Molecular Genetic Study Of PTK7 Gene Mutation In Scoliosi

BackgroundScoliosis is a three-dimensional deformity of the spine that can result from a variety of causes.According to the causes of the disease,structural scoliosis can be divided into congenital scoliosis（CS）,neuromuscular scoliosis（NMS）.neurofibromatosis scoliosis and idiopathic scoliosis（IS）.IS is a kind of disease without specific causes and congenital vertebral malformations（CVMs）.Adolescent idiopathic Scoliosis（AIS）is the most commen spinal deformity,occurs between the ages of 10 to 18 years.Due to the peak growth and development,scoliosis may develop significantly and be accompanied by changes in appearance.All types of scoliosis have different degrees of progressive potential and genetic tendency.With the continuous aggravation of scoliosis,patients may have back pain,lung dysfunction,nerve damage,disability,teratogenesis,etc.,which seriously affect the physical and mental health.Clarifying the etiology of scoliosis is not only conducive to the early accurate diagnosis and treatment of various types of scoliosis and the prediction of disease progression,but also has important clinical significance for the early intervention of some patients and the reduction of surgery and related complications.At present,the exploration of various early diagnosis methods and etiological intervention targets of scoliosis is the focus of international research.In recent years,with the application and development of gene detection technology,especially the Next Generation Sequencing（NGS）,more candidate pathogenic genes of scoliosis have been discovered and genetic factors are considered to be the important etiology.Protein tyrosine kinase（PTK7）,also known as colon carcinomakinase-4（CCK4）,is an evolutionarily conserved atypical receptor tyrosine kinase.The human PTK7 protein consists of six extracellular immunoglobulin（Ig）-like domains,one transmembrane domain and one catalytically inactive kinase domain.PTK7 plays an important role in vertebrate canonical and non-canonical Wnt（planar cell polarity,PCP）,Sema-phorin/Plexin and vascular endothelial growth factor（VEGF）signaling pathways.These signaling pathways are important for embryonic developmental processes,including tissue specification,axial morphogenesis,formation of the cardiovascular,endocrine and immune systems,and regulation of neural crest migration and tumorigenesis.Zebrafish models depleted of ptk7 presented various spinal curve phenotypes.Maternal-zygotic ptk7（MZptk7）and zygotic ptk7（Zptk7）mutant zebrafish develop spinal curvatures that model CS and AIS,respectively,due to differential timing of ptk7 loss-of-function（Lof）.Meanwhile,a novel sequence variant PTK7P545A has been reported in a patient with AIS,but without further in vitro investigation.The association between human PTK7 variants and scoliotic phenotypes in a mixed cohort of CS and AIS continue to be understudied.ObjectsTo systematically investigate the genetic etiology of PTK7 variants based on Deciphering Disorders Involving Scoliosis and COmorbidities（DISCO,http://www.discostudy.org/）cohort of PUMCH,including the filter process of candidate variants,phenotypic characteristics analysis and in vitro functional experiments.MethodsAccording to strict inclusion and exclusion criteria,based on 583 patients with CS and 302 patients with AIS in the DISCO（http://www.discostudy.org/）cohort of PUMCH,we performed whole-exome sequencing（WES）and data analysis to evaluate and screen for pathogenic variants of PTK7.In vitro experiments,the changes of PTK7 mRNA and protein expression were evaluated by quantitative PCR and western blot assay,the changes of subcellular localization of PTK7 protein were evaluated by immunofluorescence assay,and the effects of mutation on Wnt/β-catenin signaling pathway were detected by double luciferase reporter gene assay.ResultsAmong the 583 patients with CS and 302 patients with AIS in this study,four relatively high pathogenicity PTK7 gene mutations were screened out from CS cohort by WES and preliminary analysis,including one case of frameshift variant and 3 cases of missense variants.Four missense variants of PTK7 gene with pathogenic potential were detected from AIS cohort.According to ACMG variants interpretation standards and guidelines,the PTK7 gene frameshift variant c.464₄65delAC（p.His155Pfs*16）from CS cohort was judged as pathogenic（P）.Three PTK7 gene missense variants,including c.1394A>G（p.Lys465Arg）,c.1879G>A（p.Gly627Arg）,c.1955G>T（p.Arg652Leu）from CS group and four PTK7 gene missense variants sites c.49c>T（p.Leu17Phe）,c.353C>T（p.Ser118Phe）,c.2290G>A（p.Asp764Asn）,and c.2384G>A（p.Arg795His）were determined as variants uncertain significance（VUS）.The CADD scores of seven missense variants were all greater than 15.Summarize the clinical characteristics of five patients in the CS cohort and four patients in the AIS cohort carried PTK7 gene variants in this study.The involved segments of patients with CVMs in the CS cohort were distributed in the middle and lower thoracic vertebrae and lumbar vertebrae（T7-L2）,two patients had other skeletal malformations in addition to CVMs,and one patient had congenital patent ductus arteriosa（PDA）.One patient from the AIS cohort had a congenital ventricular septal defect（VSD）besides scoliosis.Based on in vitro functional experiments,we verified that PTK7 gene frameshift mutation p.His155Pfs*16 from CS cohort significantly decreased PTK7 protein expression,leading to abnormal subcellular localization and loss-of-function of PTK7 protein in Wnt/β-catenin signaling pathway.Missense variants c.353C>T and c.2290G>A from AIS cohort partially reduced the expression of PTK7 protein,but did not affect the inhibitory effect of PTK7 protein in Wnt/β-catenin signaling pathway.ConclusionPTK7 gene is an important candidate gene for congenital scoliosis and adolescent idiopathic scoliosis.The PTK7 gene frameshift variant c.464₄65delAC（p.His155Pfs*16）may affect the role of PTK7 in Wnt/β-catenin signaling pathway by significantly reducing the expression of PTK7 mutant protein,and thus lead to CVMs.Partial decrease of PTK7 protein expression did not affect its function in Wnt/β-catenin signaling pathway.Our study expanded the pathogenicity phenotype of PTK7 gene and initially explained the pathogenicity mechanism of PTK7 gene variants in CS and AIS.