Screening And Research Of Biological Function On TSHR Mutations Of Patients With Congenital Hypothyroidism In Northwestern Region

Congenital hypothyroidism is the most common endocrine disease in infants with a high incidence.Previous studies showed that thyroid hormone receptor(TSHR)is a key factor for development of thyroid gland.This study had two sections.Part I: We enrolled 227 patients with CH and 200 healthy subjects from the northwest region.DNA samples were sequenced by high-throughput sequencing to screen the TSHR mutations.The pathogenicity assessment were analyzed by bioinformatics.Part II: The experiment of in vitro were performed to explore the molecular mechanism of CH.(1)Analysis of TSHR gene mutation in CH patients in northwest of China: This study showed that a total of 16 CH patients with TSHR mutations were identified in 227 CH patients,accounting for 7%(16/227),which was higher than data reportrd in china.The 16 mutations contained 15 missense mutations and 1 nonsense mutation.12 of these mutations located on exon 10,which indicated that the rate of TSHR gene mutation on 10 exon was higher in the northwest region.Exon 10 encoded both the transmembrane and intramembrane regions in the TSHR protein,which was essential for the function of the TSHR protein.Of the 16 mutations,8 have been reported in public databases(p.N432 S,p.R450 H,p.F525 S,p.A533 T,p.I155 L,p.A204 V,p.E727 D,p.H32Y).As a hotspot mutation,p.F525 S had been found in five CH patients and its frequency was 2.2%(5/227),whereas p.R450 H appeared only once in this study.Meanwhile,8 novel mutations(p.V433 I,p.M527 K,p.R528 S,p.A579 V,p.K618*,p.C176 R,p.K338 E,p.N372T)were also detected.We also screened compound heterozygote mutations(p.R528S/ p.R450H、p.C176R/ p.K618*、p.M527K/ p.A553T)in three patients.Parents of the patients also had a pedigree validation and found that there was a mutation in each of the three patients.The analysis of all variants were performed by five softwares to predict the protein function.The results showed that two known mutations(p.R450 H,p.N432S)and two novel mutations(p.C176 R,p.K618*)could impact the protein function,while p.E727 D and p.K338 E were predicted to be likely benign for CH.According to the ACMG classification criteria of pathogenic grade and guidelines,3 novel mutations are classified as pathogenic or likely pathogenic(p.R528 S,p.C176 R,p.K618*),and the remaining 5 mutations were classified as ambiguous(VUS)due to insufficient evidence(p.V433 I,p.M527 K,p.A579 V,p.K338 E,p.N372T).5 known mutations(p.F525 S,p.A553 T,p.N432 S,p.R450 H,p.A204V)were classified as pathogenic or likely pathogenicand 2 mutations(p.H32 Y,p.I155L)which had no relevant evidence to support were classified ambiguous(VUS),p.E727 D mutation as benign.(2)Molecular pathogenesis study: we selected 4 mutation(p.R528 S,p.C176 R,p.K618*,p.R450H)for functional verification experiments in vitro.All four variants had pedigree verification and the result of the ACMG pathogenesis classification were pathogenic or likely pathogenic.The results of functional verification experiments in vitro showed that mRNA and proteins were expressed in the four mutants,and there were no difference between the mutants and wild type TSHR.The result of flow cytometry exhibited that p.C176R(c.526T>C)and p.K618*(c.1852A>T)affected the TSHR expression on the cell surface,but p.R450H(c.1349G>A)and p.R528S(c.1582 C>A)can be expressed on the cell surface.