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Molecular Analysis of Mutated Thyroid Peroxidase Detected in Patients with Total Iodide Organification Defects¹

Academic Medical Center, University of Amsterdam, Emma Children’s Hospital, Academic Medical Center, Pediatric Endocrinology (H.B., J.J.M.d.V.) and Department of Neurology (F.B.), Amsterdam

Address all correspondence and requests for reprints to: Hennie Bikker, Academic Medical Center, University of Amsterdam, Emma Children’s Hospital AMC, Experimental Pediatric Endocrinology H2-255, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail: h.bikker{at}UVA.AMC.NL

Wild-type and mutant thyroid peroxidase (TPO) was expressed in a Semliki Forest Virus (SFV)-based transient expression system in Chinese hamster ovary-K1 cells. Twenty four hours after transfection, proteins immunoreactive with TPO antibodies could be detected on a Western blot. Peroxidase activity was assayed using both the guaiacol and the I₃^- assay. Addition of hematin was necessary to obtain enzymatic active TPO. Thyroid peroxidase complementary DNA constructs containing mutations originally found in patients with hereditary congenital hypothyroidism caused by total iodide organification defects were analyzed using these techniques. In all cases TPO was expressed as shown by Western blotting and immunostaining. Enzymatic activity (measured by guaiacol and iodide oxidation assay) was below the detection level in four out of five mutants. The only mutant yielding TPO with enzymatic activity was G 1858 A (Gly 590 Ser). However, the mutation could affect splicing of TPO messenger RNA, leading to inactive TPO, because it is located at the exon 10/intron 10 border.

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