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Metabolism is intertwined with various cellular processes, including controlling cell fate, influencing tumorigenesis, participating in stress responses and more. Metabolism is a complex, interdependent network, and local perturbations can have indirect effects that are pervasive across the metabolic network. Current analytical and technical limitations have long created a bottleneck in metabolic data interpretation. To address these shortcomings, we developed Metaboverse, a user-friendly tool to facilitate data exploration and hypothesis generation. Here we introduce algorithms that leverage the metabolic network to extract complex reaction patterns from data. To minimize the impact of missing measurements within the network, we introduce methods that enable pattern recognition across multiple reactions. Using Metaboverse, we identify a previously undescribed metabolite signature that correlated with survival outcomes in early stage lung adenocarcinoma patients. Using a yeast model, we identify metabolic responses suggesting an adaptive role of citrate homeostasis during mitochondrial dysfunction facilitated by the citrate transporter, Ctp1. We demonstrate that Metaboverse augments the user’s ability to extract meaningful patterns from multi-omics datasets to develop actionable hypotheses.

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl–coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation (OXPHOS). Metabolic inflexibility, defined as an impaired ability to switch between these fuels, is implicated in a number of metabolic diseases. Here we explore the mechanism by which physical inactivity promotes metabolic inflexibility in skeletal muscle. We developed a mouse model of sedentariness by small mouse cage (SMC) that, unlike other classic models of disuse in mice, faithfully recapitulates metabolic responses that occur in humans. Bioenergetic phenotyping of mitochondria displayed metabolic inflexibility induced by physical inactivity, demonstrated by a reduction in pyruvate-stimulated respiration (JO2) in absence of a change in palmitate-stimulatedJO2. Pyruvate resistance in these mitochondria was likely driven by a decrease in phosphatidylethanolamine (PE) abundance in the mitochondrial membrane. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) was sufficient to induce metabolic inflexibility measured at the whole-body level, as well as at the level of skeletal muscle mitochondria. Low mitochondrial PE was sufficient to increase glucose flux towards lactate. We further implicate that resistance to pyruvate metabolism is due to attenuated mitochondrial entry via mitochondrial pyruvate carrier (MPC). These findings suggest a novel mechanism by which mitochondrial PE directly regulates MPC activity to modulate metabolic flexibility.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both through inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of the mitochondrial membrane potential is also observed in primary and immortalized mammalian cells as well as inDrosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial functions even with defective mitochondria.

The fate of pyruvate is a defining feature in many cell types. One major fate is mitochondrial entry via the mitochondrial pyruvate carrier (MPC). We found that diffuse large B cell lymphomas (DLBCLs) consume mitochondrial pyruvate via glutamate-pyruvate transaminase 2 to enable α-ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid cycle in DLBCLs. As a result, MPC inhibition led to decreased glutaminolysis in DLBCLs, opposite to previous observations in other cell types. We also found that MPC inhibition or genetic depletion decreased DLBCL proliferation in an extracellular matrix (ECM)–like environment and xenografts, but not in a suspension environment. Moreover, the metabolic profile of DLBCL cells in ECM is markedly different from cells in a suspension environment. Thus, we conclude that the synergistic consumption and assimilation of glutamine and pyruvate enables DLBCL proliferation in an extracellular environment-dependent manner.

SUMMARYThe fate of pyruvate, which is modulated by the activity of the mitochondrial pyruvate carrier (MPC), is a defining metabolic feature in many cancers. Diffuse large B-cell lymphomas (DLBCLs) are a genetically and metabolically heterogeneous cancer. Although MPC expression and activity differed between DLBCL subgroups, mitochondrial pyruvate was uniformly consumed by glutamate pyruvate transaminase 2 (GPT2) to support α-ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid (TCA) cycle in DLBCLs. Furthermore, we found that MPC inhibition unexpectedly leads to decreased glutaminolysis, which is contrary to previous observations in other cell types. We also discovered that MPC inhibition and depletion only decreased DLBCL proliferation in an extracellular matrix (ECM) environment and in vivo xenografts, but not in the typical DLBCL suspension environment. We also have found that the metabolic profile of DLBCL cells in ECM is markedly different from cells in suspension environment. Thus, we report that besides the canonical glutamate dehydrogenase (GDH)-mediated glutaminolysis, the non-canonical GPT2 mediated consumption and assimilation of glutamine and pyruvate in DLBCLs enables their proliferation in an extracellular environment-dependent manner.HIGHLIGHTSGlutamine, but not glucose, is a major carbon source for the tricarboxylic acid cycle in diffuse large B-cell lymphomas.Mitochondrial pyruvate supports glutaminolysis in diffuse large B-cell lymphomas by supplying pyruvate for glutamate pyruvate transaminase 2-mediated α -ketoglutarate production.Mitochondrial pyruvate carrier inhibition leads to decreased glutaminolysis in diffuse large B-cell lymphomas.α -ketoglutarate production is important for diffuse large B-cell lymphoma proliferation in a solid extracellular matrix environment.Mitochondrial pyruvate carrier activity supports diffuse large B-cell lymphoma proliferation in a solid extracellular matrix environment and in mouse xenografts.

