Long-term Evaluation Parameters in GNE Myopathy: A Five-year Observational Follow-up Natural History Study

A number of clinical trials targeting GNE myopathy patients have been conducted. However, useful clinical parameters for post-marketing surveillance and long-term clinical observation have not yet been established. Objective We conducted a 5-year observational follow-up natural history study to identify evaluation parameters which may be useful for the long-term observation of GNE myopathy patients. Methods Thirty-three genetically-conrmed GNE myopathy patients were recruited and evaluated at study entry (baseline) and yearly in a 5-year follow-up. Hand-held dynamometer measurements of knee extension strength, grip power, and pinch power, summed Manual Muscle Testing (MMT) score of 17 muscles, Gross Motor Function Measure (GMFM), 6-minute walk test, percent vital capacity and percent force vital capacity (%FVC), lean body mass (whole body, arms, and legs), creatine kinase (CK), Barthel Index, modied Rankin Scale, and SF-36 national standard scores were examined. Of the 33 patients, 22 (66%) completed evaluations for the entire 5-year follow-up period. These patients had a signicant reduction in summed MMT score (p=0.001), GMFM (p=0.001), grip power (p=0.013), pinch power (p<0.001), CK (p=0.030), %FVC (p<0.001), leg lean body mass (p=0.040), and the Physical Functioning subscale score of the SF-36 (p=0.015) at the 5th year evaluation relative to baseline. Among these parameters, summed MMT score, GMFM, pinch power, and %FVC showed signicant changes even in non-ambulant patients. MMT, GMFM, pinch power, CK, %FVC, lean body mass, and Physical Functioning subscale score of the SF-36 are useful parameters for the long-term evaluation of GNE myopathy patients. 20 non-ambulant patients. On the hand, no signicant change in grip power was observed in comparison with which were signicantly No signicant were detected

Oral sialic acid metabolite treatment can prevent muscle atrophy and weakness in a mouse GNE myopathy model [8]. Although a recent clinical trial (Phase 3 Randomized, Double-Blind, Placebo-Controlled Study to Evaluate Sialic Acid; ClinicalTrials.gov; identi er: NCT02377921) failed to demonstrate the e cacy of sialic acid to treat GNE myopathy, another clinical trial is currently underway in the United States to test ManNAc, an uncharged precursor of sialic acid (Multi-Center Study of ManNAc for GNE Myopathy (MAGiNE); ClinicalTrials.gov; identi er: NCT04231266). One of our research interests is the identi cation of clinically useful parameters for evaluation both in clinical trials and for long-term follow-up after new medications become available for GNE myopathy. We previously published a 1-year natural history study of 27 Japanese GNE myopathy patients and detected signi cant progression of the disease using Manual Muscle Testing (MMT), grip power, and % forced vital capacity (FVC) [9,10]. On the other hand, the 6-m walk test (6MWT), Gross Motor Function Measure (GMFM), hand-held dynamometer (HHD) measurements of quadriceps strength, pinch power, lean body mass, creatine kinase (CK), and activities of daily living (ADL) (e.g., as assessed by the modi ed Rankin scale (mRS) and Barthel Index (BI)) failed to detect signi cant changes during the 1-year period, possibly due to the small sample size or relatively short observation period.
The present study followed the progress of GNE myopathy patients for a longer period of 5 years to assess changes in clinical parameters with the aim of identifying evaluation parameters which could be useful for post-marketing surveys and long-term clinical observation.

Study population and design
The present study used prospective data from genetically-con rmed Japanese GNE myopathy patients who were evaluated at least twice (at baseline and at least one of the annual follow-up evaluations during the 5 year follow-up period) at the National Center of Neurology and Psychiatry (NCNP) Hospital. Genetic information was acquired from available medical records. Inclusion criteria included the ability to perform repeat testing. Data from patients who were able to attend at least one annual follow-up evaluation were included in the analysis. Patients who attended the 5th year evaluation were requested to answer the 36-item short form survey (SF-36) and provide updates on their ADL and ambulation status. The rst patients were enrolled in April 2009, and the last data analyzed were from November 30, 2019.

Data analysis
Data were summarized using descriptive statistics and presented as mean ± standard deviation (SD), median, range, frequency, or percentage. The Mann-Whitney U test and Kruskal-Wallis test were used for continuous data, and Fisher's exact test was used for binary data. The paired t-test was used to compare differences between baseline and follow-up data. Spearman's rank correlation coe cients were used to examine correlations between variables. Data of patients for whom meaningful measurements could not be made at baseline were excluded from the analysis. All analyses were performed using SPSS for Macintosh (Version 23; SPSS Inc., Chicago, IL).

