Glutamine-fructose-6-phosphate Transaminase 1 Research Articles

GFPT1 accelerates immune escape in breast cancer by modifying PD-L1 via O-glycosylation

BackgroundImmune escape is one of the causes of poor prognosis in breast cancer (BC). Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first speed-limiting enzyme of the hexosamine biosynthesis pathway (HBP) and is essential for the progression of BC. Nevertheless, the mechanism of the influence of GFPT1 in BC immune escape is not clear.MethodsFirst, the level of GFPT1 in BC was analyzed by starbase, and GFPT1 expression in BC tissues was measured by qRT-PCR, western blot and IHC. Then, the O-GlcNAc levels were detected by western blot. Thereafter, Co-IP was applied to examine the relationship between GFPT1 and PD-L1. At last, a mouse model was constructed for validation in vivo.ResultsFirstly, we discovered that GFPT1 was obviously strengthened in BC. Knockdown or introduction of GFPT1 correspondingly degraded and elevated O-GlcNAc levels in cells. Further researches revealed that there was a reciprocal relationship between GFPT1 and PD-L1. Mechanistically, we disclosed that GFPT1 enhanced PD-L1 protein stability through O-glycosylation. More interestingly, GFPT1 accelerated BC cell immune escape via upregulation of O-glycosylation-modified PD-L1. In vivo, silencing of GFPT1 attenuated immune escape of BC cells by reducing PD-L1 levels.ConclusionGFPT1 promoted BC progression and immune escape via O-glycosylation-modified PD-L1. GFPT1 may be a potential target for BC therapy.

Critical Role of Preoperative Hyperglycemia in Association with GFAT-1 Expression in Resected Pancreatic Cancer.

Hyperglycemia is involved in malignant transformation of pancreatic cancer via the hexosamine biosynthetic pathway (HBP). However, few studies have verified this mechanism based on clinical data. This study investigated the complementary effects of hyperglycemia and HBP on pancreatic cancer prognosis using detailed clinical data. The study analyzed data of 477 patients with pancreatic cancer who underwent pancreatectomy between 2006 and 2020. The patients were divided into normoglycemia and hyperglycemia groups based on their HbA1c levels. Immunostaining for glutamine fructose-6-phosphate transaminase-1 (GFAT-1), the rate-limiting enzyme in HBP, CD4, CD8, and Foxp3, was performed to evaluate the association between survival outcomes, HBP, and local tumor immunity. Overall survival (OS) was significantly poorer in the hyperglycemia group than in the normoglycemia group (mean survival time [MST]: 35.0 vs. 47.9months; p = 0.007). The patients in the hyperglycemia group with high GFAT-1 expression had significantly poorer OS than those with low GFAT-1 expression (MST, 49.0 vs. 27.6months; p < 0.001). However, the prognosis did not differ significantly between the patients with high and low GFAT-1 expression in the normoglycemia group. In addition, the patients with hyperglycemia and high GFAT-1 expression had fewer CD4+ (p = 0.015) and CD8+ (p = 0.017) T cells and a lower CD8+/Foxp3+ ratio (p = 0.032) than those with hyperglycemia and low GFAT-1 expression. The patients with hyperglycemia and high GFAT-1 expression levels had an extremely poor prognosis. Furthermore, the tumors in these patients were characterized as immunologically cold tumors.

Overexpression of Fatty Acid Synthase Upregulates Glutamine-Fructose-6-Phosphate Transaminase 1 and O-Linked N-Acetylglucosamine Transferase to Increase O-GlcNAc Protein Glycosylation and Promote Colorectal Cancer Growth.

Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.

Agenesis of Pectoralis Major Muscle in Late-Onset GFPT1-Related Congenital Myasthenic Syndrome: A Case Report.

The objective of this study was to expand the phenotypic spectrum of glutamine-fructose-6-phosphate transaminase 1 (GFPT1)-related congenital myasthenia syndrome (CMS). A 61-year-old man with agenesis of the left pectoralis major muscle presented with progressive muscle weakness for a decade that transiently improved after exertion. His examination revealed proximal and distal muscle weakness in upper extremities and proximal muscle weakness in lower extremities. Muscle enzymes were elevated. An electromyogram revealed a myopathic pattern; however, a muscle biopsy of deltoid muscle and genetic testing for limb-girdle muscular dystrophies were nondiagnostic. A 3-Hz repetitive nerve stimulation of the spinal accessory nerve recording from trapezius muscle demonstrated a >20% drop in amplitude of the 5th compound motor action potential relative to 1st at both baseline and after 45-second exercise. Acetylcholine receptor binding, lipoprotein-related protein 4, muscle-specific kinase, and voltage-gated calcium channel P/Q antibodies were negative. Genetic testing targeting CMS revealed 2 likely pathogenic variants within GFPT1: novel c.7+2T>G (intron 1) that was predicted to result in a null allele and known c*22 C>A (exon 19) associated with reduced GFPT1 expression. His muscle strength dramatically improved after pyridostigmine initiation. In addition to other reported neurodevelopmental abnormalities, pectoralis major muscle agenesis (or Poland syndrome) may be a clinical manifestation of GFPT1-related CMS.

