This study included 44 patients (19 males and 25 females) with homozygous or compound heterozygous GNE mutations, most of which were previously reported as pathogenic ones and others were predicted as being ‘damaging’ by in silico analyses, and also classified as ‘pathogenic’ or ‘likely pathogenic’ according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guideline.10 All of the recruited patients agreed to provide their medical records, complete clinical questionnaires, and provide written informed consent. The Institutional Review Board of Pusan National University Hospital approved this study (IRB No. H-1712-002-061).

All of the included patients had confirmed pathogenic GNE mutations in nine different hospitals with neuromuscular specialists prior to recruitment to this study. We extracted DNA from the whole blood of 15 of the patients who directly visited our neuromuscular center, which was used in PCR amplification and direct sequence analyses in our laboratory. The PCR amplification covered all 12 exons and the immediately flanking intronic regions of the GNE gene. The PCR products were sequenced in both the forward and reverse directions. Sequences were analyzed and compared with the reference sequence (GenBank accession number NM_001128227.2). Seven of the patients who were genetically diagnosed in other hospitals personally provided their medical records, including mutation data. For the remaining 22 patients who could not acquire their mutational reports from their hospitals, GNE tests were performed at the laboratory of EGL Genetics (Tucker, GA, USA).

We applied a questionnaire-based survey to determine the functional status of the patients. Most of the information was collected using a self-reporting questionnaire, but we also retrospectively reviewed any available hospital records for further information. The questionnaire included inquiries on the following features: age at disease onset, initial presentation symptoms, mode of ambulation, and time of losing ambulation. In addition, we asked how many of the following eight major joint movements of the body could be performed against gravity: shoulder abduction, elbow flexion, wrist flexion/extension, hip flexion, knee extension, ankle dorsiflexion, neck flexion when supine, and trunk flexion when supine. Patients were asked whether they were independent or dependent on caregivers when performing major activities of daily living, including standing from a squatting position, getting up from the bed, eating, cleaning the face, going to the toilet, and changing clothes.

Linear regression analysis was applied to the relationships between disease duration and joints with mobility against gravity, and between disease duration and independent daily activities. Subjects capable of either all or none of the functional movements and daily activities were excluded from the analysis in order to prevent the ceiling effect. We further evaluated the disease progression depending on the initial symptoms or the domains of GNE mutations. These subgroup analyses were performed using Student's t-test or one-way ANOVA. A p value of <0.05 was considered to be indicative of statistically significant data.

The clinical information of the 44 Korean patients from 41 families is provided in Table 1. Their age at the time of data collection was 36.0±10.7 years (mean±SD). Their age at onset ranged from 14 to 51 years (26.3±9.2 years), and patient 16-II was an asymptomatic brother of patient 16-I. The disease duration from onset to diagnosis was 1–41 years (10.0±8.8 years). Eleven of the patients had affected siblings compatible with an autosomal recessive inheritance pattern. None of the patients with a detailed family history reported obvious consanguinity.

All of the recruited patients harbored homozygous or compound heterozygous GNE mutations. The types of mutations and their locations and frequencies are presented in Table 1 and Fig. 1. The most common mutation was c.1807G>C (p.Val603Leu), which was found in 45 alleles (51.1%), followed by c.131G>C (p.Cys44Ser) found in 20 alleles (22.7%). Homozygosity was also related to these two common mutations, and was found in 11 patients with p.Val603Leu and 3 with p.Cys44Ser. Two of the 15 different mutations have not been reported elsewhere. One of the novel mutations (c.1619A> C, p.His540Pro) was predicted to be ‘damaging’ and ‘disease causing’ by in silico analyses implemented using PROVEAN (http://provean.jcvi.org) and MutationTaster (http://mutationtaster.org), and classified as ‘likely pathogenic’ according to the ACMG/AMP guideline, since a change in the same amino acid by a different nucleotide change (c.1618C>T, p.His540Tyr) is reportedly pathogenic.11 The second one (c.2046delT, p.Val 684Leufs*2) can be classified as ‘pathogenic’ since it is expected to cause premature termination by frameshift. The other 13 mutations were first reported in Korean, Japanese, or Chinese patients.6 Except for 3 frameshift mutations involving small deletions, 12 were all missense mutations. No case had truncating mutations in both alleles. The mutations were scattered throughout the exons. Eight of the mutations were located in the epimerase domain (ED) and the other seven were in the kinase domain (KD), as presented in Fig. 1. Both mutations were present in KD in 17 of the patients (38.6%), while both were in ED in 5 patients (11.4%). The other 22 patients (50.0%) had one mutation in each domain (ED/KD).

