Function Study Of NKX2-1 Gene Mutation, Which Caused Congenital Hypothyroidism And Central Nervous System Disorders

ObjectiveCongenital hypothyroidism （CH） is a group of diseases which always caused by different kinds of genetic variations, and NK2 homeobox 1 （NXK2-1） is a relatively common responsible gene among them. As a specific transcriptional factor, NKX2-1 plays a very important role in the development of thyroid, lung and central nervous system （CNS）. NKX2-1 mutation will classically cause hypothyroidism, chorea and neonatal respiratory distress. We had managed a pair of twin boys who had short stature and then made the diagnosis of CH. Whole genome sequencing of the affected patients revealed a heterozygous missense mutation, c.799G>T （NM₀01079668.2） of NKX2-1 （p.Va1235Phe, NP₀01073136.1）. NKX2-1 mutations had been widely reported in previous studies, but the relationship between genotype and phenotype of the disease remains unclear. Why the patients who have c.799G>T mutation of NKX2-1 have such symptoms, we have no idea. Our study aims to discover the molecular mechanisms of pathology by means of expression and transcription function analysis.MethodWe introduced the mutation c.799G>T in an NKX2-1 expression vector, and then analyzed the protein expression differences in HEK293 cells between wild type and mutant vectors through transfection experiments and Western blot. Aiming to discover the transcription activity of the mutant protein, we co-transfected the expression vectors and reporter vectors harboring the TG, TPO and SP-B gene promoters, then performed dual-luciferase assay to evaluate the function of NKX2-1 c.799G>T heterozygous mutation. Furthermore, we also anylized local structure variations of mutant NKX2-1 p.V235F using bioinformatics softwares.Results1. Obtain wildtype and mutant NKX2-1 expression vectors c.799G>T mutation was successfully induced in expression vector pENTER-wtNKX2-1 through mutagenesis PCR. Sequencing of pENTER-mutNKX2-1 confirmed the mutation.2. Expression evaluation of wildtype and mutant NKX2-1 protein Compared with wildtype NKX2-1, the expression of mutant NKX2-1 p.V235F was significantly reduced in HEK293 cells. The expression amount of mutNKX2-l p.V235F is just 0.3 times of that of wildtype NKX2-1 （p<0.05）.3. Transcription evaluation of wildtype and mutant NKX2-1 protein In dual-luciferase assay, transcriptional activities of groups co-transfected with pENTER-wtNKX2-1 and reporter vectors harboring TG, TPO, SP-B promoters are 24.47,35.19 and 44.63 times of that of groups co-transfected with pENTER empty vector and reporter vectors （p<0.05）, showing that all TG, TPO and SP-B promoters could be activated by wildtype NXK2-1. Besides, TG promoter was activated in a dose-dependent pattern, while TPO and SP-B promoters were not. Transcription activities of groups co-transfected with pENTER-mutNKX2-1 and reporter vectors harboring TG, TPO, SP-B promoters are 0.022,0.027 and 0.11 times of that of groups co-transfected with pENTER-wtNKX2-1 vector and reporter vectors （p<0.05）, indicating that mutant NKX2-1 p.V235F failed to adequately activate TG, TPO or SP-B promoters. Interestingly, mutant NKX2-1 p.V235F could weakly activate SP-B promoter, which was different from that of TG and TPO promoters.4. Transcription activity influences of mutant NKX2-1 p.V235F protein When pENTER-mutNKX2-1 and pENTER-wtNKX2-1 were mixedly transfected, dose changes of mutant NKX2-1 p.V235F didn’t change the transcription activating ability of TG, TPO or SP-B promoters induced by wildtype NKX2-1.5.Local structure analysis of mutant NKX2-1 p.V23SF protein Local structure analysis of mutant Phe235 showed abmornal contacts with nearby amino acids locaed in Helix III and Arg221 located in Helix II. Surface charge distribution also changed in this mutant model.ConclusionsCompared with wildtype NKX2-1, the expression of mutant NKX2-1 p.V235F in HEK239 cells is significantly reduced. Wildtrpe NKX2-1 could effectively activate TG, TPO and SP-B promoters. TG promoter is activated in a dose-dependent pattern, while TPO or SP-B promoters are not. All TG, TPO or SP-B promoters are failed to be adequately activated by mutant NKX2-1 p.V235F, and their transcription activities induced by wiltype NKX2-1 cannot be influenced by mutant NKX2-1 p.V235F. However, compared to TG and TPO promoters, SP-B promoter could be weakly activated by mutant NKX2-1 p.V235F. In conclusion, NKX2-1 haploinsufficiency in TG promoter activation and decreased TG transcriptional activity induced by mutant NKX2-1 p.V235F may cause hypothyroidism in NXK2-1 c.799G>T patients. Our study discovered part of the pathologic mechanisms of NXK2-1 c.799G>T mutation.