Metabolism forms a complex, interdependent network, and perturbations can have indirect effects that are pervasive. Identifying these patterns and their consequences is difficult, particularly when the effects occur across canonical pathways, and these difficulties have long acted as a bottleneck in metabolic data analysis. This challenge is compounded by technical limitations in metabolomics approaches that garner incomplete datasets. Current network-based tools generally utilize pathway-level analysis lacking the granular resolution required to provide context into the effects of all perturbations, regardless of magnitude, across the metabolic network. To address these shortcomings, we introduce algorithms that allow for the real-time extraction of regulatory patterns and trends from user data. To minimize the impact of missing measurements within the metabolic network, we introduce methods that enable complex pattern recognition across multiple reactions. These tools are available interactively within the user-friendly Metaboverse app (https://github.com/Metaboverse) to facilitate exploration and hypothesis generation. We demonstrate that expected signatures are accurately captured by Metaboverse. Using public lung adenocarcinoma data, we identify a previously undescribed multi-dimensional signature that correlated with survival outcomes in lung adenocarcinoma patients. Using a model of respiratory deficiency, we identify relevant and previously unreported regulatory patterns that suggest an important compensatory role for citrate during mitochondrial dysfunction. This body of work thus demonstrates that Metaboverse can identify and decipher complex signals from data that have been otherwise difficult to identify with previous approaches.

Summary: Metabolic pathways regulate T cell development and function, but many remain understudied. Recently, the mitochondrial pyruvate carrier (MPC) was identified as the transporter that mediates pyruvate entry into mitochondria, promoting pyruvate oxidation. Here we find that deleting Mpc1, an obligate MPC subunit, in the hematopoietic system results in a specific reduction in peripheral αβ T cell numbers. MPC1-deficient T cells have defective thymic development at the β-selection, intermediate single positive (ISP)-to-double-positive (DP), and positive selection steps. We find that early thymocytes deficient in MPC1 display alterations to multiple pathways involved in T cell development. This results in preferred escape of more activated T cells. Finally, mice with hematopoietic deletion of Mpc1 are more susceptible to experimental autoimmune encephalomyelitis. Altogether, our study demonstrates that pyruvate oxidation by T cell precursors is necessary for optimal αβ T cell development and that its deficiency results in reduced but activated peripheral T cell populations. : Ramstead et al. show that mitochondrial pyruvate carrier 1, which mediates mitochondrial uptake of pyruvate, is necessary for proper development of αβ T cells. Consequently, deletion of MPC1 in early thymic development results in reduced numbers and abnormal activation of peripheral αβ T cells.

Reliance on glutamine has long been considered to be a hallmark of cancer cell metabolism. However, some recent studies have challenged this notion in vivo , prompting a need for further clarification of the role of glutamine metabolism in cancer. We find that there is ample evidence of an essential role for glutamine in tumors, and that a variety of factors, including tissue type, the underlying cancer genetics, the tumor microenvironment, and other variables such as diet and host physiology collectively influence the role of glutamine in cancer. Thus the requirements for glutamine in cancer are overall highly heterogeneous. In this review we discuss the implications both for basic science and for targeting glutamine metabolism in cancer therapy.

The metabolic rewiring of cardiomyocytes is a widely accepted hallmark of heart failure (HF). These metabolic changes include a decrease in mitochondrial pyruvate oxidation and an increased export of lactate. We identify the Mitochondrial Pyruvate Carrier (MPC) and the cellular lactate exporter Monocarboxylate Transporter 4 (MCT4), as pivotal nodes in this metabolic axis. We observed that cardiac assist device-induced myocardial recovery in chronic HF patients was coincident with increased myocardial expression of the MPC. Moreover, the genetic ablation of the MPC in cultured cardiomyocytes and in adult murine hearts was sufficient to induce hypertrophy and HF. Conversely, MPC overexpression attenuated drug-induced hypertrophy in a cell-autonomous manner. We also introduced a novel, highly potent MCT4 inhibitor that mitigated hypertrophy in cultured cardiomyocytes and in mice. Together, we find that alteration of the pyruvate-lactate axis is a fundamental and early feature of cardiac hypertrophy and failure.

