viability. Live/dead assay discriminated live and dead populations. Lipids were detected via Oil Red O staining. Results: A strong dysregulation in AHDS patients was highlighted by transcriptomic profiling, identifying 396 DEGs when comparing AHDS patients to controls. Moreover, MTT and Live/Dead assays demonstrated a reduced cell viability in AHDS_1 patient with a splicing variant [c.1690G>A (p.Gly564Arg)]. The C-terminal missense variant [c.1690G>A (p.Gly564Arg)] did not affect fibroblasts viability, challenging a personalized in vitro behavior. Target genes expression resulted upregulated in both patients. Furthermore, myelin related genes were significatively reduced. The lipid staining revealed an increasing presence of lipid droplets in AHDS fibroblasts. Conclusion: Our preliminary data emphasize a mutation-specific impairment in patients’ specific primary fibroblasts, used as pre-clinical experimental model of the disease.