Further insights into Allan-Herndon-Dudley syndrome: a novel SLC16A2 splice site variant

Background Genetics variants in SLC16A2 gene encoding for the monocarboxylate transporter 8 (MCT8) cause a severe X-linked leukoencephalopathy known as Allan-Herndon-Dudley syndrome (AHDS). MCT8 promotes cellular uptake and efflux of thyroid hormone. Iodothyronine deiodinases (DIO) 1 and 2 are implicated in the conversion of T4 into biologically active T3, while DIO3 converts T4 into the inactive hormone reverse T3 (rT3). 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. Aim of the study The aim of this work is to investigate the impact of a novel SLC16A2 splice site variant on the pathogenesis of AHDS. Materials and Methods In silico prediction tools, such as Mutation Taster, were used to assess the pathogenic score of the identified variant, while splicing prediction tools, such as Fruit Fly Splice Predictor, were used to analyze the effect of variant on the splicing mechanism. Fibroblasts were obtained from skin biopsies of both AHDS patient and a matched control. To evaluate MCT8 and thyroid hormone signaling pathway related genes expression, RNA was extracted with TRIzoLTM and assessed by Real-Time PCR. Protein expression was evaluated via western blot and immunofluorescence. MTT assay was used to compare cell viability. Live and dead assay was used to discriminate live and dead populations. Lipid droplets were detected via oil red o staining. Results The identified variant in the SLC16A2 gene causes the breakup of the wild type donor splice site, possibly leading to exon 1 skipping, affecting cell viability. SLC16A2 RNA expression in our AHDS patient was extremely reduced in comparison with total RNA from healthy control. DIO2, progastricsin, HR and KLF9 RNA expression resulted upregulated, whilst DIO1, DIO2-AS1, DIO3 and TH were downregulated influencing T3 cell entrance. Myelin related genes were significatively reduced. The lipid staining revealed an increasing presence of lipid droplets in AHDS patients. Conclusions Our preliminary data emphasize an impairment in AHDS fibroblasts in relation to SLC16A2 splice site variant, increasing our understanding in the pathogenic mechanism of patients affected by AHDS.