Transcriptional profiling and functional characterization of 3 patient-derived skin fibroblasts affected by Allan-Herndon-Dudley syndrome

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 SLC16A2 variations on the pathogenetic mechanisms of AHDS. 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 Sequencer. 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. A strong dysregulation in AHDS patients was highlighted by transcriptomic profiling, when comparing AHDS patients to controls. Moreover, MTT and Live/Dead assays demonstrated a reduced cell viability in AHDS_1 with a splicing variant [c.1690G>A (p.Gly564Arg)] and in AHDS_2 with a missense variant [NM:006517-5: c.623G>A; p.Gly208Asp]. The C-terminal missense variant [c.1690G>A (p.Gly564Arg)] did not affect fibroblasts viability, challenging a personalized in vitro fibroblast phenotype. Target genes expression resulted upregulated in both patients (AHDS_1 and AHDS_2). Furthermore, myelin related genes were significantly reduced in all investigated patients. The lipid staining revealed an increasing presence of lipid droplets in AHDS fibroblasts. Our preliminary data emphasize a mutation-specific impairment in patients’ specific primary fibroblasts, that can be used as pre-clinical experimental model of this rare disease.