Fasciculation potentials are related to the prognosis of amyotrophic lateral sclerosis

Keiko Ohnari; Kosuke Mafune; Hiroaki Adachi

doi:10.1371/journal.pone.0313307

. 2024 Nov 8;19(11):e0313307. doi: 10.1371/journal.pone.0313307

Fasciculation potentials are related to the prognosis of amyotrophic lateral sclerosis

Keiko Ohnari ^1,^*, Kosuke Mafune ², Hiroaki Adachi ^1,^*

Editor: Aditya Kumar Padhi³

PMCID: PMC11548741 PMID: 39514515

Abstract

Some prognostic biomarkers of amyotrophic lateral sclerosis (ALS) have been described; however, they are inadequate for satisfactorily predicting individual patient outcomes. Fasciculation potentials (FPs) on electromyography (EMG) are useful for the early diagnosis of ALS, and complex FPs are associated with shorter survival in ALS. In this study, we investigated the relationship between the proportion of muscles with FPs, biochemical markers, and the prognosis of ALS. 89 Patients with ALS were retrospectively classified into three groups based on the interval from onset to death or tracheostomy (less than 1 year: fast progression; from 1 year to less than 3 years: average progression; 3 years or more: slow progression). We performed statistical analysis of the electrophysiological findings, including the percentage of examined muscles with FPs, and biochemical markers evaluated on admission. Patients with fast ALS progression had a higher percentage of muscles with FPs (93.1% vs. 37.9%, P<0.001) and lower uric acid (UA) levels (male: 4.19 mg/dl vs 5.55 mg/dl, P<0.001; female: 3.71 mg/dl vs 5.41 mg/dl, P<0.001) than patients with slow progression. Survival curves demonstrated a relationship between these factors and the survival time in patients with ALS. Furthermore, UA levels were correlated with the percentage of muscles with FPs. Our electrophysiological findings suggest that ALS presents with multisystem neurological manifestations, and these manifestations differed among the groups classified by disease progression. The percentage of muscles with FPs on EMG and serum UA levels were especially associated with the prognosis of ALS.

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by upper and lower neuronal manifestations. However, its pathogenesis remains unclear. The average survival time is 2–4 years, but disease progression varies among patients [1]. The cumulative time-dependent survival rates at 1, 5, and 10 years after diagnosis have been reported to be 76.2%, 23.4% and 11.8%, respectively [2]. Many clinical trials for ALS have been conducted and are ongoing; hence, the features and prognostic factors of ALS need to be adequately clarified to assess the effectiveness of treatment modalities. Based on data collected from patients with ALS across Europe, bulbar onset, autonomic dysfunction, diagnosis of definite ALS according to the revised El Escorial criteria, diagnostic delays, reduced forced vital capacity, progression rate, and presence of frontotemporal dementia have been reported as poor prognostic factors [3, 4].

The prognosis of ALS has been studied in terms of not only clinical symptoms but also electrophysiological findings and biochemical markers [5–7]. The correlation between the prognosis of ALS and electrophysiological findings in the central and peripheral nervous systems has been reported [8]. In particular, needle electromyography (EMG) findings, including motor unit potential morphology and the localization and frequency of fasciculation potentials (FPs), have been reported to be important for diagnosing ALS [5]. The methods for detecting FPs include conventional EMG and muscle ultrasound (MUS) [9]. MUS is not an invasive examination and can facilitate real-time detection of FPs from a large body surface area. FPs analyzed using MUS have been reported to show prognostic significance for ALS [10, 11]. However, MUS does not allow analysis of the morphology or firing characteristics of the motor units involved [12]. Moreover, MUS findings are not included in the Awaji and Gold Coast criteria [13–15].

The prognostic relationship between neurofilaments and biochemical markers such as creatinine, uric acid (UA), and lipid levels has also been reported previously [16–18]. and neurofilaments have been reported to be diagnostic biomarkers [18]. However, compared with neurofilament analyses, the analyses of creatinine and UA are cheaper and can be used as prognostic biomarkers of ALS.

Given the aforementioned limitations of MUS and prospects of creatinine and UA as prognostic markers, this study aimed to clarify the prognosis of ALS using needle EMG, and also to study the association between electrophysiological findings and biochemical markers for the prognosis of ALS.

Methods

Patients

We studied 89 patients with ALS who were treated at the Hospital of the University of Occupational and Environmental Health, Japan, between May 11, 2000 and December 31, 2023. All patients were diagnosed with sporadic, definite ALS according to the revised El Escorial and Awaji criteria [13, 14]. and presented with progressive disease. The median survival duration of the patients with ALS was 3 years, and the 1-year survival rate was 76.2% [2]. Based on the interval between symptom onset and death or tracheostomy, the patients were retrospectively classified into three groups (less than 1 year: fast progression; from 1 year to less than 3 years: average progression; and 3 years or more: slow progression). We also investigated the period from onset to first evaluation and the symptoms at onset. Biochemical tests, including the measurement of UA, albumin, total cholesterol, creatine kinase, triglyceride, and creatinine levels, and neutrophil count were performed at diagnosis. The study design was approved by the Ethics Committee of Medical Research at the University of Occupational and Environmental Health, Japan (UOEHCRB20-021). The study conforms with World Medical Association Declaration of Helsinki. Informed consent was not obtained from study participants as the study did not involve a prospective evaluation. The Ethics Committee of the University of Occupational and Environmental Health granted a permission to use the retrospective data in the study without individual informed consent. The authors had access to information that could identify individual participants during and after data collection. The data for our study was accessed on January 1, 2024.

