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Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder, causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO) account for ~25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first-ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (a) show advanced retinal degeneration in a male patient (68 years of age) harboring four transcriptionally active intact copies of rhodopsin, (b) recapitulated the clinical phenotypes using retinal organoids, and (c) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (quantitative real-time PCR [qRT-PCR] and bulk RNA sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300 days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that RHO-CNV associated adRPdevelops as a result of protein overexpression, thereby overloading the photoreceptor post-translational modification machinery.

Glycogen storage disease type‐Ia (GSD‐Ia), characterized by impaired blood glucose homeostasis, is caused by a deficiency in glucose‐6‐phosphatase‐α (G6Pase‐α or G6PC). Using the G6pc‐R83C mouse model of GSD‐Ia, we explored a CRISPR/Cas9‐based double‐strand DNA oligonucleotide (dsODN) insertional strategy that uses the nonhomologous end‐joining repair mechanism to correct the pathogenic p.R83C variant in G6pc exon‐2. The strategy is based on the insertion of a short dsODN into G6pc exon‐2 to disrupt the native exon and to introduce an additional splice acceptor site and the correcting sequence. When transcribed and spliced, the edited gene would generate a wild‐type mRNA encoding the native G6Pase‐α protein. The editing reagents formulated in lipid nanoparticles (LNPs) were delivered to the liver. Mice were treated either with one dose of LNP‐dsODN at age 4 weeks or with two doses of LNP‐dsODN at age 2 and 4 weeks. The G6pc‐R83C mice receiving successful editing expressed ~4% of normal hepatic G6Pase‐α activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed normalized blood metabolite profile. The outcomes are consistent with preclinical studies supporting previous gene augmentation therapy which is currently in clinical trials. This editing strategy may offer the basis for a therapeutic approach with an earlier clinical intervention than gene augmentation, with the additional benefit of a potentially permanent correction of the GSD‐Ia phenotype. [ABSTRACT FROM AUTHOR]

The photoreceptor outer segment is a highly specialized primary cilium that is essential for phototransduction and vision. Biallelic pathogenic variants in the cilia-associated gene CEP290 cause non-syndromic Leber congenital amaurosis 10 (LCA10) and syndromic diseases, where the retina is also affected. While RNA antisense oligonucleotides and gene editing are potential treatment options for the common deep intronic variant c.2991+1655A>G in CEP290, there is a need for variant-independent approaches that could be applied to a broader spectrum of ciliopathies. Here, we generated several distinct human models of CEP290-related retinal disease and investigated the effects of the flavonoid eupatilin as a potential treatment. Eupatilin improved cilium formation and length in CEP290 LCA10 patient-derived fibroblasts, in gene-edited CEP290 knockout (CEP290 KO) RPE1 cells, and in both CEP290 LCA10 and CEP290 KO iPSCs-derived retinal organoids. Furthermore, eupatilin reduced rhodopsin retention in the outer nuclear layer of CEP290 LCA10 retinal organoids. Eupatilin altered gene transcription in retinal organoids by modulating the expression of rhodopsin and by targeting cilia and synaptic plasticity pathways. This work sheds light on the mechanism of action of eupatilin and supports its potential as a variant-independent approach for CEP290-associated ciliopathies. [ABSTRACT FROM AUTHOR]

Neutropenia and neutrophil dysfunction are common in many diseases, although their etiology is often unclear. Previous views held that there was a single ER enzyme, glucose-6-phosphatase-[alpha] (G6Pase-[alpha]), whose activity--limited to [...]

A locus marked by genetic markers that span a 3-cM region on chromosome 11q23 have been linked to glycogen-storage disease type 1 (GSD-1). GSD-1, also known as von Gierke disease, is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase) activity. Only GSD-1b patients, of patients with the four types of the autosomal recessive disorder, suffer infectious complications, the result of the heritable neutropenia and deficiencies in functions of neutrophils and monocytes. The GSD-1b patients have complications not easily blamed on deficiency of G6Pase. In all the subgroups other clinical signs and symptoms are the same and caused by abnormalities in metabolism of glucose-6-phosphate (G6P). More research will give insight into genetic links of G6P metabolism and neutrophil-monocyte dysfunction. Consanguineous families and one nonconsanguineous family of Bedouin, North African, Iranian Jewish, Israeli Arab, Pakistani and European origins were studied.

