Characterization based on genotype–biochemical phenotype association in fructose-1,6-bisphosphatase deficiency

Characterization based on genotype–biochemical phenotype association in fructose-1,6-bisphosphatase deficiency | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Characterization based on genotype–biochemical phenotype association in fructose-1,6-bisphosphatase deficiency Tomoaki Tanaka, Ikki Sakuma, Hidekazu Nagano, Naoko Hashimoto, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2185039/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jul, 2023 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Purpose Fructose-1,6-bisphosphatase (FBPase) deficiency, caused by an FBP1 mutation, is an autosomal recessive disorder characterized by hypoglycemic lactic acidosis. The mechanism by which the mutations cause enzyme activity loss is uncertain. Methods We performed whole-exome sequencing in an adult patient with severe hypoglycemic lactic acidosis and identified that the patient carried compound heterozygous missense mutations of FBP1 with c.491G > A (p.G164D) and c.581T > C (p.F194S). Results Biochemical analysis revealed that FBP1 mutant (G164D or F194S) decreased protein expression and enzyme activity loss. The interactome analysis for binding partners demonstrated that G164D and F194S mutants interact with the proteins involved in unfolded protein response. Additionally, G164D and F194S mutants aggregated in the endoplasmic reticulum, suggesting the involvement of protein misfolding in its pathogenesis. All FBP1 missense mutations previously reported were classified into three functional categories: Type 1 mutations, located at pivotal residues in enzyme activity motifs with no effects on protein expression; Type 2 mutations, which mediate changes in amino acid hydrophobicity and structurally cluster around the substrate-binding pocket, are associated with aggregation in the endoplasmic reticulum, and decreased protein expression; and Type 3 mutations, which are likely non-pathogenic mutations. Conclusion Protein misfolding contributes to FBPase deficiency pathogenesis, particularly in Type 2 mutations. Biological sciences/Genetics/Mutation Biological sciences/Cell biology/Protein folding/Chaperones fructose-1 6-bisphosphatase deficiency FBP1 hypoglycemia protein misfolding heat shock protein Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Fructose-1,6-bisphosphatase (FBPase) is a key regulatory enzyme in gluconeogenesis that catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate 1 . Baker and Winegrad first reported FBPase deficiency in 1970 2 , and Kikawa et al. subsequently identified three FBP gene ( FBP1 ) mutations associated with FBPase deficiency in 1997 3 . FBPase deficiency is an autosomal recessive disorder that generally courses with hypoglycemia and metabolic acidosis 4 . Although fasting and febrile infections are known to trigger life-threatening episodes of hypoglycemia and lactic acidosis in infancy, these episodes rarely occur in adults due to increased glycogen storage 4 . Several harmful mutations in the coding region of FBP1 have been reported 5 . Nevertheless, with incidence rates estimated to range between 1/350,000 and 1/900,000, FBPase deficiency remains a very rare inherited disease 5 , 6. Due to its low incidence, elucidating the molecular mechanism by which the identified mutations cause the loss of enzyme activity is still challenging, particularly in terms of genotype phenotype correlation and adult-onset cases. Here, we present the case of a 22-year-old female patient who was diagnosed with FBPase deficiency and exhibited a novel compound heterozygous mutation of the FBP1 gene. Our results demonstrate the underlying mechanism by which the identified mutations caused FBPase deficiency involves protein misfolding, and we examine and categorize the biochemical phenotypes of all previously reported FBP1 missense mutations into three functional groups. Material And Methods Mutation analysis DNeasy Blood and Tissue Kits (QIAGEN, Hilden, Germany) were used to extract the genomic DNA from the patient’s blood. Then, all 7 exons of the FBP1 gene were amplified by polymerase chain reaction (PCR) and sequenced using a 3130 Genetic Analyzer (Applied Biosystems, Massachusetts, USA). Table S1 (Additional file 1) describes the primer information and PCR conditions. Whole-exome Sequencing The whole-exome sequencing involved the targeted capture of all the exon sequences using SureSelect Human All Exon v6 (Agilent Technologies, California, USA), followed by massive parallel sequencing of the enriched exon fragments on the HiSeq 2500 platform (Illumina) using the 125-bp paired-end mode as per manufacturer’s protocol 7 . The sequenced reads were aligned to the human genome reference (GRCh37) using the default parameter settings in Burrows-Wheeler Aligner version 0.7.10, while the PCR duplicates were eliminated using Picard-tools version 1.39 ( http://picard.sourceforge.net/ ). Candidate mutations with (i) depths ≥ 8; (ii) Number of variant reads ≥ 4; and (iii) variant allele frequency (VAF) 0.4–0.6, 1 were adopted, as VAFs of pathogenic germline mutations are estimated to be approximately 0.5 in heterozygous state and 1 in homozygous state. Mutations were further filtered by excluding (i) variants presenting only in unidirectional reads; (ii) insertions and deletions in simple repeat regions; (iii) synonymous SNVs; and (iv) known variants listed in the 1000 Genomes Project (Nov 2010 release), Exome Sequencing Project (ESP) 6500, Human Genome Variation Database (HGVD; October 2013 release), and ExAC database with frequencies > 0.001 in order to exclude non-pathogenic variants. Construction Of Fbp1 Expression Vectors The total RNA was extracted from the patient’s blood using RNeasy Kits (QIAGEN, Hilden, Germany) and reverse-transcribed using SuperScript 2 reverse transcriptase (Thermo Scientific, Massachusetts, USA) and oligo (dT) primers. The amplification of the coding region of the FBP1 gene was carried out using the forward primer 5’-CACCATGGCTGACCAGGCGCCCTTCG-3’ and the reverse primer 5’-TCACTGGGCAGAGTGCTTCTCATAC-3’. The PCR procedure consisted of the following steps: a) denaturing at 98°C for 2 min, followed by 94°C for 15 secs, and b) annealing for 30 secs at 58°C and extension at 72°C for 1 min for 30 cycles. The PCR products were subcloned into pGEM-T Easy Vector (Promega, Wisconsin, USA). Then, the FBP1 fragments (WT and 2 mutants) were cut from the pGEM-T Easy Vector using EcoRI and subcloned into a pCMV-Myc-N Vector (Clontech, California, USA). A corresponding pCMV-Myc-N Vector was used as a negative control. Mutagenesis Of Fbp1 Plasmids D119N, P120L, R158W, G164S, A177D, G207R, N213K, G260R, E281K, P284R, G294E, G294V, and V325A mutations were introduced by site-directed mutagenesis (Quick Change Lightning Site-Directed Mutagenesis KIT, Agilent Technologies) using the primers listed in Table S2 (Additional file 1). XL10-Gold ultracompetent cell DNA was isolated from cultured single clones and by sequencing to confirm successful mutagenesis. Generation Of Fbp1 Knockout Hepg2 Cells The protocol used for the CRISPR/Cas9 system was consistently based on that reported by Cong et al., Science 2013 8 . The backbone vectors pX459 pSpCas9(BB)-2A-Puro and pX462 pSpCas9n(BB)-2A-Puro were obtained from Addgene (Massachusetts, USA). The target guide RNA sequences were designed at exon 5 including c.491G, exon 6 including c.581T, and exon 8 including the Japanese common mutation site (c.960-961insG) of the FBP1 genome (5’-GCAGCCGGCTACGCACTGTA-3’, 5’-GCACCAAAATGAACTCCCCGA-3’ and 5’-GTCGGGGGATCCCAAGATCAC-3’) 3 . To clone the exon 5 and exon 6 target sequences into pX462 and the exon 8 target sequence into the pX459 backbone, the oligos were synthesized using Eurofins genomics (Tokyo, Japan) (Additional file 1: Table S3). Then, these oligos were submitted to annealing and phosphorylation by means of a T4 DNA Ligase Reaction Buffer and a T4 Polynucleotide Kinase (New England Biolabs, Massachusetts, USA) used at 37℃ for 30 min and 95℃ for 5 min. pX459 and pX462 were digested using BbsI (Thermo Scientific, Massachusetts, USA) at 37℃ for 30 min, with gel purification being performed by use of a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). Ligation reactions of pX459, pX462 and the annealed oligos were performed for 10 min at room temperature using a Quick Ligation Kit (New England Biolabs, Massachusetts, USA). Then, the ligated oligos were purified using PlasmidSafe exonuclease (Cambio, Cambridge, UK) at 37℃ for 30 min. The plasmids were transfected into Stbl3, and the appropriate transfectants were amplified and collected using NucleoBond Xtra Midi (Takara, Kusatsu, Japan). Three plasmids (pX462-exon5 gRNA, pX462-exon6 gRNA and pX459-exon8 gRNA) were cotransfected into HepG2 cells using lipofectamine 3000 (Thermo Scientific, Massachusetts, USA) as per manufacturer’s protocol. The plasmid-expressing HepG2 cells were selected by puromycin (Wako, Osaka, Japan), and the limiting dilution method was used to establish the monoclonal cell line. Cell Culture And Transient Transfection The human hepatocarcinoma cell line FBP1 -KO HepG2 culture was conducted using Dulbecco’s modified Eagle’s medium containing antibiotics and 10% fetal bovine serum. The FPB1-KO HepG2 cells were plated on 6-well plates and then transfected with plasmid DNA (2.5 µg) complexed with Lipofectamine 3000 reagent (7.5 µl) and P3000 reagent (5 µl) in 250 µl of Opti-MEM (Thermo Scientific, Massachusetts, USA). Subsequently, the FBPase activity of HepG2 cells was measured 48 hours after transfection using a nicotinamide adenine dinucleotide phosphate (NADP)-coupled spectrophotometric assay. Fbpase Activity Assay The FBPase activity was calculated from an NADP-coupled spectrophotometric assay as described by Kikawa et al 9 . The assay mixture (300 µl) was composed of 40 µg protein of cell lysate, 50 mmol/L Tris-HCl buffer (pH 7.5), 2.0 mmol/L MgCl 2 , 1.0 mmol/L EDTA, 0.2 mmol/L NADP, 3.5 U/mL glucose-6-phosphate dehydrogenase, 1.5 U/mL glucose-6-phosphate isomerase, and 100 µmol/L FBP, which served as the substrate. A 96-well plate reader was used to record the rate of NADPH formation. Real-time Rt-pcr Analysis Real-time RT-PCR analysis RT-qPCR experiments were performed as previously described 10 – 12 . The FBP1 gene-specific mRNA expression values were determined and normalized to those of β-actin as an internal control. Briefly, the total RNA (4 µg) was extracted using an RNeasy kit (Qiagen, Valencia, CA, USA) and reverse-transcribed using a ReverTra Ace qPCR RT Kit (Toyobo, Tokyo, Japan). The cDNA products were subjected to RT-PCR using a Step One Plus Real-Time PCR system (Applied Biosystems, Massachusetts, USA). All the primer information is provided in Table S4 (Additional file 1). Immunoblot Analysis Immunoblot analyses were performed as previously described 13 . The antibodies included FBP1 (SIGMA rabbit polyclonal, clone: HPA005857), c-Myc (Santa Cruz mouse monoclonal, clone: 9E10), actin (SIGMA rabbit polyclonal, clone: A2066), ATF6 (Novus Biologicals mouse monoclonal, clone: 70B1413.1), HSP70 (StressMarq mouse monoclonal, clone: N27F3-4), and HSP90 (Santa Cruz mouse monoclonal, clone: sc-13119), HSP60 (Abcam rabbit polyclonal, clone: ab46798) and TCP1 (Bethyl laboratories rabbit polyclonal, clone: A303-444A). Er Fractionation The extraction of the ER was performed using an Endoplasmic Reticulum Enrichment Kit (Novus Biologicals, Colorado, USA), as per manufacturer’s instructions. Immunofluorescence Analysis Immunofluorescence analysis was performed to examine the cellular expression of the WT and mutant FBP1. The FBP1 -KO HepG2 cells were cultured in 4-well chamber slides (2 × 10 4 cells/well) and then transfected with plasmids (0.5 µg). Twenty-four hours after the transfection, the cells were treated with CellLight® ER-GFP (12 µl/well; Thermo Scientific, Massachusetts, USA), which is a marker of ER, followed by fixation in 100% ethanol at − 20℃ for 10 min and incubation with blocking solution and primary antibodies against FBP1 (SIGMA rabbit polyclonal, clone: HPA005857), c-Myc (Santa Cruz mouse monoclonal, clone: 9E10) and GFP (MBL rabbit polyclonal, clone: 598) 48 hours after the transfection. A confocal laser microscope (LSM710, Carl Zeiss, Germany) was used to obtain fluorescence images. Immunoprecipitation Assay The commercially available kit c-Myc-tagged Protein Mild Purification Kit (Medical & Biological Laboratories, Aichi, Japan) was used to perform the immunoprecipitation assay. HepG2 cell extracts containing different Myc-tagged FBP1 variants were incubated with anti-Myc beads at 4℃ for 1 hour. The beads were then rinsed and eluted using a wash solution and elution peptides. Mass Spectrometry Sample Preparation The FPB1-KO HepG2 cells were transfected with Myc-tagged FBP1-WT, Myc-tagged FBP1-G164D, Myc-tagged FBP1-F194S containing plasmid DNA. These cells were treated with the mannosidase inhibitor kifunensine (200 µM) for 48 hours. Myc-tagged FBP1 HepG2 cells solubilization was obtained with the following buffer: 50 mM Tris-HCl (pH 7.5), 1.0 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM phenylmethannesulphonyl fluoride, 2.5 µg/mL leupeptin and 1.0 µg/mL antipain. Immunoprecipitation with anti-Myc beads (Medical & Biological Laboratories, Aichi, Japan) was performed at 4℃ for 1 hour. The beads were then rinsed and eluted using a wash solution and elution peptides. Immune complexes were separated by SDS-PAGE. Bands were exsected from the gel and examined by mass spectrometry to find corresponding proteins. Respective gel pieces were rinsed two times using 100 mM bicarbonate in acetonitrile with subsequent protein digestion by trypsin. 0.1% formic acid was then added to the supernatant, and the peptides were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a LTQ Mass Spectrometer (Thermo Scientific). Analysis of MS/MS data set results was conducted using the Mascot software program (Matrix Science). Proteomic Analysis And Database Search Proteomic analysis and database search were performed as previously described 14 . Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation and Gene Ontology (GO) were performed using the STRING interaction database. Protein Structure Based On Protein Data Bank Ke et al. revealed the detailed crystal structure of Sus scrofa FBP1 complexed with fructose 6-phosphate, AMP, and magnesium 15 ; Fig. 1 g shows the structure of FBP1 dimer based on their report (DOI: 10.2210/pdb1FBP/pdb ). The FBP1 amino acid sequences between Homo sapiens and Sus scrofa show approximately 90% similarity. Particularly, the functional motifs directly associated with FBPase activity are highly conserved. Statistics The results are presented as mean ± SD. Student’s t -test was used to analyze the continuous variables, and P-values < 0.05 were considered statistically significant. Study Approval In accordance with the protocols issued by the Chiba University Hospital, written informed consent for the genetic studies was obtained from the patient and her family. Results Case presentation and whole-exome sequencing We accompanied the case of a 22-year-old Japanese female patient who presented with a severe hypoglycemic attack and acidosis induced by prolonged fasting. Blood exams, including a blood gas analysis, showed very low levels of plasma glucose (12 mg/dL) with high levels of lactate (110 mg/dL) and severe metabolic acidosis (pH 6.85, PaCO 2 19 mm Hg, HCO 3 − 2.5 mmol/L). The patient reported experiencing similar episodes triggered by fasting or febrile illness during her early childhood (Additional file 1: Table S5). We suspected the case to be an inherited metabolic issue, such as fatty acid oxidation or gluconeogenesis disorders causing hypoglycemic attacks with lactic acidosis, and further examinations were performed in order to establish a definitive diagnosis. Radiological examination of the abdomen by computerized tomography (CT) revealed a fatty liver, and the liver biopsy showed hepatic steatosis without other alterations (Additional file 3: Figure S1b). The acylcarnitine profile exhibited no specific pattern, suggesting a normal β-oxidation pathway. However, the urinary organic acid profile exhibited significantly elevated glycerol, lactate, and pyruvate levels, indicating a possible FBPase deficiency. The oral fructose tolerance test demonstrated a rapid decrease in blood glucose levels and an increase in lactate concentrations (Fig. 1 a). Additionally, fructose loading increased glycerol levels in the urinary organic acid profile (Fig. 1 b). Although clinical findings up to this point strongly indicated a case of FBPase deficiency, a differential diagnosis from other metabolic disorders that course with hypoglycemic attacks and lactic acidosis was difficult due to overlapping clinical features 6 . As previous studies have shown that genetic analyses using next-generation sequencing is useful for the molecular diagnosis of complex metabolic diseases including FBPase deficiency 16 , we performed the whole-exome sequencing in samples from the proband and her family in order to identify potential underlying genetic defects. This approach revealed the 111 filtered variants in the proband (Additional file 2: Table S6). Two heterozygous variants were identified in a region of FBP1 among them, whereas there was no variants associated to mitochondrial fatty acid oxidation disorders and gluconeogenesis such as OCTN2, CACT, CPT1, CPT2, LCHAD, MCAD, SCAD, MTP, VLCAD, ACAD9, ETFDH, ETFA, ETFB, HMGCS2, HMGCL, PC, PCK1, PCK2, G6PC and PGM1 . The identified variants in FBP1 were missense mutations (II-1 in Fig. 1 c and 1 e) with c.491G > A p.G164D, which is a novel mutation, and c.581T > C p.F194S, which has been previously reported 17 . According to the exome sequencing results of family members, her parents carried single mutation respectively: G164D mutation from father (I-1 in Fig. 1 c and 1 e) and F194S mutation from her mother (I-2 in Fig. 1 c and 1 e). Then we confirmed that both of FBP1 variants with G164D and F194S in the proband were heterozygous missense mutations (Fig. 1 d) using Sanger sequencing, suggesting that it could be in a compound heterozygous state. Based on these findings, we made a definitive diagnosis of FBPase deficiency and advised the patient to avoid both prolonged fasting and intake of fructose-rich foods under a fasted state. The G164 and F194 are located in the β-strand structure (Fig. 1 g, 3 a), and both of identified mutations (G164D and F194S) could lead conformational changes due to the substitution of hydrophobic with hydrophilic amino acids (Table 1). In general, FBPase catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate in the presence of divalent cations, such as magnesium, manganese, or zinc 18 , whereas AMP acts as an allosteric inhibitor. The human liver FBPase comprises four identical polypeptide chains each containing 338 amino acid residues, which are assembled as relatively flat tetramers with subunits conventionally labeled C1–C4 18 . These homotetramers consist of two intimate dimers. Figure 1 g shows the structure of FBP1 dimer. We found that neither G164D nor F194S corresponded to the pivotal amino acid residues within functional motifs such as AMP binding sites, metal binding sites, or substrate binding sites (Fig. 1 g, 3 a). Thus, we decided to examine if and how these mutations affect FBPase enzymatic activity. Protein expression and enzymatic activity of the FBP1 mutations were lower than those in the wild-type We established FBP1 mutants in hepatocytes to analyze molecular function. In order to eliminate the influence of endogenous FBPase, we generated FBP1 -KO HepG2 cells using the CRISPR/Cas9 system (Additional file 3: Figure S1a). Next, Myc-tagged FBP1 cDNA constructs of wild-type (WT), mutant clones (G164D or F194S), or cotransfects (G164D and F194S) were transduced in the FBP1 -KO HepG2 cells. NADP-coupled spectrophotometric assays were used to examine the enzymatic activity of mutant FBPase. As a result, the FBPase activities of all of mutants (G164D; 0.73 ± 0.34, F194S; 0.80 ± 0.56, and cotransfected clone; 0.40 ± 0.41 mmol/min/mg protein) were significantly lower than WT clone (4.82 ± 1.61 mmol/min/mg protein) (Fig. 1 f). We validated the transcription level of FBP1 was not greater in WT clone, but immunoblot analysis revealed that the protein levels of FBP1 mutants were markedly reduced (Fig. 2 a). To evaluate the enzymatic activity in FBP1 protein, we additionally performed an in vitro NADP-coupled spectrophotometric assay with precipitated FBP1 protein by Myc-tag. The result represented that enzyme activity has completely diminished in FBP1 protein with both G164D and F194S (Additional file 3: Figure S2a and S2b). Subsequently, immunofluorescence analysis was used to evaluate the intracellular localization of the FBP1 gene product. We found that the mutant FBP1 with G164D or F194S aggregated in the cytoplasm, whereas FBP1 protein in WT clone was diffusely localized in the cytoplasm (Fig. 2 b). Importantly, this aggregated distribution of FBP1 protein was also found in the liver biopsy from the presented patient (Fig. 2 c), therefore we concluded that FBP1 mutations with G164D and F194S cause protein aggregation and the pathogenic loss in enzymatic activity. FBP1 mutations with G164D and F194S aggregated in the endoplasmic reticulum due to protein misfolding To address underlying mechanisms of aggregation and reduction in protein expression, we surveyed the interaction partners of FBP1 using Liquid chromatography-tandem MS (LC-MS/MS) analysis for the proteins pulled down by immunoprecipitation with anti-Myc tag. In total, 408 proteins were detected with LC-MS/MS analysis for WT, G164D mutant and F194S mutant (Fig. 2 d). Next, we evaluated the molecular functionalities of those proteins interacting with FBP1 and its G164D or F194S mutated variants, including common binding partner among WT and mutants, by Ingenuity Pathway Analysis (IPA) (Fig. 2 e). The analysis indicated that FBP1-interacting proteins can be mainly involved in protein production and degradation such as translation, post-transcriptional regulation, ubiquitination pathway and unfolded protein response. To test whether FBP1, especially its mutants, has such a connection to protein production and degradation as predicted, we additionally investigated the FBP1-interactome based on the STRING interaction database 19 . Notably, the interactome analysis revealed that the pathways and gene ontology terms connected to unfolded protein binding, heat shock protein binding, protein processing in endoplasmic reticulum and proteasome were highly enriched (Fig. 2 f). It is important that the FBP1 mutants (G164D and F194S) more strongly interacted with almost all these proteins compared to FBP1 WT (Additional file 1: Table S7), suggesting that these molecular pathways are involved in the pathogenesis of FBPase deficiency. Inherited mutations can break native protein folding, resulting in the formation of misfolded proteins that are consequently retained in the endoplasmic reticulum (ER). These unfolded proteins undergo mannose trimming by ER associated mannosidase and degradation by proteasome (endoplasmic reticulum-associated degradation; ERAD), which can be suppressed via the inhibition of ER associated mannosidase activity using kifunensine 20 . According to previous studies, protein expression of FBP1 is regulated by the ubiquitin proteasome and autophagy pathways 21 . Thus, to address the involvement of ERAD or protein degradation pathways in FBP1 mutant protein expression, we examined the effects of various inhibitors, such as a proteasome inhibitor (MG-132), an autophagy inhibitor (3-methyladenine), and a potent inhibitor of the ER mannosidase I (kifunensine), on the protein expression of the FBP1 mutants with G164D and F194S. Immunoblot analysis revealed that MG-132 and 3-methyladenine had no impact on the protein expression of these mutants (Additional file 3: Figure S2c). By contrast, kifunensine upregulated the protein expression of these FBP1 mutants with G164D and F194S, suggesting that these mutants underwent ERAD (Fig. 2 i). For further confirmation this notion, we performed confocal microscopy analysis to determine the intracellular localization of the FBP1 mutants with G164D and F194S, particularly in relation to ER markers. These mutants were primarily, but not completely, found to be colocalized in the ER (Fig. 2 g). In addition, immunoblot analyses using the ER fraction revealed that the protein expression of FBP1 mutants with G164D and F194S compared with WT was markedly high in the ER compared to that in the whole cell lysate (Fig. 2 h). The heat-shock proteins (HSPs) are a family of molecular chaperones, which collectively form a network that is critical for protein folding 22 . Moreover, missense mutations were shown to shift the folding equilibrium toward a partially folded state, thus increasing the cellular fraction of HSPs relative to the WT protein 23 . Based on these findings, we evaluated the binding of HSP70, HSP90, HSP60 and TCP1 to the FBP1 proteins using anti-Myc immunoprecipitation with subsequent immunoblot analysis (Fig. 2 j). The FBP1 mutants with G164D and F194S had significantly increased interactions with HSP70, HSP90, HSP60 and TCP1, to a greater extent than the WT (Fig. 2 k), indicating that these mutants affect the folding equilibrium. Taken together, the FBP1 variants with G164D and F194S mutations caused protein misfolding, which led to the reduction of protein expression and aggregation via the ERAD system. Examination of mutation genotype and functional phenotype in all FBP1 missense mutants To date, various FBP1 mutations (14 missense mutations, 12 deletion mutations, four nonsense mutations, four insertions/duplications, two splices, and one indel) have been reported 5 . The reported missense mutations are D119N 16 , P120L 5 , R158W 6 , G164S 3 , A177D 3 , F194S 17 , G207R 5 , N213K 24 , G260R 25 , E281K 26 , P284R 17 , G294E 27 , G294V 24 , and V325A 3 . We examined the relationship between the mutated site and the substitution of hydrophobicity in these 14 types of FBP1 missense mutations and G164D mutation we newly identified in this patient (Fig. 3 a). Eight types of mutation (G164D, G164S, A177D, F194S, G207R, G260R, P284R, and G294E) exhibited substitutions of hydrophobic amino acids with hydrophilic amino acids, and one mutation (R158W) exhibited a substitution of a hydrophilic amino acid with a hydrophobic amino acid. These nine mutations, defined as mutations with changing hydrophobicity, were not located at key amino acid residues (Fig. 3 a, indicated by arrowheads) within functional motifs of the substrate binding site, metal binding site, or AMP binding site. Six mutations (D119N, P120L, N213K, E281K, G294V, and V325A) did not generate any change to amino acid hydrophobicity, and four of these mutations (D119N, P120L, N213K, and E281K) were directly located at important amino acid residues of the enzyme activity [D119N and E281K were located at key residues in the metal binding site; P120L was located in the linker lesion between the metal binding site (D119 and L121) and the substrate binding site (D122); and N213K was located in the substrate binding region (N213-Y216)] (Fig. 3 a, indicated by arrowheads). Based on this information, we assessed how that the alteration of hydrophobicity affects the enzyme activitiy and the protein aggregation. We generated the mutant HepG2 cell line on FBP1-KO background as used previously. Then, these clones were separated into two groups: 6 mutants without change in hydrophobicity (indicated in blue) and 9 mutants with change in hydrophobicity (indicated in red) (Fig. 3 ). We firstly validated the similar gene transduction in each clone by qPCR (Additional file 3: Figure S3). Although two of mutants, G207R and V325A, kept the enzyme activity compared to WT clone, most of clones demonstrated a loss of FBPase activity (Fig. 3 b). In terms of protein level, there were several exceptions, but FBP1 expression tended to be different depending on the substitution of hydrophobicity. The mutant group without changes in hydrophobicity (indicated in blue), except G294V, maintained a similar level of protein expression as WT, whereas the mutants group with changes in hydrophobicity (indicated in red), except G207R, exhibited decreased protein expression (Fig. 3 c, 3 d). Consistent with the protein expression, immunofluorescence staining demonstrated that all mutants with protein expression similar to WT were diffusely localized in the cytoplasm, whereas mutants with decreased protein expression aggregated in the cytoplasm (Fig. 4 a, 4 b). Subsequently, we observed a strong negative correlation between the number of cells with FBP1 aggregates and its protein expression among the FBP1 missense mutants (Fig. 4 c). These data suggested that the protein aggregation could be linked to the substitution of hydrophobicity, and FBPase activities has more broadly affected by the missense mutations independent of hydrophobicity status. As there were several exceptions including G207R and V325A, the other constructive feature could be associated to enzymatic function. FBP1 missense mutations were categorized into three functional phenotypes Based on these results, we categorized the FBP1 missense mutations into three functional groups (Table 1, Fig. 5 ). The Type 1 mutations (D119N, P120L, N213K, and E281K) are direct substitution of pivotal amino acid residues inside enzyme activity site (Fig. 5 a, 5 b). These mutations did not change their amino acid hydrophobicity and protein expression and were diffusely localized in the cytoplasm similar to WT (Table 1). Therefore, these mutations cause a primary loss of FBPase enzymatic activity through mutations of key amino acid residues in the functional motif of enzymatic activity (indicated by arrowheads) without affecting protein expression and cytoplasmic localization. Indeed, Type 1 mutations are characterized by no change in hydrophobicity. Type 2 mutations (R158W, G164D, G164S, A177D, F194S, G260R, P284R, G294E, and G294V) are likely to be out of the important amino acid residues in the functional motif (Fig. 5 a) and appear to cluster around the substrate binding pocket (Fig. 5 c). In line with the changes in amino acid hydrophobicity (except for G294V), Type 2 mutations decreased protein expression and caused aggregation in the cytoplasm, possibly due to protein misfolding. Type 3 mutations (G207R and V325A) were structurally distant from sites associated with the enzyme activity motif and substrate