Screening And In Vitro Functional Study Of SLC5A5 Mutation In Patients With Congenital Hypothyroidism

Congenital hypothyroidism(CH),referred to as hypothyroidism or hypothyroidism,is a more common endocrine disease in infancy,and severe patients will have physical retardation and irreversible mental development disorders.Sodium Iodide Symporter(NIS),encoded by the SLC5A5(Solute carrier family 5 member 5)gene,is an indispensable plasma membrane glycoprotein associated in connection with the thyroid gland,and NIS mediated active transport of I into thyroid follicular cells is the crucial first step of thyroid hormone biosynthesis.The SLC5A5 gene mutation may cause Iodine transport deficiency(ITD),which in turn leads to hypothyroidism.In this study,mutation cases were screened by high-throughput sequencing of all exons and adjacent regions of the SLC5A5 gene in 227 patients with hypothyroidism,then bioinformatics analysis was performed on the mutation sites of these mutation cases.Second,a series of functional experiments were carried out,the purpose of which was to analyze whether these new mutations had an impact on the expression and function of NIS protein.It was found that NIS mutations were detected in four patients,accounting for 1.7%(4/227);one of the patients carried a compound heterozygous P58fs/G511 E mutation,and the other three patients each carried a heterozygous mutation(p.G75 A,p.P438 Q and p.Q639 *).The function of these 5 mutations is unclear because they are new mutations.The five mutant and wild-type SLC5A5 genes were expressed in vitro in HEK-293 T cells,and their m RNA levels and protein levels were determined by fluorescence quantitative PCR,Western-blotting(WB),and flow cytometry.Compared with wild-type cell lines,we found that only the P58 fs mutation significantly reduced the m RNA level and total protein(glycosylated and non-glycosylated)expression;however,the Q639 * mutation only significantly impaired glycosylated protein expression.In summary,our results suggest that three mutations(p.G75 A,p.P438 Q,p.G511E)had little effect on the NIS protein;while the mutation of P58 fs resulted in the early appearance of terminator,which greatly damaged the translation and maturation of the protein;Q639 * mutation had little effect on protein translation,but the mature glycosylated protein was almost not expressed.This study is not only helpful to clarify the role of SLC5A5 gene mutation in the pathogenesis of Chinese patients with hypothyroidism and to clarify the effect of gene mutation on NIS protein function,but also of great significance to explore the role of gene detection in the prediction,diagnosis and prognosis of hypothyroidism.