Molecular and Functional Characterization of the Novel Mutation c.2335-1G>C in the Human DUOX2 Gene Responsible of Iodide Organification Defects

RIVOLTA, CARINA M.; BELFORTE, FIORELLA S.; TESTA, GRACIELA; SOBRERO, GABRIELA; TARGOVNIK , HECTOR M.; MIRAS, MIRTA B.

Puerto Varas

Congreso; XXV Annual Meeting, SLEP; 2015

Sociedad Latinoamericana de Endocrinología Pediátrica

Introduction: Iodide Organification defects (IOD) represent 10% of cases of congenital hypothyroidism (CH) being the main genes affected that of thyroperoxidase (TPO) and Dual Oxidasa2 (DUOX2).Subjects and Methods: From a population of 20 patients with clinical and biochemical criteria suggestive of CH associated with IOD (high serum TG and high levels of serum TSH with simultaneous low levels of circulating thyroid hormones) TPO and DUOX2 genes were analyzed by means of PCR-SSCP and sequencing. Splicing mutations were analysed by bioinformatics using the NNSplice program and were functionally characterizated by means of minigenes.Results: A novel heterozygous compound to the mutations c.2335-1G>C (intron 17) and c.3264-3267delCAGC (exon 24) was identified. Exon 18 of DUOX2 gene was amplified together with the intron flanking regions from genomic ADN of our patient and cloning, both alleles (WT and mutant) in pSPL3 vector. HeLa cells were transfected with wild-type, mutant, and control pSPL3 and the resulting fragments were evaluated by RT-PCR and sequencing. The mutation c.2335-1G>C created a new or activated an existing unusual cryptic donor splice site in intron 17 located at position ?14 of the authentic intron 17-exon 18 junction site. Aditionally, exon skipping and cryptic 5activation in exon 18 were determinated. Conclusions: A novel heterozygous compound was characterized being responsible of IOD. Cryptic splicing sites have been identified in DUOX2 for the first time. The use of molecular biology techniques is a valuable tool for understanding the molecular pathophysiology of this type of thyroid defects.