Homozygous or compound heterozygous mutations in either the epimerase or kinase domain of GNE cause decreased UDP-GlcNAc 2-epimerase and ManNAc kinase activities in the cells of HIBM patients [16,17]. However, the pathogenic mechanisms responsible for the myopathy of HIBM have not been determined. Measurements of the sialic acid content of glycoproteins have given variable results [17,21-24,43-45]. Using sialylated α-dystroglycan-specific antibodies, Huizing et al. first reported decreased muscle α-dystroglycan sialylation in four HIBM patients, each having one mutation in the epimerase domain and one in the kinase domain of GNE . Noguchi et al. followed with findings of fiber-to-fiber variability in the hyposialylation of α-dystroglycan in the muscles of patients with DMRV, allelic with HIBM . Saito et al. reported hyposialylation of muscle glycoproteins in a single patient with DMRV , but Hinderlich et al. and Salama et al. showed no decrease in sialylation of proteins in myoblasts cultured from HIBM patients homozygous for the Persian Jewish GNE mutation, i.e., M712T [43,44]. Broccolini et al. recently reported hyposialylated muscle α-dystroglycan in 4 of 5 HIBM patients, with no effect on binding to laminin ; they surmised that the sialylation status of α-dystroglycan is not critical to the protein's function. Ricci et al. used NCAM as an indicator of the extent of muscle glycoprotein sialylation; in HIBM, muscle NCAM was found to be hyposialylated . The divergence of these findings allows for the possibility that sialylation abnormalities represent epiphenomena in HIBM, and GNE mutations could cause disease by affecting cellular functions not influenced by sialylation.
Indeed, while we verified the hyposialylation of muscle NCAM in our HIBM patients (Figure 4A,B), we found that decreased sialylation of glycoproteins is not a constant finding in HIBM. Isoelectric focusing studies revealed normal N-linked glycosylation of serum transferrin and O-linked glycosylation of Apo C-III [see Addional file 1]. In addition, sialylated α-dystroglycan was not decreased in the muscle of our four HIBM patients prior to IVIG administration. This could be because patients vary with respect to their specific GNE mutations, the extent of their sialic acid deficiency, and the degree to which their muscle proteins are sialylated, even when sampling the same muscle. Conditions of the immunohistochemical staining could differ, as could the specificity and sensitivity of the antibodies.
With normal baseline glycosylation of serum glycoproteins and muscle α-dystroglycan in HIBM, we had no opportunity to demonstrate improvement in these parameters after IVIG treatment. Muscle NCAM was hyposialylated before IVIG administration, but we could not discern consistent improvements in NCAM sialylation, either by immunohistochemistry or by immunoblotting, after IVIG treatment. One possible explanation is that the amount of sialic acid provided by IVIG was inadequate to increase glycoprotein sialylation. We estimate that normal adult urinary free sialic acid, a reasonable measure of daily production, approximates 0.3 mmol . Our IVIG loading doses (2 g/kg) should provide 8 μmol/g or ~1 mmol of sialic acid, roughly 3 days' worth of normal sialic acid production. This may or may not be sufficient to achieve a detectable or functional increase in the glycosylation of muscle protein.
We would expect a decrease in muscle NCAM to accompany the decrease in muscle degeneration hoped for with IVIG treatment, and this occurred in patients 1 and 2. The increased amount of hyposialylated NCAM in patient 4 after IVIG treatment contradicts this finding and reflects variability either in response to treatment or in muscle sampling.
In contrast to the absence of consistent effects on histological and immunological markers of sialylation, IVIG treatment improved objective and subjective measures of muscle strength and function in HIBM patients. Muscles that had greater strength at baseline, such as the quadriceps, appeared to benefit more than muscles that were already extensively damaged; results were variable but, on average, the improvement in strength exceeded 20% in the quadriceps and shoulders. Even though the acquisition of functional abilities was short-lived, all the patients were pleased, and two of them wished to pursue IVIG treatments independently. It is not surprising that acute clinical improvements were not accompanied by histological changes, since morphological changes should require long-term treatments and, perhaps, muscle regeneration.
There are several possible explanations for the beneficial clinical effects of IVIG despite the absence of evidence for increased glycoprotein sialylation. The salutary responses to IVIG could have been due to its anti-inflammatory actions [47,48]; patient 3 had previous evidence of central nervous system inflammation and patient 4 had a muscle biopsy read as possible polymyositis. Nevertheless, the myopathy in HIBM is not considered to be inflammatory, and muscle biopsies performed at the NIH on all four patients lacked signs of inflammation. A second possibility is that minor increases in sialylated muscle glycoproteins, responsible for improved muscle function, were present transiently after IVIG treatment, but were undetectable in our assay. A third possibility is that the muscle we biopsied, the quadriceps, had minimal baseline reduction in glycoprotein sialylation so that any increased sialylation after therapy could not be appreciated. Finally, the beneficial clinical effects might not be at all related to sialylation.
Ours was clearly a pilot study, clarifying some issues for future therapeutic investigations. For example, we documented that muscle strength testing provides an objective outcome parameter, with fixed dynamometry detecting small and large changes in strength, complemented by more subjective measures of muscle function . We also determined that muscle histology, immunohistology and sialylated glycoprotein quantitation by immunoblotting do not provide reliable measures of improvement, at least in the short term. These parameters require further investigation using longer treatment regimens and greater amounts of sialic acid supplementation. One possibility would be to administer only the Fc portion of immune globulin, which contains the bulk of the sialic acid residues and has ten times the anti-inflammatory efficacy of the complete molecule . Even so, IVIG is not likely to be the treatment of choice for HIBM, because it must be administered repeatedly, its mode of delivery requires hospitalization, its cost is considerable, and it has side effects. However, the finding of definitive improvements, albeit moderate and transient, attributable to IVIG suggests that the provision of sialic acid holds therapeutic promise.
Our next pursuit will involve administration of ManNAc, an uncharged sugar situated in the sialic acid synthetic pathway after the rate-limiting UDP-GlcNAc 2-epimerase step. Residual ManNAc kinase activity in HIBM patients, or ancillary kinases such as GlcNAc kinase , might convert ManNAc into ManNAc-6P for subsequent synthesis of sialic acid. In fact, hyposialylated, GNE-deficient mouse embryonic stem cells became resialylated after their growth medium was supplemented with ManNAc . Furthermore, ManNAc supplementation has resulted in prolonged survival of a mouse model of HIBM , and could yield increased glycoprotein sialylation in the tissues of humans with HIBM.