Objective To identify new susceptibility gene variants in Chinese papillary thyroid carcinoma（PTC） patients by whole exome sequencing（WES） combined with targeted analysis. Methods A total of 286 PTC patients who underwent surgery at Beijing Hospital from 2018 to 2020 were enrolled to identify mutation sites in PTC candidate genes，and 214 PTC patients who underwent surgery at Beijing Hospital from 2021 to 2022 were enrolled to calculate the mutation frequencies of these mutation sites. WES was used to determine peripheral blood leukocyte genomic DNA of all the subjects. Rare variants in protein-coding regions and flanking selective splicing sequences of 28 PTC candidate genes identified by genome-wide association study（GWAS） were identified using targeted analysis. The allele expression of mutation sites in tumor tissues and adjacent tissues was determined. Case-control association analysis was performed to assess the effect of mutation sites on PTC risk. Genotyping data for the controls were obtained from gnomAD，TOPMed，ChinaMAP and HUABIAO databases. Results Two extremely rare missense mutations were identified on the erythrocyte membrane protein band 4.1 like 4A（EPB41L4A） gene（rs1455421289 site c.163G>C：p.D55H and rs761977647 site c.855G>C：p.W285C），which could potentially affect protein function. Among the 286 PTC patients used to identify candidate gene mutation sites，3 patients carried rs1455421289 site c.163G>C：p.D55H mutation，and 1 patient carried the rs761977647 site c.855G>C：p.W285C mutation. The carry frequency of these 2 sites was <0.05% in all 4 control groups. Among the 214 PTC patients used to calculate mutation site mutation frequencies，1 patient carried rs1455421289 site c.163G>C：p.D55H mutation，and no rs761977647 site c.855G>C：p.W285C mutation was determined. The mutation frequency of EPB41L4A rs1455421289 was 0.8%（4/500），and rs761977647 had a mutation frequency of 0.2%（1/500）. Moreover，the EPB41L4A rs1455421289 mutation resulted in significantly increased abundance of mutant mRNA compared to wild mRNA（P<0.01）. The rs1455421289 site C.163G>C：p.D55H mutation resulted in significantly increased PTC risk compared to the 4 control groups [odds ratios（OR） were 40.3，85.4，213.5 and 541.1，95% confidence intervals（CI） were 4.5-361.5，9.5-765.4，57.2-797.2 and 98.7-2 960.8]. Conclusions Based on WES，a new susceptibility gene variant EPB41L4A rs1455421289 results in significantly increased risk of PTC.