Electrophysiological studies

All patients underwent nerve conduction studies (NCS) and needle EMG (Neuropack; Nihon Kouden, Tokyo, Japan). Motor conduction studies were performed in the median, ulnar, tibial, and peroneal nerves, for which recordings were obtained from the abductor pollicis, abductor digiti minimi, extensor digitorum brevis, and abductor hallucis, respectively. Sensory conduction studies were performed in the median, ulnar, and sural nerves. Recordings were obtained from the proximal interphalangeal joint of the index finger, proximal interphalangeal joint of the little finger, and area just behind the lateral malleolus. We ruled out polyradiculoneuropathy using NCS. Needle EMG was performed on the muscles of the upper extremities (UEs) and lower extremities (LEs), and the paraspinal and trapezius muscles. As tongue relaxation was difficult and fibrillation potentials, positive sharp waves (Fib/PSWs), and FPs were more frequently observed in the trapezius muscle than in the tongue in patients with ALS [19]. we performed EMG in the trapezius muscle. The most frequently examined muscles of the extremities in different segments were the deltoid, biceps brachii, triceps brachii, first dorsalis interossei, rectus femoris, anterior tibialis, and gastrocnemius. The number of muscles examined was determined based on the diagnostic implications. We examined the occurrence of Fib/PSWs and FPs at rest. FPs were defined as spontaneous and random motor unit potentials showing a highly complex morphology with reproducibility over observation for up to 90 seconds [20]. Therefore, we analyzed the proportion of the examined muscles that had Fib/PSWs and FPs.

Statistical analysis

Age, interval from onset to first evaluation, biochemical test results, percentage of muscles with Fib/PSWs and FPs, and NCS results were compared among the groups using analysis of variance. The variables in the NCS include motor nerve conduction velocities, distal latencies, compound muscle action potentials, sensory nerve conduction velocities, and sensory nerve conduction potentials. Sex and symptoms at onset were analyzed using Fisher’s exact test. The relationships between these variables and percentage of FPs were analyzed using correlation coefficient and a multiple linear regression model. Survival analysis was performed using Kaplan–Meier method with a log-rank test, the variables being the percentage of FPs and biochemical markers. Furthermore, Cox proportional hazards regression models were used to adjust for other factors affecting the percentage of FPs and UA levels. The covariates included in the model were age, sex, and symptoms at disease onset. The percentage of FPs was classified into three groups: less than 50%, 50–99%, and 100%. UA level was classified into three groups for both sexes: for males, <4.7, 4.7–5.4, and >5.4 mg/dl; for females, <3.7, 3.7–4.4, >4.4 mg/dL. The upper limit of the average survival time was defined as 48 months. Linear regression was used to examine the correlation between the percentage of FPs and UA levels. P-value <0.05 indicated statistically significant difference among the three groups.

Results

Based on the interval from ALS onset to death or tracheostomy, the total number of patients with ALS was 89. There were 29, 28, and 32 patients with fast, average, and slow progression, respectively (Table 1). The three groups showed no significant differences in sex distribution or rate of onset of extremity weakness. The age at onset did not differ between patients with fast progression and those in the other two groups; however, slow progression was more frequently observed among young patients. The percentage of patients with bulbar onset in the fast progression group was higher than that in the average progression group; however, there was no significant difference from the slow progression group. The time from symptom onset to the first evaluation in patients with fast progression was significantly shorter than in the other two groups (5.0±2.3, 11.3±7.3, and 19.5±10.9, respectively, P <0.001) (Table 1).

Table 1. Clinical findings in patients with ALS classified according to clinical course.

	Fast progression	Average progression	Slow progression	P-value	P-value, Fast progression vs Average progression	P-value, Fast progression vs Slow progression	P-value, Average progression vs Slow progression
Number of patients	29	28	32
Sex (men/women)	19/10	15/13	15/17	0.337
Age at onset (years)	67.8 ± 7.9	69.2 ± 9.0	62.4 ± 12.5	0.024 ^*	0.855	0.099	0.028
Time from symptom onset to the first evaluation (months)	5.0 ± 2.3	11.3 ± 7.3	19.5 ± 10.9	<0.001 ^*	0.009 ^*	<0.001 ^*	<0.001 ^*
Bulbar onset	15	5	12	0.035 ^*	0.049 ^*	0.390	0.243
UE onset	10	16	10	0.091
LE onset	7	8	10	0.825

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* statistically significant

ALS, amyotrophic lateral sclerosis; UE, upper extremity; LE, lower extremity

The EMG findings at rest are summarized in Table 2. The percentages of muscles with FPs in the fast, average, and slow progression groups were 93.1%, 62.9%, and 37.9%, respectively. Shorter disease period was significantly associated with more frequent FPs (P <0.001) (Table 2). Moreover, the survival curves obtained using the Kaplan–Meier method and Cox proportional hazards regression models demonstrated a relationship between the percentage of muscles with FPs and survival time in patients with ALS (P<0.000) (Fig 1A and 1B). In contrast, Fib/PSWs did not differ significantly among the three groups (S1 Fig). During the NCS, the motor conduction velocity (MCV) of the ulnar and tibial nerves in patients with fast progression was significantly lower than that in the other two groups (Table 3). The distal latencies of the tibial nerve in patients with fast progression were longer than those in the other two groups. Furthermore, the sensory conduction velocities (SCV) of the ulnar and sural nerves in patients with fast progression were significantly lower than those in the other two groups. The other components of the NCS showed no significant differences, and we found milder disturbances in patients with slow progression compared to the other groups (Table 3).

Table 2. EMG findings in patients with ALS classified according to clinical course.

	Fast progression	Average progression	Slow progression	P-value	P-value, Fast progression vs Average progression	P-value, Fast progression vs Slow progression	P-value, Average progression vs Slow progression
Muscles with FPs (%)	93.1 ± 11.6	62.9 ± 26.4	37.9 ± 25.9	<0.001 ^*	<0.001 ^*	<0.001 ^*	<0.001 ^*
Muscles with Fib/PSWs (%)	34.1 ± 31.4	51.9 ± 27.6	42.9 ± 33.2	0.101

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* statistically significant

ALS, amyotrophic lateral sclerosis; EMG, electromyography; FPs: fasciculation potential; Fib/PSWs: fibrillation potential and positive sharp waves

Fig 1 — Comparison of the survival curves after disease onset, stratified by the percentage of muscles with fasciculation potentials (FPs) in patients with amyotrophic lateral sclerosis by using the Kaplan-Meier method (A) and Cox proportional hazard regression models (B). The survival curves obtained using Cox proportional hazard regression model showed a relationship between the percentage of muscles with FPs and survival time (B). Comparison of the survival curves after disease onset stratified by uric acid levels using Kaplan-Meier method (C) and Cox proportional hazard regression models (D). The survival curves of the Cox proportional hazard regression model showed significant differences between patients with low uric acid levels and the other groups (D).

Table 3. Nerve conduction studies of patients with ALS classified according to the clinical course.