Acetate is a major nutrient that supports acetyl-coenzyme A (Ac-CoA) metabolism and thus lipogenesis and protein acetylation. Its source however has been unclear. Here we report that pyruvate, the end product of glycolysis and key node in central carbon metabolism, quantitatively generates acetate in mammals. This phenomenon becomes more pronounced in contexts of nutritional excess such as during hyperactive glucose metabolism. Conversion of pyruvate to acetate occurs through two mechanisms: 1) coupling to reactive oxygen species (ROS), and 2) neomorphic enzyme activity from keto acid dehydrogenases that enable function as pyruvate decarboxylases. Further, we demonstrate that de novo acetate production sustains Ac-CoA pools and cell proliferation in limited metabolic environments such as during mitochondrial dysfunction or ATP citrate lyase (ACLY) deficiency. De novo acetate production occurs in mammals and is further coupled to mitochondrial metabolism providing possible regulatory mechanisms and links to pathophysiology.

CHC22 clathrin plays a key role in intracellular membrane trafficking of the insulin-responsive GLUT4 glucose transporter, and so in post-prandial clearance of glucose from human blood. We performed population genetic and phylogenetic analyses of the CLTCL1 gene, encoding CHC22, to understand its variable presence in vertebrates and gain insight into its functional evolution. Analysis of ~50 complete vertebrate genomes showed independent loss of CLTCL1 in nine lineages after it arose from a gene duplication during the emergence of jawed vertebrates. All vertebrates considered here retained the parent CLTC gene encoding CHC17 clathrin, which mediates endocytosis and other housekeeping membrane traffic pathways, as performed by the single clathrin gene product in nonvertebrate eukaryotes. Statistical analysis provides evidence of strong purifying selection over phylogenetic timescales for CLTCL1, as well as for CLTC, supporting preserved functionality of CHC22 in those species that have retained CLTCL1. In population genetic analyses of humans and chimpanzees, extensive allelic diversity was observed for CLTCL1 compared to CLTC. In all human populations, two variants of CLTCL1 segregate at high frequency, resulting in CHC22 protein with either methionine or valine at position 1316. The V1316 variant occurs only in humans, but the same site is polymorphic in non-human primates as well. Analysis of archaic and ancient humans assigned the appearance of the derived V1316 allele to 500-50 KYA. Balancing selection on the two high-frequency CHC22 variants is inferred, with V1316 being more frequent in farming, as compared to hunter-gatherer populations. Together these analyses suggest that CHC22 clathrin is undergoing selection in humans related to its role in nutrient metabolism. Consistent with this conclusion, we observed functional differences between the two CHC22 variants in their ability to control GLUT4 membrane traffic, as predicted by structural modeling and differences in cellular dynamics of the two variants.

A key strategy targeting the metabolic changes underlying tumorigenesis involves inhibiting the glutaminase isozyme GLS to disrupt glutamine catabolism. However, identifying patient groups for this approach remains a challenge. Here, we define the roles of GLS and its less studied counterpart liver-type glutaminase (GLS2) in breast cancer. We find that GLS2 is upregulated and essential for TCA cycle anaplerosis, cell proliferation, and tumorigenesis in luminal-subtype breast cancers, which account for >70% of disease incidence, and that it mediates resistance to GLS-selective inhibitors. Although GLS is overexpressed in basal-subtype breast cancers, sensitivity to GLS inhibitors depends on glutamate efflux through the xCT antiporter. These findings establish an important role for GLS2 in tumorigenesis and suggest patient stratification approaches for glutaminase-targeted therapy.

