Transcriptional profiling and functional characterization of three genetic variants in SLC16A2 gene

Background 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 (RXRs) can form heterodimer complexes which bind to hormone response elements (HREs) leading to activation or repression of transcription. Aim of the study Our aim is to investigate the impact of SLC16A2 variations on the pathogenetic mechanisms of AHDS. Materials and Methods Fibroblasts were obtained from skin biopsies of 3 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 Platform. Protein expression was assessed 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 gene expression was highlighted by transcriptomic profiling in AHDS patients to controls. Moreover, MTT and Live/Dead assays demonstrated a reduced cell viability in both AHDS_1 with a splicing variant [NM_006517: c.430+1G>A] and in AHDS_3 with a missense variant [NM:006517-5: c.623G>A; (p.Gly208Asp)]. In AHDS_2, the C-terminal missense variant [c.1690G>A (p.Gly564Arg)] did not affect fibroblasts viability, suggesting a differential phenotype based on mutation. Target genes expression resulted upregulated in all cell lines. Furthermore, myelin related genes were significantly reduced in all investigated patients. The lipid staining revealed an increasing presence of lipid droplets in AHDS fibroblasts. Conclusions Our preliminary data emphasize a mutation-specific impairment in patients’ primary fibroblasts which can be used as pre-clinical experimental model of this rare disease.