Inositol Pyrophosphates as Versatile Metabolic Messengers.

Discovered in 1993, inositol pyrophosphates are evolutionarily conserved signaling metabolites whose versatile modes of action are being increasingly appreciated. These include their emerging roles as energy regulators, phosphodonors, steric/allosteric regulators, and G protein-coupled receptor messengers. Through studying enzymes that metabolize inositol pyrophosphates, progress has also been made in elucidating the various cellular and physiological functions of these pyrophosphate-containing, energetic molecules. The two main forms of inositol pyrophosphates, 5-IP7 and IP8, synthesized respectively by inositol-hexakisphosphate kinases (IP6Ks) and diphosphoinositol pentakisphosphate kinases (PPIP5Ks), regulate phosphate homeostasis, ATP synthesis, and several other metabolic processes ranging from insulin secretion to cellular energy utilization. Here, we review the current understanding of the catalytic and regulatory mechanisms of IP6Ks and PPIP5Ks, as well as their counteracting phosphatases. We also highlight the genetic and cellular evidence implicating inositol pyrophosphates as essential mediators of mammalian metabolic homeostasis.

Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast.

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.

Assembly mechanism of Integrator's RNA cleavage module.

The modular Integrator complex is a transcription regulator that is essential for embryonic development. It attenuates coding gene expression via premature transcription termination and performs 3'-processing of non-coding RNAs. For both activities, Integrator requires endonuclease activity that is harbored by an RNA cleavage module consisting of INTS4-9-11. How correct assembly of Integrator modules is achieved remains unknown. Here, we show that BRAT1 and WDR73 are critical biogenesis factors for the human cleavage module. They maintain INTS9-11 inactive during maturation by physically blocking the endonuclease active site and prevent premature INTS4 association. Furthermore, BRAT1 facilitates import of INTS9-11 into the nucleus, where it is joined by INTS4. Final BRAT1 release requires locking of the mature cleavage module conformation by inositol hexaphosphate (IP6). Our data explain several neurodevelopmental disorders caused by BRAT1, WDR73, and INTS11 mutations as Integrator assembly defects and reveal that IP6 is an essential co-factor for cleavage module maturation.

Calcium-release channels: structure and function of IP3 receptors and ryanodine receptors.

Ca2+-release channels are giant membrane proteins that control the release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. The two members, ryanodine receptors (RyRs) and inositol-1,4,5-trisphosphate receptors (IP3Rs), are evolutionarily related and are both activated by cytosolic Ca2+. They share a common architecture, but RyRs have evolved additional modules in the cytosolic region. Their massive size allows for the regulation by tens of proteins and small molecules, which can affect the opening and closing of the channels. In addition to Ca2+, other major triggers include IP3 for the IP3Rs and depolarization of the plasma membrane for a particular RyR subtype expressed in skeletal muscle. Their size has made them popular targets for study via electron microscopic methods, with current structures culminating near 3 Å. The available structures have provided many new mechanistic insights into the binding of auxiliary proteins and small molecules, how these can regulate channel opening, and the mechanisms of disease-associated mutations. They also help scrutinize previously proposed binding sites, as some of these are now incompatible with the structures. Many questions remain around the structural effects of posttranslational modifications, additional binding partners, and the higher order complexes these channels can make in situ. This review summarizes our current knowledge about the structures of Ca2+-release channels and how this informs on their function.

Inositol serves as a natural inhibitor of mitochondrial fission by directly targeting AMPK.

Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. Here, we show that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Inositol decline by IMPA1/2 deficiency elicits AMPK activation and mitochondrial fission without affecting ATP level, whereas inositol accumulation prevents AMPK-dependent mitochondrial fission. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Inositol directly binds to AMPKγ and competes with AMP for AMPKγ binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKγ for AMP binding. Hence, AMPK is an inositol sensor, whose inactivation by inositol serves as a mechanism to restrict mitochondrial fission.

Oxidative bursts of single mitochondria mediate retrograde signaling toward the ER.

