Multidimensional analyses of the pathomechanism caused by the non-catalytic GNE variant, c.620A>T, in patients with GNE myopathy

Figure 1. Two distinct haplotypes of patients carrying c.620A>T. (a) Two distinct haplotypes carrying the c.620A>T allele. IBD analysis of c.620A>T homozygotes. An IBD stretch spanning 1780 kb (Chr9: 36,118,198–37,898,880), including the whole of GNE, was identified in P1 and P2. A different IBD stretch, spanning 680 kb (Chr9: 36,137,298–36,818,382), and also including the whole of GNE, was identified in P3 and H1. (b) Age at onset of patients homozygous for c.1807G>C and c.620A>T. Haplotype α was shared by P1 and P2. Haplotype β was shared by P3 and H1. Age at onset of c.620A>T/c.1807G>C compound heterozygous patients (hapα: n = 19, hapβ: n = 7) was later than in those homozygous for c.1807G>C (n = 84). There was no significant difference between patients carrying Haplotype α and Haplotype β. The ages at onset of P1, P2, and P3 were 37, 34, and 60 years, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2. CNV analysis of GNE and structure of the GNE transcript in skeletal muscles from c.620A>T homozygotes. (a) XHMM plot of chr9: 36,000,000–38,000,000. No large deletions or insertions were found in GNE in P1–3 or H1. (b) Sashimi plots of GNE transcripts in skeletal muscle showing that there were no structural variants in P1–P3.

Figure 3. SA level and skeletal muscle transcript expression levels of genes involved in the SA metabolic pathway in c.620A>T homozygotes. (a) Serum SA concentrations in patients with GNE myopathy carrying other than c.620A>T homozygous mutation (n = 47) were significantly lower than those in healthy individuals (n = 19) (p < 0.0001). Serum SA concentration of P3 was in the upper range of that in patients with GNE myopathy carrying other than c.620A>T homozygous mutation, while that of H1 was in the range of healthy controls. (b) SA levels in skeletal muscles from patients with GNE myopathy carrying other than c.620A>T homozygous mutation (n = 6) were significantly lower than those of the disease controls (n = 4) (p = 0.0041), who were suspected to have muscle disease but whose muscle biopsy specimens exhibited no changes, while that of c.620A>T homozygous patients (n = 3, P1–3) was higher than those of other GNE myopathy patients (p = 0.0066) and lower than those of controls (p = 0.19) although the difference was not significant. (c) SA concentrations in fibroblasts from patients with GNE myopathy carrying other than c.620A>T homozygous mutation (n = 13) were significantly lower than…

Figure 4. Location of aspartic acid 176 in a GNE protein structural model. (a) Structure of the epimerase domain of GNE according to Protein Data Bank (4ZHT). Aspartic acid 176 is located outside of the catalytic site (at 28 Å within the same domain and 30 Å from the other monomer). (b) Full-length GNE protein structure predicted using AlphaFold2. Left: Illustration of a GNE dimer consisting of monomer 1 (light blue) and monomer 2 (light green). Aspartic acid 176 (red) is located on the surface of the epimerase domain, which may be involved in intramolecular ManNAc transfer (yellow arrow in monomer 1). Side view of the GNE complex rotated by 60°. Aspartic acid 176 is located near the dimer-dimer interface but does not appear to be directly involved in the interaction between the two monomers.

Figure 5. Model of ManNAc 6-phosphate production by mutated GNE. ManNAc 6-phosphate production ability was predicted as the sum of production route scores, which were calculated by multiplying the UDP-GlcNAc 2-epimerase reaction, ManNAc transfer, and ManNAc kinase reactions in the GNE dimer. Enzymatic reactions mediated by UDP-GlcNAc 2-epimerase and ManNAc kinase, and intramolecular and intermonomer ManNAc transfer in normal GNE dimer: Score 1. In GNE molecule with D207V, intramolecular ManNAc transfer may be affected: Score 0.5. Intermonomer transfer in GNE dimer with D207V is variable, due to failure of oligomerization: Score 0.3–0.5. In mutant GNE with catalytic mutations or V603L, both enzymatic reactions are affected: Score 0.1 and 0.2, respectively.