Glycogen storage disease type‐Ia (GSD‐Ia), caused by a deficiency in glucose‐6‐phosphatase‐α (G6Pase‐α or G6PC), is characterized by impaired glucose homeostasis with a hallmark hypoglycemia, following a short fast. We have shown that G6pc‐deficient (G6pc−/−) mice treated with recombinant adeno‐associated virus (rAAV) vectors expressing either wild‐type (WT) (rAAV‐hG6PC‐WT) or codon‐optimized (co) (rAAV‐co‐hG6PC) human (h) G6Pase‐α maintain glucose homeostasis if they restore ≥3% of normal hepatic G6Pase‐α activity. The co vector, which has a higher potency, is currently being used in a phase I/II clinical trial for human GSD‐Ia (NCT 03517085). While routinely used in clinical therapies, co vectors may not always be optimal. Codon‐optimization can impact RNA secondary structure, change RNA/DNA protein‐binding sites, affect protein conformation and function, and alter posttranscriptional modifications that may reduce potency or efficacy. We therefore sought to develop alternative approaches to increase the potency of the G6PC gene transfer vectors. Using an evolutionary sequence analysis, we identified a Ser‐298 to Cys‐298 substitution naturally found in canine, mouse, rat, and several primate G6Pase‐α isozymes, that when incorporated into the WT hG6Pase‐α sequence, markedly enhanced enzymatic activity. Using G6pc−/− mice, we show that the efficacy of the rAAV‐hG6PC‐S298C vector was 3‐fold higher than that of the rAAV‐hG6PC‐WT vector. The rAAV‐hG6PC‐S298C vector with increased efficacy, that minimizes the potential problems associated with codon‐optimization, offers a valuable vector for clinical translation in human GSD‐Ia. [ABSTRACT FROM AUTHOR]

Hepatocellular adenoma/carcinoma (HCA/HCC) is a long‐term complication of glycogen storage disease type‐Ia (GSD‐Ia), which is caused by a deficiency in glucose‐6‐phosphatase‐α (G6Pase‐α or G6PC), a key enzyme in gluconeogenesis. Currently, there is no therapy to address HCA/HCC in GSD‐Ia. We have previously shown that a recombinant adeno‐associated virus (rAAV) vector‐mediated G6PC gene transfer to 2‐week‐old G6pc−/− mice prevents HCA development. However, it remains unclear whether G6PC gene transfer at the tumor developing stage of GSD‐Ia can prevent tumor initiation or abrogate the pre‐existing tumors. Using liver‐specific G6pc‐knockout (L‐G6pc−/−) mice that develop HCA/HCC, we now show that treating the mice at the tumor‐developing stage with rAAV‐G6PC restores hepatic G6Pase‐α expression, normalizes glucose homeostasis, and prevents de novo HCA/HCC development. The rAAV‐G6PC treatment also normalizes defective hepatic autophagy and corrects metabolic abnormalities in the nontumor liver tissues of both tumor‐free and tumor‐bearing mice. However, gene therapy cannot restore G6Pase‐α expression in the HCA/HCC lesions and fails to abrogate any pre‐existing tumors. We show that the expression of 11 β‐hydroxysteroid dehydrogenase type‐1 that mediates local glucocorticoid activation is downregulated in HCA/HCC lesions, leading to impairment in glucocorticoid signaling critical for gluconeogenesis activation. This suggests that local glucocorticoid action downregulation in the HCA/HCC lesions may suppress gene therapy mediated G6Pase‐α restoration. Collectively, our data show that rAAV‐mediated gene therapy can prevent de novo HCA/HCC development in L‐G6pc−/− mice at the tumor developing stage, but it cannot reduce any pre‐existing tumor burden. [ABSTRACT FROM AUTHOR]