	Fast progression	Average progression	Slow progression	P-value	P-value, Fast progression vs Average progression	P-value, Fast progression vs Slow progression	P-value, Average progression vs Slow progression
Median
MCV (m/s)	51.8±5.9	49.9±5.7	53.3±4.4	0.075
DL (ms)	4.4±0.9	4.7±1.3	4.3±0.7	0.202
CMAP (mV)	4.4±3.0	3.1±3.3	5.1±3.7	0.072
SCV (m/s)	48.8±5.1	50.0±8.1	53.1±6.2	0.087
SNAP (μV)	20.4±8.9	20.3±12.0	25.5±10.3	0.144
Ulnar
MCV (m/s)	49.9±6.5	50.8±6.9	57.2±5.5	<0.001 ^*	0.879	0.001 ^*	0.001 ^*
DL (ms)	3.6±0.5	3.6±1.0	3.2±0.3	0.056
CMAP (mV)	5.6±2.9	4.2±2.9	5.4±2.9	0.174
SCV (m/s)	50.1±4.2	52.3±5.8	55.3±6.9	0.012 ^*	0.420	0.010 ^*	0.144
SNAP (μV)	17.6±9.0	17.2±9.3	21.5±10.6	0.212
Tibial
MCV (m/s)	42.4±3.7	44.3±5.3	46.6±4.3	0.007 ^*	0.303	0.005 ^*	0.162
DL (ms)	4.9±0.9	4.2±0.9	4.0±0.6	<0.001 ^*	0.010 ^*	<0.001 ^*	0.457
CMAP (mV)	7.0±4.5	9.2±5.1	8.0±5.4	0.296
Peroneal
MCV (m/s)	41.9±4.5	41.6±4.6	44.5±5.4	0.082
DL (ms)	5.1±1.2	5.5±1.2	5.0±1.0	0.327
CMAP (mV)	2.0±2.4	1.7±1.9	1.8±1.9	0.891
Sural
SCV (m/s)	45.5±4.6	47.4±5.8	50.8±4.3	0.001 ^*	0.359	0.001 ^*	0.037 ^*
SNAP (μV)	12.4±6.5	12.5±6.6	13.3±6.9	0.861

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* statistically significant

MCV, motor nerve conduction velocity; DL, distal latency; CMAP, compound muscle action potential; SCV, sensory nerve conduction velocity; SNAP, sensory nerve conduction velocity

Table 4 presents a comparison of the laboratory data among the three groups. The UA levels in both male and female patients with fast progression were lower than those in the average and slow progression groups (male: 4.19, 4.89, and 5.55, P = 0.000; female: 3.71, 4.00, and 5.41, P<0.001). Furthermore, the survival curve obtained using the Kaplan-Meier method demonstrated a relationship between UA levels and survival time in patients with ALS (P<0.001) (Fig 1C). The survival curves of the Cox proportional hazards regression model showed significant differences between patients with low UA levels and patients in the other two groups (P<0.000) (Fig 1D). The UA levels were significantly associated with FP (Table 5, Fig 2); this tendency was also observed in multiple regression analysis (Table 5). Laboratory parameters other than UA levels did not differ among the three groups (S2–S7 Figs). The triglyceride and creatine kinase levels of patients with fast progression were lower than those of patients with average progression, but not significantly lower than those of patients with slow progression.

Table 4. Laboratory data of patients with ALS classified according to clinical course.

	Fast progression	Average progression	Slow progression	P-value	P-value, Fast progression vs Average progression	P-value, Fast progression vs Slow progression	P-value, Average progression vs Slow progression
Albumin (g/dl)	4.04 ± 0.37	4.12 ± 0.35	4.07 ± 0.36	0.702
T-cholesterol (mg/dl)	195.57 ± 40.44	204.32 ± 30.21	193.86 ± 36.4	0.537
Males	188.56 ± 38.09	194.79 ± 28.15	188.36 ± 31.87	0.847
Females	208.20 ± 43.49	218.00 ± 30.63	199.69 ± 39.57	0.503
CK (U/L)	148.96 ± 180.06	227.43 ± 238.69	203.93 ± 154.79	0.319
Males	189.17 ± 210.16	317.07 ± 283.82	282.36 ± 173.41	0.254
Females	68.56 ± 24.51	124.00 ± 112.14	135.31 ± 97.47	0.219
TG (mg/dl)	87.81 ± 30.65	120.15 ± 58.22	106.77 ± 49.60	0.049 ^*	0.040 ^*	0.276	0.591
Uric acid (mg/dl)	4.05 ± 0.90	4.89 ± 1.38	5.55 ± 1.17	<0.001 ^*	0.026 ^*	<0.001 ^*	0.114
Males	4.19 ± 0.77	5.60 ± 1.11	5.71 ± 0.81	<0.001 ^*	<0.001 ^*	<0.001 ^*	0.956
Females	3.71 ± 1.14	4.00 ± 1.17	5.41 ± 1.42	0.006 ^*	0.875	0.014 ^*	0.022 ^*
Creatinine (mg/dl)	0.59 ± 0.21	0.61 ± 0.17	0.56 ± 0.19	0.678
Males	0.63 ± 0.19	0.67 ± 0.17	0.61 ± 0.23	0.719
Females	0.50 ± 0.24	0.53 ± 0.14	0.53 ± 0.16	0.921
Neutrophils (/μl)	3526.04 ± 1508.33	3719.32 ± 1096.84	3118.79 ± 1330.28	0.248

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* statistically significant

ALS, amyotrophic lateral sclerosis; T-cholesterol, total cholesterol; CK, creatine kinase; TG, triglyceride

Table 5. Factors related to the frequency of fasciculation potential.

	Univariate analysis				Multivariate analysis
	B^§	95% CI	β ^¶	P value	B^§	95% CI	β ^¶	P value
UA^†	-7.465	59.072–71.933	-0.321	0.001^*	-6.608	-13.766–0.551	-0.279	0.070^†
Sex					-14.020	-26.146 –-1.894	-0.229	0.024^*
Age at onset					0.507	-0.069–1.084	0.168	0.083^†
Bulbar onset					14.384	-7.936–36.704	0.225	0.203
UE onset					15.772	-5.092–36.637	0.253	0.136
LE onset					-1.565	-22.517–19.386	-0.024	0.882
Time from symptom onset to the first evaluation					-0.334	-1.893 –-0.449	-0.334	0.002^*
Interaction
UA × Sex					0.049	-5.857–13.734	0.049	0.730

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^§ partial regression coefficient

^¶ standardized partial regression coefficient

* statistically significant <0.05 ^† statistically significant <0.1

^† mean uric acid level

UA, uric acid level; UE, upper extremity; LE, lower extremity; CI: confidence interval

Sex is a dummy variable labeled 0 = men and 1 = women.