The emerging role of mitochondria as signaling organelles raises the question of whether individual mitochondria can initiate heterotypic communication with neighboring organelles. Using fluorescent probes targeted to the endoplasmic-reticulum-mitochondrial interface, we demonstrate that single mitochondria generate oxidative bursts, rapid redox oscillations, confined to the nanoscale environment of the interorganellar contact sites. Using probes fused to inositol 1,4,5-trisphosphate receptors (IP3Rs), we show that Ca2+ channels directly sense oxidative bursts and respond with Ca2+ transients adjacent to active mitochondria. Application of specific mitochondrial stressors or apoptotic stimuli dramatically increases the frequency and amplitude of the oxidative bursts by enhancing transient permeability transition pore openings. Conversely, blocking interface Ca2+ transport via elimination of IP3Rs or mitochondrial calcium uniporter channels suppresses ER-mitochondrial Ca2+ feedback and cell death. Thus, single mitochondria initiate local retrograde signaling by miniature oxidative bursts and, upon metabolic or apoptotic stress, may also amplify signals to the rest of the cell.

Biochemical and structural characterization of an inositol pyrophosphate kinase from a giant virus.

Kinases that synthesize inositol phosphates (IPs) and pyrophosphates (PP-IPs) control numerous biological processes in eukaryotic cells. Herein, we extend this cellular signaling repertoire to viruses. We have biochemically and structurally characterized a minimalist inositol phosphate kinase (i.e., TvIPK) encoded by Terrestrivirus, a nucleocytoplasmic large ("giant") DNA virus (NCLDV). We show that TvIPK can synthesize inositol pyrophosphates from a range of scyllo- and myo-IPs, both in vitro and when expressed in yeast cells. We present multiple crystal structures of enzyme/substrate/nucleotide complexes with individual resolutions from 1.95 to 2.6 Å. We find a heart-shaped ligand binding pocket comprising an array of positively charged and flexible side chains, underlying the observed substrate diversity. A crucial arginine residue in a conserved "G-loop" orients the γ-phosphate of ATP to allow substrate pyrophosphorylation. We highlight additional conserved catalytic and architectural features in TvIPK, and support their importance through site-directed mutagenesis. We propose that NCLDV inositol phosphate kinases may have assisted evolution of inositol pyrophosphate signaling, and we discuss the potential biogeochemical significance of TvIPK in soil niches.

The COP9 signalosome: A versatile regulatory hub of Cullin-RING ligases.

The COP9 signalosome (CSN) is a universal regulator of Cullin-RING ubiquitin ligases (CRLs) - a family of modular enzymes that control various cellular processes via timely degradation of key signaling proteins. The CSN, with its eight-subunit architecture, employs multisite binding of CRLs and inactivates CRLs by removing a small ubiquitin-like modifier named neural precursor cell-expressed, developmentally downregulated 8 (Nedd8). Besides the active site of the catalytic subunit CSN5, two allosteric sites are present in the CSN, one of which recognizes the substrate recognition module and the presence of CRL substrates, and the other of which can 'glue' the CSN-CRL complex by recruitment of inositol hexakisphosphate. In this review, we present recent findings on the versatile regulation of CSN-CRL complexes.

Disorder-to-order active site capping regulates the rate-limiting step of the inositol pathway.

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD+-dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus Thermochaetoides thermophila. By resolving the native structure at 2.48 Å (FSC = 0.143), we revealed a fully populated active site. Utilizing 3D variability analysis, we uncovered conformational states of MIPS, enabling us to directly visualize an order-to-disorder transition at its catalytic center. An acyclic intermediate of G6P occupied the active site in two out of the three conformational states, indicating a catalytic mechanism where electrostatic stabilization of high-energy intermediates plays a crucial role. Examination of all isomerases with known structures revealed similar fluctuations in secondary structure within their active sites. Based on these findings, we established a conformational selection model that governs substrate binding and eventually inositol availability. In particular, the ground state of MIPS demonstrates structural configurations regardless of substrate binding, a pattern observed across various isomerases. These findings contribute to the understanding of MIPS structure-based function, serving as a template for future studies targeting regulation and potential therapeutic applications.

MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

The human milk component myo-inositol promotes neuronal connectivity.

Effects of micronutrients on brain connectivity are incompletely understood. Analyzing human milk samples across global populations, we identified the carbocyclic sugar myo-inositol as a component that promotes brain development. We determined that it is most abundant in human milk during early lactation when neuronal connections rapidly form in the infant brain. Myo-inositol promoted synapse abundance in human excitatory neurons as well as cultured rat neurons and acted in a dose-dependent manner. Mechanistically, myo-inositol enhanced the ability of neurons to respond to transsynaptic interactions that induce synapses. Effects of myo-inositol in the developing brain were tested in mice, and its dietary supplementation enlarged excitatory postsynaptic sites in the maturing cortex. Utilizing an organotypic slice culture system, we additionally determined that myo-inositol is bioactive in mature brain tissue, and treatment of organotypic slices with this carbocyclic sugar increased the number and size of postsynaptic specializations and excitatory synapse density. This study advances our understanding of the impact of human milk on the infant brain and identifies myo-inositol as a breast milk component that promotes the formation of neuronal connections.

ß-lapachone regulates mammalian inositol pyrophosphate levels in an NQO1- and oxygen-dependent manner.

Inositol pyrophosphates (PP-InsPs) are energetic signaling molecules with important functions in mammals. As their biosynthesis depends on ATP concentration, PP-InsPs are tightly connected to cellular energy homeostasis. Consequently, an increasing number of studies involve PP-InsPs in metabolic disorders, such as type 2 diabetes, aspects of tumorigenesis, and hyperphosphatemia. Research conducted in yeast suggests that the PP-InsP pathway is activated in response to reactive oxygen species (ROS). However, the precise modulation of PP-InsPs during cellular ROS signaling is unknown. Here, we report how mammalian PP-InsP levels are changing during exposure to exogenous (H2O2) and endogenous ROS. Using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that PP-InsP levels decrease upon exposure to oxidative stressors in HCT116 cells. Application of quinone drugs, particularly ß-lapachone (ß-lap), under normoxic and hypoxic conditions enabled us to produce ROS in cellulo and to show that ß-lap treatment caused PP-InsP changes that are oxygen-dependent. Experiments in MDA-MB-231 breast cancer cells deficient of NAD(P)H:quinone oxidoreductase-1 (NQO1) demonstrated that ß-lap requires NQO1 bioactivation to regulate the cellular metabolism of PP-InsPs. Critically, significant reductions in cellular ATP concentrations were not directly mirrored in reduced PP-InsP levels as shown in NQO1-deficient MDA-MB-231 cells treated with ß-lap. The data presented here unveil unique aspects of ß-lap pharmacology and its impact on PP-InsP levels. The identification of different quinone drugs as modulators of PP-InsP synthesis will allow the overall impact on cellular function of such drugs to be better appreciated.

Insights into the roles of inositol hexakisphosphate kinase 1 (IP6K1) in mammalian cellular processes.

Inositol phosphates and their metabolites play a significant role in several biochemical pathways, gene expression regulation, and phosphate homeostasis. Among the different inositol phosphates, inositol hexakisphosphate (IP6) is a substrate of inositol hexakisphosphate kinases (IP6Ks), which phosphorylate one or more of the IP6 phosphate groups. Pyrophosphorylation of IP6 leads to the formation of inositol pyrophosphates, high-energy signaling molecules that mediate physiological processes through their ability to modify target protein activities, either by directly binding to their target protein or by pyrophosphorylating protein serine residues. 5-diphosphoinositol pentakisphosphate, the most abundant inositol pyrophosphate in mammals, has been extensively studied and found to be significantly involved in a wide range of physiological processes. Three IP6K (IP6K1, IP6K2, and IP6K3) isoforms regulate IP7 synthesis in mammals. Here, we summarize our current understanding of IP6K1's roles in cytoskeletal remodeling, trafficking, cellular migration, metabolism, gene expression, DNA repair, and immunity. We also briefly discuss current gaps in knowledge, highlighting the need for further investigation.