Various initial symptoms at disease onset were reported by the patients, as indicated in Table 1. Foot drop and/or big-toe extension weakness indicating affected anterior lower leg muscles was reported in 19 of the patients (43.2%). Thirteen patients (29.5%) reported running or climbing difficulty suggestive of hip-girdle weakness as their first symptom. Six of the patients (13.6%) described difficulty in toe gait or calf atrophy corresponding to the posterior lower legs being affected. One exceptional patient reported weak thumb flexion as his first symptom. Patient 16-II remained asymptomatic until the last follow-up at an age of 19 years. The remaining four patients recalled experiencing nonspecific symptoms (e.g., buttock pain or gait disturbance) at the beginning of their disease.

Among 20 patients for whom medical records on the initial symptoms were available, 12 described having more prominent weakness in ankle dorsiflexion. Meanwhile, disturbed toe gait and/or calf muscle atrophy was reported in two of the patients, while proximal dominant leg weakness was described in the other four.

We considered a set of joints to be functional when the patient could flex or extend the joint against gravity. Fig. 2A depicts the pattern of loss of functional joint movement. Ankle dorsiflexion was the most commonly affected movement when the patient had just begun to lose a single functional set of joints. On the other hand, wrist or neck flexion was most likely to be the last functional movement that was lost.

All 8 sets of joint movements remained functional in 12 of the patients, and their disease duration was 2–7 years (4.2±2.2 years). Seven patients had no functionality in any of the eight considered joint movements, and their disease duration was 11–41 years (23.3±10.7 years). The number of functional joint sets was strongly correlated with the disease duration, as shown by the results of the linear regression analysis in Fig. 2B (r²=0.5228, p<0.0001) reflecting the progression of GNE myopathy. The mean yearly rate of decrease in the number of functional joint sets was −0.2581, with a 95% CI of −0.1517 to −0.3645.

Sixteen patients (36.4%) had to use wheelchairs throughout the day, 20 patients (45.5%) were still capable of completely independent ambulation, and 8 patients (18.1%) used walking aids when walking longer distances. The self-reported disease duration to a wheelchair-bound state was 10.6±3.3 years (5–15 years). There was a clear difference in the disease duration between ambulatory and wheelchair-bound patients, at 4.5±0.7 years (1–13 years) and 17.4±2.2 years (9–41 years), respectively (Fig. 3A).

The daily activities that each patient could perform independently are listed in Fig. 3B, sorted in decreasing numerical order. Among 41 patients who provided information on their independent daily activities, 21 (51.2%) were could still independently perform all six kinds of the activities surveyed. Standing from a squatting position was disturbed at the earliest time point, which was lost in all five patients who could still perform the other five activities. The disease duration of these patients was 6–10 years (7.8±1.6 years).

After standing from a squatting position, the order of independent activities lost were getting up from the bed, changing clothes, and going to the toilet, all of which require movements of both the upper and lower extremities. Cleaning the face and eating, which mainly involve the upper extremities, were the last activities to be disturbed. Eight patients (19.5%) could perform none of the activities at all, and they had a disease duration of 11–41 years (24.0±10.1 years). The relationship between disease duration and independent activities is depicted in Fig. 3C (r²=0.4841, p=0.012).

The age at onset was markedly younger in the subgroup with limb-girdle phenotype (23.2±1.6 years) than in that with foot drop onset (31.2±2.0 years, p=0.006; Fig. 4A). The duration from the onset to the diagnosis was significantly longer in the subgroup with limb-girdle (5.1±0.9 vs. 11.1±2.6 years, p=0.0186), as shown in Fig. 4B.

The yearly rates of loss of functional joints were −0.3008 (r²=0.5827, p=0.0015) and −0.2901 (r²=0.8893, p=0.0162) in the subgroups of foot drop and limb-girdle respectively, with no significant difference (p=0.5731) (Fig. 4C and D).

We compared the disease severity among subgroups according to domains where the mutations were located; that is, the KD, ED/KD, and ED subgroups. The age at onset did not differ significantly among these subgroups (p=0.387) (Fig. 5A). On the other hand, the decrease ratios of functional joints did differ significantly among the three subgroups (p=0.0062, Fig. 5B–D), with a mean decrease ratio of -0.3617 in KD (r²=0.7234, p=0.0037), −0.2601 in ED/KD (r²=0.4403, p=0.0186), and −0.1402 in ED (r²=0.8760, p=0.0641). The disease duration among wheelchair-bound patients did not differ significantly between the two subgroups of KD (17.5±3.6 years, n=8) and ED/KD (15.9±3.4 years, n=8; p=0.7455). The ED subgroup was excluded from this analysis due to the small number of wheelchair-dependent subjects (n=3).