Fig 2 — The percentage of muscles with fasciculation potentials was negatively correlated with uric acid level (R = -0.321, P = 0.004).

Discussion

This study aimed to clarify the prognosis of ALS by using needle EMG and assessing the association between electrophysiological findings and biochemical markers. Our electrophysiological study of the peripheral and central nervous systems demonstrated significantly different findings among the groups classified according to the prognosis of ALS. We found that patients with rapidly progressing ALS had higher percentage of muscles with FPs and lower UA levels. UA level as a biochemical marker is related to survival in patients with ALS. In particular, the percentage of muscles with FPs on EMG was associated with ALS prognosis, and the percentage of muscles with FPs and serum UA level showed a significant negative relationship. We also found that the MCV of the ulnar and tibial nerves and the SCV of the ulnar and sural nerves in patients with fast progression were significantly lower than those in the other two groups. Our present data confirmed that the percentage of muscles with FPs on EMG and serum UA levels are important biomarkers for predicting the prognosis of ALS.

This study demonstrated that higher percentage of muscles with FPs is associated with shorter disease duration in patients with ALS. The source generator of FPs is the motor neuron or axon prior to its terminal branches [21]. FPs are associated with numerous disease processes affecting the lower motor neurons, such as ALS [21]. FPs detected with needle EMG were the core findings for diagnosing ALS since they reflect ongoing denervation in the affected muscles. Therefore, FPs and Fib/PSWs are equally important [14]. The rate and distribution of FPs have been reported to be specific findings in patients with ALS, in contrast to the findings of previous studies on polio, spinal and bulbar muscular atrophy, cervical spondylotic radiculopathy or myelopathy, lumbosacral radiculopathy, and multifocal motor neuropathy [22, 23]. In a previous study, the EMG findings in the trapezius muscle in cervical spondylosis showed neither Fib/PSWs nor FPs [19]. Moreover, the mean amplitude and duration of FPs increased with disease progression [24]. In contrast, the appearance of FPs is an extremely early marker of ALS, and FPs have been reported to occur in ALS without weakness and muscle atrophy [25].

The use of MUS for noninvasive diagnosis of ALS has been developed recently, and MUS and EMG have been reported to have nearly the same detection rates of FPs [10]. Several studies on MUS have shown that FPs are associated with the process and prognosis of ALS [6, 11, 12]. The rate of disease progression has been correlated with the number of fasciculations detected using MUS [11]. Furthermore, higher frequency of FPs in the biceps brachii and brachialis muscles with FPs on MUS was reported to be associated with shorter disease duration and faster decline in the ALS Functional Rating Scale-Revised (ALSFRS-R) score [6]. The features of FPs obtained using EMG in the ALS criteria have been compared to those obtained using MUS [5, 12, 26, 27]. EMG is more sensitive than MUS in detecting fibrillation [26]. Moreover, MUS does not allow the analysis of morphology or firing characteristics of the motor units involved [12]. The morphology of FPs in EMG has been reported to be important for determining the diagnosis and stage of ALS [5, 12] and the presence of complex FPs was associated with shorter survival [5]. Small FPs may be undetectable with MUS because of the significantly lower mean amplitude of FPs in patients with EMG-detected FPs alone than in those with both FPs and MUS fasciculations (0.39 ± 0.25 mV and 1.22 ± 0.92 mV, respectively: P < 0.0001) [27]. In this study, using EMG, we found a higher percentage of muscles with FPs in patients showing fast progression than in the other two groups. In accordance with previous reports on MUS, these results indicate that FPs are related to the prognosis of patients with ALS [6, 11, 12].

In addition to needle EMG, electrophysiological studies, including NCS have also been performed for ALS diagnosis [14]. The results of NCS have been reported to be related to the prognosis of ALS [28]. Compound muscle action potential (CMAP) and sensory nerve action potential (SNAP) amplitudes of the median nerve were considered independent prognostic factors of ALS [28]. In our study, we did not find significant differences in the CMAP and SNAP amplitudes of the median nerves among the three groups, but the MCV, CMAP, SCV, and SNAP amplitudes of the median nerve in patients with fast progression tended to be lower than the corresponding values in patients with slow progression. The MCV of the ulnar and tibial nerves and the SCV of nerves other than the median nerve in patients with fast progression were shorter than those in the other two groups. These differences are related to poor prognosis in patients with ALS. Sensory abnormalities have also been reported to be common in NCS performed in patients with ALS [29, 30].

Although patients with ALS usually do not present with sensory disturbances, they may show subclinical dysfunction in the sensory nervous system [8]. The peak-to-peak amplitude between N20 and P25 in the median SEP has been associated with shorter survival in ALS [31]. In addition to needle EMG, NCS showed significantly different results between patients with ALS with fast progression and patients in the other two groups. These findings indicate that ALS is a neurodegenerative disease that affects not only the upper and lower neurons but also the multisystem of neurons, as reported previously [8]. The pathological findings obtained in the sural nerve biopsy of patients with ALS have been reported previously [32]. Pathological sensory disturbance detected through skin biopsies has also been reported for ALS, with observations including, a significant loss of intraepidermal nerve fiber (IFNF) and Meissner corpuscle density in ALS compared with healthy controls, and increasing IENF density over time were associated with a poorer prognosis [33]. Furthermore, the 43-kDa TAR DNA-binding protein identified to be related to ALS pathogenesis is found widely in the nervous system, including the upper and lower neurons [34]. Thus, based on its neuropathology, ALS can be considered a form of multisystem neurodegeneration rather than a pure motor neuron disease.

