Abstract
GNE myopathy (GNEM), also known as hereditary inclusion body myopathy (HIBM), is a late- onset, progressive myopathy caused by mutations in the GNE gene encoding the enzyme responsible for the first regulated step in the biosynthesis of sialic acid (SA). The disease is characterized by distal muscle weakness in both the lower and upper extremities, with the quadriceps muscle relatively spared until the late stages of disease. To explore the role of SA synthesis in the disease, we conducted a comprehensive and systematic analysis of both free and total SA levels in a large cohort of GNEM patients and a mouse model. A sensitive LC/MS/MS assay was developed to quantify SA in serum and muscle homogenates. Mean serum free SA level was 0.166 μg/mL in patients and 18% lower (p<0.001) than that of age-matched control samples (0.203 μg/mL). In biopsies obtained from patients, mean free SA levels of different muscles ranged from 0.046–0.075 μg/μmol Cr and were markedly lower by 72–85% (p<0.001) than free SA from normal controls. Free SA was shown to constitute a small fraction (3–7%) of the total SA pool in muscle tissue. Differences in mean total SA levels in muscle from patients compared with normal controls were less distinct and more variable between different muscles, suggesting a small subset of sialylation targets could be responsible for the pathogenesis of GNEM. Normal quadriceps had significantly lower levels of free SA (reduced by 39%) and total SA (reduced by 53%) compared to normal gastrocnemius. A lower SA requirement for quadriceps may be linked to the reported quadriceps sparing in GNEM. Analysis of SA levels in GneM743T/M743T mutant mice corroborated the human study results. These results show that serum and muscle free SA is severely reduced in GNEM, which is consistent with the biochemical defect in SA synthesis associated with GNE mutations. These results therefore support the approach of reversing SA depletion as a potential treatment for GNEM patients.
Highlights
GNE myopathy (GNEM), known as hereditary inclusion body myopathy (HIBM), distal myopathy with rimmed vacuoles (DMRV) or Nonaka disease, is a rare, late-onset, progressivePLOS ONE | DOI:10.1371/journal.pone.0173261 March 7, 2017Free sialic acid deficiency in GNE myopathy was done internally at Ultragenyx and samples sent to Intertek
Materials and methods Acquisition of GNEM patient serum samples and muscle biopsies Serum samples and muscle biopsies were collected from 47 GNEM patients who enrolled in a phase 2 randomized, double-blind, placebo-controlled, parallel group study to evaluate the dose and pharmacodynamic efficacy of aceneuramic acid extended release (AceER) tablets (ClinicalTrials.gov: NCT01517880)
To precisely quantify free sialic acid (SA) in biological samples, we have developed a sensitive and specific LC/MS/MS assay for measuring NANA in serum and muscle tissue that was based in part from a previous method [35]
Summary
GNE myopathy (GNEM), known as hereditary inclusion body myopathy (HIBM), distal myopathy with rimmed vacuoles (DMRV) or Nonaka disease, is a rare, late-onset, progressive. A transgenic mouse model (DMRV-hIBM) overexpressing human GNE D176V mutation in null GNE background recapitulated many of the pathological changes in skeletal muscles reminiscent of GNEM in terms of rimmed vacuole and inclusion body formation [26] In this mouse model, total SA levels in serum and muscle membrane-bound fractions were decreased compared with control littermates. Free sialic acid deficiency in GNE myopathy equivalence of homoallelic M743T mutation, oral treatment of SA in adult mutant mice for 12 weeks improved muscle sialylation as demonstrated by lectin histochemistry [30, 31] These animal studies indicated that SA deficiency as measured by total SA in muscle is a component of the disease and provided the rationale for developing oral SA treatment for GNEM patients.
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