Background: In conditions of hyperactive cellular metabolism, excessive cellular nutrient uptake results in incomplete metabolism and excretion of intermediates. These intermediates may serve as unconventional fuel sources satisfy metabolic demands during nutrient scarcity. Interestingly, acetate metabolism provides a parallel pathway for acetyl-CoA production and allows for protein acetylation and lipogenesis independent of citrate conversion to acetyl-CoA. This pathway is important in tumorigenesis, immune alertness, neural plasticity, and other diverse contexts but the origin of acetate has been unclear. Thus, we have conducted a re-evaluation of endogenous acetate generation and the biological relevance. Method: Cancer cells were cultured in RPMI medium with 13C labelled nutrients in the presence of 18O2. Mouse models of soft tissue sarcoma were generated in a mixed 129/SVJae and C57BL/6 background. A jugular vein catheter was surgically implanted and exteriorized via a vascular access port, which allows infusion of [13C6]-glucose via the venous catheter. Acetate in medium and blood is quantified using [2H3] labelled acetate as the standard after 2-hydrazinoquinoline (HQ) derivatization, and other polar metabolites were directly analyzed after cold methanol extraction. All metabolites were measured using liquid chromatography coupled with high resolution mass spectrometer. Results: By employing multiple-isotope tracing technology, quantitative proteomics, and mouse genetics tools, we demonstrated that acetate is quantitatively generated from pyruvate, the end product of glycolysis and key node in central carbon metabolism in cancer cells and tumor. One reaction mechanism found to generate acetate occur through altered enzyme activity of thiamine-dependent keto acid dehydrogenases, which transforms their activity to keto acid decarboxylases. The other reaction mechanism to generate acetate occur by reaction with reactive oxygen species (ROS), a finding which potentially links this pathway to numerous physiological and pathophysiological processes. Thiamine starvation and the addition of exogeneous ROS greatly stimulated the ROS contribution to acetate production, which can be used to replenish intracellular acetyl groups. Thus, increased acetate production and release could potentially favor the neighboring cells deficient in cytosolic acetyl-CoA, as demonstrated by co-culturing ACLY KO cells with HCT116 cells. Conclusion: We have not only provided direct evidence that acetate arises from endogenous metabolism of glucose in mammalian cells, but also identified the regulatory mechanisms, which involve ROS and mitochondrial functions. Note: This abstract was not presented at the meeting. Citation Format: Xiaojing Liu, Daniel E. Cooper, Ahmad A. Cluntun, Marc O. Warmoes, Steven Zhao, Michael A. Reid, Juan Liu, Peder J. Lund, Mariana Lopes, Benjamin A. Garcia, Kathryn E. Wellen, David G. Kirsch, Jason W. Locasale. Acetate production from glucose and coupling to mitochondrial metabolism in mammals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 792.

The ability of a cell to exhibit anchorage independent growth is a hallmark of cancer, and has been extensively investigated in hopes of finding new therapeutic strategies. In particular, the processes that give rise to and support this dysregulated growth phenotype, which allows cells to proliferate in the absence of survival signals resulting from their attachment to a basement membrane, has been of interest. The contribution of oncogenes and tumor suppressors on anchorage independent growth has been well studied; however, the alterations in cellular metabolism that support this process are much less defined. Changes in cellular metabolism are among the most notable hallmarks of solid tumors, where glucose is imported at high rates and exported from cancer cells as lactate, instead of being utilized within the citric acid cycle (TCA cycle) (1,2). This process, known as the Warburg effect, represents the primary metabolic phenotype associated with cancer cells. In order to adapt to this altered metabolism, cancer cells have been shown to rely on other primary metabolic pathways to support their increased requirements for biosynthesis and redox control to allow for their sustained proliferation. In particular, glutamine metabolism has been shown to supplement the decrease in glucose-derived carbon entering the TCA cycle in 2D cultured cancer cells (Figure 1, compare quiescent versus proliferating cells). However, it remains unclear how expendable glutaminkfueled anaplerosis is for cells grown in 3D. In their recent Nature article, DeBerardinis and coworkers [2016] set out to describe the metabolic alterations that accompany the anchorage-independent growth of human cancer cells when cultured as a 2D monolayer or as a 3D spheroid, thereby mimicking the detachment from the extracellular matrix that often occurs during tumorigenesis (3). They find that the same cancer cells exhibit drastically different metabolic phenotypes when grown as spheroids than they do in a monolayer culture.

Background Glucose metabolism links metabolic status to protein acetylation. However, it remains poorly understood to what extent do features of glucose metabolism contribute to protein acetylation and whether the process can be dynamically and quantitatively regulated by differing rates of glycolysis. Results Here, we show that titratable rates of glycolysis with corresponding changes in the levels of glycolytic intermediates result in a graded remodeling of a bulk of the metabolome and resulted in gradual changes in total histone acetylation levels. Dynamic histone acetylation levels were found and most strongly correlated with acetyl coenzyme A (ac-CoA) levels and inversely associated with the ratio of ac-CoA to free CoA. A multiplexed stable isotopic labeling by amino acids in cell culture (SILAC)-based proteomics approach revealed that the levels of half of identified histone acetylation sites as well as other lysine acylation modifications are tuned by the rate of glycolysis demonstrating that glycolytic rate affects specific acylation sites. Conclusions We demonstrate that histone acylation is directly sensed by glucose flux in a titratable, dose-dependent manner that is modulated by glycolytic flux and that a possible function of the Warburg Effect, a metabolic state observed in cancers with enhanced glucose metabolism, is to confer specific signaling effects on cells.

