ESPE Abstracts

Introduction: Thyroid dyshormonogenesis is a heterogeneous group of hereditary diseases produced by the total/partial blockage of the biochemical processes involved in thyroid-hormone synthesis and secretion. Many genes are involved in this process. PAX8 is a transcription factor essential for thyroid-gland morphogenesis and synthesis of thyroid hormones, since it activates TG, TPO and TSHR gene transcription. More than 50 PAX8 variants were reported, but most not functionally tested. We identified 4 PAX8 variants in 5 patients with congenital hypothyroidism (CH).

Objective: To determine if these PAX8 variants are pathogenic with in-vitro functional studies.

Patients and Methods: Patients Five patients (4 males, 1 female), from the Catalan-CH-screening program (n = 70), with an eutopic thyroid. Screening TSH was 68.3±58.4 mU/l and confirmation TSH was 264±168.4 mU/l and T4L 0.94±0.38 ng/dL. Scintigraphy in 4 patients showed an uptake absence in 1, a hypocaptating gland in 2 and a normocaptating gland in another. Four patients were reevaluated at 4.5±2.3 years and diagnosed with permanent hypothyroidism: 2 mild (subclinical) and 2 severe. The fifth had neonatal hyperthyrotropinemia, started levothyroxine (22 months, TSH=13.8 mU/l) and was reevaluated (4 years) confirming permanent mild HC. The discharge test (perchlorate) was negative in 2 and compatible with partial iodine-organization deficit in 3. Massive-sequencing techniques identified PAX8 heterozygous variants in these patients (3 missense, one deletion): c.117C>A/p.(S59R); c.196_198delTAC; c.397C>T/p.(R133W) and c.398G>A/p.(R133Q) (in 2 patients).

Methods: In-vitro functional studies based on transactivating activity of PAX8 on a TG-gene promoter were performed. We obtained a PAX8 wild-type (WT) expression vector and perfomed site-directed mutagenesis to obtain mutant-expression constructs of the variants. A TG promoter was inserted into a reporter. These vectors (PAX8-WT/PAX8-mutant + TG-reporter) were transiently transfected into HEK293 cells and dual-luciferase assay was performed. Experiments were performed ≥3 times. The transactivating activity of the mutants was compared to WT.

Results: PAX8 variants had an impaired transcriptional activity of TG promoter: c.177C>A retained a 76% activity, c.196_198delTAC a 69%; variant c.397C> T a 52%, and c.398G>A a 71%. Activities were statistically lower than the WT or almost. Therefore, the variants were considered pathogenic and to be causing the CH in these patients.

Conclusions: PAX8-in-vitro functional studies have shown that our patients’ variants are pathogenic. These studies have proven their effectiveness in evaluating these types of variants and will continue to be used to analyze the rest of the variants detected in PAX8 in our patients.