A Novel Variant In The FBP1 Gene Causes Fructose-1,6-bisphosphatase Deficiency Through NEDD4-2-mediated Ubiquitination

Objective:Fructose-1,6-diphosphatase(FBPase)deficiency is an autosomal recessive genetic disorder caused by mutations in the fructose-1,6-diphosphatase 1(FBP1)gene.Fructose-1,6-diphosphatase is a key catalytic enzyme in the gluconeogenic pathway.The defect of this enzyme leads to the failure of fructose 1,6-diphosphate to be converted into fructose 6-phosphate,which affects the conversion of gluconeogenic substrates such as pyruvate,lactic acid and glycerol to glucose,leading to glucagon-resistant hypoglycemia.Children with reduced glycogen storage and large consumption of glycogen such as hunger,infection and stress are prone to hypoglycemia,hyperlactemia and metabolic acidosis.Due to the variety of clinical manifestations and lack of specificity of symptoms,the disease is easily confused with other genetic metabolic diseases.In recent years,the in-depth development of gene detection technology makes the timely and effective diagnosis of the disease possible.We report the clinical phenotype of recurrent hypoglycemia and seizures in a Chinese boy with FBPase deficiency.We identified a complex heterozygous mutation in the FBP1 gene c.761A>G(H254R)and c.962C>T(S321F).The molecular mechanism of enhanced ubiquitination and proteasome degradation of mutant FBP1 mediated by NEDD4-2 was elucidated.Methods:1.Nedd4-2 knockout mice have been constructed in the previous experiment of our research group.2.Urine samples of children at the acute stage were collected,and glycerol,lactic acid and glycerol triphosphate were detected by GC/MS.The medical history of the children was reviewed,and the brain MRI results and the values of sodium bicarbonate,lactic acid and glucose at multiple acute episodes were collected.3.Verify mutation sites by whole exon sequencing and sanger sequencing for children and parents to screen potential pathogenic mutations;SIFT,Mutation Taster and polyphen2 were used to score the harmfulness of the mutant sites.4.Mega version 7 software was used to analyze the species conservation of mutant sites in children.5.The FBP1 gene mutant spectrum was analyzed and summarized by literatures,ClinVar and HGMD databases.6.Western blot was used to detect FBP1 protein levels in HepG2 and U251 cells transfected with wild type,S321F,H254R or blank control.7.To detect if the mutant FBPase activity is reduced,NADP-coupled spectrophotometry was used to detect FBP1 mutant transfected cell lines and peripheral blood leukocytes of patients and his parents.8.In order to detect the instability and degradation of mutant FBP1,wild type,S321F and H254R FBP1 were transfected into HepG2 and U251 cells and treated with actinomycin(100μg/mL)for 0,2 and 4 h.Western blot was used to detect the expression of FBP1.9.In order to detect the increased ubiquitination level of mutant FBP1,wild type,S321F and H254R mutant FBP1 were transfected into HepG2 and U251 cells,and Co-IP was used to detect the binding of FBP1 to Ub in cell lines.10.In order to confirm the mutant FBP1 degradation pathway,HepG2 cells and U251 cells were treated with proteasome inhibitor MG132 for 24 h,and the expression changes of wild type,S321F and H254R mutant FBP1 were detected by Western blot.11.The ubiquitin E3 ligase of FBP1 was predicted by UbiBrowser.12.Protein-protein docking,NEDD4-2 was interacted with FBP1 by using bioinformatics analysis software,and the docking results were displayed in 3D with PyMol.13.In order to confirm the negative regulatory effect of Nedd4-2 on Fbp1 in vivo and in vitro,Western blot was used to detect Nedd4-2 and Fbp1 protein levels in Nedd4-2+/-mice and cultured cells.14.To confirm Fbp1 as a new ubiquitination substrate for Nedd4-2 in vivo and in vitro,Co-IP detected the binding of Nedd4-2 to Fbp1 in Nedd4-2+/-mouse models and cultured cells.Results:1.GC/MS analysis of organic acid metabolites in the urine of children in the acute stage showed that the contents of glycerol,lactic acid and ketones were increased;A retrospective analysis of glucose,sodium bicarbonate and lactic acid in the acute stage of previous episodes found that the levels of glucose and sodium bicarbonate were reduced,while the levels of lactic acid were increased;Cerebral MRI showed lateral ventricle dilation,corpus callosum thinning and frontal lobe atrophy;Abdominal