Aerobic glycolysis or the Warburg Effect (WE) is characterized by the increased metabolism of glucose to lactate. It remains unknown what quantitative changes to the activity of metabolism are necessary and sufficient for this phenotype. We developed a computational model of glycolysis and an integrated analysis using metabolic control analysis (MCA), metabolomics data, and statistical simulations. We identified and confirmed a novel mode of regulation specific to aerobic glycolysis where flux through GAPDH, the enzyme separating lower and upper glycolysis, is the rate-limiting step in the pathway and the levels of fructose (1,6) bisphosphate (FBP), are predictive of the rate and control points in glycolysis. Strikingly, negative flux control was found and confirmed for several steps thought to be rate-limiting in glycolysis. Together, these findings enumerate the biochemical determinants of the WE and suggest strategies for identifying the contexts in which agents that target glycolysis might be most effective. DOI: http://dx.doi.org/10.7554/eLife.03342.001, eLife digest Cells generate energy from a sugar called glucose via a process called glycolysis. This process involves many enzymes that catalyze 10 different chemical reactions, and it essentially converts glucose step-by-step into a simpler chemical called pyruvate. Pyruvate is then normally transported into structures within the cell called mitochondria, where it is further broken down using oxygen to release more energy. However, in cells that are rapidly dividing, pyruvate is converted into another chemical called lactate—which releases energy more quickly, but releases less energy overall. Cancer cells often convert most of their glucose into lactate, rather than breaking down pyruvate in their mitochondria: an observation known as the ‘Warburg effect’. And while many factors affect how a cell releases energy from pyruvate, it remains unclear what regulates which of these biochemical processes is most common in a living cell. In this study, Shestov et al. have developed a computational model for the process of glycolysis and used this to investigate the causes of the Warburg Effect. The model was based on the known characteristics of the enzymes and chemical reactions involved at each step. It predicted that the activity of the enzyme called GAPDH, which carries out the sixth step in glycolysis, in many cases affects how much lactate is produced. This suggests that this enzyme represents a bottleneck in the pathway. Next, Shestov et al. performed experiments where they used drugs to block different stages of the glycolysis pathway, and confirmed that the GAPDH enzyme is important for regulating this pathway in living cancer cells too. In these treated cells, the levels of a chemical called fructose-1,6-biphosphate (which is made in a step in the pathway between glucose and pyruvate) were either very high or very low. Shestov et al. proposed that the flow of chemicals through the glycolysis pathway is controlled by the GAPDH enzyme when the chemicals used by the enzymes upstream of GAPDH in the pathway (which includes fructose-1,6-biphosphate) are plentiful. However, if these chemicals are limited, other enzymes that are involved in earlier steps of the pathway regulate the process instead. The findings of Shestov et al. reveal that the regulation of glycolysis is more complex than previously thought, and is also very different when cells are undergoing the Warburg Effect. In the future, these findings might help to identify the types of cancer that could be effectively treated using drugs that target the glycolysis process, which are currently being tested in pre-clinical studies. DOI: http://dx.doi.org/10.7554/eLife.03342.002

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously. Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Deficiency of fructose-1,6-bisphosphatase (FBP) results in impaired gluconeogenesis, which is characterized by episodes of hyperventilation, apnea, hypoglycemia, and metabolic and lactic acidosis. This autosomal recessive disorder is caused by mutations in the FBP1 gene, which encodes for fructose-1,6-bisphosphatase 1 (FBP1). Although FBP1 gene mutations have been described in FBP-deficient individuals of various ethnicities, there has been limited investigation into the genetics of this disorder in Arab patients. This study employed five consanguineous Arab families, in which 17 patients were clinically diagnosed with FBP deficiency. Seven patients and six carrier parents were analyzed for mutations in the FBP1 gene. DNA sequencing of the FBP1 gene identified two novel mutations in these families. A novel six nucleotide repetitive insertion, c114_119dupCTGCAC, was identified in patients from three families. This mutation encodes for a duplication of two amino acids (p.Cys39_Thr40dup) in the N-terminal domain of FBP1. A novel nonsense c.841G>T mutation encoding for a p.Glu281X truncation in the active site of FBP1 was discovered in patients from two families. The newly identified mutations in the FBP1 gene are predicted to produce FBP1 deficiency. These mutations are the only known genetic causes of FBP deficiency in Arab patients. The p.Cys39_Thr40dup is the first reported amino acid duplication in FBP deficiency patients. This study provides a strong rationale for genetic testing of FBP deficient patients of Arab ethnicity for recurrent or novel mutations in the FBP1 gene.