Sialylation of Thomsen–Friedenreich Antigen is a Noninvasive Blood-Based Biomarker for GNE Myopathy

Abstract

GNE myopathy is an adult-onset progressive myopathy, resulting from mutations in GNE, the key enzyme of sialic acid synthesis. The pathomechanism of GNE myopathy likely involves aberrant sialylation, since administration of sialic acid itself, or its precursor, N-acetylmannosamine (ManNAc), rescued hyposialylation of GNE myopathy mice. Recently, clinical trials for GNE myopathy patients were initiated. A robust, noninvasive biomarker is highly desirable for diagnosis of GNE myopathy and for evaluating response to therapy. Since muscle biopsies of patients with GNE myopathy demonstrated hyposialylation of predominantly O-linked glycans, we analyzed the O-linked glycome of patients’ plasma proteins using mass spectrometry. Most patients showed increased plasma levels of the core 1 O-linked glycan, Thomsen-Friedenreich (T)-antigen and/or decreased amounts of its sialylated form, ST-antigen. In addition, compared to unaffected individuals, all analyzed patients had a consistently increased ratio of T-antigen to ST-antigen. Importantly, the T/ST ratios were in the normal range in a GNE myopathy patient treated with intravenous immunoglobulins as a source of sialic acid, indicating response to therapy. Natural history and clinical trial data will reveal whether T/ST ratios can be correlated to muscle function. These findings not only highlight plasma T/ST ratios as a robust blood-based biomarker for GNE myopathy, but may also help explain the pathology and course of the disease.

Quantification of lectin fluorescence in GNE myopathy muscle biopsies

“…Statistical significance was absent likely to the limited number of biopsies in each group. These findings are in line with previously published, sometimes subtle, qualitative GNE myopathy lectin imaging results …”

“…It is often at the discretion of the investigator performing the imaging to capture representative fields of the tissue, determine microscope settings and qualitatively analyze the results, i.e., score the fields by eye. While this is an accepted practice, quantitation of the fluorescence signal in the entire biopsy may be more suitable if diagnosis/outcome or clinical trial decisions are based on such results. A few other studies have explored quantitation of fluorescence signals in muscle biopsy slides, mostly for dystrophin‐expression related studies …”

“…This software program has previously been used in muscle image analysis for other purposes . Previous imaging demonstrated that SNA‐reactive glycans are expressed at muscle membranes and all muscle fibers appear to express SNA‐reactive glycans equally, Therefore, we imaged entire muscle sections and included each intact muscle cell (stained by caveolin) in our analysis.…”

“…Lectin histochemistry demonstrated that sialic acid residues in a α(2,6)‐linkage with either galactose or with GalNAc, present on sarcolemmal glycans appeared to be absent or decreased in human and mouse GNE myopathy muscle sections (Supplementary Fig. S1, which is available online) . In addition, GNE myopathy mice receiving 12 weeks of oral therapy with the sialic acid precursor N‐acetylmannosamine (ManNAc) showed re‐sialylation of sarcolemmal membranes by lectin histochemistry (Supplementary Fig.…”

“…Lectins are sugar‐binding proteins with ligand specificities for defined carbohydrate sequences . Staining of GNE myopathy human and mouse muscle sections with sialic acid binding lectins or lectins binding to desialylated sugar moieties demonstrated hyposialylation of sarcolemmal membranes . In particular, use of the lectin SNA ( Sambucus nigra agglutinin) that predominantly recognizes terminal sialic acid (Neu5Ac) in an α(2,6)‐linkage with either galactose or with N‐acetylgalactosamine (GalNAc), was previously proven informative for GNE myopathy muscle sialylation status …”