E41K mutation activates Bruton's tyrosine kinase by stabilizing an inositol hexakisphosphate-dependent invisible dimer.

Bruton's tyrosine kinase (BTK) regulates diverse cellular signaling of the innate and adaptive immune system in response to microbial pathogens. Downregulation or constitutive activation of BTK is reported in patients with autoimmune diseases or various B-cell leukemias. BTK is a multidomain protein tyrosine kinase that adopts an Src-like autoinhibited conformation maintained by the interaction between the kinase and PH-TH domains. The PH-TH domain plays a central role in regulating BTK function. BTK is activated by binding to PIP3 at the plasma membrane upon stimulation by the B-cell receptor (BCR). The PIP3 binding allows dimerization of the PH-TH domain and subsequent transphosphorylation of the activation loop. Alternatively, a recent study shows that the multivalent T-cell-independent (TI) antigen induces BCR response by activating BTK independent of PIP3 binding. It was proposed that a transiently stable IP6-dependent PH-TH dimer may activate BTK during BCR activation by the TI antigens. However, no IP6-dependent PH-TH dimer has been identified yet. Here, we investigated a constitutively active PH-TH mutant (E41K) to determine if the elusive IP6-dependent PH-TH dimer exists. We showed that the constitutively active E41K mutation activates BTK by stabilizing the IP6-dependent PH-TH dimer. We observed that a downregulating mutation in the PH-TH domain (R28H) linked to X-linked agammaglobulinemia impairs BTK activation at the membrane and in the cytosol by preventing PH-TH dimerization. We conclude that the IP6 dynamically remodels the BTK active fraction between the membrane and the cytoplasm. Stimulating with IP6 increases the cytosolic fraction of the activated BTK.

The yeast AMP-activated protein kinase Snf1 phosphorylates the inositol polyphosphate kinase Kcs1.

The yeast Snf1/AMP-activated kinase (AMPK) maintains energy homeostasis, controlling metabolic processes and glucose derepression in response to nutrient levels and environmental cues. Under conditions of nitrogen or glucose limitation, Snf1 regulates pseudohyphal growth, a morphological transition characterized by the formation of extended multicellular filaments. During pseudohyphal growth, Snf1 is required for wild-type levels of inositol polyphosphate (InsP), soluble phosphorylated species of the six-carbon cyclitol inositol that function as conserved metabolic second messengers. InsP levels are established through the activity of a family of inositol kinases, including the yeast inositol polyphosphate kinase Kcs1, which principally generates pyrophosphorylated InsP7. Here, we report that Snf1 regulates Kcs1, affecting Kcs1 phosphorylation and inositol kinase activity. A snf1 kinase-defective mutant exhibits decreased Kcs1 phosphorylation, and Kcs1 is phosphorylated in vivo at Ser residues 537 and 646 during pseudohyphal growth. By in vitro analysis, Snf1 directly phosphorylates Kcs1, predominantly at amino acids 537 and 646. A yeast strain carrying kcs1 encoding Ser-to-Ala point mutations at these residues (kcs1-S537A,S646A) shows elevated levels of pyrophosphorylated InsP7, comparable to InsP7 levels observed upon deletion of SNF1. The kcs1-S537A,S646A mutant exhibits decreased pseudohyphal growth, invasive growth, and cell elongation. Transcriptional profiling indicates extensive perturbation of metabolic pathways in kcs1-S537A,S646A. Growth of kcs1-S537A,S646A is affected on medium containing sucrose and antimycin A, consistent with decreased Snf1p signaling. This work identifies Snf1 phosphorylation of Kcs1, collectively highlighting the interconnectedness of AMPK activity and InsP signaling in coordinating nutrient availability, energy homoeostasis, and cell growth.

Rice myo-inositol-3-phosphate synthase 2 (RINO2) alleviates heat injury-induced impairment in pollen germination and tube growth by modulating Ca2+ signaling and actin filament cytoskeleton.

