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Molecular pathogenesis of a novel thyroid hormone resistance syndrome caused by mutations in thyroid receptor alpha 1

Final Report Summary - TRACTION (Molecular pathogenesis of a novel thyroid hormone resistance syndrome caused by mutations in thyroid receptor alpha 1)

Objectives
The overall scientific aim of the project is to unravel the molecular mechanisms leading to the clinical phenotype of patients with TRα1 mutations. The key objectives are:
(1) to study the functional properties of TRa1 mutations in reporter gene assays;
(2) to investigate the effect of TRa1 mutations on downstream target genes by transcriptome profiling of mutation-containing patient primary cells studied ex vivo;
(3) to identify altered interaction of the receptor mutants with cofactor proteins;
(4) to establish a genotype-phenotype correlation by integrating the clinical phenotype with the results obtained from objectives i-iii.

Work performed
To achieve the objective 2 and 3, we have started to carefully phenotype all known patients with TRα1 mutations. Extensive clinical, biochemical and imaging tests enabled to delineate the clinical phenotype of these patients in detail. Also, primary cells (fibroblasts and PMBCs) were obtained from all patients.
Subsequently, we have introduced a new molecular biology method to (high-throughput GST pull-down) achieve objective 3. In addition, design of different assays to study protein-protein interactions were designed to achieve objectives 1 and 3.

Main results
All TRa1 mutations have been studied together in a reporter gene assay, indicating that most mutants are inactive and display dominant negative activity on the WT TRa1 (objective 1).
PBMCs, T-cells and fibroblasts from TRa1 patients and controls with different T3 concentrations have been cultured. Analysis of important T3-target genes shows disturbed regulation of specific genes (objective 2).
The altered interaction with co-activator and co-repressor proteins has been studied by different assays. A high-throughput GST pull down assay enabled us to delineate which domains of co-repressor proteins show enhanced binding to the mutant TRa1 receptors (objective 3).
The molecular phenotype of most patients studied appears very similar. Therefore, the mild variation in clinical phenotype is likely to be attributed to modifier genes Further, we have identified the first patients harbouring a mutation to both TRa1 and its splice variant TRa2. Clinical, biochemical and molecular testing revealed that their phenotype is largely similar to patients with mutations in TRa1 alone. Importantly, this mutant has retained the capacity to bind T3, albeit at a lower affinity. Such observations may underlie differences in phenotype and, importantly, can have implications for treatment with thyroxine (objective 4).

Final results and impact
The extensive clinical and imaging tests have largely refined the phenotype. This will be of benefit for the the identification of new patients, which is of great impact for patients and families.
Our focus on neurocognitive impairments will largely contribute to the understanding of the mild mental delays in the patients. Obviously, if a therapy is available, early recognition is important to prevent neurocognitive impairments. Indeed, our description of a family whose affected members were treated from early childhood on with thyroxine shows that several features of this disease can be (partially) rescued.
Since thyroid hormone binding to TRa1 mutants is absent, co-repressor proteins are not released, resulting in abberent downstream signalling. We have clarified which receptor-interacting domains are involved in binding to the TRa1 mutants. Subsequent studies will investigate if small molecule drugs, which disrupt corepressor-TRa1 binding will be beneficial in these patients. Early treatment may prevent and/or recover clinical featurs such as neurocognitive impairments and growth abnormalities.