Screening And Functional Analysis Of TPO Gene Mutations In A Cohort Of Congenital Hypothyroidism Patients

Congenital hypothyroidism（CH）is a common endocrine disease in neonatal period.If the treatment is not timely,accompanied by hypothyroidism,it is very likely to affect the intellectual development of children.As an important gene of thyroid hormone synthesis,TPO（thyroid peroxidase）gene mutation is considered to be one of the main genetic factors leading to CH.In this study,219 cases of CH were collected from Shaanxi and Xinjiang.The exons and adjacent intron fragments of TPO gene were sequenced by high-throughput sequencing.Combined with bio-informatics analysis,suspected pathogenic variants were screened and identified,and then to determine the mutation spectrum of TPO gene in Northwest China.The biological function of newly found mutations in this study was evaluated by in vitro experiments.Mutations were detected in 17 of 219 patients（7.8%）.Among the 19 variants detected,there were 1 nonsense mutation,1 splicing defect mutation and 17missense mutations.Seven mutations were found for the first time in this study,including p.Ser309Pro,p.Ala443Val,p.Asn592Ser,p.Asn674Ser,p.Asn798Lys,p.Ser853Leu and IVS7-1 G>A.The majority（n=17）of detected mutations were found only in one different case,while two mutations p.Pro883Ser and p.Ala846Trp were respectively detected in two cases,whose clinical phenotype were different.All cases with TPO mutation（s）were demonstrated a normal-sized thyroid gland or goiter.15 patients only carried one heterozygous mutation,2patients were detected with 2 or more mutations,whom were all severe CH,with a normal thyroid gland.The hot spot mutation c.2268dup T reported in the literature was only detected in one patient in this study.In this study,the biological functions of the six novel variants were studied in vitro.Using real-time quantitative PCR and western blotting,the results found that the six mutations did not affect the m RNA and protein expression levels of TPO gene.The enzymatic activity and enzyme kinetics of six mutants and wild type protein were detected by spectrophotometer with guaiacol as substrate,and the enzymatic activity of all mutant proteins was decreased to varying degrees.Of these mutant proteins,proteins having p.Ser309Pro and p.Asn674Ser showed the lowest enzymatic activity,with V_max/K_m values only were 24%and 12%of that of wild type protein（P<0.05）.These data were in accordance with 3-D structures homology modeling of proteins having p.Ser309Pro and p.Asn674Ser,which showed a dramatic change in the TPO protein structure,indicating these two mutations would alter the catalytic domain of TPO protein,thus to reduce the binding affinity of protein and substrate.In conclusion,through screening and identification of TPO mutations in CH patients in Northwest China,the overall detection rate of TPO mutations was low（7.8%）,and the proportion of patients with multiple TPO mutations was less than1%,indicating that TPO mutation may not be the key pathogenic gene leading to CH in China.The preliminary study on the biological function of the novel mutations may help to clarify the pathogenesis of CH and the relationship between the structure and function of TPO protein.