A deficiency in glucose-6-phosphatase-α (G6Pase-α) in glycogen storage disease type Ia (GSD-Ia) leads to impaired glucose homeostasis and metabolic manifestations including hepatomegaly caused by increased glycogen and neutral fat accumulation. A recent report showed that G6Pase-α deficiency causes impairment in autophagy, a recycling process important for cellular metabolism. However, the molecular mechanism underlying defective autophagy is unclear. Here we show that in mice, liver-specific knockout of G6Pase-α (L-G6pc-/-) leads to downregulation of sirtuin 1 (SIRT1) signaling that activates autophagy via deacetylation of autophagy-related (ATG) proteins and forkhead box O (FoxO) family of transcriptional factors which transactivate autophagy genes. Consistently, defective autophagy in G6Pase-α-deficient liver is characterized by attenuated expressions of autophagy components, increased acetylation of ATG5 and ATG7, decreased conjugation of ATG5 and ATG12, and reduced autophagic flux. We further show that hepatic G6Pase-α deficiency results in activation of carbohydrate response element-binding protein, a lipogenic transcription factor, increased expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), a lipid regulator, and suppressed expression of PPAR-α, a master regulator of fatty acid β-oxidation, all contributing to hepatic steatosis and downregulation of SIRT1 expression. An adenovirus vector-mediated increase in hepatic SIRT1 expression corrects autophagy defects but does not rectify metabolic abnormalities associated with G6Pase-α deficiency. Importantly, a recombinant adeno-associated virus (rAAV) vector-mediated restoration of hepatic G6Pase-α expression corrects metabolic abnormalities, restores SIRT1-FoxO signaling, and normalizes defective autophagy. Taken together, these data show that hepatic G6Pase-α deficiency-mediated down-regulation of SIRT1 signaling underlies defective hepatic autophagy in GSD-Ia. [ABSTRACT FROM AUTHOR]

FDA-cleared ovarian cancer biomarkers are limited to CA-125 and HE4 for monitoring and recurrence and OVA1, a multivariate panel consisting of CA-125 and four additional biomarkers, for referring patients to a specialist. Due to relatively poor performance of these tests, more accurate and broadly applicable biomarkers are needed. We evaluated the dysregulation of 259 candidate cancer markers in serum samples from 499 patients. Sera were collected prospectively at 11 monitored sites under a single well-defined protocol. All stages of ovarian cancer and common benign gynecological conditions were represented. To ensure consistency and comparability of biomarker comparisons, all measurements were performed on a single platform, at a single site, using a panel of rigorously calibrated, qualified, high-throughput, multiplexed immunoassays and all analyses were conducted using the same software. Each marker was evaluated independently for its ability to differentiate ovarian cancer from benign conditions. A total of 175 markers were dysregulated in the cancer samples. HE4 (AUC = 0.933) and CA-125 (AUC = 0.907) were the most informative biomarkers, followed by IL-2 receptor α, α1-antitrypsin, C-reactive protein, YKL-40, cellular fibronectin, CA-72-4 and prostasin (AUC>0.800). To improve the discrimination between cancer and benign conditions, a simple multivariate combination of markers was explored using logistic regression. When combined into a single panel, the nine most informative individual biomarkers yielded an AUC value of 0.950, significantly higher than obtained when combining the markers in the OVA1 panel (AUC 0.912). Additionally, at a threshold sensitivity of 90%, the combination of the top 9 markers gave 88.9% specificity compared to 63.4% specificity for the OVA1 markers. Although a blinded validation study has not yet been performed, these results indicate that alternative biomarker combinations might lead to significant improvements in the detection of ovarian cancer. [ABSTRACT FROM AUTHOR]

Blood glucose homeostasis between meals depends upon production of glucose within the endoplasmic reticulum (ER) of the liver and kidney by hydrolysis of glucose-6-phosphate (G6P) into glucose and phosphate (Pi). This reaction depends on coupling the G6P transporter (G6PT) with glucose-6-phosphatase-α (G6Pase-α). Only one G6PT, also known as SLC37A4, has been characterized, and it acts as a Pi-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:Pi exchangers, have not been characterized functionally. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-α. G6PT- and mock-proteoliposomes are used as positive and negative controls, respectively. We show that SLC37A1 and SLC37A2 are ER-associated, Pi-linked antiporters, that can transport G6P. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-α. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis. [ABSTRACT FROM AUTHOR]