UA has been identified as a natural antioxidant and free radical scavenger in the processes underlying oxidative stress [35]. UA is also reportedly related to some neurodegenerative diseases with underlying pathogenesis involving oxidative stress, such as Parkinson’s disease [36]. The pathogenesis of ALS is related to not only electrophysiological findings but also biomarkers such as UA [7, 37]. Oxidative stress has been proposed to play a role in the pathogenesis of ALS [38]. In one study, administration of edaravone, a potent radical and peroxynitrite scavenger, significantly delayed disease progression compared to what was observed in untreated patients [39]. Furthermore, the plasma levels of UA have been reported to increase in treated ALS patients [39]. Treatment with inosine was shown in another study to elevate the serum urate levels and slow down the progression of ALS, which was characterized using ALSFRS-R total scores [40]. The serum UA levels of patients with ALS were previously reported to be lower than those in healthy individuals [37]. and shown to be associated with prolonged survival in ALS [41, 42]. Similar to previous reports, we found that serum UA levels were associated with prolonged survival in ALS. Moreover, univariate analysis showed a significant negative correlation between the percentage of muscles with FPs and UA levels, whereas multivariate analysis showed a significant relationship. No reports on the relationship between FPs and serum UA levels s in ALS have been published to date. However, serum UA levels and FPs were negatively correlated in this study. This suggests that serum UA levels s may be involved in the mechanism underlying electrophysiological changes that lead to FPs in skeletal muscle and may modify the pathology of ALS. Although the association between prognosis and UA level was only reported in male patients previously [43]. UA level was subsequently reported to be a prognostic factor without significant differences between the sexes [44]. Albumin and creatinine levels have also been reported to be prognostic biomarkers of ALS. In contrast, lipid levels and neutrophil counts have recently been reported to be unrelated to disease progression [45, 46]. Our findings did not show any association between lipid levels, neutrophil counts, and ALS prognosis. Thus, UA level can be presumed to be an important prognostic factor for ALS. Sporadic ALS is an intractable disease, and its mechanism of onset is still unknown. The discovery of several factors involved in the pathogenesis or modification of the disease progression of ALS may lead to the elucidation of the mechanism of onset. These factors, which include electrophysiological and biochemical markers, are also expected to facilitate the early diagnosis of diseases, prediction of prognosis, and development of treatments.

Regarding clinical symptoms and disease course, the interval from symptom onset to the first evaluation was a significant factor influencing prognosis. One recent study reported that the disease duration at entry is one of the variables that can be used to discriminate slow, average, and fast progressors [47]. Among the onset symptoms, although bulbar onset has been reported to be related to shorter survival [48]. it is not an independent predictor of outcome by multivariable analyses [49]. We found that the onset symptoms did not differ significantly between patients with fast and slow progression.

Our study had several limitations. We did not examine the firing frequency of the FPs in each muscle. Differences in the firing frequency of FPs between ALS and benign fasciculations are valuable [12], but diagnosis may be difficult when using the firing frequency of FPs. However, because the firing frequency of FPs has been reported to correlate with disease progression, evaluation of FPs in a fixed period may be required. Furthermore, the examined muscles were determined according to the revised El Escorial criteria and Awaji criteria, however, because this was a retrospective study, the examined muscles and numbers differed among the patients. In our study, we evaluated the prognosis of patients using the severity of ALS as an index, which indicated whether they would die or develop respiratory failure. This study was also retrospective. Therefore, the standard ALSFRS-R, which is a clinically useful functional assessment scale, was not suitable for this study. In addition, it was not possible to incorporate evaluation items based on the presence or absence of muscle weakness. Further, the sample was small, and the findings of the study may not be generalizable. The retrospective nature of the study may have also introduced bias. While it is important to include data from the healthy control group for comparative analysis, we were unable to analyze the relationship between FPs and uric acid levels by adding data from the healthy control group. FPs can be observed in healthy individuals (benign fasciculation), but they are distinguished by the absence of accompanying muscle weakness and the simple waveform [14, 21]. When we performed needle electromyography, FPs were rarely observed in healthy individuals. Additionally, it has been reported that uric acid concentrations are significantly lower in patients with ALS than in individuals without it [37], but they varied within the normal range in this study.

Conclusions

The electrophysiological findings indicated degeneration from the peripheral to the central nervous system in ALS, and they were different among the groups classified by prognosis. Although this was a small study, the subjects were classified according to their real survival durations. This study revealed that the percentage of muscles with FPs and UA levels are reliable and useful prognostic factors. Particularly, high percentage of FPs observed on EMG is related to rapid progression of ALS. As described in previous studies, UA levels are negatively correlated with ALS prognosis. Surprisingly, our results demonstrated that the percentage of muscles with FPs had a significant negative relationship with UA levels. Collectively, our findings indicate that electrophysiological tests with needle EMG and laboratory data can predict the prognosis of ALS precisely, and will be beneficial for guiding therapeutic strategies for ALS.

Supporting information

S1 Fig. Comparison of the survival curves after disease onset stratified by rate of muscle with fibrillation potentials and positive sharp waves (Fib/PSWs) in ALS patients by using Kaplan–Meier method.

(DOCX)

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S2 Fig. The survival curves for ALS patients with albumin < 4 g/dL vs. albumin > 4.1 g/dL using Kaplan–Meier method.

(DOCX)

pone.0313307.s002.docx^{(70.3KB, docx)}

S3 Fig

The survival curves for male ALS patients with total cholesterol < 189 mg/dL vs. total cholesterol > 190 mg/dL using Kaplan–Meier method (A). The survival curves for female ALS patients with total cholesterol < 199 mg/dL vs. total cholesterol > 200 mg/dL using Kaplan–Meier method (B).

(DOCX)

pone.0313307.s003.docx^{(135.7KB, docx)}

S4 Fig

The survival curves for male ALS patients with creatine kinase < 180 U/L vs. creatine kinase > 181 U/L using Kaplan–Meier method (A). The survival curves for female ALS patients with creatine kinase < 80 U/L vs. creatine kinase > 81 U/L using Kaplan–Meier method (B).

(DOCX)

pone.0313307.s004.docx^{(127.8KB, docx)}

S5 Fig. The survival curves for ALS patients with triglyceride < 90 mg/dL vs. triglyceride > 91 mg/dL using Kaplan–Meier method.

