Background:The transcription factor paired box 8 (PAX8) was associated with type 2 congenital non-goitrous hypothyroidism (CHNG2),a clinical phenotype of congenital hypothyroidism (CH).Though studied in a few regions with different ethnicities,the incidence of PAX8 mutations varied,even among Chinese cohorts in different regions.This study aimed to identify and characterize PAX8 mutations and explore the prevalence of its mutations in another cohort of CH.Methods:The 105 unrelated Chinese patients with CH were collected from four major hospitals.Exomes of the 105 samples were sequenced by Hiseq 2000 platform to identify mutations of PAX8 on genomic DNAs extracted from peripheral blood samples.Luciferase reporter assays were used to assess the effects of mutations on the transcription of thyroid peroxidase (TPO).Results:Three PAX8 mutations in four subjects were identified in 105 samples.One variant,rs189229644,was detected in two subjects,and categorized as uncertain significance.The other two missense mutations (275T＞C/lle92Thr and 398G＞A/Arg133Gln)were not detected in three large-scale genotyping projects,namely 1000 Genome Project,Exome Aggregation Consortium and GO Exome Sequencing Project.Functional studies for the two mutations revealed that they could impair the transcription ability of PAX8 on one of its target genes,TPO.Therefore,the two mutations were causative for the pathogenesis of CHNG2.After combining the studies of PAX8 mutations,an average frequency of 1.74％ (21/1209) could be obtained in Chinese patients with CH.Conclusion:The study specifically demonstrates the role of two mutations in impairing the transcription ability of PAX8,which should be considered as pathogenic variants for CH.