binding pocket (Fig. 5 d). These mutations exhibited normal FBPase enzyme activity and their protein expression and cytoplasmic localization were similar to those of WT regardless of amino acid hydrophobicity, indicating that Type 3 mutations are likely non-pathogenic in terms of biochemical phenotype of FBPase deficiency. In fact, the previous reports suggested that the cases with V325A had no functional defect 3 . As expected, when we examined the binding ability of all mutants to HSP, only Type 2 mutants increased the interaction with HSP70 and HSP90 to a greater extent than those seen with WT, Type 1, and Type 3 mutants (Fig. 5 e and Additional file 3: Figure S4). Furthermore, the binding ability of FBP1 WT and mutants to either HSP70 or HSP90 was significantly correlated with the number of cells with FBP1 aggregates (%) (Fig. 5 f). Thus, we postulate that decreased protein expression due to protein misfolding in association with HSP recognition and protein aggregation via ERAD system is involved in the pathogenesis of FBPase deficiency, particularly in Type 2 mutations. Discussion FBPase deficiency is sometimes misdiagnosed or diagnosed in delay following initial suspicion of other energy-related deficiencies. Our patient’s case and the observations we made in the present study illustrate how FBPase deficiency can be confused with disorders of fatty acid oxidation. While acylcarnitine profiles are useful for the diagnosis of the latter, we advocate that fructose tolerance test and genetic analyses would be beneficial and provide informative confirmation to the diagnosis of patients with FBPase deficiency. In general, fructose loads can lead to large, rapid expansions in the hexose- and triose-phosphate pools, potentially providing increased substrate for all central carbon metabolic pathways, including glycolysis, glycogenesis, gluconeogenesis, lipogenesis, and oxidative phosphorylation. In FBPase deficiency, marked decrease in intracellular free phosphate due to hepatic accumulation of fructose 1,6-bisphosphate can inhibit glycogenolysis, leading to fructose induced hypoglycemia. To date, several harmful mutations in the coding region of FBP1 have been reported. Santer et al . reviewed 35 different mutations, including 14 missense mutations, 12 deletion mutations, 4 nonsense mutations, 4 insertions/duplications, two splices, and one indel 5 . Although the FBP1 c.581T > C (which results in the missense mutation F194S) and c.490G > A (which affects the neighboring nucleotide) mutations have been previously demonstrated 3 , 17 , the c.491G > A mutation (responsible for the missense mutation G164D) demonstrated in this study is novel. The G164 and F194 mutations, which are located in the β-strand (Fig. 1 g and 3 a), demonstrated the substitution of hydrophobic amino acids with hydrophilic amino acids (Table 1), suggesting certain conformational changes. The examinations carried out in this study confirmed that the FBP1 mutants with G164D and F194S decreased protein expression and resulted in a loss of FBPase enzyme activity. The interactome analysis based on Liquid chromatography-tandem MS data for binding partners demonstrated that FBP1, particularly in its mutant forms, interacts with the proteins involved in the molecular chaperone related to unfolded protein response including heat shock protein (HSP). Protein misfolding has been recognized as an important pathophysiological cause of protein deficiency in some genetic disorders, such as Fabry disease, Pompe disease, and Gaucher disease 28 . Inherited mutations can break native protein folding, resulting in the formation of misfolded proteins that are consequently retained in the endoplasmic reticulum (ER). The heat-shock proteins (HSPs) are a family of molecular chaperones, which collectively form a network that is critical for protein folding 22 . It has been suggested that HSP70 recognizes unfolded proteins, and HSP90 recognizes partially folded proteins, but fully folded proteins do not bind with either HSP70 or HSP90 23 . Additionally, missense mutations have been shown to shift protein folding equilibrium toward a partially folded state, thus increasing the cellular fraction of HSP70 and HSP90 relative to WT proteins 23 . Some proteins can only be partially folded by HSP70 and therefore require additional assistance from HSP60 (chaperonin) in order to acquire a folded functional conformation 22 . Aberrant protein aggregation was found to be controlled by chaperonins containing TCP-1. Based on the findings that FBP1 variants with G164D and F194S mutations have increased interactions with HSP70, HSP90, HSP60 and TCP1 and are partially aggregated and trapped in the ER, we conclude that the decrement in protein expression detected in FBPase deficiency is, at least in part, a result of protein misfolding. Finally, 15 FBP1 missense mutations (Figs. 3 , 4 and 5 ) were reviewed and classified into three categories. Type 1 causes loss of enzyme activity due to mutations in functional domain with unchanged protein expression and diffuse cytoplasmic localization. Type 1 mutations do not change their amino acid hydrophobicity. Type 2 causes loss of enzyme activity with reduction in protein expression and ER aggregation due to protein misfolding. Type 2 mutations change amino acid hydrophobicity, except for G294V. It is noted that G294V from the Type 2 category exhibits unchanges in its amino acid hydrophobicity, suggesting that G294 plays a role in FBP1 protein folding. Type 3 mutations are likely non-pathogenic mutations in regard to obvious phenotype of FBPase deficiency, whereas the relationship between these mutations and disease onset are obscure. This finding is consistent with clinical characteristics, but the further investigation could be needed to uncover the role of our defined sites. Conclusions In the context of genotype biochemical phenotype associations, previous in vitro studies evaluated only two missense mutations (D119N and G164S) and, consistent with our findings, D119N decreased enzymatic activity with no impact on protein expression 16 , whereas G164S decreased protein expression 29 . Thus, G207R and V325A were defined as Type 3 mutations. The G207R mutation, wherein the second allele exhibited a deletion of exon 8, was present in FBPase deficiency 5 . This variant has been previously reported in the heterozygous state with an allele frequency of 0.0001498 in 10 European (non-Finnish) individuals 5 , although its pathological effects have not yet been elucidated. By contrast, the V325A mutation is not recorded in the dbSNP database. Although this mutation was observed in patients with FBPase deficiency, it was found to harbor the same mutant alleles as G164S. Based on an examination of the chimeric V325A mutation, Kikawa et al. suggested that this mutation does not play a pathogenic role in FBPase deficiency 3 , which is consistent with the findings of our study. Besides missense mutations, several types of FBP1 mutations (12 deletion mutations, four nonsense mutations, four insertions/duplications, two splices, and one indel) have been reported 5 . Previous in vitro studies reported that c.704delC, c.838delT, and c.960dupG all decreased truncated FBP1 protein expression 16 , 29 , 30 . However, the mechanism that leads to a decrease in truncated FBP1 protein expression has not been sufficiently elucidated. Considering our findings, protein misfolding may be involved in the underlying disease pathophysiology in these truncated types of FBP1 mutations, similar to Type 2 mutations. The clinical applications of pharmacological chaperone therapy for Fabry disease have become increasingly popular. The chaperone molecules support the folding of mutated enzymes and increase their stability and activity, which may be broadly applicable to other protein deficiencies. Life-threatening episodes of hypoglycemia and lactic acidosis are sometimes triggered by fasting and febrile infections during childhood in cases of FBPase deficiency. Yet, to date, no preventive agent has been developed for these patients and their symptoms. Our findings indicate the possibility that certain patients with Type 2 mutation may respond to such kind of chaperone molecules, although Type 1 mutations are likely to be untreatable cases of FBPase deficiency. At any rate, further studies are warranted in order to fully understand the pathophysiology of this rare disease as well as to clarify the usefulness and efficacy of pharmacological chaperone therapy. Abbreviations ER endoplasmic reticulum FBPase Fructose-1,6-bisphosphatase FBP1 FBP gene HSP heat-shock proteins PCR polymerase chain reaction VAF variant allele frequency WT wild-type Declarations Competing interests The authors declare no competing financial interests. References Rahil JF, de Maine MM, Benkovic SJ. Rapid-quench and isotope-trapping studies on fructose-1,6-bisphosphatase. Biochemistry . 1982;21(14):3358-3363. Baker L, Winegrad AI. Fasting hypoglycaemia and metabolic acidosis associated with deficiency of hepatic fructose-1,6-diphosphatase activity. Lancet . 1970;2(7662):13-16. Kikawa Y, Inuzuka M, Jin BY, et al. Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency. American journal of human genetics . 1997;61(4):852-861. Nyhan W BB, Al-Aqeel A. Fructose-1,6-diphosphatase deficiency. In: Atlas of Inherited Metabolic Diseases . London: Hodder Arnold; 2012:354-358. Santer R, du Moulin M, Shahinyan T, et al. A summary of molecular genetic findings in fructose-1,6-bisphosphatase deficiency with a focus on a common long-range deletion and the role of MLPA analysis. Orphanet journal of rare diseases . 2016;11:44. Lebigot E, Brassier A, Zater M, et al. Fructose 1,6-bisphosphatase deficiency: clinical, biochemical and genetic features in French patients. Journal of inherited metabolic disease . 2015;38(5):881-887. Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature . 2011;478(7367):64-69. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science . 2013;339(6121):819-823. Kikawa Y, Shin YS, Inuzuka M, Zammarchi E, Mayumi M. Diagnosis of fructose-1,6-bisphosphatase deficiency using cultured lymphocyte fraction: a secure and noninvasive alternative to liver biopsy. Journal of inherited metabolic disease . 2002;25(1):41-46. Sakuma I, Higuchi S, Fujimoto M, et al. Cushing Syndrome Due to ACTH-Secreting Pheochromocytoma, Aggravated by Glucocorticoid-Driven Positive-Feedback Loop. The Journal of clinical endocrinology and metabolism . 2016;101(3):841-846. Tamura A, Ogasawara T, Fujii Y, et al. Glucagonoma With Necrolytic Migratory Erythema: Metabolic Profile and Detection of Biallelic Inactivation of DAXX Gene. The Journal of clinical endocrinology and metabolism . 2018;103(7):2417-2423. Tanaka T, Ohkubo S, Tatsuno I, Prives C. hCAS/CSE1L associates with chromatin and regulates expression of select p53 target genes. Cell . 2007;130(4):638-650. Suzuki S, Tanaka T, Poyurovsky MV, et al. Phosphate-activated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive oxygen species. Proceedings of the National Academy of Sciences of the United States of America . 2010;107(16):7461-7466. Hosokawa H, Romero-Wolf M, Yui MA, et al. Bcl11b sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2 and Zbtb16. Nat Immunol . 2018;19(12):1427-1440. Ke HM, Zhang YP, Lipscomb WN. Crystal structure of fructose-1,6-bisphosphatase complexed with fructose 6-phosphate, AMP, and magnesium. Proceedings of the National Academy of Sciences of the United States of America . 1990;87(14):5243-5247. Li N, Chang G, Xu Y, et al. Clinical and Molecular Characterization of Patients with Fructose 1,6-Bisphosphatase Deficiency. International journal of molecular sciences . 2017;18(4). Matsuura T, Chinen Y, Arashiro R, et al. Two newly identified genomic mutations in a Japanese female patient with fructose-1,6-bisphosphatase (FBPase) deficiency. Molecular genetics and metabolism . 2002;76(3):207-210. Kaur R, Dahiya L, Kumar M. Fructose-1,6-bisphosphatase inhibitors: A new valid approach for management of type 2 diabetes mellitus. European journal of medicinal chemistry . 2017;141:473-505. Szklarczyk D, Franceschini A, Kuhn M, et al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic acids research . 2011;39(Database issue):D561-568. Wang F, Song W, Brancati G, Segatori L. Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases. The Journal of biological chemistry . 2011;286(50):43454-43464. Brown CR, Chiang HL. A selective autophagy pathway that degrades gluconeogenic enzymes during catabolite inactivation. Communicative & integrative biology . 2009;2(2):177-183. Johnston CL, Marzano NR, van Oijen AM, Ecroyd H. Using Single-Molecule Approaches to Understand the Molecular Mechanisms of Heat-Shock Protein Chaperone Function. Journal of molecular biology . 2018;430(22):4525-4546. Karras GI, Yi S, Sahni N, et al. HSP90 Shapes the Consequences of Human Genetic Variation. Cell . 2017;168(5):856-866 e812. Herzog B, Morris AA, Saunders C, Eschrich K. Mutation spectrum in patients with fructose-1,6-bisphosphatase deficiency. Journal of inherited metabolic disease . 2001;24(1):87-88. Herzog B, Wendel U, Morris AA, Eschrich K. Novel mutations in patients with fructose-1,6-bisphosphatase deficiency. Journal of inherited metabolic disease . 1999;22(2):132-138. Afroze B, Yunus Z, Steinmann B, Santer R. Transient pseudo-hypertriglyceridemia: a useful biochemical marker of fructose-1,6-bisphosphatase deficiency. European journal of pediatrics . 2013;172(9):1249-1253. Asberg C, Hjalmarson O, Alm J, Martinsson T, Waldenstrom J, Hellerud C. Fructose 1,6-bisphosphatase deficiency: enzyme and mutation analysis performed on calcitriol-stimulated monocytes with a note on long-term prognosis. Journal of inherited metabolic disease . 2010. Parenti G. Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics. EMBO molecular medicine . 2009;1(5):268-279. Moon S, Kim JH, Han JH, et al. Novel compound heterozygous mutations in the fructose-1,6-bisphosphatase gene cause hypoglycemia and lactic acidosis. Metabolism: clinical and experimental . 2011;60(1):107-113. Kikawa Y, Inuzuka M, Jin BY, et al. Identification of a genetic mutation in a family with fructose-1,6- bisphosphatase deficiency. Biochemical and biophysical research communications . 