Pathogenicity Of Novel REEP6 Variants Associated With Retinitis Pigmentosa Type 77

BackgroundRetinitis pigmentosa type 77 is an autosomal recessive inherited retinal degeneration,caused by mutations of Receptor expression-enhancing protein 6（REEP6[MIM:609346]）.REEP6 encodes a protein for the development of photoreceptors,involved in intracellular trafficking by controlling cargo capacity at the endoplasmic reticulum.PurposeTen causative variants in REEP6 associated with retinitis pigmentosa have been reported,and only one of them was from China.Our study was to identify the causative gene variants leading to retinitis pigmentosa in three Chinese families and study the pathogenicity by means of molecular biology.Method:Clinical data collection:Comprehensive ophthalmic examinations were performed on three probands from three independent families.Targeted next-generation sequencing:DNA was extracted from venous blood for genotyping.Genomic DNA of the three probands were subjected to NGS based on a custom-designed panel（PS400）,containing 376 genes associated with posterior segment eye diseases.Raw reads were mapped to the human genome reference（UCSC hg19）via XYGeneRanger 2.0,TGex,and Efficient Genosome Intepration System（EGIS）.Sanger sequencing was performed to confirm the variants,and family segregation was performed in family members when available.Pathogenicity analysis:Allele frequencies of the variants in different populations were determined using Exome Aggregation Consortium（ExAC）,1000 Genomes Project（1000G）,Genome Aggregation Database（gnomAD）,and dbSNP.Functional consequences through different in silico algorithms were available via VarCards including CADD（model:GRCh38-v1.6）,Mutation Taster,SIFT,Poly Phen2,GERP++,phyloP,phastCons,et cetera.Splice site changing was predicted by Net Gene2 and NNSplice.Nucleotide conservation was predicted by GERP,phyloP,Phast Cons,and SiPhy via VarCards.Amino acid sequences between species were con-trasted by ClustalOmega.The protein structure was predicted by I-TASSER online 3D structure prediction server.Domains were predicted by CDD online software.Project HOPE and ProtParam were used to predict the structural changes by the variants.Stability of proteins encoded by the variants was predicted by MUpro and I-Mutant v2.0.Pathogenicity of the variants was assessed according to the standards and guidelines published by the American College of Medical Genetics and Genomics（ACMG）.In vitro experiment:Wild type REEP6 gene was synthesized,mutant REEP6 gene was constructed.REEP6 gene was linked to plasmid pcDNA3.1-HA.HEK293T cells were transfected with pcDNA3.1,pcDNA3.1-REEP6^WT,pcDNA3.1-REEP6^268G>C and pcDNA3.1-REEP6^468delC,respectively.The mRNA expression level of REEP6 in each group was detected by qRT-PCR.Western Bolt was performed to detect the protein stability.Immunofluorescence was performed to determine the localization and difference at the protein expression level between the wild type group and mutant groups.Result:All three probands were diagnosed as retinitis pigmentosa.They all presented with night blindness and tunnel vision.Fundus showed typical retinitis pigmentosa alters.Electroretinogram（ERG）response of proband A:II-3 were undetectable,while that of proband B:II-4 were significantly reduced.By targeted next-generation sequencing,three patients were found to carry a homozygous variant and two heterozygous variants of REEP6,respectively,which were all unreported variants,including a missense variant c.268G>C,a frameshift deletion variant c.468delC,and a splicing variant c.598+1G>C.The allele frequency of REEP6 c.268G>C was 0.0012 in all populations in gnomAD database and 0.0111 in East Asian population.REEP6 c.468delC and c.598+1G>C are not found in gnomAD database.Bioinformatics analysis showed that these variants were deleterious and conserved in nucleotide and amino acid sequences.All three variants are defined as likely pathogenic variants according to the ACMG guidelines.Experimental data indicated that mRNA expression levels of REEP6 c.268G>C and c.468delC were significantly increased compared to the wild type.REEP6 p.Vau90Leu and p.Asn156Lysfs*14 resulted in decreased protein stability and shortened half-life.Immunofluorescence showed that REEP6 p.Vau90Leu and p.Asn156Lysfs*14 formed inclusions in HEK293T cells.Conclusion:We identified three novel REEP6 variants in three Chinese RP families,including one missense variant c.268G>C,one frameshift variant c.468delC,and one splicing variant c.598+1G>C.According to the ACMG guidelines,the three variants are defined as likely pathogenic variants.REEP6 c.268G>C and c.468delC destabilized the REEP6 protein.Immunofluorescence showed that REEP6 c.268G>C and c.468delC formed inclusion bodies in HEK293T cells.