Low phytic acid (lpa) crop is considered as an effective strategy to improve crop nutritional quality, but a substantial decrease in phytic acid (PA) usually has negative effect on agronomic performance and its response to environment adversities. Myo-inositol-3-phosphate synthase (MIPS) is the rate-limiting enzyme in PA biosynthesis pathway, and regarded as the prime target for engineering lpa crop. In this paper, the rice MIPS gene (RINO2) knockout mutants and its wild type were performed to investigate the genotype-dependent alteration in the heat injury-induced spikelet fertility and its underlying mechanism for rice plants being imposed to heat stress at anthesis. Results indicated that RINO2 knockout significantly enhanced the susceptibility of rice spikelet fertility to heat injury, due to the severely exacerbated obstacles in pollen germination and pollen tube growth in pistil for RINO2 knockout under high temperature (HT) at anthesis. The loss of RINO2 function caused a marked reduction in inositol and phosphatidylinositol derivative concentrations in the HT-stressed pollen grains, which resulted in the strikingly lower content of phosphatidylinositol 4,5-diphosphate (PI (4,5) P2) in germinating pollen grain and pollen tube. The insufficient supply of PI (4,5) P2 in the HT-stressed pollen grains disrupted normal Ca2+ gradient in the apical region of pollen tubes and actin filament cytoskeleton in growing pollen tubes. The severely repressed biosynthesis of PI (4,5) P2 was among the regulatory switch steps leading to the impaired pollen germination and deformed pollen tube growth for the HT-stressed pollens of RINO2 knockout mutants.

Carbon usage in yellow-fleshed Manihot esculenta storage roots shifts from starch biosynthesis to cell wall and raffinose biosynthesis via the myo-inositol pathway.

Cassava is a crucial staple crop for smallholder farmers in tropical Asia and Sub-Saharan Africa. Although high yield remains the top priority for farmers, the significance of nutritional values has increased in cassava breeding programs. A notable negative correlation between provitamin A and starch accumulation poses a significant challenge for breeding efforts. The negative correlation between starch and carotenoid levels in conventional and genetically modified cassava plants implies the absence of a direct genomic connection between the two traits. The competition among various carbon pathways seems to account for this relationship. In this study, we conducted a thorough analysis of 49 African cassava genotypes with varying levels of starch and provitamin A. Our goal was to identify factors contributing to differential starch accumulation. Considering carotenoid levels as a confounding factor in starch production, we found that yellow- and white-fleshed storage roots did not differ significantly in most measured components of starch or de novo fatty acid biosynthesis. However, genes and metabolites associated with myo-inositol synthesis and cell wall polymer production were substantially enriched in high provitamin A genotypes. These results indicate that yellow-fleshed cultivars, in comparison to their white-fleshed counterparts, direct more carbon toward the synthesis of raffinose and cell wall components. This finding is underlined by a significant rise in cell wall components measured within the 20 most contrasting genotypes for carotenoid levels. Our findings enhance the comprehension of the biosynthesis of starch and carotenoids in the storage roots of cassava.

Duf89 abets lncRNA control of fission yeast phosphate homeostasis via its antagonism of precocious lncRNA transcription termination.

Fission yeast phosphate homeostasis gene pho1 is actively repressed during growth in phosphate-rich medium by transcription in cis of a long noncoding (lnc) RNA from the 5' flanking prt(nc-pho1) gene. Pho1 expression is: (i) derepressed by genetic maneuvers that favor precocious lncRNA 3'-processing and termination, in response to DSR and PAS signals in prt; and (ii) hyperrepressed in genetic backgrounds that dampen 3'-processing/termination efficiency. Governors of 3'-processing/termination include the RNA polymerase CTD code, the CPF (cleavage and polyadenylation factor) complex, termination factors Seb1 and Rhn1, and the inositol pyrophosphate signaling molecule 1,5-IP8 Here, we present genetic and biochemical evidence that fission yeast Duf89, a metal-dependent phosphatase/pyrophosphatase, is an antagonist of precocious 3'-processing/termination. We show that derepression of pho1 in duf89Δ cells correlates with squelching the production of full-length prt lncRNA and is erased or attenuated by: (i) DSR/PAS mutations in prt; (ii) loss-of-function mutations in components of the 3'-processing and termination machinery; (iii) elimination of the CTD Thr4-PO4 mark; (iv) interdicting CTD prolyl isomerization by Pin1; (v) inactivating the Asp1 kinase that synthesizes IP8; and (vi) loss of the putative IP8 sensor Spx1. The findings that duf89Δ is synthetically lethal with pho1-derepressive mutations CTD-S7A and aps1Δ-and that this lethality is rescued by CTD-T4A, CPF/Rhn1/Pin1 mutations, and spx1Δ-implicate Duf89 more broadly as a collaborator in cotranscriptional regulation of essential fission yeast genes. The duf89-D252A mutation, which abolishes Duf89 phosphohydrolase activity, phenocopied duf89 +, signifying that duf89Δ phenotypes are a consequence of Duf89 protein absence, not absence of Duf89 catalysis.

