Since mutations in the GNE gene are associated with HIBM, the presumed mechanism of pathophysiology would be: GNE mutations (mostly missense, ) lead to decreased GNE/MNK enzymatic activities resulting in decreased production of sialic acid. The decrease in intracellular sialic acid content would then lead to the muscle degeneration in HIBM. Although excellent experimental work has been done in pursuit of supportive evidence of this hypothesis, the exact cellular mechanisms behind the development of the myopathy in HIBM has remained elusive.
The effects of GNE
mutations on the enzymatic properties of GNE/MNK were assessed by assays of both GNE-epimerase and MNK-kinase activities, which were reduced, but not absent, in HIBM muscle biopsies, as well as in cultured HIBM fibroblasts, lymphoblasts, and myoblasts [5
]. In vitro
studies, in which specific human GNE
mutations were expressed in Sf6 insect cells [38
], in COS-7 cells [45
], or in a cell-free in vitro
transcription-translation system [30
], revealed that the reduction in GNE and MNK enzymatic activity is mutation-dependent. Moreover, mutations in one enzymatic domain affect not only that domain’s enzyme activity but also the activity of the other domain. Compared with enzyme activities in a cell-free system, fibroblasts exhibited higher residual activities of both GNE and MNK, suggesting the presence in fibroblasts of additional sugar epimerases and kinases with overlapping substrate specificity [30
These experiments revealed that the mechanism of pathology is not that of a typical autosomal recessive disorder with low enzyme activity in the gene product. Rather the total functional activity of GNE/MNK activity in a cell may be dependent both on the location or domain of the mutation in the GNE/MNK protein as well as the activity of other enzymes in metabolically interconnected pathways.
Equally enigmatic are the results of several investigators analyzing sialic acid levels in tissue from individuals affected with HIBM. Hinderlich et al. demonstrated normal membrane bound sialic acid levels in lymphoblastoid lines with the p.M712T mutation [49
]. Yet, cultured muscle cells from patients with a variation of GNE
mutations, showed variable sialylation, ranging from the normal range to significantly decreased [45
]. These studies suggested that muscle cells with a strong reduction in epimerase activity, below 35% of normal, resulting from at least one GNE
mutation in the epimerase domain, consistently showed measurable decreased sialyation [45
]. However, isoelectric focusing studies of serum transferrin, which contains only N-GlcNAc linked glycans, and serum apolipoprotein CIII, which contains only O-GalNAc linked glycans, appeared normal in all HIBM patients tested so far. This suggests that unaffected serum N-GlcNAc linked and O-GalNAc linked glycosylation in hepatically derived serum glycoproteins in individuals with HIBM [51
]. Hyposialylation of specific glycosylated proteins in HIBM muscle was reported for PSA, polysialic acid, on NCAM [14
] and for α-dystroglycan [33
], but was reported to be unaffected in other studies [46
]. Hyposialylation of O-linked glycans in HIBM muscle cells was also demonstrated by use of specific lectins [45
]. Importantly, Noguchi et al. showed that the hyposialylation of these cells can be reversed by the addition of ManNAc, a substrate in sialic acid synthesis, or sialic acid itself to the media of the cells [45
Since HIBM is an adult onset disease, and patients have residual GNE/MNK enzymatic activity, the effects of sialic acid deficiency may appear gradually. Some glycoconjugates, for example N-linked, might be more readily sialylated than others, for example O-linked or PSA linked. Thus, when a shortage of sialic acid occurs, specific proteins may be inadequately glycosylated, such as PSA-NCAM or alpha-dystroglycan, contributing to the pathology of HIBM.
Apart from hyposialylation, other hypotheses have arisen for the role of mutated GNE/MNK in the pathology of HIBM. These include the unusual compartmentalization of GNE/MNK in cells [43
], leading to speculation of additional GNE/MNK enzymatic activities in cells. Exploration of this phenomenon showed no difference in the compartmentalization of GNE/MNK in either skeletal muscle or primary myoblasts from individuals affected with HIBM [55
]. In addition, two novel isoforms of GNE/MNK (GNE1) were identified, which have extended (GNE2) or partially deleted N-termini (GNE3), and display tissue-specific expression [56
], which may contribute to the pathology of HIBM. Furthermore, impaired apoptotic signaling in HIBM cells was reported, implicating involvement of apoptotic pathways in HIBM pathophysiology [57
]. Another intriguing finding is that GNE/MNK may control sialyltransferase expression, ganglioside production and modulation of proliferation and apoptosis, independent of sialic acid production [58
]. In another study, microarray RNA expression and muscle morphology analysis indicated that mitochondrial processes may be affected in HIBM muscle [59
]. And recently, co-immunoprecipitation assays identified alpha-actinin 1, an actin binding and crosslinking protein, as a ligand of GNE/MNK [60
]. The relevance of α-actinin 1 function in skeletal muscle and its role in HIBM pathophysiology remains elusive.
The above findings underscore that there is more to be learned about the cellularsite of pathology and the mechanism of muscle cell degeneration in HIBM. To further analyze these pathways on a whole animal, as well as explore potential treatment methods, efforts to study HIBM mouse models are ongoing.