Screening Of TSHR Gene Mutation In Children With Congenital Hypothyroidism And Pathogenesis Of DUOX2 Gene Mutation

Congenital hypothyroidism (CH) is one of the most common neonatal endocrine disorders,presenting with abnormal growth and intellectual impairment due to loss of thyroid function at different levels.All the CH patients can be divided into2groups:about85%of cases are caused by thyroid dysgenesis (TD),whereas the left15%are associated with dyshormonogenesis.A variety of gene mutations have been proven to affect the thyroid hormone synthesis,such as thyroglobulin(TG),thyroperoxidase(TPO),dual oxidase2(DUOX2) and DUOX maturation factor2(DUOXA2).Part I:Objective:We studied mutations from exon10of TSHR gene in patients with congenital hypothyroidism secondary to ectopy,and identified genetic characteristics of TSHR gene mutations,which plays an important role in gene diagnosis and prenatal diagnosis. Methods:After collecting blood samples from89babies with congenital hypothyroidism, we extracted genomic DNA and detected exon10mutations by PCR as well as direct sequencing. Results:A missense mutation c.1269G>A(p.V424I)and a SNP(rs1991517,c.2181G>C)were found according to direct sequencing of89subjects. From the NCBI website,we obtained the TSHR family protein-sequence of various species, including Homo sapiens, Mus musculus, Rattus norvegicus, Danio rerio, Felis catus, Sus scrofa and Bos taurus. Using DNAMAN software, we achieved multiple sequence alignment of the TSHR family of different species. We found that codon424where the mutation (p.V424I) was identified, was located in highly conserved region of TSHR. Conclusion:TSHR gene exonlO mutation rate is relatively low,probably not a main cause of congenital hypothyroidism with ectopy in Shandong province.Part II:Context:Mutations in dual oxidase2(DUOX2) gene, which results in an impairment of the hydrogen peroxidase-generating system and dyshormonogenesis, have been proposed as a cause of autosomal recessive congenital goiter with hypothyroidism. Objective:To identify mutations in DUOX2as a cause of congenital hypothyroidism (CH) due to organification defect and explore the effects of these mutations on DUOX2function. Methods:After collecting blood samples of10babies from unrelated families with congenital hypothyroidism and goiter, we extracted genomic DNA and detected all the exons by PCR as well as direct sequencing. H2O2generation was measured48hours after transfection with the indicated expression vectors, using a calibration curve to convert changes in fluorescence intensity into absolute nanomoles. Cycloheximide (CHX) was added to the medium of Hela transfected with DUOXA2and either WT or one of the mutations DUOX2expression plasmids, and DUOX2protein half-life was detected by western blot. Results:Analysis of DUOX2revealed2novel mutations from unrelated families:c.1060OT (p.R354W) and c.G3616A(p.A1206T).H2O2production assay indicated that both mutations displayed a complete deficiency phenotype in generating H2O2, and CHX chase experiments demonstrated mature WT DUOX2proteins appeared to display a higher stability than mutant ones, meaning prolonged half-life.Conclusions: Our study indicated that WT DUOX2displayed complete H2O2-generating activity only with DUOXA2expressed together. Two new mutations in DUOX2were responsible for the deficit in the organification process. Co-transfected with DUOXA2, mutant DUOX2tended to degrade easier than WT ones.