Results
General characteristics at study entry Patient characteristics are summarized in Table 1. A total of 33 Japanese GNE myopathy patients (12 males and 22 females) participated in this study. Two female patients were siblings, and all other patients were unrelated to each other. Mean age at the time of data collection was 43.2 ± 13.4 years (mean ± SD), and mean age at disease onset was 26.6 ± 11.3 years. Thirty percent (11/33) of patients were ambulant and completed the 6MWT without assistance, 18.2% (6/33) required assistance (e.g., cane and/or brace), and 60.6% (20/33) had lost ambulation. Mean age at loss of ambulation was 34.1 ± 11.7 years. Table 1, 2)
Patient characteristics during and at end of follow-up period ( Table 2)  Follow-up was disrupted for 5 ambulant patients due to their participation in Phase II/III clinical trials of SA, and 9 patients due to personal reasons (mainly di culty visiting the hospital due to disease progression). The remaining 22 patients were followed for 5 years, although 9 of these patients missed some of the annual visits. For patients unable to attend all annual visits, we requested that they prioritize attending the 1st year and 5th year evaluations. Of patients who completed the 5th year evaluation, only 4 were able to complete the 6MWT. Two patients lost ambulation, and one patient newly developed multiple sclerosis and nephrotic syndrome, during the follow-up period.
Annual changes in physical status and measurements (Table 2) Measurement results are shown in Table 2. A total of 30 patients participated in the 1st year follow-up visit. For physical evaluation, 2, 21, 10, 10, and 5 patients were unable to complete the GMFM, 6MWT, HHD, grip power, and pinch power tests at baseline and thus were excluded from the analysis. For 6MWT, HHD, grip power, and pinch power tests, 1, 2, 2, and 6 patients, respectively, scored 0 for these measurements during the 5-year follow-up period. Summed MMT score, 6MWT, GMFM, %FVC, %VC, and grip power signi cantly differed at the 1st year evaluation compared to baseline. ADL and QOL scores ( Table 2, Table 3) No signi cant changes were observed in mRS and BI during the 5-year follow-up period. Among the subscales of SF-36, only scores for Physical Functioning were signi cantly reduced at the 3rd, 4th, and 5th year evaluations compared to baseline (Table 2).
National standard scores for SF-36 were used to evaluate QOL. All assessed subscales had signi cantly lower scores than the national standard index (score=50). Motor function measures (summed MMT, GMFM, 6MWT, grip power, and pinch power), respiratory function (%VC and FVC), whole body and leg lean body mass, and ADL scores (BI and mRS) were signi cantly correlated with the Physical Functioning subscale (Table 3).
Correlation between quantitative items and simpli ed items, DEXA, and CK (Supplementary table 4) Summed MMT and GMFM were well correlated with pinch power, grip power, %VC or %FVC, CK, and DEXA (legs). These items were also correlated with certain muscle MMT scores (e.g., elbow exion and knee extension).

Discussion
To our knowledge, this study is the rst to assess the 5-year natural history of GNE myopathy. In our previous 1-year observational study, not all parameters assessed at the 1st year evaluation were signi cantly different compared to baseline [8]. Thus, in the present study, the follow-up period was extended to 5 years. MMT, GMFM, grip power, pinch power, %FVC, %VC, leg lean body mass, CK, and the Physical Functioning subscale of the SF-36 showed signi cant changes at the 5th year evaluation compared to baseline. On the other hand, no signi cant changes were observed for 6MWT, HHD, arm and truncal body mass, BI, mRS, and other SF-36 subscales.
The lack of change in the 6MWT during the follow-up period could be explained by the small number of patients who could be tested and the exclusion of patients due to their participation in clinical trials. Among the muscles evaluated by MMT, shoulder girdle muscles showed continuous signi cant changes, even as early as at the 1st year evaluation, suggesting that MMT of shoulder girdle muscles may be useful to include in evaluations for clinical trials and natural history studies.
While a previous observational study of GNE myopathy patients assessed HHD over the course of 3 years [15], the quantitative measurement of many muscles is not realistic for routine clinical visits. In contrast, our study clearly demonstrated the utility of summed MMT score as well as shoulder muscle MMT scores, in addition to other laboratory parameters. Importantly, these items are easy to measure during clinical visits and do not overly burden evaluators and patients.
For severely affected, non-ambulant patients, walking parameters and grip power are not always useful. We evaluated clinical parameters in non-ambulant patients and found signi cant reductions in all items, except for grip power, which showed signi cant reductions in the analysis of the entire population at the 5th year evaluation. As 43% of the Japanese patients were non-ambulant, items for non-ambulant patients were quite important. Although we did not analyze data from ambulant patients due to the small sample size, the simpli ed items identi ed in the present study may be useful in clinical practice, even in outpatient settings with limited labor force.
Physical functioning subscales of SF-36 were signi cantly correlated to other evaluation items. Therapeutic approach could be ameliorate QOL of GNE myopathy patients, and SF-36 physical functioning can be useful for self-reporting evaluation.
This study has some limitations. First, the small number of patients, especially ambulant patients, did not allow us to draw conclusions regarding the utility of 6MWT. Second, the evaluation of ADL was limited to the use of mRS and BI, which are not sensitive enough to detect temporal disease progression. Using disease-speci c scales, such as the GNE Myopathy Functional Activity Scale, may be an alternative, but it was not used from the beginning of our study period.
In This study have been performed in accordance with the Declaration of Helsinki ,and was approved by the Medical Ethics Committee of the NCNP (20-9-Ji6 and A2018-024). Study objectives, design, risks, and bene ts of participation were explained to all patients, and their written informed consent was obtained prior to enrollment.

Consent for publication
Not applicable

Availability of data and materials
This study included articles which are available via PubMed. All information analyzed in this study was collected in a dataset, which is available from the corresponding author on reasonable request.

Figure 1
Annual changes in assessed parameters Manual muscle testing at baseline and annual follow-ups. *p < 0.05