(DOCX)

pone.0313307.s005.docx^{(74.8KB, docx)}

S6 Fig

The survival curves for male ALS patients with creatinine < 0.6 mg/dL vs. creatinine > 0.61 mg/dL by using Kaplan–Meier method (A). The survival curves for female ALS patients with creatinine < 0.5 mg/dL vs. creatinine > 0.51 mg/dL by using Kaplan–Meier method (B).

(DOCX)

pone.0313307.s006.docx^{(131.8KB, docx)}

S7 Fig. The survival curves for ALS patients with neutrophils < 3499/μL vs. neutrophils > 3500/μL using Kaplan–Meier method.

(DOCX)

pone.0313307.s007.docx^{(74.4KB, docx)}

Acknowledgments

The authors express their appreciation to the laboratory technicians in the study center for their technical support in performing electrophysiological studies.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The author(s) received no specific funding for this work.

References

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PLoS One. doi: 10.1371/journal.pone.0313307.r001

Decision Letter 0

Aditya Kumar Padhi

19 Jun 2024

PONE-D-24-18589Fasciculation potentials are related to the prognosis of amyotrophic lateral sclerosisPLOS ONE

Dear Dr. Adachi,

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Reviewer #1: This article offers insights into the prognostic factors of amyotrophic lateral sclerosis (ALS). The study discovered that a higher percentage of muscles exhibiting fasciculation potentials (FPs) during EMG examinations, combined with lower uric acid (UA) levels, are strongly linked to faster disease progression and reduced survival times in ALS patients. These results underscore the potential of utilizing needle EMG and UA levels as dependable and practical biomarkers for forecasting ALS outcomes. While the findings are interesting, several issues need to be addressed for clarity and completeness.

1. Classification: The authors should better justify their classification into faster, medium and slower progressors. How did they set the time limit?

2. EMG: it seems that the authors did not follow a specific protocol with definite muscles to explore. Additionally, it is crucial to know whether the muscles examined were clinically affected or subclinically involved. As a result, the percentage of muscles exhibiting fasciculation or fibrillation potentials becomes a relative measure, undermining the reliability of the findings. This inconsistency needs to be addressed to ensure comparability and accuracy of the results across all subjects. Any association between the morphology of FPs and prognosis/survival?

3. I have concerns about the use of the trapezius muscle instead of the genioglossus muscle for EMG. Although the genioglossus is difficult for patients to relax, it is more representative of bulbar muscles, which are crucial in ALS assessment. In contrast, the trapezius muscle is more associated with the upper cervical roots rather than bulbar regions. This choice may not accurately reflect bulbar involvement and could affect the study's findings and conclusions.

4. UA: the authors should better describe the biological reason of using UA, and not another routine biomarker, in this study.

5. Sensory conduction and symptoms: the authors did not mention a very recent study describing that sensory symptoms and SNAP amplitude parallel motor disability in ALS patients, trough the classification by King’s stage. I suggest the author to perform a similar analysis, stratifying patients according to the clinical stages. Again, the sentence: “Although patients with ALS do not exhibit sensory disturbances” I don’t think is correct. Please rephrase. Pathological results on sensory dysfunction are based non only on sural nerve biopsy but also on skin biopsy as recently demonstrated, please update the literature on the topic.

Minor:

Typos: There is an error in the naming of supplementary materials.

Introduction: please note that among poor prognostic factors, the authors should also include autonomic dysfunction as recently described. Please add this info.

Reviewer #2: PONE-D-24-18589: Fasciculation potentials are related to the prognosis of amyotrophic lateral sclerosis

The manuscript presents a study investigating the relationship between the proportion of muscles with fasciculation potentials (FPs), various biochemical markers, and the prognosis of ALS. The findings are significant and well-supported by the data, demonstrating clear associations between the percentage of muscles with FPs, uric acid (UA) levels, and ALS progression.With the suggested major revisions and clarifications, the manuscript will be well-positioned to make a substantial contribution to the field of ALS research.

Below are my comments:

Major comments

1. The manuscript is missing one of the most important progressions measuring tool, ALS Functional Rating Scale-Revised (ALSFRS-R). This tool is widely used clinical tool that assesses functional abilities across four domains: bulbar function, fine motor function, gross motor function, and respiratory function. The ALS FR should be provided for all patients and the disease progression and the findings in this manuscript should be clearly correlated. Include data on functional outcomes using ALSFRS-R scores to correlate the findings with patient quality of life and functional decline. This will help validate the clinical significance of FPs and UA levels.

2. Assess the firing frequency and other characteristics of FPs. This could differentiate between benign fasciculations and those indicative of ALS progression. Such detailed analysis can add depth to the understanding of FPs in ALS.

3. Is the genetic data available for these patients? Most importantly the commonly associated mutations with ALS (e.g., SOD1, C9orf72) to see if there is any correlation with FPs and UA levels. This could provide insights into the underlying mechanisms.

4. The control data is missing. Include a healthy control group for comparison to clearly delineate the differences in FPs and UA levels between ALS patients and healthy individuals. This will strengthen the argument for the specificity of these markers in ALS. Comparisons should be made between healthy controls and patients for making any associations between findings.

5. The discussion needs more details of the potential mechanisms underlying the associations found.

Minor comments

1. Consider providing a brief overview of ALS, its pathophysiology, and the significance of identifying prognostic markers.

2. The rationale for focusing on FPs and UA levels is well-articulated, but it might benefit from a brief explanation of how these markers have been studied in previous studies.

3. Clearly classify the patients are possible, probable and definite ALS.

4. Mention the specific criteria used for defining FPs in more detail, including any relevant thresholds or cut-off values.

5. Address the study's limitations more comprehensively, including potential biases and generalizability issues.

6. Suggest directions for future research, particularly regarding the clinical application of your findings.

Reviewer #3: 1. Time from symptom onset to the first evaluation significantly differed between the groups. However, the difference was not taken into account in the analysis shown in Table 5.

2. The authors reported the correlation between the FP rate and the UA level. However, biological relevance of this finding is unclear. It appears to be spurious corrlation.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2024 Nov 8;19(11):e0313307. doi: 10.1371/journal.pone.0313307.r002

Author response to Decision Letter 0

3 Sep 2024

General comments to the Reviewers:

We thank the three expert Reviewers for their valuable feedback on our manuscript and sincerely appreciate their time and efforts. Their comments have been invaluable. In response to the Reviewer’s comments and suggestions, we have thoroughly revised our manuscript. For clarity, the revised portions of the manuscript are indicated in red. We've addressed each of your specific comments separately. We appreciate your continued consideration and look forward to any further insights.

