Study On GLI-Similar3 Mutations In Patients With Congenital Hypothyroidism

Objective To investigate the features and characteristics of GLIS3 gene mutation in patients with congenital hypothyroidism?CH?from Shandong province,and to investigate the relationship between GLIS3 and CH.We explore the pathogenic mechanisms of CH caused by the two mutations found in our study and help to establish theoretical basis for gene diagnosis and prenatal diagnosis of CH.Methods Genomic DNA was extracted from peripheral blood leukocytes of 50 TD patients.We detected if the thyroid glands of children were normal with B-ultrasound.The exon 2 to 11 of GLIS3 were amplified with 11 pairs of sequence specific primers designed by Primer5.0.PCR and the first generation of sequencing method?Sanger sequencing?were used to detect mutation.Comparing the sequencing results with the GLIS3 reference sequenc[National Center for Biotechnology Information?NCBI?Reference Sequence: NC₀00009.12]helps to screen gene mutations.We constructed the the wild-type expression vector and mutative-type expression vector of GLIS3 to investigate the effects of GLIS3 mutation on its function.q-PCR was carried out to detect the m RNA expression levels of GLIS3.Furthermore,dual-luciferase expression vector was constructed and cotransfected with the wild-type expression vector or mutative-type expression vector of GLIS3 into the HEK293-T cells.We explore the effects of GLIS3 mutation on activating promoter by the Dual-Luciferase Reporter Assay System.Results The 50 CH cases included 22 boys and 28 girls,and the sex ratio was 1.0:1.3.The mean age was?2.5±0.5?years.The B-ultrasound showed 6 cases?12%?with thyroid gland hypoplasia,23 cases?46%?with thyroid gland agenesis and 21 cases?42%?present with ectopic thyroid gland.One mutant?rs150310830,c.G2710A?was detected that is a missense mutation?p.G904R?in one thyroid gland agenesis child.C2507 A missense mutation was found in exon 10 of GLIS3 in another thyroid gland agenesis case,which may result in proline to glutamine substitution at codon 836.Functional studies show that the two mutations decrease the expression of m RNA.G904 R decreases by 59.95% while P836 Q decreases by 31.23% compared with WT.The Dual-Luciferase Reporter Assay System shows that the two mutations decrease the ability of transactivation?P<0.05?and neither has dominant negative effect on the WT protein.Conclusion Functional studies indicate that GLIS3 mutation may lead to CH.The mutation rate of GLIS3 gene is very low in CH children of Shandong province.Further studies are needed to investigate the relationship between GLIS3 genotypes and clinical phenotypes.