Based On Exome Sequencing Identified Pathogenic Gene NARFL For DPAVMs

Background Pulmonary arteriovenous malformations?PAVMs?are abnormal vessels diseases and are fistulous connections between pulmonary arteries and veins,which are usually diagnosis conducted by CT or enhance CT.When PAVMs are involved in every subsegmental artery within at least one lobe,diffuse PAVMs are recorded as.Hereditary hemorrhagic telangiectasia?HHT?is the most common cause of pulmonary arterovenous malformations.About 80%to 90%of PAVMs were caused by HHT.Up to now,4 genes have been identified by using the candidate gene screening,and exome sequencing methods.Nevertheless,about 10%to 20%of PAVMs were caused by NOT-HHT and they could not identify the candidate genes.Therefore,PAVMs is a disease with high genetic heterogeneity,remained many novel genes to be found in consanguineous families.The aim of the study was to identify the pathogenic gene and mutation in a Chinese consanguineous family pedigree with dPAVMs using whole exome sequencing and to investigate the underlying molecular mechanism.Methods Blood samples of all family members and 500 controls were collected and lung tissues were also collected from the proband.Genomic DNA was extracted and ENG,ACVRL1,SMAD4,and RASA1 were sequencing using Sanger sequencing.Then we used the whole exome sequencing and array-based comparative genome hybridization technology to localize the causative genes and mutations.Then,causative gene and mutation of dPAVMs was identified by bioinformatics analysis such as hereditary model,Exac database,SIFT and Polyphen-2.Subsequently,microvascular malformations were detected using CD31 and other indicators in the lung tissues of proband.The expressions of NARFL and VEGF were analyzed by immunohistochemical staining.The levels of Fe3+ were analyzed by prussian blue staining.Transfections were done using Iipo2000.Eukaryotic expression plasmid of wide type and mutant type were constructed using pCMV-HA.Recombinant wild type and S161I mutation type plasmid were transfected into HEK93T cell lines to investigate the impact on mRNA stability.shRNA were constructed using pSUPER and they were transfected into HEK293T to observed the effects on cytosolic aconitase.CRISPR/Cas9 was used to gene editing and knock-out the gene expression.The genetypes of zebrafish were maped using PAGE and Sanger sequencing.Then alkaline phosphatase staining was used to evaluate the abnormal phenotypes of blood vessels.Subepithelial veins of zebrafish were examined using confocal fluorescent microscopy.The expression levels of SOD and CAT were measured using Q-PCR.Results After excluded the mutation of known ENG,ACVRL1,SMAD4,and RASA1 gene,we identified a novel causative gene,NARFL gene with its homozygous missence mutation?hg19 NM₀22493 c.482G>T,p.Ser161Ile?.All family members carried this mutation but it was absent in proband's grandfather and 500 representative controls.This mutation was cosegregated with clinical phenotypes.Vascular malformations were significant confirmed by CD31 staining and others.The higher levels of NARFL and Fe3+ were observed in the proband,but lower in VEGF.When plasmids were transfected into HEK293T,mutant NARFL mRNAs were decreased more sharply than wide-type.NARFL knock-down lead to decreased activity of cytosolic aconitase.Using the zebrafish model,vascular malformations of subepithelial veins were observed in narfl knock-out embryos.There were lower expressions of SOD and CAT in mutant zebrafish than those in wide-type.Conclusions In a consanguineous family with diffuse pulmonary arteriovenous malformations?dPAVMs?,we identified a novel pathogenic gene,NARFL gene with its homozygous missence mutation?hg19 NM₀22493 c.482G>T,p.Ser161Ile?.MutantNARFL were decreased more sharply than wide-type.NARFL knock-down lead to decreased activity of cytosolic aconitase.narfl knock-out zebrafish embryos had abnormal aberrant ventral-vein plexus and upregulated vascular endothelial growth factor?VEGF?,and decreased levels of SOD and CAT.In conclusion,NARFL gene is associated with the occurrence of dPAVMs.