1995;210(3):797-804. Tables Table 1. Categories of FBP1 missense mutations Mutation Location Enzymatic Activity Protein Expression Intracellular Aggregation Chaperone Binding Ability Type of Amino Acid Hydropathy Index Structure Amino Acid Change Compared to Wild-type Compared to Wild-type Localization (%) HSP70 HSP90 Wild-type Mutant Wild-type Mutant Type 1: Loss of enzyme activity due to mutations in functional domain with unchanged protein expression and diffuse cytoplasmic localization D119N Metal binding site decrease no change 1.4 Diffuse (cytoplasm) 9.4 0.1 0.3 hydrophilic- acidic hydrophilic- neutral -3.5 -3.5 β-strand P120L Linker lesion at metal and substrate binding site decrease no change 1.3 Diffuse (cytoplasm) 8.5 1.1 0.5 hydrophobic- aliphatic hydrophobic- aliphatic -1.6 3.8 β-strand N213K Substrate binding site decrease no change 1.1 Diffuse (cytoplasm) 13.3 1.5 0.5 hydrophilic- neutral hydrophilic- basic -3.5 -3.9 ND E281K Metal binding site decrease no change 0.9 Diffuse (cytoplasm) 33.6 2.4 0.5 hydrophilic- acidic hydrophilic- basic -3.5 -3.9 turn Type 2: Loss of enzyme activity with decreased protein expression and ER aggregation in association with hydrophobicity change R158W decrease decrease 0.5 Aggregated (in ER) 69.6 7.7 7.8 hydrophilic- basic hydrophobic- aromatic -4.5 -0.9 α-helix G164D decrease decrease 0.4 Aggregated (in ER) 71.4 13.7 15.8 hydrophobic- aliphatic hydrophilic- acidic -0.4 -3.5 β-strand G164S decrease decrease 0.6 Aggregated (in ER) 42.7 6.4 12.2 hydrophobic- aliphatic hydrophilic- neutral -0.4 -0.8 β-strand A177D decrease decrease 0.2 Aggregated (in ER) 73.9 24.0 37.5 hydrophobic- aliphatic hydrophilic- acidic 1.8 -3.5 β-strand F194S decrease decrease 0.4 Aggregated (in ER) 62.6 15.4 19.8 hydrophobic- aromatic hydrophilic- neutral 2.8 -0.8 β-strand G260R decrease decrease 0.4 Aggregated (in ER) 73.5 18.9 28.2 hydrophobic- aliphatic hydrophilic- basic -0.4 -4.5 ND P284R decrease decrease 0.3 Aggregated (in ER) 57.7 41.2 47.2 hydrophobic- aliphatic hydrophilic- basic -1.6 -4.5 α-helix G294E decrease decrease 0.4 Aggregated (in ER) 64.1 64.9 89.9 hydrophobic- aliphatic hydrophilic- acidic -0.4 -3.5 ND G294V decrease decrease 0.6 Aggregated (in ER) 65.9 6.1 3.5 hydrophobic- aliphatic hydrophobic- aliphatic -0.4 4.2 ND Type 3: Likely non-pathogenic mutations in regards to biochemical phenotype G207R no change no change 1.0 Diffuse (cytoplasm) 12.7 3.1 3.9 hydrophobic- aliphatic hydrophilic- basic -0.4 -4.5 ND V325A no change no change 1.0 Diffuse (cytoplasm) 25.4 2.3 1.3 hydrophobic- aliphatic hydrophobic- aliphatic 4.2 1.8 α-helix Abbreviations: ND, no data; ER, endoplasmic reticulum Additional Declarations There is NO Competing Interest. Supplementary Files FBPaseDSupFiglegend.pdf SupplementalTable.docx SupplementalTable6.xlsx Supplemental Table 6 Cite Share Download PDF Status: Published Journal Publication published 28 Jul, 2023 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2185039","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":148263782,"identity":"4f77f537-0df2-4f12-9166-6788fe133d99","order_by":0,"name":"Tomoaki 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Miki","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Miki","suffix":""}],"badges":[],"createdAt":"2022-10-20 04:40:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2185039/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2185039/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-023-05160-y","type":"published","date":"2023-07-28T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":28579676,"identity":"687be4b4-f87f-46fb-84fd-b20ce6ec037c","added_by":"auto","created_at":"2022-11-02 19:37:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":342969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFBP1 mutations identified in an adult patient with severe hypoglycemic acidosis and its clinico-endocrinological profiles\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePanels \u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003eb \u003c/strong\u003edemonstrate results of the oral fructose tolerance test. Oral fructose loading resulted in hypoglycemia accompanied by increased lactate levels. Additionally, the 3-hydroxybutyrate levels mildly increased, whereas the free fatty acid (FFA) levels exhibited a dramatic elevation. The patient and a healthy volunteer are shown in red and black, respectively.\u003c/p\u003e\n\u003cp\u003ePanels \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed,\u003c/strong\u003eand \u003cstrong\u003ee\u003c/strong\u003e show the results of the genetic analyses of \u003cem\u003eFBP1\u003c/em\u003e gene. \u003cstrong\u003e(c)\u003c/strong\u003eWhole-exome sequencing results of the patient and her family. IGV browser visualization of the whole-exome sequencing results revealed a novel compound heterozygous missense mutation in the \u003cem\u003eFBP1\u003c/em\u003egene with c.491G \u0026gt; A p.G164D and c.581T \u0026gt; C p.F194S. The patient inherited the G164D and F194S mutations from her father and mother, respectively.\u003cstrong\u003e (d) \u003c/strong\u003eCompound heterozygous missense mutations in the \u003cem\u003eFBP1\u003c/em\u003egene with G164D and F194S in the patient were validated using a Sanger sequence analysis. \u003cstrong\u003e(e)\u003c/strong\u003e Pedigree and genotypes of the family. Solid symbols indicate the allele of G164D, while half solid symbols indicate the allele of F194S. Squares denote males, and circles denote females. Arrow indicates the patient. \u003cstrong\u003e(f) \u003c/strong\u003eThe FBPase enzyme activity of G164D, F194S, and cotransfected constructs markedly decreased compared to that in the WT. The data are shown as mean ± SD. *P \u0026lt; 0.01 versus the WT (Student’s \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003e(g)\u003c/strong\u003e Structure of the FBP1 dimer based on the protein data bank (DOI: 10.2210/pdb1FBP/pdb) is shown. Cyan sphere: substrate binding site; magenta sphere: metal binding site; blue sphere: AMP binding site; G164D and F194S are shown as a smudge and in lemon color, respectively.\u003c/p\u003e","description":"","filename":"FBPaseDFigure1combio.png","url":"https://assets-eu.researchsquare.com/files/rs-2185039/v1/2acc464189713b59e78bc3cf.png"},{"id":28579672,"identity":"63e33983-f9fd-47c0-81ef-53916445b4d3","added_by":"auto","created_at":"2022-11-02 19:37:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":798952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBiochemical consequences of FBP1 mutations with G164D and F194S in association with decreased protein expression and aggregation in ER due to protein misfolding.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e functional analysis of the FBP1 mutants with G164D and F194S in \u003cem\u003eFBP1\u003c/em\u003e-KO HepG2 cells. FBP1 protein expression was examined. \u003cstrong\u003e(a)\u003c/strong\u003e Protein expression analysis demonstrated that Myc-tagged FBP1 decreased in G164D, F194S, and cotransfected constructs compared with that in WT. \u003cstrong\u003e(b)\u003c/strong\u003e Immunofluorescence analysis showed that FBP1 mutants with G164D and F194S aggregated in the cytoplasm. RT-qPCR analysis detected sufficient quantities of mRNA among the constructs. \u003cstrong\u003e(c)\u003c/strong\u003e Immunofluorescence analysis of a liver biopsy specimen from the patient. FBP1 aggregated in the cytoplasm. \u003cstrong\u003e(d) \u003c/strong\u003eVenn diagram of the proteins identified in the FBP1 interactome based on MS analysis. \u003cstrong\u003e(e)\u003c/strong\u003e The main function of FBP1 interactome proteins based on Ingenuity Pathway Analysis (IPA). \u003cstrong\u003e(f)\u003c/strong\u003eClusters of the enriched biological processes in the FBP1 interactome. Data from STRING interaction database were visualized using Cytoscape. \u003cstrong\u003e(g)\u003c/strong\u003e Intracellular localization of endoplasmic reticulum (green) and Myc-tagged FBP1 (red). FBP1 mutants with G164D and F194S colocalized primarily, but not completely, in the endoplasmic reticulum. \u003cstrong\u003e(h)\u003c/strong\u003e Immunoblot analyses of FBP1 and ATF6. ATF6 is an endoplasmic reticulum, stress-regulated, trans-membrane transcription factor. It was enriched in the endoplasmic reticulum fractions. FBP1 protein was detected in the whole cell lysate and endoplasmic reticulum fractions. Protein expression of FBP1 mutants of G164D and F194S was relatively high in the endoplasmic reticulum compared to the whole cell lysate. \u003cstrong\u003e(i)\u003c/strong\u003e HepG2 cells were treated with the mannosidase inhibitor kifunensine (200 µM) for 48 hours, which increased the protein expression of the FBP1 mutants with G164D and F194S. \u003cstrong\u003e(j)\u003c/strong\u003e Immunoprecipitation with anti-Myc and immunoblot analysis of FBP1, HSP70, HSP90, HSP60 and TCP1. \u003cstrong\u003e(k)\u003c/strong\u003e FBP1 mutants with G164D and F194S exhibited greater interaction with HSP70, HSP90, HSP60 and TCP1 than did the wild-type (WT). The data are presented as the mean ± SD of four independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus WT (Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"FBPaseDFigure2combio.png","url":"https://assets-eu.researchsquare.com/files/rs-2185039/v1/2d5fafaa38533b73f2a4f068.png"},{"id":28580308,"identity":"2b785acc-1032-455e-83d5-1b5614a5fc23","added_by":"auto","created_at":"2022-11-02 19:45:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":230676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCharacterization of all previously reported FBP1 missense mutations in enzyme activity and protein expression\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Schematic distribution of all previously reported \u003cem\u003eFBP1\u003c/em\u003e missense mutations. Substrate binding site, metal binding site, and AMP binding sites are shown according to the NCBI and UniProt database. \u003cstrong\u003e(b)\u003c/strong\u003eFBPase enzyme activity of all FBP1 missense mutants. All these mutants except for G207R and V325A exhibited a loss in enzymatic activity. The data are presented as mean ± SD. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus WT (Student’s \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003e(c)\u003c/strong\u003e and \u003cstrong\u003e(d)\u003c/strong\u003e Protein expression of all FBP1 missense mutants in \u003cem\u003eFBP1\u003c/em\u003e-KO HepG2 cells. All the mutants that did not change their hydrophobicity except for G294V exhibited sufficient protein expression compared with that of the WT, whereas all the mutants that changed their hydrophobicity except for G207R exhibited decreased protein expression. The data are presented as the mean ± SD of three independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus WT (Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"FBPaseDFigure3combio.png","url":"https://assets-eu.researchsquare.com/files/rs-2185039/v1/2ba2894e287586875e1b317a.png"},{"id":28579677,"identity":"90e6b21f-fad4-4ee7-8d76-7970acfe0794","added_by":"auto","created_at":"2022-11-02 19:37:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":266021,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIntracellular localization of FBP1 missense mutations in FBP1-KO HepG2 cells\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e and (\u003cstrong\u003eb)\u003c/strong\u003e Immunofluorescence studies of FBP1 missense mutants are shown. We counted the number of cells in two groups with intracellular FBP1 with diffuse localization and with aggregation in the cytoplasm. Then, the ratio of cells with FBP1 aggregates (%) was calculated. All the mutants that did not change their hydrophobicity except for G294V diffusely localized in the cytoplasm similar to the WT, whereas all the mutations that changed their hydrophobicity except for G207R aggregated in the cytoplasm. The data are presented as mean ± SD of three independent experiments. *P \u0026lt; 0.05; **P \u0026lt; 0.01 versus WT (Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e A negative correlation was observed between the number of cells with FBP1 aggregates (%) and FBP1 protein expression (mutant/WT). The correlation was determined by the Pearson coefficient of determination R\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"FBPaseDFigure4combio.png","url":"https://assets-eu.researchsquare.com/files/rs-2185039/v1/89a61ceed9e31cd109b8a18e.png"},{"id":28579675,"identity":"b0cc39fe-f4be-4c40-8ee2-70b9e126d927","added_by":"auto","created_at":"2022-11-02 19:37:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":402792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCategorization of FBP1 missense mutations based on the genotype functional phenotype association\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Schematic distribution of all previously reported \u003cem\u003eFBP1\u003c/em\u003e missense mutations. Type 1 mutations without change in amino acid hydrophobicity are direct substitutions of key residues within an enzymatically active site. Type 2 mutations are those with change of hydrophobicity and Type 3 mutations are those that not located at pivotal residues in functional motifs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)–(d),\u003c/strong\u003e Structure of the FBP1 dimer based on the protein data bank (DOI: 10.2210/pdb1FBP/pdb). Locations of the mutations are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Type 1 mutations include D119N (red), P120L (green), N213K (yellow), and E281K (orange). These mutations are directly located at pivotal residues in functional sites of substrate or metal binding site.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e Type 2 mutations include R158W (salmon), G164D/S (smudge), A177D (light blue), F194S (lemon), G260R (light pink), P284R (teal), and G294E/V (yellow orange). These mutations are likely to be out of the important amino acid residues in the functional motif and appear to cluster around the substrate binding pocket.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e Type 3 mutations include G207R (white) and V325A (white), and these mutations are structurally distant from the sites associated with enzyme activity motif and substrate binding pocket.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Binding ability of HSP70 and HSP90 to FBP1 mutant proteins. Type 2 mutants showed increased HSP70 and HSP90 interactions compared to WT, Type 1 or Type 3 mutants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e Panel shows a scatterplot of the signal intensity ratios of HSP70 (upper) or HSP90 (lower) binding ability (mutant/WT) and the number of cells with FBP1 aggregates (%). Both HSP70 and HSP90 binding ability was significantly correlated with FBP1 aggregates.\u003c/p\u003e","description":"","filename":"FBPaseDFigure5combio.png","url":"https://assets-eu.researchsquare.com/files/rs-2185039/v1/d41daaf4a268a84a54a1d56c.png"},{"id":40752413,"identity":"030881fa-da39-4cbe-970f-ba9bc0dd1dcb","added_by":"auto","created_at":"2023-07-29 07:07:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3223984,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2185039/v1/14ac9932-8447-4fa4-b192-6538fcb62875.pdf"},{"id":28579679,"identity":"42e940ad-5648-46bf-b971-569fbf78e88b","added_by":"auto","created_at":"2022-11-02 19:37:01","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14365287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"FBPaseDSupFiglegend.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2185039/v1/427f2beab68092cdc99b73bb.pdf"},{"id":28579678,"identity":"ccebc8e6-f2fb-4e0f-aed8-0304350c38a1","added_by":"auto","created_at":"2022-11-02 19:37:00","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":30618,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-2185039/v1/8312ed87f824432c91dc2a68.docx"},{"id":28579673,"identity":"37591444-3b11-42e3-a72e-b4298828a174","added_by":"auto","created_at":"2022-11-02 19:37:00","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":23125,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Table 6\u003c/p\u003e","description":"","filename":"SupplementalTable6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2185039/v1/3989a7bb3526c186ea1d76d8.