Association of embryonic inositol status with susceptibility to neural tube defects, metabolite profile, and maternal inositol intake.

Maternal nutrition contributes to gene-environment interactions that influence susceptibility to common congenital anomalies such as neural tube defects (NTDs). Supplemental myo-inositol (MI) can prevent NTDs in some mouse models and shows potential for prevention of human NTDs. We investigated effects of maternal MI intake on embryonic MI status and metabolism in curly tail mice, which are genetically predisposed to NTDs that are inositol-responsive but folic acid resistant. Dietary MI deficiency caused diminished MI in maternal plasma and embryos, showing that de novo synthesis is insufficient to maintain MI levels in either adult or embryonic mice. Under normal maternal dietary conditions, curly tail embryos that developed cranial NTDs had significantly lower MI content than unaffected embryos, revealing an association between diminished MI status and failure of cranial neurulation. Expression of inositol-3-phosphate synthase 1, required for inositol biosynthesis, was less abundant in the cranial neural tube than at other axial levels. Supplemental MI or d-chiro-inositol (DCI) have previously been found to prevent NTDs in curly tail embryos. Here, we investigated the metabolic effects of MI and DCI treatments by mass spectrometry-based metabolome analysis. Among inositol-responsive metabolites, we noted a disproportionate effect on nucleotides, especially purines. We also found altered proportions of 5-methyltetrahydrolate and tetrahydrofolate in MI-treated embryos suggesting altered folate metabolism. Treatment with nucleotides or the one-carbon donor formate has also been found to prevent NTDs in curly tail embryos. Together, these findings suggest that the protective effect of inositol may be mediated through the enhanced supply of nucleotides during neural tube closure.

A recurrent missense variant in ITPR3 causes demyelinating Charcot-Marie-Tooth with variable severity.

Charcot-Marie-Tooth (CMT) disease is a neuromuscular disorder affecting the peripheral nervous system. The diagnostic yield in demyelinating CMT (CMT1) is typically ∼80-95%, of which at least 60% is due to the PMP22 gene duplication. The remainder of CMT1 is more genetically heterogeneous. We used whole exome and whole genome sequencing data included in the GENESIS database to investigate novel causal genes and mutations in a cohort of ∼2,670 individuals with CMT neuropathy. A recurrent heterozygous missense variant p.Thr1424Met in the recently described CMT gene ITPR3, encoding IP3R3 (inositol 1,4,5-trisphosphate receptor 3) was identified. This previously reported p.Thr1424Met change was present in 33 affected individuals from nine unrelated families from multiple populations, representing an unusual recurrence rate at a mutational hotspot, strengthening the gene-disease relationship (GnomADv4 allele frequency 1.76e-6). Sanger sequencing confirmed the co-segregation of the CMT phenotype with the presence of the mutation in autosomal dominant and de novo inheritance patterns, including a four-generation family with multiple affected second-degree cousins. Probands from all families presented with slow nerve conduction velocities, matching the diagnostic category of CMT1. Remarkably, we observed a uniquely variable clinical phenotype for age at onset and phenotype severity in p.Thr1424Met carrying patients, even within families. Finally, we present data supportive of a dominant-negative effect of the p.Thr1424Met mutation with associated changes in protein expression in patient-derived cells.