Splicing regulation of GFPT1 muscle-specific isoform and its roles in glucose metabolisms and neuromuscular junction

Glutamine:fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP). A 54-bp exon 9 of GFPT1 is specifically included in skeletal and cardiac muscles to generate a long isoform of GFPT1 (GFPT1-L). We showed that SRSF1 and Rbfox1/2 cooperatively enhance, and hnRNP H/F suppresses, the inclusion of human GFPT1 exon 9 by modulating recruitment of U1 snRNP. Knockout (KO) of GFPT1-L in skeletal muscle markedly increased the amounts of GFPT1 and UDP-HexNAc, which subsequently suppressed the glycolytic pathway. Aged KO mice showed impaired insulin-mediated glucose uptake, as well as muscle weakness and fatigue likely due to abnormal formation and maintenance of the neuromuscular junction. Taken together, GFPT1-L is likely to be acquired in evolution in mammalian striated muscles to attenuate the HBP for efficient glycolytic energy production, insulin-mediated glucose uptake, and the formation and maintenance of the neuromuscular junction.

Tisp40 prevents cardiac ischemia/reperfusion injury through the hexosamine biosynthetic pathway in male mice

The hexosamine biosynthetic pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to facilitate O-linked GlcNAc (O-GlcNAc) protein modifications, and subsequently enhance cell survival under lethal stresses. Transcript induced in spermiogenesis 40 (Tisp40) is an endoplasmic reticulum membrane-resident transcription factor and plays critical roles in cell homeostasis. Here, we show that Tisp40 expression, cleavage and nuclear accumulation are increased by cardiac ischemia/reperfusion (I/R) injury. Global Tisp40 deficiency exacerbates, whereas cardiomyocyte-restricted Tisp40 overexpression ameliorates I/R-induced oxidative stress, apoptosis and acute cardiac injury, and modulates cardiac remodeling and dysfunction following long-term observations in male mice. In addition, overexpression of nuclear Tisp40 is sufficient to attenuate cardiac I/R injury in vivo and in vitro. Mechanistic studies indicate that Tisp40 directly binds to a conserved unfolded protein response element (UPRE) of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) promoter, and subsequently potentiates HBP flux and O-GlcNAc protein modifications. Moreover, we find that I/R-induced upregulation, cleavage and nuclear accumulation of Tisp40 in the heart are mediated by endoplasmic reticulum stress. Our findings identify Tisp40 as a cardiomyocyte-enriched UPR-associated transcription factor, and targeting Tisp40 may develop effective approaches to mitigate cardiac I/R injury.

GFPT1 promotes the proliferation of cervical cancer via regulating the ubiquitination and degradation of PTEN.

Cervical cancer demonstrates the fourth incidence and death rate in females worldwide. Glutamine--fructose-6-phosphate transaminase 1 (GFPT1), the first rate-limited enzyme of the hexosamine biosynthesis pathway, has been reported to promote the progression of cancers. However, the prognostic value and roles of GFPT1 in cervical cancer are largely unknown. Transcription expression data for cervical cancer were downloaded from public databases. GFPT1 overexpressed and knockdown cell lines were constructed. Colony formation assays, Edu assays and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to measure the proliferation capabilities of cervical cancer cells. Western blot, Immunofluorescence and co-immunoprecipitation assays were performed to verify the interaction between GFPT1and Phosphatase and tensin homolog (PTEN). Animal assays were applied to verify the results in vivo. GFPT1 expression was higher in cervical cancer cell lines. The proliferation capabilities of cervical cancer cells were suppressed in GFPT1 knockdown cells and GFPT1 inhibitor L-DON treated cells. And overexpression of GFPT1 promoted cell proliferation. PTEN was up-regulated in GFPT1 knockdown cells and downregulated in GFPT1 overexpression cells. Immunofluorescence and co-immunoprecipitation results showed that GFPT1 was co-localized and interacted with PTEN. GFPT1 promoted the ubiquitination and degradation of PTEN. Silence of PTEN offsets the growth inhibition of cervical cancer caused by GFPT1 knockdown. Animal assays showed that GFPT1 promoted the proliferation of cervical cancer in vivo. Our study revealed that GFPT1 could promote the progression of cervical cancer by regulating PTEN expression. Our study highlights the GFPT1-PTEN regulation as a potential therapy target for cervical cancer. .