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Characterization based on genotype–biochemical phenotype association in fructose-1,6-bisphosphatase deficiency","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFructose-1,6-bisphosphatase (FBPase) is a key regulatory enzyme in gluconeogenesis that catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Baker and Winegrad first reported FBPase deficiency in 1970 \u003csup\u003e2\u003c/sup\u003e, and Kikawa \u003cem\u003eet al.\u003c/em\u003e subsequently identified three \u003cem\u003eFBP\u003c/em\u003e gene (\u003cem\u003eFBP1\u003c/em\u003e) mutations associated with FBPase deficiency in 1997 \u003csup\u003e3\u003c/sup\u003e. FBPase deficiency is an autosomal recessive disorder that generally courses with hypoglycemia and metabolic acidosis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Although fasting and febrile infections are known to trigger life-threatening episodes of hypoglycemia and lactic acidosis in infancy, these episodes rarely occur in adults due to increased glycogen storage \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Several harmful mutations in the coding region of \u003cem\u003eFBP1\u003c/em\u003e have been reported \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Nevertheless, with incidence rates estimated to range between 1/350,000 and 1/900,000, FBPase deficiency remains a very rare inherited disease 5\u003csup\u003e,\u003c/sup\u003e6. Due to its low incidence, elucidating the molecular mechanism by which the identified mutations cause the loss of enzyme activity is still challenging, particularly in terms of genotype phenotype correlation and adult-onset cases. Here, we present the case of a 22-year-old female patient who was diagnosed with FBPase deficiency and exhibited a novel compound heterozygous mutation of the FBP1 gene. Our results demonstrate the underlying mechanism by which the identified mutations caused FBPase deficiency involves protein misfolding, and we examine and categorize the biochemical phenotypes of all previously reported FBP1 missense mutations into three functional groups.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMutation analysis\u003c/h2\u003e \u003cp\u003eDNeasy Blood and Tissue Kits (QIAGEN, Hilden, Germany) were used to extract the genomic DNA from the patient\u0026rsquo;s blood. Then, all 7 exons of the \u003cem\u003eFBP1\u003c/em\u003e gene were amplified by polymerase chain reaction (PCR) and sequenced using a 3130 Genetic Analyzer (Applied Biosystems, Massachusetts, USA). Table S1 (Additional file 1) describes the primer information and PCR conditions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWhole-exome Sequencing\u003c/h3\u003e\n\u003cp\u003eThe whole-exome sequencing involved the targeted capture of all the exon sequences using SureSelect Human All Exon v6 (Agilent Technologies, California, USA), followed by massive parallel sequencing of the enriched exon fragments on the HiSeq 2500 platform (Illumina) using the 125-bp paired-end mode as per manufacturer\u0026rsquo;s protocol \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The sequenced reads were aligned to the human genome reference (GRCh37) using the default parameter settings in Burrows-Wheeler Aligner version 0.7.10, while the PCR duplicates were eliminated using Picard-tools version 1.39 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://picard.sourceforge.net/\u003c/span\u003e\u003cspan address=\"http://picard.sourceforge.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Candidate mutations with (i) depths\u0026thinsp;\u0026ge;\u0026thinsp;8; (ii) Number of variant reads\u0026thinsp;\u0026ge;\u0026thinsp;4; and (iii) variant allele frequency (VAF) 0.4\u0026ndash;0.6, 1 were adopted, as VAFs of pathogenic germline mutations are estimated to be approximately 0.5 in heterozygous state and 1 in homozygous state. Mutations were further filtered by excluding (i) variants presenting only in unidirectional reads; (ii) insertions and deletions in simple repeat regions; (iii) synonymous SNVs; and (iv) known variants listed in the 1000 Genomes Project (Nov 2010 release), Exome Sequencing Project (ESP) 6500, Human Genome Variation Database (HGVD; October 2013 release), and ExAC database with frequencies\u0026thinsp;\u0026gt;\u0026thinsp;0.001 in order to exclude non-pathogenic variants.\u003c/p\u003e\n\u003ch3\u003eConstruction Of Fbp1 Expression Vectors\u003c/h3\u003e\n\u003cp\u003eThe total RNA was extracted from the patient\u0026rsquo;s blood using RNeasy Kits (QIAGEN, Hilden, Germany) and reverse-transcribed using SuperScript 2 reverse transcriptase (Thermo Scientific, Massachusetts, USA) and oligo (dT) primers. The amplification of the coding region of the \u003cem\u003eFBP1\u003c/em\u003e gene was carried out using the forward primer 5\u0026rsquo;-CACCATGGCTGACCAGGCGCCCTTCG-3\u0026rsquo; and the reverse primer 5\u0026rsquo;-TCACTGGGCAGAGTGCTTCTCATAC-3\u0026rsquo;. The PCR procedure consisted of the following steps: a) denaturing at 98\u0026deg;C for 2 min, followed by 94\u0026deg;C for 15 secs, and b) annealing for 30 secs at 58\u0026deg;C and extension at 72\u0026deg;C for 1 min for 30 cycles. The PCR products were subcloned into pGEM-T Easy Vector (Promega, Wisconsin, USA). Then, the \u003cem\u003eFBP1\u003c/em\u003e fragments (WT and 2 mutants) were cut from the pGEM-T Easy Vector using EcoRI and subcloned into a pCMV-Myc-N Vector (Clontech, California, USA). A corresponding pCMV-Myc-N Vector was used as a negative control.\u003c/p\u003e\n\u003ch3\u003eMutagenesis Of Fbp1 Plasmids\u003c/h3\u003e\n\u003cp\u003eD119N, P120L, R158W, G164S, A177D, G207R, N213K, G260R, E281K, P284R, G294E, G294V, and V325A mutations were introduced by site-directed mutagenesis (Quick Change Lightning Site-Directed Mutagenesis KIT, Agilent Technologies) using the primers listed in Table S2 (Additional file 1). XL10-Gold ultracompetent cell DNA was isolated from cultured single clones and by sequencing to confirm successful mutagenesis.\u003c/p\u003e\n\u003ch3\u003eGeneration Of Fbp1 Knockout Hepg2 Cells\u003c/h3\u003e\n\u003cp\u003eThe protocol used for the CRISPR/Cas9 system was consistently based on that reported by Cong et al., Science 2013 \u003csup\u003e8\u003c/sup\u003e. The backbone vectors pX459 pSpCas9(BB)-2A-Puro and pX462 pSpCas9n(BB)-2A-Puro were obtained from Addgene (Massachusetts, USA). The target guide RNA sequences were designed at exon 5 including c.491G, exon 6 including c.581T, and exon 8 including the Japanese common mutation site (c.960-961insG) of the \u003cem\u003eFBP1\u003c/em\u003e genome (5\u0026rsquo;-GCAGCCGGCTACGCACTGTA-3\u0026rsquo;, 5\u0026rsquo;-GCACCAAAATGAACTCCCCGA-3\u0026rsquo; and 5\u0026rsquo;-GTCGGGGGATCCCAAGATCAC-3\u0026rsquo;) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. To clone the exon 5 and exon 6 target sequences into pX462 and the exon 8 target sequence into the pX459 backbone, the oligos were synthesized using Eurofins genomics (Tokyo, Japan) (Additional file 1: Table S3). Then, these oligos were submitted to annealing and phosphorylation by means of a T4 DNA Ligase Reaction Buffer and a T4 Polynucleotide Kinase (New England Biolabs, Massachusetts, USA) used at 37℃ for 30 min and 95℃ for 5 min. pX459 and pX462 were digested using BbsI (Thermo Scientific, Massachusetts, USA) at 37℃ for 30 min, with gel purification being performed by use of a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). Ligation reactions of pX459, pX462 and the annealed oligos were performed for 10 min at room temperature using a Quick Ligation Kit (New England Biolabs, Massachusetts, USA). Then, the ligated oligos were purified using PlasmidSafe exonuclease (Cambio, Cambridge, UK) at 37℃ for 30 min. The plasmids were transfected into Stbl3, and the appropriate transfectants were amplified and collected using NucleoBond Xtra Midi (Takara, Kusatsu, Japan). Three plasmids (pX462-exon5 gRNA, pX462-exon6 gRNA and pX459-exon8 gRNA) were cotransfected into HepG2 cells using lipofectamine 3000 (Thermo Scientific, Massachusetts, USA) as per manufacturer\u0026rsquo;s protocol. The plasmid-expressing HepG2 cells were selected by puromycin (Wako, Osaka, Japan), and the limiting dilution method was used to establish the monoclonal cell line.\u003c/p\u003e\n\u003ch3\u003eCell Culture And Transient Transfection\u003c/h3\u003e\n\u003cp\u003eThe human hepatocarcinoma cell line \u003cem\u003eFBP1\u003c/em\u003e-KO HepG2 culture was conducted using Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium containing antibiotics and 10% fetal bovine serum. The FPB1-KO HepG2 cells were plated on 6-well plates and then transfected with plasmid DNA (2.5 \u0026micro;g) complexed with Lipofectamine 3000 reagent (7.5 \u0026micro;l) and P3000 reagent (5 \u0026micro;l) in 250 \u0026micro;l of Opti-MEM (Thermo Scientific, Massachusetts, USA). Subsequently, the FBPase activity of HepG2 cells was measured 48 hours after transfection using a nicotinamide adenine dinucleotide phosphate (NADP)-coupled spectrophotometric assay.\u003c/p\u003e\n\u003ch3\u003eFbpase Activity Assay\u003c/h3\u003e\n\u003cp\u003eThe FBPase activity was calculated from an NADP-coupled spectrophotometric assay as described by Kikawa et al \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The assay mixture (300 \u0026micro;l) was composed of 40 \u0026micro;g protein of cell lysate, 50 mmol/L Tris-HCl buffer (pH 7.5), 2.0 mmol/L MgCl\u003csub\u003e2\u003c/sub\u003e, 1.0 mmol/L EDTA, 0.2 mmol/L NADP, 3.5 U/mL glucose-6-phosphate dehydrogenase, 1.5 U/mL glucose-6-phosphate isomerase, and 100 \u0026micro;mol/L FBP, which served as the substrate. A 96-well plate reader was used to record the rate of NADPH formation.\u003c/p\u003e\n\u003ch3\u003eReal-time Rt-pcr Analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eReal-time RT-PCR analysis\u003c/div\u003e \u003cp\u003eRT-qPCR experiments were performed as previously described \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eFBP1\u003c/em\u003e gene-specific mRNA expression values were determined and normalized to those of β-actin as an internal control. Briefly, the total RNA (4 \u0026micro;g) was extracted using an RNeasy kit (Qiagen, Valencia, CA, USA) and reverse-transcribed using a ReverTra Ace qPCR RT Kit (Toyobo, Tokyo, Japan). The cDNA products were subjected to RT-PCR using a Step One Plus Real-Time PCR system (Applied Biosystems, Massachusetts, USA). All the primer information is provided in Table S4 (Additional file 1).\u003c/p\u003e\n\u003ch3\u003eImmunoblot Analysis\u003c/h3\u003e\n\u003cp\u003eImmunoblot analyses were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The antibodies included FBP1 (SIGMA rabbit polyclonal, clone: HPA005857), c-Myc (Santa Cruz mouse monoclonal, clone: 9E10), actin (SIGMA rabbit polyclonal, clone: A2066), ATF6 (Novus Biologicals mouse monoclonal, clone: 70B1413.1), HSP70 (StressMarq mouse monoclonal, clone: N27F3-4), and HSP90 (Santa Cruz mouse monoclonal, clone: sc-13119), HSP60 (Abcam rabbit polyclonal, clone: ab46798) and TCP1 (Bethyl laboratories rabbit polyclonal, clone: A303-444A).\u003c/p\u003e\n\u003ch3\u003eEr Fractionation\u003c/h3\u003e\n\u003cp\u003eThe extraction of the ER was performed using an Endoplasmic Reticulum Enrichment Kit (Novus Biologicals, Colorado, USA), as per manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence Analysis\u003c/h3\u003e\n\u003cp\u003eImmunofluorescence analysis was performed to examine the cellular expression of the WT and mutant FBP1. The \u003cem\u003eFBP1\u003c/em\u003e-KO HepG2 cells were cultured in 4-well chamber slides (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) and then transfected with plasmids (0.5 \u0026micro;g). Twenty-four hours after the transfection, the cells were treated with CellLight\u0026reg; ER-GFP (12 \u0026micro;l/well; Thermo Scientific, Massachusetts, USA), which is a marker of ER, followed by fixation in 100% ethanol at \u0026minus;\u0026thinsp;20℃ for 10 min and incubation with blocking solution and primary antibodies against FBP1 (SIGMA rabbit polyclonal, clone: HPA005857), c-Myc (Santa Cruz mouse monoclonal, clone: 9E10) and GFP (MBL rabbit polyclonal, clone: 598) 48 hours after the transfection. A confocal laser microscope (LSM710, Carl Zeiss, Germany) was used to obtain fluorescence images.\u003c/p\u003e\n\u003ch3\u003eImmunoprecipitation Assay\u003c/h3\u003e\n\u003cp\u003eThe commercially available kit c-Myc-tagged Protein Mild Purification Kit (Medical \u0026amp; Biological Laboratories, Aichi, Japan) was used to perform the immunoprecipitation assay. HepG2 cell extracts containing different Myc-tagged FBP1 variants were incubated with anti-Myc beads at 4℃ for 1 hour. The beads were then rinsed and eluted using a wash solution and elution peptides.\u003c/p\u003e\n\u003ch3\u003eMass Spectrometry Sample Preparation\u003c/h3\u003e\n\u003cp\u003eThe FPB1-KO HepG2 cells were transfected with Myc-tagged FBP1-WT, Myc-tagged FBP1-G164D, Myc-tagged FBP1-F194S containing plasmid DNA. These cells were treated with the mannosidase inhibitor kifunensine (200 \u0026micro;M) for 48 hours. Myc-tagged FBP1 HepG2 cells solubilization was obtained with the following buffer: 50 mM Tris-HCl (pH 7.5), 1.0 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.1 mM EDTA, 0.5 mM phenylmethannesulphonyl fluoride, 2.5 \u0026micro;g/mL leupeptin and 1.0 \u0026micro;g/mL antipain. Immunoprecipitation with anti-Myc beads (Medical \u0026amp; Biological Laboratories, Aichi, Japan) was performed at 4℃ for 1 hour. The beads were then rinsed and eluted using a wash solution and elution peptides. Immune complexes were separated by SDS-PAGE. Bands were exsected from the gel and examined by mass spectrometry to find corresponding proteins. Respective gel pieces were rinsed two times using 100 mM bicarbonate in acetonitrile with subsequent protein digestion by trypsin. 0.1% formic acid was then added to the supernatant, and the peptides were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a LTQ Mass Spectrometer (Thermo Scientific). Analysis of MS/MS data set results was conducted using the Mascot software program (Matrix Science).\u003c/p\u003e\n\u003ch3\u003eProteomic Analysis And Database Search\u003c/h3\u003e\n\u003cp\u003eProteomic analysis and database search were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation and Gene Ontology (GO) were performed using the STRING interaction database.\u003c/p\u003e\n\u003ch3\u003eProtein Structure Based On Protein Data Bank\u003c/h3\u003e\n\u003cp\u003eKe \u003cem\u003eet al.\u003c/em\u003e revealed the detailed crystal structure of \u003cem\u003eSus scrofa\u003c/em\u003e FBP1 complexed with fructose 6-phosphate, AMP, and magnesium \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg shows the structure of FBP1 dimer based on their report (DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2210/pdb1FBP/pdb\u003c/span\u003e\u003cspan address=\"10.2210/pdb1FBP/pdb\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The FBP1 amino acid sequences between \u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eSus scrofa\u003c/em\u003e show approximately 90% similarity. Particularly, the functional motifs directly associated with FBPase activity are highly conserved.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eThe results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to analyze the continuous variables, and P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\n\u003ch3\u003eStudy Approval\u003c/h3\u003e\n\u003cp\u003eIn accordance with the protocols issued by the Chiba University Hospital, written informed consent for the genetic studies was obtained from the patient and her family.