Below, we provide a point-by-point response to each of the Reviewers’ comments:

1. Classification: The authors should better justify their classification into faster, medium and slower progressors. How did they set the time limit?

Response: We thank the Reviewer for this constructive feedback on classification. The median survival duration of the patients with ALS was 3 years, and the 1-year survival rate was 76.2% (Reference 2). Therefore, we considered 3 years as the average survival duration and 1 year as short duration associated with rapid progression of the disease. We classified the survival durations into 3: less than 1 year: fast progression; from 1 year to less than 3 years: average progression; and 3 years or more: slow progression. We have added the following sentences to the first paragraph of the Methods section accordingly (lines 86–87): "The median survival duration of the patients with ALS was 3 years, and the 1-year survival rate was 76.2%."

Response: We thank the Reviewer for this constructive criticism of our manuscript. We selected muscles from four body regions according to the diagnostic criteria for ALS. However, this study was retrospective, as you pointed out. The problem was that different muscles were used for different cases, and the influence of the presence or absence of muscle weakness was not considered. We have added the following sentences to the limitations section of the study (lines 354-356): " In addition, it was not possible to incorporate evaluation items based on the presence or absence of muscle weakness.” Further, since it has been reported that the morphology of FP is related to prognosis, we evaluated polyphasic FP. The methods are described (lines 118–121).

Response: We are grateful to the Reviewer for the valuable comments. As you pointed out, the trapezius muscle is also controlled by the upper cervical roots, so it may not be appropriate to evaluate it as a cranial nerve muscle. However, on the other hand, active denervation findings for the trapezius muscle are highly specific to ALS. This study focused on the trapezius muscle because needle electromyography is easy to perform on it and the burden on patients is light. We have included text regarding the selection of the muscles to be tested in the methods section (lines 112–115).

4. UA: the authors should better describe the biological reason of using UA, and not another routine biomarker, in this study.

Response: This point is one that the Reviewer would naturally have doubts about. As pointed out by the Reviewer, we also investigated the effects of albumin, triglycerides, total cholesterol, creatinine, neutrophil count, and creatine kinase, among others. These have been reported to be related to the prognosis of ALS. We also investigated the relationship between these markers and prognosis. The results are shown in Figures S2–S7. However, only uric acid demonstrated an association with prognosis, and we further investigated it.

Response: We would like to thank the Reviewer for these constructive comments. As pointed out, there have been important reports on the relationship between the stage of ALS and SNAP amplitude recently. Therefore, the sentence “Although patients with ALS do not exhibit sensory disturbances” has been corrected to “Although patients with ALS do not usually show sensory disturbances” (line 290). In addition, sensory neuropathy in ALS has been pathologically proven using skin biopsy tissue. We have added the following sentences to the discussion section (lines 298-300): "Pathological sensory disturbance detected through skin biopsies has also been reported for ALS, with observations including axonal swellings as the foremost morphological changes and small fiber regeneration insufficiency."

Minor:

Typos: There is an error in the naming of supplementary materials.

Response: We thank the Reviewer for pointing this out. We have corrected the error in the naming of supplementary materials.

Introduction: please note that among poor prognostic factors, the authors should also include autonomic dysfunction as recently described. Please add this info.

Response: We thank the Reviewer for this suggestion. We have added autonomic dysfunction as a prognostic factor for ALS in the Introduction (line 55).

Reviewer #2: PONE-D-24-18589: Fasciculation potentials are related to the prognosis of amyotrophic lateral sclerosis

The manuscript presents a study investigating the relationship between the proportion of muscles with fasciculation potentials (FPs), various biochemical markers, and the prognosis of ALS. The findings are significant and well-supported by the data, demonstrating clear associations between the percentage of muscles with FPs, uric acid (UA) levels, and ALS progression. With the suggested major revisions and clarifications, the manuscript will be well-positioned to make a substantial contribution to the field of ALS research.

Below are my comments:

Major comments

Response: We thank the Reviewer for the insightful comments and suggestions. As pointed out, the ALSFRS-R is a clinically useful functional evaluation scale and is considered an essential scale in clinical trials for evaluating the therapeutic effects of drugs and the differences in functional prognosis due to related factors. On the other hand, in our current research, we evaluated the prognosis of ALS using the severity of the disease, which indicated whether a patient would die or develop respiratory failure. Therefore, it is considered slightly different from the adaptation of ALSFRS-R, which can sensitively detect gradual changes in function. Additionally, this study was retrospective, and it would be difficult to evaluate ALSFRS-R retrospectively. Therefore, we have added the following sentences related to the adaptation of ALSFRS-R to the research limitations (lines 351-354): “In our study, we evaluated the prognosis of patients using the severity of ALS as an index, which indicated whether they would die or develop respiratory failure. This study was also retrospective. Therefore, the standard ALSFRS-R, which is a clinically useful functional assessment scale, was not suitable for this study.”

Response: We thank the Reviewer for the insightful comments and valuable suggestions. As pointed out, the firing frequency and morphology of FPs were related to the progression of ALS. However, it is very difficult to accurately measure the firing frequency of FPs in muscles of a certain size, and we do not usually measure the firing frequency in clinical practice. However, regarding morphology, electromyography is performed to exclude benign fasciculations and to not count FPs that show only a single phase. These points are discussed in the Methods and Limitations section (lines 119–121 and 344-348).

Response: We thank the Reviewer for the important comments. Examining whether mutations commonly associated with ALS (e.g., SOD1, C9orf72) influence the correlation between FPs and UA levels and prognosis will yield clinically important data about the underlying mechanisms. Unfortunately, we targeted patients who were thought to have sporadic ALS and were negative for commonly associated mutations in our study, and patients with a family history were excluded. We have added that we targeted patients with sporadic ALS (line 85).

