Endocrine Abstracts

Mutations in THRA, a ligand-inducible transcription factor, cause Resistance to Thyroid Hormone α (RTHα). RTHα manifests with features of hypothyroidism (skeletal dysplasia and growth retardation, neurocognitive impairment, low metabolic rate, reduced intestinal transit) reflecting hormone resistance in TRα-expressing tissues, but associated paradoxically with near-normal circulating TH levels. The lack of clear-cut, abnormal thyroid function tests together with variable phenotype in RTHα, makes diagnoses of this disorder by linking relatively nonspecific clinical features (e.g., growth retardation and neurocognitive abnormalities) with THRA variants with unknown significance (VUS) identified by WES and NGS immensely challenging. In this work, we analyzed 20 different THRA VUS identified by whole exome sequencing belonging the 100K project, using zebrafish as a model system. The THRA mutant-injected zygotes exhibited, at vary degrees, several developmental defects, consisting of abnormal morphology index (AMI) or skeletal malformation (SMI). Head size, pericardial edema, body length, curved tail, and jaw malformations were used to calculate the AMI (0 = unaffected, 1-3 = moderate, 3-5 = severe). The A263S were only mildly affected, as most of the embryos were comparable with the WT injected zygotes. The R228C, T275M, T273A, R384C, V282L, P224L, I299T, M256T, L287P, R266L, G278R, and D268N exhibited moderate phenotype, whereas the remaining variants (H381Q, G291S, P399S, E403K, L400Tfs*7 and del268-273) severely affected embryo morphology. Interestingly, the exposure of high T3 concentration was able to significantly rescue the AMI in zygotes injected in most of the THRA mutant mRNAs, except for the G291S, E403K, L400Tfs*7 and del268-273 transcripts. Skeletal abnormalities were then investigated visualizing cranial cartilage development and bone mineralization in WT or THRA mutant larvae at 120 hpf. Malformations of ceratohyal, ceratobranchial arches and pectoral fins cartilages, and the absent mineralization of hyomandibular, operculum, and cleithrum were quantified to compute SMI (0 = normal, 1-4= moderate, 4-6 = severe). Consistently with AMI, A263S was similar of those of WT larvae, whereas R228C, T275M, T273A, R384C, V282L, P224L, I299T, M256T, L287P, R266L, G278R, D268N, H381Q and P399S moderately affected skeletal development and benefited from the exposure to high T3 dosage. Lastly, zygotes injected with G291S, E403K, L400Tfs*7 and del268-273 mRNAs were those who showed the most severe SMI at 120 hpf, which were also insensitive to T3 treatment. All these findings correlate with the variable clinical spectrum reported in the patients or the biochemical in vitro data and structural model. We propose zebrafish as a rapid and reliable in vivo model for VUS screening, representing a strong tool to stratify THRA variants pathogenicity.