GFAT1 is highly expressed in cancer stem cells of pancreatic cancer.

BackgroundCancer stem cells (CSCs) play pivotal roles in the growth, invasion, metastasis, and chemoresistance of pancreatic cancer (PC). The current characterization of CSCs in PC is not complete. Glutamine-fructose-6-phosphate transaminase 1 (GFAT1) is a key enzyme that regulates the hexosamine pathway (HP), in which it not only controls glucose influx but also catalyzes the reaction to form glucosamine 6-phosphate. Recently, it was reported that GFAT1 is highly expressed in PC. However, the relevance of this high expression of GFAT1, especially its association with cancer stemness, has not been well defined and is thus addressed in the current study.MethodsGFAT1 levels were determined in PC from a public database and assessed by bioinformatics tools. GFAT1 expression in CD133+ and CD44+ CSCs in PC was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunostaining on cytospun cells after flow cytometry-based cell sorting. GFAT1+ cells were separated from GFAT1− cells by transfection of PC cell lines with a designed plasmid that expressed red fluorescent protein (RFP) under a GFAT1 promoter. Tumor sphere formation, tumor growth, tumor cell invasion, cell migration, and the resistance to gemcitabine-induced apoptosis were determined in GFAT1+ vs. GFAT1− PC cells.ResultsGFAT1 levels were significantly upregulated in PC compared to the adjacent non-PC tissue. There were significantly more GFAT1+ cells in the CD133+ population than in the CD133− population, and similarly, there were significantly more GFAT1+ cells in the CD44+ population than in the CD44− population. Compared to GFAT1− PC cells, GFAT1+ PC cells generated significantly more tumor spheres in culture, appeared to be more invasive and migratory, and were significantly more resistant to gemcitabine-induced cell apoptosis.ConclusionsGFAT1 is highly expressed in CSCs among PC cells and may be crucial for PC growth, metastasis, and chemoresistance.

LncRNA ELFN1-AS1 promotes esophageal cancer progression by up-regulating GFPT1 via sponging miR-183-3p.

Accumulating studies highlight the critical role of long non-coding RNAs (lncRNAs) in the development of various human cancers. Extracellular leucine rich repeat and fibronectin type III domain containing 1-antisense RNA 1 (ELFN1-AS1) was shown to be a newly found lncRNA that abnormally expressed in human tumors. However, till now the specific function of this lncRNA in esophageal cancer (ESCA) remains unknown. In this study, we discovered that higher ELFN1-AS1 expression indicated shorter patient survival in pan-cancer, including ESCA, using online The Cancer Genome Atlas (TCGA) tools. The lncRNA ELFN1-AS1 was significantly up-regulated in ESCA tissues and cell lines when compared with the counterparts. Down-regulation of ELFN1-AS1 restrained cell proliferation, migration, and invasion of ESCA in vitro. In addition, we found that the expression of microRNA-183-3p (miR-183-3p) and ELFN1-AS1 or glutamine-fructose-6-phosphate transaminase 1 (GFPT1) were inversely correlated in ESCA. Both ELFN1-AS1 and GFPT1 are direct targets of miR-183-3p in ESCA. The effects of ELFN1-AS1 knockdown on ESCA progression were partially rescued by inhibition of miR-183-3p or over-expression of GFPT1. In summary, the results of this study suggest that the lncRNA ELFN1-AS1 facilitates the progression of ESCA by acting as a competing endogenous RNA (ceRNA) to promote GFPT1 expression via sponging miR-183-3p.

