Study On Mutations Of Nicotinamide Nucleotide Transhydrolase In Patients With Congenital Hypothyroidism And Thyroid Dysplasia

Objective: Congenital hypothyroidism(CH)is the most common endocrine disease in newborns.In recent years,the defect of nicotinamide nucleotide transhydrolase(NNT)had been reported in CH patients,but whether it is pathogenic or not is still a matter of study.In this research,the NNT in patients with CH and TD from Shandong,China was screened to explore the relationship between the gene and CH and find new candidate pathogenicity genes.Besides,further functional analysises of the mutations were carried out to explore the possible abnormalities caused by the mutations,reveal the pathogenesis,and ultimately to serve the clinic.Methods: In this study,100 patients with TD and 120 healthy persons from different regions of Shandong were selected for screening.The abnormality of classical CH pathogenic genes,such as thyroid stimulating hormone receptor(TSH),thyroid transcription factors including NKX2.1,FOXE1,NKX2.5,PAX8,were excluded in all the patients.Genomic DNA which was extracted from peripheral blood clots of all220 patients was qualitatively examined.21 pairs of primers were designed to amplify21 coding regions of NNT.After amplification,the products were examined by agarose gel electrophoresis,and the qualified individuals were recovered and sequenced by Sanger sequencing.After that,the results were compared with the corresponding sequence from NCBI(NM012343.3)to receive mutations of NNT.Then the sequence homology of the discovered mutations were accomplished to obtain more informations.In functional analysis,the wild-type NNTpc DNA3.1recombinant plasmid was synthesized and transfected into Hela cells respectively as will as the mutant NNTpc DNA3.1 recombinant plasmids.After 48 hours of culture,the expression of NNT protein was detected by Western Blot.At the same time,Amplex Red method was used to detect the absorbance of the pores at 560 nm after 48 h of transfection.Finally,the hydrophobicity of the mutant sites w analyzed to predict the effect of mutations on protein stability.Results: In this study,we found 3 NNT mutations: c.811G>T,p.A271S;c.2078G>A,p.R693H;c.2581G>A,p.V861 M.All of them were missense mutations.The homology analysis of the three mutations showed that they all located in the conserved region of the protein.Western Blot results showed that the three mutations did not change the expression of NNT.Amplex Red results showed that the three mutations did not influence the hydrogen peroxide produced by the cells.But hydrophobicity analysis showed that mutations had changed the hydrophobicity of some regions of the protein.Conclusion: This study was the first time to screen NNT in CH patients with TD in China and three unreported missense mutations were detected.The three unrelated host patients had shown significant hypothyroidism,suggesting that NNT may be a candidate virulence gene for CH with TD.In addition,compared with cells carrying the normal NNT,cells carrying three mutations showed no change in hydrogen peroxide.However,these three mutations had changed the stability of NNT,so the specific pathogenetic mechanism of TD and Ch caused by the mutations still needs to be further studied.