Molecular characterization of thyroglobulin variants identified in patients with goitrous hypothyroidism. Analysis of the splicing mechanism.

Abstract

Thyroglobulin (TG) is a homodimeric glycoprotein synthesized by the thyroid gland. To date, two hundred twenty-seven variations of the TG gene have been identified in humans. Thyroid dyshormonogenesis due to TG gene mutations have an estimated incidence of approximately 1 in 100,000 newborns. The clinical spectrum ranges from euthyroid to mild or severe hypothyroidism. Splicing mutations represent a major cause of human disease, between 15?50% of all human disease. Variants at the level of the splice site culminate in important defects at the level of the pre-mRNA splicing process. The splicing process is quite complex whose molecular bases and interactions with underlying elements are still not entirely clear, resulting as we show in the present work, a rare phenotype involving mechanisms of such processing of those pre-RNAS from a variant founded for our group in a hypothyroid patient.Thepurpose of the present studywas to identifyand characterizenew variants in the TG gene. Wereport an Argentine patient with congenital hypothyroidism,enlarged thyroid gland and low levels of serum TG. Sequencing of DNA,expression of chimeric minigenes as well as bioinformatics analysis were performed.DNA sequencing identified the presence of compound heterozygousvariant in the TG gene: the maternal mutationconsists of a c.3001+5G>A, whereasthe paternal mutation consists of p.R296*.Minigen analysis of the variant c.3001+5G>A performed in HeLa, CV1 and Hek93T cell lines, shows a total miss of transcript expression. So, in order to validate that the lack of expressionwas caused by such variation, site-directed mutagenesis was performed on the mutated clone,who had a pSPL3 vector change, to give rise to a wild-type clone c.3001+5G and to indorsing that the mutation c.3001+5G>A is the cause of the total lack of expression. These results open up new perspectives in the knowledge of the mechanism of splicingforthe TG pre-mRNA.