Genetic Validation And Pathogenesis Of Candidate Gene NKX6-2 In Children With Congenital Thyroid Dysgenesis

Objective: Congenital hypothyroidism(CH)is the most common metabolic disorder in infants.The patients,if do not get early diagnosis and timely treatment,can present irreversible growth retardation and intellectual disability in childhood.The common type of CH is thyroid dysgenesis(TD).Although genetic factors play an important role in the pathogenesis of TD,only a few TD pathogenic genes have been identified.Based on the preliminary screening of NKX6-2 as a novel candidate pathogenic gene for TD,genetic amplification of the candidate pathogenic gene was performed by targeted sequencing technology to determine whether NKX6-2 is the pathogenic gene of TD.Then,the pathogenic mechanism of the four identified NKX6-2 heterozygous missense mutations was studied to clarify the molecular pathogenic mechanism of TD caused by the functional changes of the missense mutations.Methods: In the early stage,a total of 290 patients with CH were recruited for whole exome sequencing,and were found that NKX6-2 may be one of the candidate genes related to thyroid development after advanced bioinformatics analysis and literature review.In this study,the initially identified novel TD candidate pathogenic gene NKX6-2 was genetically verified on the selected 479 TD patients by targeted sequencing of Next generation sequencing(NGS),and the relationships between genotype and phenotype were studied combined with the NKX6-2 mutations found in previous work.Then,four heterozygous missense mutations c.32T>C/p.L11 P,c.280G>A/p.G94 R,c.609G>T/p.W203 C and c.782C>T/p.S261 L,which were found in previous work,were functionally studied.First,after NKX6-2 wild type eukaryotic expression vector was constructed,the mutant eukaryotic expression vectors were induced by point mutation kit.The wild type and mutant NKX6-2eukaryotic expression vectors were transfected into human normal thyroid cell line Nthy-ori3-1.After 24 h and 48 h,the total RNA and total protein were extracted respectively.The mRNA expression of NKX6-2 in thyroid cells was detected by quantitative real-time PCR(qPCR),and the protein expression of NKX6-2 was detected by Western blotting.The cell proliferation assay(CCK-8),scratch assay and transwell assay were used to evaluate the effects of wild type and mutant NKX6-2 on the proliferation and migration of the thyroid cells.The effects of wild type and mutant NKX6-2 on Wnt/β-catenin signaling pathway were determined by dual luciferase reporter assay.Results: NKX6-2 was sequenced in 479 TD patients by using targeted sequencing technology.The result of Fisher’s exact test was P=1.958e-08 compared with the China MAP controls(N=10588).Four different mutations c.196C>T/p.R66 W,c.436G>C/p.G146 R,c.58C>T/p.H20 Y and c.354G>A/p.W118 X were identified in 5 TD patients(2 patients with ectopy and 3 patients with hypoplasia).The four mutations were predicted to be deleterious by Poly Phen-2,SIFT,Mutation Taster and PROVEAN softwares.H20,R66 and G146 were located in the conserved regions of the protein,while W118 was located in the non-conserved region.Comprehensive analysis revealed no NKX6-2 mutation hotspots or clear genotypic and phenotypic associations in combination with one homozygous NKX6-2missense mutation c.388C>A/p.P130 T and four different heterozygous missense mutations c.32T>C/p.L11 P,c.280G>A/p.G94 R,c.609G>T/p.W203 C and c.782C>T/p.S261 L identified in previous work.Cellular functional studies of four heterozygous missense mutations detected earlier showed that there were no significant differences in the proliferation and migration of Nthy-ori 3-1 cells between the empty vector group and WT group,or between WT group and mutant groups L11 P,G94R,W203 C,S261L.Although there was no significant difference in the expression of NKX6-2 between L11 P group and WT group,the luciferase activity was significantly decreased in the L11 P group;The mRNA and protein expressions of NKX6-2 were decreased in G94 R group,while the luciferase activity was not statistically significant compared with WT group;The mRNA expression of NKX6-2 in W203 C and S261 L groups was not changed compared with WT group,but the protein levels were significantly decreased,and the luciferase activity revealed no statistically significant difference in comparison with WT group.Conclusions: This study revealed that NKX6-2 may be a novel candidate pathogenic gene for TD and described NKX6-2 mutations found in TD patients.However,no clear relationships between genotype and phenotype were found in our study.Cellular functional studies of four NKX6-2 heterozygous missense mutations showed that NKX6-2 gene may not involve in the proliferation and migration of thyroid cells during thyroid development.Although these four different mutations have impacts on the expression or transcriptional activation ability of NKX6-2,further experiments are needed to verify the effects of the four mutants on other physiological processes of thyroid development,such as cell differentiation and apoptosis.