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCase presentation and whole-exome sequencing\u003c/h2\u003e \u003cp\u003eWe accompanied the case of a 22-year-old Japanese female patient who presented with a severe hypoglycemic attack and acidosis induced by prolonged fasting. Blood exams, including a blood gas analysis, showed very low levels of plasma glucose (12 mg/dL) with high levels of lactate (110 mg/dL) and severe metabolic acidosis (pH 6.85, PaCO\u003csub\u003e2\u003c/sub\u003e 19 mm Hg, HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e 2.5 mmol/L). The patient reported experiencing similar episodes triggered by fasting or febrile illness during her early childhood (Additional file 1: Table S5). We suspected the case to be an inherited metabolic issue, such as fatty acid oxidation or gluconeogenesis disorders causing hypoglycemic attacks with lactic acidosis, and further examinations were performed in order to establish a definitive diagnosis. Radiological examination of the abdomen by computerized tomography (CT) revealed a fatty liver, and the liver biopsy showed hepatic steatosis without other alterations (Additional file 3: Figure S1b). The acylcarnitine profile exhibited no specific pattern, suggesting a normal β-oxidation pathway. However, the urinary organic acid profile exhibited significantly elevated glycerol, lactate, and pyruvate levels, indicating a possible FBPase deficiency. The oral fructose tolerance test demonstrated a rapid decrease in blood glucose levels and an increase in lactate concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Additionally, fructose loading increased glycerol levels in the urinary organic acid profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Although clinical findings up to this point strongly indicated a case of FBPase deficiency, a differential diagnosis from other metabolic disorders that course with hypoglycemic attacks and lactic acidosis was difficult due to overlapping clinical features \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs previous studies have shown that genetic analyses using next-generation sequencing is useful for the molecular diagnosis of complex metabolic diseases including FBPase deficiency \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, we performed the whole-exome sequencing in samples from the proband and her family in order to identify potential underlying genetic defects. This approach revealed the 111 filtered variants in the proband (Additional file 2: Table S6). Two heterozygous variants were identified in a region of \u003cem\u003eFBP1\u003c/em\u003e among them, whereas there was no variants associated to mitochondrial fatty acid oxidation disorders and gluconeogenesis such as \u003cem\u003eOCTN2, CACT, CPT1, CPT2, LCHAD, MCAD, SCAD, MTP, VLCAD, ACAD9, ETFDH, ETFA, ETFB, HMGCS2, HMGCL, PC, PCK1, PCK2, G6PC\u003c/em\u003e and \u003cem\u003ePGM1\u003c/em\u003e. The identified variants in \u003cem\u003eFBP1\u003c/em\u003e were missense mutations (II-1 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) with c.491G\u0026thinsp;\u0026gt;\u0026thinsp;A p.G164D, which is a novel mutation, and c.581T\u0026thinsp;\u0026gt;\u0026thinsp;C p.F194S, which has been previously reported \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. According to the exome sequencing results of family members, her parents carried single mutation respectively: G164D mutation from father (I-1 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) and F194S mutation from her mother (I-2 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Then we confirmed that both of \u003cem\u003eFBP1\u003c/em\u003e variants with G164D and F194S in the proband were heterozygous missense mutations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) using Sanger sequencing, suggesting that it could be in a compound heterozygous state. Based on these findings, we made a definitive diagnosis of FBPase deficiency and advised the patient to avoid both prolonged fasting and intake of fructose-rich foods under a fasted state.\u003c/p\u003e \u003cp\u003eThe G164 and F194 are located in the β-strand structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), and both of identified mutations (G164D and F194S) could lead conformational changes due to the substitution of hydrophobic with hydrophilic amino acids (Table\u0026nbsp;1). In general, FBPase catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate in the presence of divalent cations, such as magnesium, manganese, or zinc \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, whereas AMP acts as an allosteric inhibitor. The human liver FBPase comprises four identical polypeptide chains each containing 338 amino acid residues, which are assembled as relatively flat tetramers with subunits conventionally labeled C1\u0026ndash;C4 \u003csup\u003e18\u003c/sup\u003e. These homotetramers consist of two intimate dimers. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg shows the structure of FBP1 dimer. We found that neither G164D nor F194S corresponded to the pivotal amino acid residues within functional motifs such as AMP binding sites, metal binding sites, or substrate binding sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Thus, we decided to examine if and how these mutations affect FBPase enzymatic activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eProtein expression and enzymatic activity of the FBP1 mutations were lower than those in the wild-type\u003c/span\u003e \u003c/p\u003e \u003cp\u003eWe established FBP1 mutants in hepatocytes to analyze molecular function. In order to eliminate the influence of endogenous FBPase, we generated \u003cem\u003eFBP1\u003c/em\u003e-KO HepG2 cells using the CRISPR/Cas9 system (Additional file 3: Figure S1a). Next, Myc-tagged \u003cem\u003eFBP1\u003c/em\u003e cDNA constructs of wild-type (WT), mutant clones (G164D or F194S), or cotransfects (G164D and F194S) were transduced in the \u003cem\u003eFBP1\u003c/em\u003e-KO HepG2 cells. NADP-coupled spectrophotometric assays were used to examine the enzymatic activity of mutant FBPase. As a result, the FBPase activities of all of mutants (G164D; 0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34, F194S; 0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56, and cotransfected clone; 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 mmol/min/mg protein) were significantly lower than WT clone (4.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61 mmol/min/mg protein) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). We validated the transcription level of FBP1 was not greater in WT clone, but immunoblot analysis revealed that the protein levels of FBP1 mutants were markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). To evaluate the enzymatic activity in FBP1 protein, we additionally performed an \u003cem\u003ein vitro\u003c/em\u003e NADP-coupled spectrophotometric assay with precipitated FBP1 protein by Myc-tag. The result represented that enzyme activity has completely diminished in FBP1 protein with both G164D and F194S (Additional file 3: Figure S2a and S2b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, immunofluorescence analysis was used to evaluate the intracellular localization of the \u003cem\u003eFBP1\u003c/em\u003e gene product. We found that the mutant FBP1 with G164D or F194S aggregated in the cytoplasm, whereas FBP1 protein in WT clone was diffusely localized in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Importantly, this aggregated distribution of FBP1 protein was also found in the liver biopsy from the presented patient (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), therefore we concluded that \u003cem\u003eFBP1\u003c/em\u003e mutations with G164D and F194S cause protein aggregation and the pathogenic loss in enzymatic activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFBP1 mutations with G164D and F194S aggregated in the endoplasmic reticulum due to protein misfolding\u003c/h2\u003e \u003cp\u003eTo address underlying mechanisms of aggregation and reduction in protein expression, we surveyed the interaction partners of FBP1 using Liquid chromatography-tandem MS (LC-MS/MS) analysis for the proteins pulled down by immunoprecipitation with anti-Myc tag. In total, 408 proteins were detected with LC-MS/MS analysis for WT, G164D mutant and F194S mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Next, we evaluated the molecular functionalities of those proteins interacting with FBP1 and its G164D or F194S mutated variants, including common binding partner among WT and mutants, by Ingenuity Pathway Analysis (IPA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). The analysis indicated that FBP1-interacting proteins can be mainly involved in protein production and degradation such as translation, post-transcriptional regulation, ubiquitination pathway and unfolded protein response. To test whether FBP1, especially its mutants, has such a connection to protein production and degradation as predicted, we additionally investigated the FBP1-interactome based on the STRING interaction database \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Notably, the interactome analysis revealed that the pathways and gene ontology terms connected to unfolded protein binding, heat shock protein binding, protein processing in endoplasmic reticulum and proteasome were highly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). It is important that the FBP1 mutants (G164D and F194S) more strongly interacted with almost all these proteins compared to FBP1 WT (Additional file 1: Table S7), suggesting that these molecular pathways are involved in the pathogenesis of FBPase deficiency.\u003c/p\u003e \u003cp\u003eInherited mutations can break native protein folding, resulting in the formation of misfolded proteins that are consequently retained in the endoplasmic reticulum (ER). These unfolded proteins undergo mannose trimming by ER associated mannosidase and degradation by proteasome (endoplasmic reticulum-associated degradation; ERAD), which can be suppressed via the inhibition of ER associated mannosidase activity using kifunensine \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. According to previous studies, protein expression of FBP1 is regulated by the ubiquitin proteasome and autophagy pathways \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Thus, to address the involvement of ERAD or protein degradation pathways in FBP1 mutant protein expression, we examined the effects of various inhibitors, such as a proteasome inhibitor (MG-132), an autophagy inhibitor (3-methyladenine), and a potent inhibitor of the ER mannosidase I (kifunensine), on the protein expression of the FBP1 mutants with G164D and F194S. Immunoblot analysis revealed that MG-132 and 3-methyladenine had no impact on the protein expression of these mutants (Additional file 3: Figure S2c). By contrast, kifunensine upregulated the protein expression of these FBP1 mutants with G164D and F194S, suggesting that these mutants underwent ERAD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). For further confirmation this notion, we performed confocal microscopy analysis to determine the intracellular localization of the FBP1 mutants with G164D and F194S, particularly in relation to ER markers. These mutants were primarily, but not completely, found to be colocalized in the ER (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). In addition, immunoblot analyses using the ER fraction revealed that the protein expression of FBP1 mutants with G164D and F194S compared with WT was markedly high in the ER compared to that in the whole cell lysate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eThe heat-shock proteins (HSPs) are a family of molecular chaperones, which collectively form a network that is critical for protein folding \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Moreover, missense mutations were shown to shift the folding equilibrium toward a partially folded state, thus increasing the cellular fraction of HSPs relative to the WT protein \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Based on these findings, we evaluated the binding of HSP70, HSP90, HSP60 and TCP1 to the FBP1 proteins using anti-Myc immunoprecipitation with subsequent immunoblot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). The FBP1 mutants with G164D and F194S had significantly increased interactions with HSP70, HSP90, HSP60 and TCP1, to a greater extent than the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ek), indicating that these mutants affect the folding equilibrium. Taken together, the FBP1 variants with G164D and F194S mutations caused protein misfolding, which led to the reduction of protein expression and aggregation via the ERAD system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eExamination of mutation genotype and functional phenotype in all FBP1 missense mutants\u003c/h2\u003e \u003cp\u003eTo date, various \u003cem\u003eFBP1\u003c/em\u003e mutations (14 missense mutations, 12 deletion mutations, four nonsense mutations, four insertions/duplications, two splices, and one indel) have been reported \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The reported missense mutations are D119N \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, P120L \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, R158W \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, G164S \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, A177D \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, F194S \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, G207R \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, N213K \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, G260R \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, E281K \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, P284R \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, G294E \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, G294V \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and V325A \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. We examined the relationship between the mutated site and the substitution of hydrophobicity in these 14 types of \u003cem\u003eFBP1\u003c/em\u003e missense mutations and G164D mutation we newly identified in this patient (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Eight types of mutation (G164D, G164S, A177D, F194S, G207R, G260R, P284R, and G294E) exhibited substitutions of hydrophobic amino acids with hydrophilic amino acids, and one mutation (R158W) exhibited a substitution of a hydrophilic amino acid with a hydrophobic amino acid. These nine mutations, defined as mutations with changing hydrophobicity, were not located at key amino acid residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, indicated by arrowheads) within functional motifs of the substrate binding site, metal binding site, or AMP binding site. Six mutations (D119N, P120L, N213K, E281K, G294V, and V325A) did not generate any change to amino acid hydrophobicity, and four of these mutations (D119N, P120L, N213K, and E281K) were directly located at important amino acid residues of the enzyme activity [D119N and E281K were located at key residues in the metal binding site; P120L was located in the linker lesion between the metal binding site (D119 and L121) and the substrate binding site (D122); and N213K was located in the substrate binding region (N213-Y216)] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, indicated by arrowheads).