CT showed no abnormalities in liver or other organs.2.Exon sequencing found two heterozygous variants in FBP1 gene.c.962C>T(p.S321F)can be found in NCBI dbSNP database(rs747191202)and ExAc database,with a low allele frequency of 0.000008.c.761A>G(p.H254R)is new and has not been reported in dbSNP,1000 Genomes Project,or Exome Variant Server databases.Generation sequencing revealed that the mutation was a complex heterozygote,inherited from one of his parents.SIFT,Mutation Taster and polyphen2 were used to predict the harmfulness of the two variants,and the results showed that both variants were harmful(p.H254R:SIFT score 0.001,Mutation Taster score 1,polyphen2 score 1;P.s.321f:SIFT score 0,Mutation Taster score 1,polyphen2 score 1).3.Amino acid conserved analysis revealed that the two mutation sites of the patient were highly conserved among the 11 species.4.Through literature review and analysis of HGMD and ClinVar databases,58 FBP1 gene mutations were found(excluding 8 involving large fragment deletion of FBP1 gene),and more than 20 cases of fructose-1,6-diphosphatase deficiency caused by FBP1 gene mutation were reported in Chinese population.5.Western blot results showed that the S321F mutant was significantly lower than the wild-type FBP1 protein.The H254R mutant FBP1 was almost undetectable,and the expression of the double mutant was also decreased.6.Compared with the wild type,the FBPase activity of the mutant was significantly decreased by NADP-coupled spectrophotometry,in the order of S321F>S321F+H254R>H254R,which was parallel to the protein expression level;NADPcoupled spectrophotometry confirmed a significant reduction in maternal(H254R)and paternal(S321F)compared with 20 healthy controls,consistent with the results of cell line tests.7.Western blot showed that the FBP1 level of the H254R mutant decreased sharply and that of the S321F mutant was similar to that of the wild type after 2 and 4 h CHX treatment.8.According to the results of Co-IP,H254R mutant remarkable enhance the ubiquitination of FBP1,S321F mutant was no significant difference compared with wild type.9.Western blot showed that the protein level of H254R mutant FBP1 was significantly recovered after 24 h treatment with MG132 proteasome inhibitor.10.The ubiquitin E3 ligase of FBP1 with high confidence in NEDD4-2 was predicted by UbiBrowser.11.The S321F mutation was located outside the interaction junction surface,while the H254R mutation was located at the junction surface.The H254R mutant FBP1 reduced the average distance to the NEDD4-2 molecule E57 and added a new hydrogen bond.12.Western blot analysis of liver and brain tissue of Nedd4-2+/-mice showed that Nedd4-2 expression was decreased,and the expression of Fbp1 was upregulated,which was about twice that of wild-type control.HepG2 and U251 cells were subjected to siRNA knockdown of NEDD4-2 and transfection plasmid overexpression of NEDD4-2,respectively.WB detection showed that FBP1 expression was increased in cells with low knockdown of NEDD4-2 and decreased in cells with overexpression of NEDD4-2.13.CoIP experiments confirmed that Nedd4-2 interacts with Fbp1 in the liver and brain of KO and WT mice.In addition,the binding of fbp1 to Nedd4-2 was significantly decreased in KO group compared with WT group;NEDD4-2 was co-transfected with wild type,S321F and H254R mutant FBP1 in HepG2 and U251 cells.Co-IP detection showed that the signal of the binding zone of NEDD4-2 and H254R was enhanced during NEDD4-2 immunoprecipitation and FBP1 immunoblotting.During the immunoprecipitation of FBP1 and western blotting of NEDD4-2,the binding signal of H254R to NEDD4-2 may be weakened due to protein instability.Conclusions:1.In this study,starting from clinical cases,c.761A>G(p.H254R)of FBP1 gene was identified for the first time as a new pathogenic mutation in fructose-1,6-diphosphatase deficiency.2.Ubiquitination and proteome degradation of FBP1 H254R mutant increased.FBP1 gene variation was detected to reduce FBP1 protein expression and enzyme activity by peripheral white blood cells and cell lines cultured in vitro.3.FBP1 was demonstrated to be a new ubiquitination substrate for Nedd4-2 by cell lines and NEDD4-2 knockout mouse model in vitro,and the H254R mutant FBP1 was identified as an increased instability,resulting in ubiquitination modification by binding to NEDD4-2,and degradation by the proteasome pathway.