Pathogenicity And Functional Analysis Of CFAP410 Mutations Causing Cone-Rod Dystrophy

Background:Cone-rod dystrophy（CORD）caused by mutations in CFAP410 is a very rare disease.The mechanisms by which the mutations caused the disease remained largely unknown.Objective:The aim was to identify the causative gene of CORD and to investigate the pathogenesis of the mutated gene in the study of a Chinese family containing a patient with CORD.Methods:Firstly,the ocular and other related medical examinations were performed on the patients with vision decline for 1 year（proband）and other members of the family.Next,we collected whole peripheral blood of CORD family and detected pathogenic genes involved in inherited retinal diseases in the proband using next-generation sequencing（Tg-NGS）.After screening,possible pathogenic genes were validated by Sanger sequencing.Subsequently,functional changes in the identified mutations were predicted and evaluated using bioinformatics software,and mutation pathogenicity was graded according to the American College of Medical Genetics and Genomics.Finally,wild-type and mutated plasmids were constructed and transferred to HEK293T cells,measured cell cycle and apoptosis levels using flow cytometry.Flow cytometry was used to determine the cell cycle as well as the apoptosis level,and immunofluorescence technique and cycloheximide tracking assay were used to determine the expression and stability of mutant proteins,combined with proteasome inhibitor assay and immunoprecipitation technique to understand the degradation pathway of mutant proteins to verify the pathogenicity of mutant genes.Results:The proband,a six-year-old boy,had been noticed by his parents that he had decreased vision and narrowed field of vision in both eyes since a year ago.The best-corrected visual acuity was 0.20 bilaterally.Fundus showed macular staphyloma and uneven granular pigment disorder in the periphery of the retina.SS-OCT showed thinning and atrophy of the outer retina,residual ellipsoid bands in the fovea.Scotopic and photopic ERG responses severe reduced.No abnormalities in hearing.There were no obvious abnormalities in the X-ray and heart color ultrasound image.According to the history and examination results,proband was diagnosed with CORD with macular staphyloma.Two heterozygous missense mutations（c.319T>C,p.Tyr107His;c.347C>T,p.Pro116Leu）were found in exon 4 of the CFAP410.Sanger sequencing validation revealed that the father carried mutations c.319T>C,while mother and brother carried c.347C>T,consistent with lineage co-segregation.Bioinformatics analysis revealed that multiple online software predictive mutations such as SIFT and Poly Phen-2,retrieved from the Varcard database,were harmful,and predicted mutations such as GERP and phylo P were highly conserved.The HOPE website analyzes mutations that may affect the protein structure and its function.The ACMG guidelines assess that CFAP410 c.319T>C is"pathogenic"and c.347C>T is"likely pathogenic".Experiments were performed using different plasmid constructs(CFAP410^WT,CFAP410^Y107H,and CFAP410^P116L)transfected with HEK293T cells,and flow cytometric cell cycle assays revealed that the number of cells in G1 phase was significantly higher in the mutation groups than in the CFAP410 wild-type and pc DNA3.1 groups.The results of immunofluorescence and cycloheximide tracking assays showed that the two mutant plasmid-transfected groups had reduced fluorescence and protein stability.Meanwhile,the data of proteasome inhibition assay and immunoprecipitation suggested that degradation via ubiquitin-proteasome pathway was increased in the two mutant groups.In contrast,the flow apoptosis assay increased apoptosis only in cells transfected with the c.319T>C mutant plasmid,while the other mutation had no effect.Conclusions:Compound heterozygous CFAP410 mutations c.319T>C and c.347C>T in CFAP410 caused CORD with macular staphyloma.The pathogenic mechanisms may be associated with disruption of normal cell cycle processes,alternations of protein stability and degradation through the ubiquitin-proteasome pathway.