\u003c/p\u003e \u003cp\u003eBased on this information, we assessed how that the alteration of hydrophobicity affects the enzyme activitiy and the protein aggregation. We generated the mutant HepG2 cell line on FBP1-KO background as used previously. Then, these clones were separated into two groups: 6 mutants without change in hydrophobicity (indicated in blue) and 9 mutants with change in hydrophobicity (indicated in red) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We firstly validated the similar gene transduction in each clone by qPCR (Additional file 3: Figure S3). Although two of mutants, G207R and V325A, kept the enzyme activity compared to WT clone, most of clones demonstrated a loss of FBPase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eIn terms of protein level, there were several exceptions, but FBP1 expression tended to be different depending on the substitution of hydrophobicity. The mutant group without changes in hydrophobicity (indicated in blue), except G294V, maintained a similar level of protein expression as WT, whereas the mutants group with changes in hydrophobicity (indicated in red), except G207R, exhibited decreased protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Consistent with the protein expression, immunofluorescence staining demonstrated that all mutants with protein expression similar to WT were diffusely localized in the cytoplasm, whereas mutants with decreased protein expression aggregated in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Subsequently, we observed a strong negative correlation between the number of cells with FBP1 aggregates and its protein expression among the FBP1 missense mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). These data suggested that the protein aggregation could be linked to the substitution of hydrophobicity, and FBPase activities has more broadly affected by the missense mutations independent of hydrophobicity status. As there were several exceptions including G207R and V325A, the other constructive feature could be associated to enzymatic function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eFBP1 missense mutations were categorized into three functional phenotypes\u003c/h2\u003e \u003cp\u003eBased on these results, we categorized the \u003cem\u003eFBP1\u003c/em\u003e missense mutations into three functional groups (Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The Type 1 mutations (D119N, P120L, N213K, and E281K) are direct substitution of pivotal amino acid residues inside enzyme activity site (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). These mutations did not change their amino acid hydrophobicity and protein expression and were diffusely localized in the cytoplasm similar to WT (Table\u0026nbsp;1). Therefore, these mutations cause a primary loss of FBPase enzymatic activity through mutations of key amino acid residues in the functional motif of enzymatic activity (indicated by arrowheads) without affecting protein expression and cytoplasmic localization. Indeed, Type 1 mutations are characterized by no change in hydrophobicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eType 2 mutations (R158W, G164D, G164S, A177D, F194S, G260R, P284R, G294E, and G294V) are likely to be out of the important amino acid residues in the functional motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) and appear to cluster around the substrate binding pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In line with the changes in amino acid hydrophobicity (except for G294V), Type 2 mutations decreased protein expression and caused aggregation in the cytoplasm, possibly due to protein misfolding.\u003c/p\u003e \u003cp\u003eType 3 mutations (G207R and V325A) were structurally distant from sites associated with the enzyme activity motif and substrate binding pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). These mutations exhibited normal FBPase enzyme activity and their protein expression and cytoplasmic localization were similar to those of WT regardless of amino acid hydrophobicity, indicating that Type 3 mutations are likely non-pathogenic in terms of biochemical phenotype of FBPase deficiency. In fact, the previous reports suggested that the cases with V325A had no functional defect \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs expected, when we examined the binding ability of all mutants to HSP, only Type 2 mutants increased the interaction with HSP70 and HSP90 to a greater extent than those seen with WT, Type 1, and Type 3 mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and Additional file 3: Figure S4). Furthermore, the binding ability of FBP1 WT and mutants to either HSP70 or HSP90 was significantly correlated with the number of cells with FBP1 aggregates (%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Thus, we postulate that decreased protein expression due to protein misfolding in association with HSP recognition and protein aggregation via ERAD system is involved in the pathogenesis of FBPase deficiency, particularly in Type 2 mutations.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFBPase deficiency is sometimes misdiagnosed or diagnosed in delay following initial suspicion of other energy-related deficiencies. Our patient\u0026rsquo;s case and the observations we made in the present study illustrate how FBPase deficiency can be confused with disorders of fatty acid oxidation. While acylcarnitine profiles are useful for the diagnosis of the latter, we advocate that fructose tolerance test and genetic analyses would be beneficial and provide informative confirmation to the diagnosis of patients with FBPase deficiency.\u003c/p\u003e \u003cp\u003eIn general, fructose loads can lead to large, rapid expansions in the hexose- and triose-phosphate pools, potentially providing increased substrate for all central carbon metabolic pathways, including glycolysis, glycogenesis, gluconeogenesis, lipogenesis, and oxidative phosphorylation. In FBPase deficiency, marked decrease in intracellular free phosphate due to hepatic accumulation of fructose 1,6-bisphosphate can inhibit glycogenolysis, leading to fructose induced hypoglycemia.\u003c/p\u003e \u003cp\u003eTo date, several harmful mutations in the coding region of FBP1 have been reported. Santer \u003cem\u003eet al\u003c/em\u003e. reviewed 35 different mutations, including 14 missense mutations, 12 deletion mutations, 4 nonsense mutations, 4 insertions/duplications, two splices, and one indel \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although the FBP1 c.581T\u0026thinsp;\u0026gt;\u0026thinsp;C (which results in the missense mutation F194S) and c.490G\u0026thinsp;\u0026gt;\u0026thinsp;A (which affects the neighboring nucleotide) mutations have been previously demonstrated 3\u003csup\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, the c.491G\u0026thinsp;\u0026gt;\u0026thinsp;A mutation (responsible for the missense mutation G164D) demonstrated in this study is novel. The G164 and F194 mutations, which are located in the β-strand (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), demonstrated the substitution of hydrophobic amino acids with hydrophilic amino acids (Table\u0026nbsp;1), suggesting certain conformational changes. The examinations carried out in this study confirmed that the FBP1 mutants with G164D and F194S decreased protein expression and resulted in a loss of FBPase enzyme activity.\u003c/p\u003e \u003cp\u003eThe interactome analysis based on Liquid chromatography-tandem MS data for binding partners demonstrated that FBP1, particularly in its mutant forms, interacts with the proteins involved in the molecular chaperone related to unfolded protein response including heat shock protein (HSP). Protein misfolding has been recognized as an important pathophysiological cause of protein deficiency in some genetic disorders, such as Fabry disease, Pompe disease, and Gaucher disease \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Inherited mutations can break native protein folding, resulting in the formation of misfolded proteins that are consequently retained in the endoplasmic reticulum (ER). The heat-shock proteins (HSPs) are a family of molecular chaperones, which collectively form a network that is critical for protein folding \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. It has been suggested that HSP70 recognizes unfolded proteins, and HSP90 recognizes partially folded proteins, but fully folded proteins do not bind with either HSP70 or HSP90 \u003csup\u003e23\u003c/sup\u003e. Additionally, missense mutations have been shown to shift protein folding equilibrium toward a partially folded state, thus increasing the cellular fraction of HSP70 and HSP90 relative to WT proteins \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Some proteins can only be partially folded by HSP70 and therefore require additional assistance from HSP60 (chaperonin) in order to acquire a folded functional conformation \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Aberrant protein aggregation was found to be controlled by chaperonins containing TCP-1. Based on the findings that FBP1 variants with G164D and F194S mutations have increased interactions with HSP70, HSP90, HSP60 and TCP1 and are partially aggregated and trapped in the ER, we conclude that the decrement in protein expression detected in FBPase deficiency is, at least in part, a result of protein misfolding.\u003c/p\u003e \u003cp\u003eFinally, 15 FBP1 missense mutations (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) were reviewed and classified into three categories. Type 1 causes loss of enzyme activity due to mutations in functional domain with unchanged protein expression and diffuse cytoplasmic localization. Type 1 mutations do not change their amino acid hydrophobicity. Type 2 causes loss of enzyme activity with reduction in protein expression and ER aggregation due to protein misfolding. Type 2 mutations change amino acid hydrophobicity, except for G294V. It is noted that G294V from the Type 2 category exhibits unchanges in its amino acid hydrophobicity, suggesting that G294 plays a role in FBP1 protein folding. Type 3 mutations are likely non-pathogenic mutations in regard to obvious phenotype of FBPase deficiency, whereas the relationship between these mutations and disease onset are obscure. This finding is consistent with clinical characteristics, but the further investigation could be needed to uncover the role of our defined sites.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the context of genotype biochemical phenotype associations, previous \u003cem\u003ein vitro\u003c/em\u003e studies evaluated only two missense mutations (D119N and G164S) and, consistent with our findings, D119N decreased enzymatic activity with no impact on protein expression \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, whereas G164S decreased protein expression \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Thus, G207R and V325A were defined as Type 3 mutations. The G207R mutation, wherein the second allele exhibited a deletion of exon 8, was present in FBPase deficiency \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This variant has been previously reported in the heterozygous state with an allele frequency of 0.0001498 in 10 European (non-Finnish) individuals \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, although its pathological effects have not yet been elucidated. By contrast, the V325A mutation is not recorded in the dbSNP database. Although this mutation was observed in patients with FBPase deficiency, it was found to harbor the same mutant alleles as G164S. Based on an examination of the chimeric V325A mutation, Kikawa \u003cem\u003eet al.\u003c/em\u003e suggested that this mutation does not play a pathogenic role in FBPase deficiency \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, which is consistent with the findings of our study. Besides missense mutations, several types of \u003cem\u003eFBP1\u003c/em\u003e mutations (12 deletion mutations, four nonsense mutations, four insertions/duplications, two splices, and one indel) have been reported \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Previous \u003cem\u003ein vitro\u003c/em\u003e studies reported that c.704delC, c.838delT, and c.960dupG all decreased truncated FBP1 protein expression \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, the mechanism that leads to a decrease in truncated FBP1 protein expression has not been sufficiently elucidated. Considering our findings, protein misfolding may be involved in the underlying disease pathophysiology in these truncated types of \u003cem\u003eFBP1\u003c/em\u003e mutations, similar to Type 2 mutations.\u003c/p\u003e \u003cp\u003eThe clinical applications of pharmacological chaperone therapy for Fabry disease have become increasingly popular. The chaperone molecules support the folding of mutated enzymes and increase their stability and activity, which may be broadly applicable to other protein deficiencies. Life-threatening episodes of hypoglycemia and lactic acidosis are sometimes triggered by fasting and febrile infections during childhood in cases of FBPase deficiency. Yet, to date, no preventive agent has been developed for these patients and their symptoms. Our findings indicate the possibility that certain patients with Type 2 mutation may respond to such kind of chaperone molecules, although Type 1 mutations are likely to be untreatable cases of FBPase deficiency. At any rate, further studies are warranted in order to fully understand the pathophysiology of this rare disease as well as to clarify the usefulness and efficacy of pharmacological chaperone therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eendoplasmic reticulum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBPase\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFructose-1,6-bisphosphatase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBP1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFBP gene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHSP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eheat-shock proteins\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVAF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evariant allele frequency\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewild-type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRahil JF, de Maine MM, Benkovic SJ. 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Fructose 1,6-bisphosphatase deficiency: enzyme and mutation analysis performed on calcitriol-stimulated monocytes with a note on long-term prognosis. Journal of inherited metabolic disease\u003cem\u003e. \u003c/em\u003e2010.\u003c/li\u003e\n\u003cli\u003eParenti G. Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics. EMBO molecular medicine\u003cem\u003e. \u003c/em\u003e2009;1(5):268-279.