Background: Most women with a clinical presentation consistent with ovarian cancer have benign conditions. Therefore methods to distinguish women with ovarian cancer from those with benign conditions would be beneficial. We describe the development and preliminary evaluation of a serum-based multivariate assay for ovarian cancer. This hypothesis-driven study examined whether an informative pattern could be detected in stage I disease that persists through later stages. Methodology/Principal Findings: Sera, collected under uniform protocols from multiple institutions, representing 176 cases and 187 controls from women presenting for surgery were examined using high-throughput, multiplexed immunoassays. All stages and common subtypes of epithelial ovarian cancer, and the most common benign ovarian conditions were represented. A panel of 104 antigens, 44 autoimmune and 56 infectious disease markers were assayed and informative combinations identified. Using a training set of 91 stage I data sets, representing 61 individual samples, and an equivalent number of controls, an 11-analyte profile, composed of CA-125, CA 19-9, EGF-R, C-reactive protein, myoglobin, apolipoprotein A1, apolipoprotein CIII, MIP-1α, IL-6, IL-18 and tenascin C was identified and appears informative for all stages and common subtypes of ovarian cancer. Using a testing set of 245 samples, approximately twice the size of the model building set, the classifier had 91.3% sensitivity and 88.5% specificity. While these preliminary results are promising, further refinement and extensive validation of the classifier in a clinical trial is necessary to determine if the test has clinical value. Conclusions/Significance: We describe a blood-based assay using 11 analytes that can distinguish women with ovarian cancer from those with benign conditions. Preliminary evaluation of the classifier suggests it has the potential to offer approximately 90% sensitivity and 90% specificity. While promising, the performance needs to be assessed in a blinded clinical validation study. [ABSTRACT FROM AUTHOR]

The article provides an analysis of the profiling of serum antigens and autoimmune and infectious disease molecules to identify biomarkers dysregulated in epithelial ovarian cancer in the U.S. It states that systematic evaluation of the markers in a sample set under a well-defined Gynecologic Oncology Group protocol has been undertaken. With the use of a large sample cohort, the study also showed that some of the reported ovarian cancer biomarkers are more robust than others.

Glycogen storage disease type Ib (GSD-Ib) is caused by deficiencies in the glucose-6-phosphate (G6P) transporter (G6PT) that have been well characterized. Interestingly, deleterious mutations in the G6PT gene were identified in clinical cases of GSD type Ic (GSD-Ic) proposed to be deficient in an inorganic phosphate (Pi) transporter. We hypothesized that G6PT is both the G6P and PPi transporter. Using reconstituted proteoliposomes we show that both G6P and PPi are efficiently taken up into PPi-loaded G6PT-proteoliposomes. The G6P uptake activity decreases as the internal:external PPi ratio decreases and the PPi uptake activity decreases in the presence of external G6P. Moreover, G6P or PPi uptake activity is not detectable in PPi-loaded proteoliposomes containing the p.R28H G6PT null mutant. The G6PT-proteoliposome-mediated G6P or PPi uptake is inhibited by cholorgenic acid and vanadate, both specific G6PT inhibitors. Glucose-6-phosphatase-α (G6Pase-α), which facilitates microsomal G6P uptake by G6PT, fails to stimulate G6P uptake in PPi-loaded G6PT-proteoliposomes, suggesting that the G6Pase-α-mediated stimulation is caused by decreasing G6P and increasing PPi concentrations in microsomes. Taken together, our results suggest that G6PT has a dual role as a G6P and a PPi transporter and that GSD-Ib and GSD-Ic are deficient in the same G6PT gene.--Chen, S.-Y., Pan, C.-.J, Nandigama, K., Mansfield, B., Ambudkar, S., Chou, J. The glucose-6-phosphate transporter is a phosphate-linked antiporter deficient in glycogen storage disease type Ib and and Ic. [ABSTRACT FROM AUTHOR]

The islet-specific glucose-6-phosphatase-related protein (IGRP) has no known catalytic activity, but is of interest because it is the source of the peptide autoantigen targeted by a prevalent population of pathogenic CD8+ T cells in non-obese diabetic mice. To better understand the potential roles of this protein in diabetes mellitus, we examine the subcellular localization and membrane topography of human IGRP. We show that IGRP is a glycoprotein, held in the endoplasmic reticulum by nine transmembrane domains, which is degraded in cells predominantly through the proteasome pathway that generates the major histocompatibility complex class I-presented peptides. [Copyright &y& Elsevier]

Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), a key enzyme in endogenous glucose production. This autosomal recessive disorder is characterized by impaired glucose homeostasis and long-term complications of hepatocellular adenoma/carcinoma (HCA/HCC). We have shown that hepatic G6Pase-α deficiency-mediated steatosis leads to defective autophagy that is frequently associated with carcinogenesis. We now show that hepatic G6Pase-α deficiency also leads to enhancement of hepatic glycolysis and hexose monophosphate shunt (HMS) that can contribute to hepatocarcinogenesis. The enhanced hepatic glycolysis is reflected by increased lactate accumulation, increased expression of many glycolytic enzymes, and elevated expression of c-Myc that stimulates glycolysis. The increased HMS is reflected by increased glucose-6-phosphate dehydrogenase activity and elevated production of NADPH and the reduced glutathione. We have previously shown that restoration of hepatic G6Pase-α expression in G6Pase-α-deficient liver corrects metabolic abnormalities, normalizes autophagy, and prevents HCA/HCC development in GSD-Ia. We now show that restoration of hepatic G6Pase-α expression normalizes both glycolysis and HMS in GSD-Ia. Moreover, the HCA/HCC lesions in L -G6pc−/− mice exhibit elevated levels of hexokinase 2 (HK2) and the M2 isoform of pyruvate kinase (PKM2) which play an important role in aerobic glycolysis and cancer cell proliferation. Taken together, hepatic G6Pase-α deficiency causes metabolic reprogramming, leading to enhanced glycolysis and elevated HMS that along with impaired autophagy can contribute to HCA/HCC development in GSD-Ia. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphatase-β (G6Pase-β or G6PC3) deficiency is characterized by neutropenia and dysfunction in both neutrophils and macrophages. G6Pase-β is an enzyme embedded in the endoplasmic reticulum membrane that catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. To date, 33 separate G6PC3 mutations have been identified in G6Pase-β-deficient patients but only the p.R253H and p.G260R missense mutations have been characterized functionally for pathogenicity. Here we functionally characterize 16 of the 19 known missense mutations using a sensitive assay, based on a recombinant adenoviral vector-mediated expression system, to demonstrate pathogenicity. Fourteen missense mutations completely abolish G6Pase-β enzymatic activity while the p.S139I and p.R189Q mutations retain 49% and 45%, respectively of wild type G6Pase-β activity. A database of residual enzymatic activity retained by the G6Pase-β mutations will serve as a reference for evaluating genotype–phenotype relationships. [ABSTRACT FROM AUTHOR]

Abstract: The SLC37 family consists of four sugar-phosphate exchangers, A1, A2, A3, and A4, which are anchored in the endoplasmic reticulum (ER) membrane. The best characterized family member is SLC37A4, better known as the glucose-6-phosphate (G6P) transporter (G6PT). SLC37A1, SLC37A2, and G6PT function as phosphate (Pi)-linked G6P antiporters catalyzing G6P:Pi and Pi:Pi exchanges. The activity of SLC37A3 is unknown. G6PT translocates G6P from the cytoplasm into the lumen of the ER where it couples with either glucose-6-phosphatase-α (G6Pase-α) or G6Pase-β to hydrolyze intraluminal G6P to glucose and Pi. The functional coupling of G6PT with G6Pase-α maintains interprandial glucose homeostasis and the functional coupling of G6PT with G6Pase-β maintains neutrophil energy homeostasis and functionality. A deficiency in G6PT causes glycogen storage disease type Ib, an autosomal recessive disorder characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction. Neither SLC37A1 nor SLC37A2 can functionally couple with G6Pase-α or G6Pase-β, and there are no known disease associations for them or SLC37A3. Since only G6PT matches the characteristics of the physiological ER G6P transporter involved in blood glucose homeostasis and neutrophil energy metabolism, the biological roles for the other SLC37 proteins remain to be determined. [Copyright &y& Elsevier]

Glucose is absolutely essential for the survival and function of the brain. In our current understanding, there is no endogenous glucose production in the brain, and it is totally dependent upon blood glucose. This glucose is generated between meals by the hydrolysis of glucose-6-phosphate (Glc-6-P) in the liver and the kidney. Recently, we reported a ubiquitously expressed Glc-6-P hydrolase, glucose-6-phosphatase-β (Glc-6-Paseβ), that can couple with the Glc-6-P transporter to hydrolyze Glc-6-P to glucose in the terminal stages of glycogenolysis and gluconeogenesis. Here we show that astrocytes, the main reservoir of brain glycogen, express both the Glc-6-Pase-β and Glc-6-P transporter activities and that these activities can couple to form an active Glc-6-Pase complex, suggesting that astrocytes may provide an endogenous source of brain glucose. [ABSTRACT FROM AUTHOR]