Identification And Mechanisms Of Two Novel Mutations In PAX3 Gene Associated With Waardenburg Syndrome

BackgroundWaardenburg syndrome type Ⅰ（WS1）is a rare autosomal dominant disorder characterized by sensorineural deafness,heterochromia iridis,abnormal pigmentation of hair and skin,and dystopia canthorum.The major pathogenic gene of WS1 was Paired box gene 3（PAX3）.The protein encoded by PAX3 functions as an important transcription factor to regulate the development of both the neural crest cells and its derivatives（i.e.,melanocytes）during embryogenesis.Variants or abnormal expression of PAX3 could lead to disorder in the development and distribution of melanocytes,further affecting the pigmentation on body surface and the sensorineural function.The purpose of this study was to explore pathogenic variants in two WS1 families previously enrolled in our hospital,verify their functions through in vitro experiments,and investigate their potential molecular mechanisms.Methods（1）Six patients and five unaffected individuals from two Chinese families were included in this work.A comprehensive clinical evaluation was performed on all participants.（2）Genomic DNA from probands’ blood samples were extracted,the targeted next generation sequencing（NGS）was performed to screen the potential pathogenic variants in a total of 127 deafness genes.（3）Sanger sequencing was performed to confirm the co-segregation of the candidate variants with WS1 phenotypes in their own families and their presence in 200 healthy controls.（4）Bioinformatics analysis of PAX3 variants.The pathogenicity of PAX3 variants was assessed by SIFT,Polyphen-2,and MutationTaster.The 3-dimensional（3D）molecular structure of PAX3 proteins were simulated using I-TASSER to determine the effects of PAX3 variants on their protein structure.（5）The PAX3 plasmids were transfected into HEI-OC1 cells and HEK293 cells for Western blot（WB）to determine whether the variants affected the expression of PAX3.（6）The PAX3 plasmids were transfected into HEI-OC1 cells and HEK293 cells for Immunofluorescence（IF）to determine whether the variants affected the subcellular localization of PAX3.（7）The effects of the variants on the transcription activity of PAX3 and the synergic regulation of MITF transcription with PAX3 and SOX 10 were evaluated by luciferase reporter assays.Results（1）Two families were diagnosed with WS1 based on their clinical presentation.Two patients in F139*family showed bilateral sensorineural deafness,dystopia canthorum,hypopigmentation of hair and heterochromia iris.The proband of F226*family had the same clinical manifestations as F139*,but incomplete penetrance was present in other patients.（2）Targeted NGS revealed that two heterozygous variants of PAX3,c.215T>C（p.I72 T）and c.234₂35delGT（p.V78delinsVPfs）were in F139*and F226*families respectively.（3）Sanger sequencing showed that two variants were co-segregated with WS1 phenotypes in their own families,respectively,and absent in 200 controls.（4）Bioinformatics analysis showed that both variants were pathogenic and were not reported previously;3D molecular structures showed that the p.I72T PAX3 protein structure was significantly altered and the p.V78delinsVPfs PAX3 protein structure was abnormally shortened compared to the wild type（WT）PAX3.（5）The results of WB indicated that the molecular weight of p.I72T PAX3 protein did not change compared with WT PAX3 protein,but the bands were weaker,while p.V78delinsVPfs PAX3 protein had reduced molecular weight and weaker bands.（6）IF analysis showed that the subcellular localization of p.I72T PAX3 protein was not altered,whereas the nuclear distribution of p.V78delinsVPfs PAX3 protein was impaired and its localization changed from the nucleus to the nucleus and cytoplasm.（7）Luciferase reporter assays demonstrated that the transcriptional activities of both p.I72T and p.V78delinsVPfs PAX3 were significantly reduced compared with WT PAX3;p.I72T PAX3 and p.V78delinsVPfs PAX3 proteins had no effect on the transcriptional regulatory function of WT PAX3 protein but affected the synergistic effect of SOX 10 and PAX3 on MITF.ConclusionsTwo novel PAX3 variants,c.215T>C（p.I72T）and c.234₂35delGT（p.V78delinsVPfs）,were identified as key pathogenic genetic variants in two Chinese WS1 families.The abnormal functions of the mutant PAX3 proteins indicate that these two variants are likely to be the genetic causes of the two WS1 families,while the clinical phenotypes of WS1 may be related to the haploinsufficiency of mutant PAX3.