\u003c/li\u003e\n\u003cli\u003eMoon S, Kim JH, Han JH, et al. Novel compound heterozygous mutations in the fructose-1,6-bisphosphatase gene cause hypoglycemia and lactic acidosis. Metabolism: clinical and experimental\u003cem\u003e. \u003c/em\u003e2011;60(1):107-113.\u003c/li\u003e\n\u003cli\u003eKikawa Y, Inuzuka M, Jin BY, et al. Identification of a genetic mutation in a family with fructose-1,6- bisphosphatase deficiency. Biochemical and biophysical research communications\u003cem\u003e. \u003c/em\u003e1995;210(3):797-804.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Categories of FBP1 missense mutations\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"1012\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.49802371541502%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.57707509881423%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.683794466403162%\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnzymatic Activity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.486166007905139%\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9.0909%;\" width=\"5.632411067193676%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntracellular\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.225296442687747%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAggregation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"10.57312252964427%\"\u003e\n \u003cp\u003e\u003cstrong\u003eChaperone Binding Ability\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"13.339920948616601%\"\u003e\n \u003cp\u003e\u003cstrong\u003eType of Amino Acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 11.6601%;\" width=\"10.079051383399209%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHydropathy Index\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eStructure\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmino Acid Change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\n \u003cp\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompared to Wild-type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.504950495049505%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompared to Wild-type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 9.0909%;\" width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocalization\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHSP70\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHSP90\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003eWild-type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003eWild-type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"23\" style=\"width: 54.3478%;\" width=\"53.81565906838454%\"\u003e\n \u003cp\u003e\u003cstrong\u003eType 1: Loss of enzyme activity due to mutations in functional domain with unchanged protein expression and diffuse cytoplasmic localization\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eD119N\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetal binding site\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003eno change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiffuse\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(cytoplasm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e9.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eacidic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eneutral\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-3.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-3.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;-strand\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP120L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLinker lesion at metal and substrate binding site\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003eno change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiffuse\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(cytoplasm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-1.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;-strand\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eN213K\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubstrate binding site\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003eno change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiffuse\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(cytoplasm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e13.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eneutral\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ebasic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-3.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-3.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003eND\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eE281K\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetal binding site\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003eno change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiffuse\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(cytoplasm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eacidic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ebasic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-3.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-3.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003eturn\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"23\" style=\"width: 54.3478%;\" width=\"53.81565906838454%\"\u003e\n \u003cp\u003e\u003cstrong\u003eType 2: Loss of enzyme activity with decreased protein expression and ER aggregation in association with hydrophobicity change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eR158W\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAggregated\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in ER)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e69.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ebasic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003earomatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-4.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026alpha;-helix\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eG164D\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAggregated\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in ER)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e71.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e13.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e15.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eacidic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-3.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;-strand\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eG164S\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAggregated\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in ER)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e42.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e12.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eneutral\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;-strand\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eA177D\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAggregated\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in ER)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e73.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e24.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e37.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eacidic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-3.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;-strand\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eF194S\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAggregated\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in ER)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e62.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e15.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e19.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003earomatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eneutral\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;-strand\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eG260R\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAggregated\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in ER)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e73.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e18.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e28.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ebasic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-4.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003eND\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP284R\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAggregated\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in ER)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e57.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e41.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e47.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ebasic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-1.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-4.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026alpha;-helix\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eG294E\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAggregated\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in ER)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e64.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e64.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e89.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eacidic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-3.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003eND\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eG294V\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003edecrease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAggregated\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in ER)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e65.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003eND\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"23\" width=\"38.71287128712871%\"\u003e\n \u003cp\u003e\u003cstrong\u003eType 3: Likely non-pathogenic mutations in regards to biochemical phenotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eG207R\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003eno change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003eno change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiffuse\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(cytoplasm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e12.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophilic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ebasic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-4.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003eND\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.514851485148515%\"\u003e\n \u003cp\u003e\u003cstrong\u003eV325A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.613861386138613%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"9.702970297029703%\"\u003e\n \u003cp\u003e\u003cstrong\u003eno change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003eno change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.752475247524752%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.643564356435643%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiffuse\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(cytoplasm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.237623762376238%\"\u003e\n \u003cp\u003e\u003cstrong\u003e25.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.247524752475248%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"6.633663366336633%\"\u003e\n \u003cp\u003e\u003cstrong\u003ehydrophobic-\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ealiphatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"5.445544554455446%\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.653465346534653%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.03960396039604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026alpha;-helix\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations: ND, no data; ER, endoplasmic reticulum\u003c/strong\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"fructose-1, 6-bisphosphatase deficiency, FBP1, hypoglycemia, protein misfolding, heat shock protein","lastPublishedDoi":"10.21203/rs.3.rs-2185039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2185039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eFructose-1,6-bisphosphatase (FBPase) deficiency, caused by an FBP1 mutation, is an autosomal recessive disorder characterized by hypoglycemic lactic acidosis. The mechanism by which the mutations cause enzyme activity loss is uncertain.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe performed whole-exome sequencing in an adult patient with severe hypoglycemic lactic acidosis and identified that the patient carried compound heterozygous missense mutations of FBP1 with c.491G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.G164D) and c.581T\u0026thinsp;\u0026gt;\u0026thinsp;C (p.F194S).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBiochemical analysis revealed that FBP1 mutant (G164D or F194S) decreased protein expression and enzyme activity loss. The interactome analysis for binding partners demonstrated that G164D and F194S mutants interact with the proteins involved in unfolded protein response. Additionally, G164D and F194S mutants aggregated in the endoplasmic reticulum, suggesting the involvement of protein misfolding in its pathogenesis. All FBP1 missense mutations previously reported were classified into three functional categories: Type 1 mutations, located at pivotal residues in enzyme activity motifs with no effects on protein expression; Type 2 mutations, which mediate changes in amino acid hydrophobicity and structurally cluster around the substrate-binding pocket, are associated with aggregation in the endoplasmic reticulum, and decreased protein expression; and Type 3 mutations, which are likely non-pathogenic mutations.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eProtein misfolding contributes to FBPase deficiency pathogenesis, particularly in Type 2 mutations.\u003c/p\u003e","manuscriptTitle":"Characterization based on genotype–biochemical phenotype association in fructose-1,6-bisphosphatase deficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-02 19:36:55","doi":"10.21203/rs.3.rs-2185039/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4ec78aa6-a438-4300-a543-658ec4e93542","owner":[],"postedDate":"November 2nd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":16607402,"name":"Biological sciences/Genetics/Mutation"},{"id":16607403,"name":"Biological sciences/Cell biology/Protein folding/Chaperones"}],"tags":[],"updatedAt":"2023-07-29T07:07:31+00:00","versionOfRecord":{"articleIdentity":"rs-2185039","link":"https://doi.org/10.1038/s42003-023-05160-y","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2023-07-28 04:00:00","publishedOnDateReadable":"July 28th, 2023"},"versionCreatedAt":"2022-11-02 19:36:55","video":"","vorDoi":"10.1038/s42003-023-05160-y","vorDoiUrl":"https://doi.org/10.1038/s42003-023-05160-y","workflowStages":[]},"version":"v1","identity":"rs-2185039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2185039","identity":"rs-2185039","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}