Congenital myasthenic syndrome caused by a frameshift insertion mutation in GFPT1

ObjectiveDescription of a new variant of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) gene causing congenital myasthenic syndrome (CMS) in 3 children from 2 unrelated families.MethodsMuscle biopsies, EMG, and whole-exome sequencing were performed.ResultsAll 3 patients presented with congenital hypotonia, muscle weakness, respiratory insufficiency, head lag, areflexia, and gastrointestinal dysfunction. Genetic analysis identified a homozygous frameshift insertion in the GFPT1 gene (NM_001244710.1: c.686dupC; p.Arg230Ter) that was shared by all 3 patients. In one of the patients, inheritance of the variant was through uniparental disomy (UPD) with maternal origin. Repetitive nerve stimulation and single-fiber EMG was consistent with the clinical diagnosis of CMS with a postjunctional defect. Ultrastructural evaluation of the muscle biopsy from one of the patients showed extremely attenuated postsynaptic folds at neuromuscular junctions and extensive autophagic vacuolar pathology.ConclusionsThese results expand on the spectrum of known loss-of-function GFPT1 mutations in CMS12 and in one family demonstrate a novel mode of inheritance due to UPD.

GFPT1 deficiency in muscle leads to myasthenia and myopathy in mice.

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway which yields precursors required for protein and lipid glycosylation. Mutations in GFPT1 and other genes downstream of this pathway cause congenital myasthenic syndrome (CMS) characterized by fatigable muscle weakness owing to impaired neurotransmission. The precise pathomechanisms at the neuromuscular junction (NMJ) owing to a deficiency in GFPT1 is yet to be discovered. One of the challenges we face is the viability of Gfpt1−/− knockout mice. In this study, we use Cre/LoxP technology to generate a muscle-specific GFPT1 knockout mouse model, Gfpt1tm1d/tm1d, characteristic of the human CMS phenotype. Our data suggest a critical role for muscle derived GFPT1 in the development of the NMJ, neurotransmission, skeletal muscle integrity and highlight that a deficiency in skeletal muscle alone is sufficient to cause morphological postsynaptic NMJ changes that are accompanied by presynaptic alterations despite the conservation of neuronal GFPT1 expression. In addition to the conventional morphological NMJ changes and fatigable muscle weakness, Gfpt1tm1d/tm1d mice display a progressive myopathic phenotype with the presence of tubular aggregates in muscle, characteristic of the GFPT1-CMS phenotype. We further identify an upregulation of skeletal muscle proteins glypican-1, farnesyltransferase/geranylgeranyltransferase type-1 subunit α and muscle-specific kinase, which are known to be involved in the differentiation and maintenance of the NMJ. The Gfpt1tm1d/tm1d model allows for further investigation of pathophysiological consequences on genes and pathways downstream of GFPT1 likely to involve misglycosylation or hypoglycosylation of NMJs and muscle targets.

Mutations in GFPT1-related congenital myasthenic syndromes are associated with synaptic morphological defects and underlie a tubular aggregate myopathy with synaptopathy.

Mutations in GFPT1 (glutamine-fructose-6-phosphate transaminase 1), a gene encoding an enzyme involved in glycosylation of ubiquitous proteins, cause a limb-girdle congenital myasthenic syndrome (LG-CMS) with tubular aggregates (TAs) characterized predominantly by affection of the proximal skeletal muscles and presence of highly organized and remodeled sarcoplasmic tubules in patients' muscle biopsies. We report here the first long-term clinical follow-up of 11 French individuals suffering from LG-CMS with TAs due to GFPT1 mutations, of which nine are new. Our retrospective clinical evaluation stresses an evolution toward a myopathic weakness that occurs concomitantly to ineffectiveness of usual CMS treatments. Analysis of neuromuscular biopsies from three unrelated individuals demonstrates that the maintenance of neuromuscular junctions (NMJs) is dramatically impaired with loss of post-synaptic junctional folds and evidence of denervation-reinnervation processes affecting the three main NMJ components. Moreover, molecular analyses of the human muscle biopsies confirm glycosylation defects of proteins with reduced O-glycosylation and show reduced sialylation of transmembrane proteins in extra-junctional area. Altogether, these results pave the way for understanding the etiology of this rare neuromuscular disorder that may be considered as a "tubular aggregates myopathy with synaptopathy".

Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1).

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed “limb-girdle CMS with tubular aggregates”. CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells.

Identification of Driving ALK Fusion Genes and Genomic Landscape of Medullary Thyroid Cancer.

The genetic landscape of medullary thyroid cancer (MTC) is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog (N = 14), serine/threonine kinase 11 (N = 11), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (N = 6), mutL homolog 1 (N = 4), Kiesten rat sarcoma viral oncogene homolog (N = 3) and MET proto-oncogene (N = 3). We also evaluated anaplastic lymphoma kinase (ALK) rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization (FISH). Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5’ fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1 (GFPT1)-ALK and echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular target therapy.

