Visualizing Muscle Sialic Acid Expression in the GNED207VTgGne-/- Cmah-/- Model of GNE Myopathy: A Comparison of Dietary and Gene Therapy Approaches

Background: GNE myopathy (GNEM) is a rare, adult-onset, inclusion body myopathy that results from partial loss of function mutations in the GNE gene. GNE encodes UDP-GlcNAc epimerase/Mannose-6 kinase, a protein with two enzymatic activities that comprise the committed step in biosynthesis of sialic acid (SA), an essential glycan that appears on the terminal positions of many extracellular oligosaccharide chains. These GNE mutations can cause a reduction of SA in many tissues, although pathology is restricted to skeletal muscles through a poorly understood mechanism. Objective: Despite recent advances in the field, it remains unclear which therapeutic avenue is most promising for the restoration of SA level in skeletal muscle affected by GNEM. Our objective was to assess dietary and gene therapy strategies for GNEM in Cmah-deficient GNED207VTgGne-/- mice, a model that allows for the visualization of orally delivered N-glycolylneuraminic acid (Neu5Gc), one of the two predominant SA forms in muscle. Methods: Methods included in situ physiology studies of the tibialis anterior muscle, studies of ambulation and limb grip strength, and muscle staining using MAA, SNA, and anti-Neu5Gc antibody, along with qPCR, qRT-PCR, western blot, and HPLC studies to assess virally introduced DNA, GNE gene expression, GNE protein expression, and SA expression. Results: We found that a diet enriched in Neu5Gc-containing glycoproteins had no impact on Neu5Gc immunostaining in muscles of GNEM model mice. Delivery of a single high dose oral Neu5Gc therapy, however, did increase Neu5Gc immunostaining, though to levels below those found in wild type mice. Delivery of a single dose of GNE gene therapy using a recombinant Adeno Associated Virus (rAAV) vector with a liver-specific or a muscle-specific promoter both caused increased muscle Neu5Gc immunostaining that exceeded that seen with single dose monosaccharide therapy. Conclusions: Our findings indicate that dietary loading of Neu5Gc-containing glycoproteins is not effective in increasing muscle Neu5Gc expression, while single dose oral Neu5Gc monosaccharide or GNE gene therapy are. Neu5Gc immunostaining, however, showed greater changes than did lectin staining or HPLC analysis. Taken together, these results suggest that Neu5Gc immunostaining may be more sensitive technique to follow SA expression than other more commonly used methods and that liver expression of GNE may contribute overall muscle SA content.

Dissecting role of founder mutation p.V727M in GNE in Indian HIBM cohort

GNE gene-specific c.2179G>A(p.V727M) is a key alteration reported in patients with hereditary inclusion body myopathy (HIBM) and represents an ethnic founder mutation in the Indian cohort. However, the underlying role of this mutation in pathogenesis remains largely unknown. Thus, in this study, we aimed to access possible mechanisms of V727M mutation that could be leading to myopathy. We evaluated various in silico tools to predict the effect of this mutation on pathogenicity, structural or possible interactions, that could induce myopathy. Our results propose that V727M mutation could induce deleterious effects or pathogenicity and affect the stability of GNE protein. Analysis of differential genes reported in the V727 mutant case suggests that it can affect GNE protein interaction with Myc-proto-oncogene (MYC) transcription factor. Our in silico analysis also suggests a possible interaction between GNE ManNac-kinase domain with MYC protein at the C-terminal DNA-binding domain. MYC targets reported in skeletal muscles via ChIP-seq suggest that it plays a key role in regulating the expression of many genes reported differentially expressed in V727M-mutated HIBMs. We conclude that V727M mutation could alter the interaction of GNE with MYC thereby altering transcription of sialyltransferase and neuromuscular genes, thus understanding these effects could pave the way for developing effective therapies against HIBM.

Generation and Characterization of Skeletal Muscle Cell based Model for GNE Myopathy: Implications in Actin Dynamics

UDP-N-acetyl glucosamine-2 epimerase/ N-acetyl mannosamine kinase (GNE) catalyzes key enzymatic reactions in the biosynthesis of sialic acid. Mutation in GNE gene causes GNE-Myopathy characterized by adult onset muscle weakness and degeneration. GNE is involved in different cellular processes like adhesion, apoptosis, ER stress and autophagy. Lack of appropriate model system limits drug and treatment options for GNE Myopathy as GNE knock out was found to be embryonically lethal. In the present study, we have generated L6 rat skeletal muscle cell-based model system for GNE Myopathy where GNE gene is knocked out at exon 3 using AAV mediated SEPT homology recombination. The cell line was heterozygous for GNE gene with one wild type and one truncated allele as confirmed by sequencing. The phenotype showed reduced GNE epimerase activity with little reduction in sialic acid content. In addition, the GNE knock out cell line revealed altered cytoskeletal organization with disrupted actin filament. The signaling cascade regulating actin dynamics such as Rho A, Cofilin, FAK and Src were altered leading to reduced cell migration in GNE heterozygous cells. Our study indicates possible role of GNE in regulating actin dynamics and cell migration of skeletal muscle cell. The skeletal muscle cell based system offers great potential in understanding pathomechanism and target identification for GNE Myopathy.

Sialuria-Related Intellectual Disability in Children and Adolescent of Pakistan: Tenth Patient Described has a Novel Mutation in the GNE Gene

Background: Sialuria is a rare inborn error of metabolism caused by excessive synthesis of sialic acid due to the mutation in the binding site of the cytidine monophosphate-sialic acid of UDPGlcNAc 2-Epimerase/ManNAc Kinase (GNE/MNK). Objective: This is the first study investigating the molecular basis of neuronal disorders exhibiting sialuria in Pakistani children/adolescents. Methods: The current study genotyped GNE SNPs rs121908621, rs121908622 and rs121908623 by using PCR, RFLP, and DNA sequencing methods. Socioeconomic and clinical histories were also recorded. Results: Our data suggest that clinical symptoms and financial status play a significant role in conferring sialuria related Intellectual Disability (ID). SNP: rs121908623 showed G/A substitution (R263Q) in the GNE gene. Conclusion: We have identified one case study in Pakistan, so this makes our research a leap forward towards the identification of the 10th case study worldwide.