Delayed diagnosis of Resistance to Thyroid Hormone alpha (RTH) in two patients

Supporting Data_Marelli et al. – Novel THRA Variants Associated with RTHα (Excel and GraphPad Prism files) This dataset supports the study entitled “Delayed diagnosis of Resistance to Thyroid Hormone alpha (RTHα) in two patients”, investigating the pathogenicity and functional consequences of two novel de novo THRA variants (p.D268H and p.D402_G405del) identified in unrelated Italian patients affected by Resistance to Thyroid Hormone alpha (RTHα). The study combines clinical characterization, patient-derived PBMC assays, and zebrafish in vivo modeling to evaluate thyroid hormone responsiveness, hypothalamic-pituitary-thyroid (HPT) axis function, and cardiac phenotypes associated with mutant TRα proteins. This dataset contains all raw numerical data corresponding to the graphs and supplementary tables presented in the manuscript and supplementary materials. Statistical analyses were performed using GraphPad Prism 10 software using Student’s t-test or one-way ANOVA, as appropriate. Statistically significant values are highlighted in red where applicable. Each worksheet (Excel) or subfolder (Graphpad) corresponds to a figure or supplementary table, as detailed below: FIG.1E–H Whole-body thyroid hormone measurements in zebrafish larvae at 5 dpf, including total T4, total T3, free T4, and free T3 quantified by ELISA assays (Cusabio kits). Measurements were performed on pools of approximately 200 larvae/sample. Results are normalized to wet weight and larval number and expressed as mean ± SEM from three biological replicates. FIG.1O Relative expression of the cardiac marker myl7 measured by RT-qPCR in WT- and mutant THRA-injected zebrafish embryos/larvae at 2 and 5 dpf. Expression values were normalized against eef1a and calculated using the ΔΔCt method. Results are expressed as mean ± SD from three independent biological replicates. FIG.1P Heart rate measurements (beats per minute) in zebrafish embryos/larvae injected with WT or mutant THRA RNAs under basal conditions or following chronic T3 treatment (20 μM from 6–120 hpf). Data derive from 15 larvae per group across independent injections. FIG.1Q RT-qPCR analysis of the T3-responsive gene klf9 in zebrafish larvae at 5 dpf under basal or T3-treated conditions. Expression levels were normalized against eef1a and expressed as mean ± SD from biological triplicates using pools of 20 larvae. FIG.1R RT-qPCR analysis of KLF9 expression in PBMCs isolated from controls and RTHα patients carrying THRA variants. PBMCs were cultured in RPMI supplemented with stripped FBS in basal conditions or after stimulation with 100 nM T3 for 24 h. Expression was normalized against RPLP0 and expressed as mean ± SD from triplicate experiments. FIG.S1A Relative expression levels of HPT-axis-related genes (trh, tshba, tg, nis, dio2, and dio3b) in zebrafish larvae at 5 dpf following injection of WT or mutant THRA transcripts. Data are expressed as mean ± SD from three independent experiments using pools of 20 larvae each. FIG.S1B TSH measurements in WT- and mutant THRA-injected larvae treated with vehicle or increasing T3 concentrations (5, 10, and 20 nM) from 6–120 hpf. Hormone concentrations were quantified by ELISA using pools of approximately 200 larvae/sample and expressed as mean ± SEM of biological triplicates. TAB.S1 Clinical, biochemical, and neurological characteristics of the two RTHα patients carrying the p.D268H and p.D402_G405del THRA variants. TAB.S2 Quantification of FISH fluorescence signal volumes for HPT-axis markers (trh, tshba, dio2, and tg) in WT- and mutant THRA-injected zebrafish larvae at 5 dpf. TAB.S3 Quantification of T4-positive thyroid follicles in WT- and mutant THRA-injected zebrafish larvae at 5 dpf. TAB.S4 Primer sequences used for FISH/WISH riboprobes. TAB.S5 Primer sequences used for RT-qPCR experiments. Experimental procedures Human THRA transcripts were synthesized in vitro and microinjected into zebrafish embryos at the 1–2 cell stage. Zebrafish experiments were conducted under standard husbandry conditions (28.5°C; 14:10 h light/dark cycle). Confocal image quantification was performed using Fiji/ImageJ and Volocity Software (Nikon). RT-qPCR analyses were performed using the ΔΔCt method with normalization to housekeeping genes (eef1a for zebrafish and RPLP0 for PBMCs). Ethical approval All zebrafish experiments were approved by the OPBA, University of Milan (Protocol 198283). Related publication Marelli F, Rurale G, Campi I, Pozzobon G, Vigone MC, Persani L. Delayed diagnosis of Resistance to Thyroid Hormone alpha (RTHα) in two patients. Submitted to Thyroid.