A 3'-UTR mutation creates a microRNA target site in the GFPT1 gene of patients with congenital myasthenic syndrome.

Mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) cause the neuromuscular disorder limb-girdle congenital myasthenic syndrome (LG-CMS). One recurrent GFPT1 mutation detected in LG-CMS patients is a c.*22C>A transversion in the 3'-untranslated region (UTR). Because this variant does not alter the GFPT1 open reading frame, its pathogenic relevance has not yet been established. We found that GFPT1 protein levels were reduced in myoblast cells of the patients carrying this variant. In silico algorithms predicted that the mutation creates a microRNA target site for miR-206*. Investigation of the expression of this so far unrecognized microRNA confirmed that miR-206* (like its counterpart miR-206) is abundant in skeletal muscle. MiR-206* efficiently reduced the expression of reporter constructs containing the mutated 3'-UTR while no such effect was observed with reporter constructs containing the wild-type 3'-UTR or when a specific anti-miR-206* inhibitor was added. Moreover, anti-miR-206* inhibitor treatment substantially rescued GFPT1 expression levels in patient-derived myoblasts. Our data demonstrate that the c.*22C>A mutation in the GFPT1 gene leads to illegitimate binding of microRNA resulting in reduced protein expression. We confirm that c.*22C>A is a causative mutation and suggest that formation of microRNA target sites might be a relevant pathomechanism in Mendelian disorders. Variants in the 3'-UTRs should be considered in genetic diagnostic procedures.

Genetic diseases affecting the eyelids

The molecular basis of a number of inherited diseases that affect the eyelids has been elucidated over the last two decades. Due to the vast number of these diseases, a clinician may become overwhelmed by the volume of data, making it difficult to incorporate newer information into his or her clinical practice. This article intends to review the recent developments of inherited diseases that affect the eyelids that a typical oculoplastic surgeon will encounter. This review proposes categorizing genetic diseases affecting the eyelids on rarity and whether the disease manifests itself at birth or later in life. Based on this classification system the following 10 diseases (the first five manifesting at birth, the last five later in life) are considered more likely to be encountered by the typical oculoplastic surgeon and reviewed in detail: blepharophimosis-ptosis-epicanthus inversus syndrome, congenital fibrosis of the extraocular muscles, lymphedema-distichiasis syndrome, neurofibromatosis type 1, congenital myasthenic syndrome, oculopharyngeal muscular dystrophy, chronic progressive external ophthalmoplegia, myotonic dystrophy, neurofibromatosis type 2, and basal cell nevus syndrome. The remaining known genetic disorders that affect the eyelids are considered less likely to be encountered by the typical oculoplastic surgeon and are listed in tabular form. It is prudent for the oculoplastic surgeon to be knowledgeable of inherited disorders that affect the eyelids to aid in accurate diagnosis, counseling, and treatment. The development of future therapies may at some point make treatment of these diseases no longer surgical. http://links.lww.com/COOP/A4.

Mutations in GFPT1 that underlie limb-girdle congenital myasthenic syndrome result in reduced cell-surface expression of muscle AChR

Mutations in GFPT1 underlie a congenital myasthenic syndrome (CMS) characterized by a limb-girdle pattern of muscle weakness. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is a key rate-limiting enzyme in the hexosamine biosynthetic pathway providing building blocks for the glycosylation of proteins and lipids. It is expressed ubiquitously and it is not readily apparent why mutations in this gene should cause a syndrome with symptoms restricted to muscle and, in particular, to the neuromuscular junction. Data from a muscle biopsy obtained from a patient with GFPT1 mutations indicated that there were reduced endplate acetylcholine receptors. We, therefore, further investigated the relationship between identified mutations in GFPT1 and expression of the muscle acetylcholine receptor. Cultured myotubes derived from two patients with GFPT1 mutations showed a significant reduction in cell-surface AChR expression (Pt1 P < 0.0001; Pt2 P = 0.0097). Inhibition of GFPT1 enzymatic activity or siRNA silencing of GFPT1 expression both resulted in reduced AChR cell-surface expression. Western blot and gene-silencing experiments indicate this is due to reduced steady-state levels of AChR α, δ, ε, but not β subunits rather than altered transcription of AChR-subunit RNA. Uridine diphospho-N-acetylglucosamine, a product of the hexosamine synthetic pathway, acts as a substrate at an early stage in the N-linked glycosylation pathway. Similarity between CMS due to GFPT1 mutations and CMS due to DPAGT1 mutations would suggest that reduced endplate AChR due to defective N-linked glycosylation is a primary disease mechanism in this disorder.

