Except for the five known variants, two heterozygous missenses (c.275T>C/p.Ile92Thr and c.398G>A/p.Arg133Gln) were identified in PAX8 in two individual samples [Table 2]. One patient (Sample 3) with c.275T>C was a male subject, who was born at 41 weeks of gestation from non-consanguineous parents with birth weight of 3200 g and length of 52 cm. He was diagnosed as CH by neonatal screening with a high TSH (>100 μU/mL) and low FT4 (6.2 pmol/L). The other patient (Sample 4) with c.398G>A was a female subject, who was born at 38 weeks of gestation from non-consanguineous parents with birth weight of 12,000 g and length of 85 cm. The neonatal screening revealed a high TSH (97.1 μU/mL) and low FT4 (3.1 pmol/L).
These two mutations have not been discovered by any of the three large-scale genotyping projects. The two non-synonymous variants were not detected in 347 ethnic-matched healthy controls. Therefore, they were regarded as novel SNVs. Functional predictions by Polyphen and SIFT indicated that the two SNVs were detrimental to the proper functions of PAX8 [Table 2] and should be classified as pathogenic variants. These novel variants located at the PAX domain in which the majority of reported pathogenic variants located [Figure 1]. All variants were confirmed through MassArray iPLEX MALDI-TOF platform.
In our cohort involving 105 patients with CH with Chinese origin, two novel missense mutations of PAX8 were identified and functionally characterized. The result of luciferase reporter assay revealed that the efficiency to drive TPO expression was severely impaired for PAX8 with Ile92Thr or Arg133Gln mutations.
Since Ile92Thr and Arg133Gln mutations identified in our cohort are located in the region of PAX8 responsible for the DNA binding, the amino acid substitutions might severely interfere with the expression of its target genes, such as TPO and TG. Luciferase reporter assay on TPO promoter revealed that for the two mutations could significantly reduce the expression of TPO gene.
This project was supported by a grant from the National Natural Science Foundation of China (No. 81101490).
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