Study On DUOX1 Mutation Screening And Pathogenic Mechanism Of DUOX1/DUOXA1 Mutation In CH Patients With Goiter

Objective: To explore the types and characteristics and build the relationship spectrum between genotype and phenotype of DUOX1 mutation in CH patients with thyroid goiter of Shandong Province,China.We explored the effects of novel mutation?R1307Q?in DUOX1 and the other novel mutation?R56W?in DUOXA1 which is identified in previous study on DUOX1 function,in order to clarify the pathogenic mechanism of goitrous CH caused by DUOX1/DUOXA1 mutation.Methods: 43 cases of CH patients and 100 cases of normal controls were enrolled and their genomic DNA were extracted from peripheral blood.All exons of DUOX1 were amplified by PCR and the PCR products were sequenced by Sanger sequencing.In order to explore the effects of DUOX1 and DUOXA1?identified in previous study?mutation on DUOX1 function,the wild type expression vector and mutative type expression vector of DUOX1 and DUOXA1 gene were constructed.The mRNA expression levels of DUOX1 and DUOXA1 were detected by q-PCR,and the protein expression levels of DUOX1 was detected by western-blot.The amount of H₂O₂ was measured in Hela cells after transfected for 48 h to detect the effects of DUOX1 mutation on the enzyme activity of dual oxidase 1.In addition,DUOX1 protein in different times was measured after adding the CHX for 24 h.Then the effects of DUOX1 mutation on stability of DUOX1 protein can be determined by calculating DUOX1 protein degradation rate.Results: A novel heterozygous missense mutation?c.3910G>A,p.R1307Q?in DUOX1 was identified in patient with mild transient CH,and a novel heterozygous missense mutation?c.166C>T,p.R56W?was identified in patient with permanent CH in previous study.Functional studies indicated that R1307 Q mutant decreased the DUOX1 activity but not changed the stability of DUOX1 protein.Furthermore,the amount of H₂O₂ generation in Hela cells transfected with R56 W mutant or both R56 W mutant and R1307 Q mutant decreased significantly compared with Hela cells transfected with both wild type DUOX1 and DUOXA1 expression vector.Conclusion: A novel heterozygous missense mutation?c.3910G>A,p.R1307Q?of DUOX1 was identified in one patient.It indicated that DUOX1 mutation rate was verylow,which may not be the main factor causing the occurance of CH patients with goiter in Shandong Province,China.The novel heterozygous missense mutation of DUOX1?c.3910G>A,p.R1307Q?and DUOXA1?c.166C>T,p.R56W?which identified in two different patients can lead to the absence of H₂O₂ generation partly,thyroid hormone deficiency,thyroid stimulating hormone rised,and appeared to CH and goiter.In addition,DUOXA1 was considered as a cofactor to DUOX1 in the process of H₂O₂ generation.The relationship spectrum between genotype and phenotype of DUOX1/DUOXA1 mutation can not be built due to the less reports between DUOX1/DUOXA1 mutation and the occurance of CH.