Limb‐girdle myasthenia with tubular aggregates associated with novel GFPT1 mutations

Limb-girdle myasthenia with tubular aggregates (LGM with TAs) is a subtype of congenital myasthenic syndrome caused by recessive mutations of glutamine-fructose-6-phosphate transaminase 1 (GFPT1). Clinical and neurophysiological assessment was made in a Korean boy who had proximal limb muscle weakness. Findings suggested a diagnosis of congenital myasthenic syndrome. Muscle biopsy disclosed numerous TAs in muscle fibers, and DNA sequence analysis disclosed 2 novel missense mutations (p.E256Q and p.M499T) in GFPT1. Treatment with oral cholinesterase inhibitors produced a dramatic improvement in muscle strength. GFPT1 is the key enzyme in the hexosamine biosynthesis pathway, and mutations in GFPT1 cause defective glycosylation in the proteins of the neuromuscular junction. Identification of LGM with TAs among patients with congenital myasthenic syndrome is important because treatment with cholinesterase inhibitors can produce symptomatic improvement.

Functional roles of fructose

During the periimplantation period of pregnancy, pig blastocysts undergo morphological changes and differentiation requiring secretion and transport of nutrients (histotroph) into the uterine lumen. Of these nutrients, glucose is converted to fructose, an isomer of glucose, by conceptus trophectoderm. Although glucose is an energy source for proliferation and growth of mammalian cells, the role of fructose in uterine histotroph is unclear although it is the most abundant hexose sugar in fetal blood and fluids of ungulate mammals (e.g., cows, sheep, and pigs). In this study, we used porcine trophectoderm cells to determine that fructose increased cell proliferation, as did glucose. Western blot analyses of porcine trophectoderm cell extracts revealed that fructose increased the abundance of phosphorylated-RPS6K, -EIF4EBP1, and -RPS6 over basal levels within 30 min, and those levels remained elevated to 120 min. Phosphorylation of both RPS6K and EIF4EBP1 proteins in response to fructose was inhibited by inhibitors of both PI3K and MTOR. Further, when we investigated the inhibition of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) by azaserine (an inhibitor of GFPT1) and GFPT1 siRNA, we found that MTOR-RPS6K and MTOR-EIF4EBP1 signaling in response to fructose is mediated via GFPT1 activation and the hexosamine pathway. We further demonstrated that fructose stimulates the production of hyaluronic acid via GFPT1 and the hexosamine biosynthesis pathway. Collectively, these results demonstrate critical roles for fructose that are mediated via the hexosamine biosynthesis pathway to stimulate MTOR cell signaling, proliferation of porcine trophectoderm cells, and synthesis of hyaluronic acid, a significant glycosaminoglycan in the pregnant uterus.

Glucose Metabolism Gene Variants Modulate the Risk of Pancreatic Cancer

Long-term type 2 diabetes is a known risk factor for pancreatic cancer (PC). We hypothesized that genetic variants in glucose metabolism modify individual susceptibility to PC, especially those associated with diabetes. We retrospectively genotyped 26 single-nucleotide polymorphisms of 5 glucose metabolism genes: glucokinase (GCK), glutamine-fructose-6-phosphate transaminase 1 (GFPT1), glucose phosphate isomerase (GPI), hexokinase 2 (HK2), and O-linked N-acetylglucosamine transferase (OGT) in a case-control study of PC conducted at MD Anderson during 2004 to 2010. Initial genotyping was conducted in 706 patients with PC and 706 cancer-free controls by using the Sequenom method. A HK2 genotype (R844K) with low frequency of homozygous variant was further examined in additional 948 patients and 476 controls. In the combined set of 1,654 cases and 1,182 controls, we showed a significant association of the HK2 R844K GA/AA genotype with reduced PC risk (OR = 0.78; 95% CI, 0.64-0.94; P = 0.009) and a significant interaction with diabetes (P(interaction) < 0.001). The HK2 R844K GA/AA genotype was associated with a reduced risk of PC among nondiabetic individuals (OR = 0.68; 95% CI, 0.56-0.83) but with increased risk among diabetic patients (OR = 3.69; 95% CI, 2.34-5.82). These risk associations remained statistically significant when the analysis was restricted to whites or after exclusion of recent onset diabetes. No significant main effect of other genes or significant interaction of genotype with other risk factors was observed. The findings show a potential role of HK2 gene, alone or in interaction with diabetes, in modifying the risk of PC.