Response: We thank the Reviewer for this constructive criticism of our manuscript. As pointed out, we also believe that analysis that includes comparison with a healthy control group is important. Although FPs are observed in normal people (benign fasciculation), they are distinguished by the absence of muscle weakness and simple waveforms (References 14,21). FPs are extremely rare in healthy people when we perform needle electromyography, and we were unable to provide data for the healthy control group in this study. On the other hand, it has been reported that uric acid levels are significantly lower for the ALS group than for the control group (Reference 37). In our study, the ALS group, which had rapid progression, showed lower values. However, the fluctuations in uric acid levels were within the normal range for general uric acid levels. Therefore, we did not include a comparison with a healthy control group in this analysis. We have added the following sentences to the limitations section regarding the fact that we were unable to analyze the relationship between FPs and uric acid levels by adding a comparison with a healthy control group (lines 357-365): “While it is important to include data from the healthy control group for comparative analysis, we were unable to analyze the relationship between FPs and uric acid levels by adding data from the healthy control group. FPs can be observed in healthy individuals (benign fasciculation), but they are distinguished by the absence of accompanying muscle weakness and the simple waveform [14,21]. When we performed needle electromyography, FPs were rarely observed in healthy individuals. Additionally, it has been reported that uric acid levels are significantly lower in patients with ALS than in individuals without it [37], but they varied within the normal range in this study.”

5. The discussion needs more details of the potential mechanisms underlying the associations found.

Response: We thank the Reviewer for this valuable suggestion. Unfortunately, we were unable to conduct an objective discussion using the literature, as no reports on the relationship between FPs and serum UA levels in ALS have been published. However, from our data, serum UA levels are involved in the mechanism underlying electrophysiological changes that lead to FPs in skeletal muscle and may modify the pathology of ALS. We have added the following sentences to the Discussion section (lines 320-324): “No reports on the relationship between FPs and serum UA levels in ALS have been published to date. However, serum UA levels and FPs were negatively correlated in this study. This suggests that serum UA levels s may be involved in the mechanism underlying electrophysiological changes that lead to FPs in skeletal muscle and may modify the pathology of ALS.”

Minor comments

1. Consider providing a brief overview of ALS, its pathophysiology, and the significance of identifying prognostic markers.

Response: Thank you for your valuable suggestions. We have added the following sentences to the Discussion section (lines 331-336): “Sporadic ALS is an intractable disease, and its mechanism of onset is still unknown. The discovery of several factors involved in the pathogenesis or modification of the disease progression of ALS may lead to the elucidation of the mechanism of onset. These factors, which include electrophysiological and biochemical markers, are also expected to facilitate the early diagnosis of diseases, prediction of prognosis, and development of treatments.”

2. The rationale for focusing on FPs and UA levels is well-articulated, but it might benefit from a brief explanation of how these markers have been studied in previous studies.

Response: Unfortunately, we were unable to conduct an objective discussion using the literature, as no reports on the relationship between FPs and serum UA concentrations in ALS have been published.

3. Clearly classify the patients are possible, probable and definite ALS.

Response: This study included patients who could be followed up to death or respiratory support, and they were classified as having definite ALS. We have added this information (line 85).

4. Mention the specific criteria used for defining FPs in more detail, including any relevant thresholds or cut-off values.

Response: As pointed out, we have added that FPs in needle electromyography are identified by polyphasic waves that appear spontaneously and randomly (lines 119-121).

5. Address the study's limitations more comprehensively, including potential biases and generalizability iss

Attachment

Submitted filename: Response_to_Reviewers.docx

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PLoS One. doi: 10.1371/journal.pone.0313307.r003

Decision Letter 1

Aditya Kumar Padhi

22 Sep 2024

PONE-D-24-18589R1Fasciculation potentials are related to the prognosis of amyotrophic lateral sclerosisPLOS ONE

Dear Dr. Adachi,

Please submit your revised manuscript by Nov 06 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have done a commendable job with the revisions, but I would like to point out a minor issue in the following sentence: 'Pathological sensory disturbance detected through skin biopsies has also been reported for ALS, with observations including axonal swellings as the foremost morphological changes and small fiber regeneration insufficiency.' The authors cite an older study that does not clearly highlight the extent of cutaneous denervation or the progression of SNAP amplitudes across different disease stages. I would kindly request that the authors revise this sentence, citing a more appropriate and recent study about skin innervation across amyotrophic lateral sclerosis clinical stages.

Reviewer #2: All my concerns and comments have been answered. The manuscript seems to have considerably improved after inclusion of author response.

Reviewer #3: The review comments at the previous round have been addressed. The reviewer has no additional comments.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2024 Nov 8;19(11):e0313307. doi: 10.1371/journal.pone.0313307.r004

Author response to Decision Letter 1

6 Oct 2024

Response: We thank the Reviewer for this constructive feedback on pathological sensory disturbance detected through skin biopsies in ALS. As you pointed out, we have cited other, more recent study and revised the following sentence in the fifth paragraph of the discussion part: “Pathological sensory disturbance detected through skin biopsies has also been reported for ALS, with observations including, a significant loss of intraepidermal nerve fiber (IFNF) and Meissner corpuscle density in ALS compared with healthy controls, and increasing IENF density over time were associated with a poorer prognosis” (line 298-301).

Attachment

Submitted filename: Response_to_Reviewer.docx

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PLoS One. doi: 10.1371/journal.pone.0313307.r005

Decision Letter 2

Aditya Kumar Padhi

23 Oct 2024

Fasciculation potentials are related to the prognosis of amyotrophic lateral sclerosis

PONE-D-24-18589R2

Dear Dr. Adachi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

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Reviewer #1: The authors have adequately addressed all of my previous comments and concerns. I have no further requests or suggestions to submit.

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PLoS One. doi: 10.1371/journal.pone.0313307.r006

Acceptance letter

Aditya Kumar Padhi

30 Oct 2024

PONE-D-24-18589R2

PLOS ONE

Dear Dr. Adachi,

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

(DOCX)

pone.0313307.s001.docx^{(75.2KB, docx)}

S2 Fig. The survival curves for ALS patients with albumin < 4 g/dL vs. albumin > 4.1 g/dL using Kaplan–Meier method.

(DOCX)

pone.0313307.s002.docx^{(70.3KB, docx)}

S3 Fig

(DOCX)

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S4 Fig

(DOCX)

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S5 Fig. The survival curves for ALS patients with triglyceride < 90 mg/dL vs. triglyceride > 91 mg/dL using Kaplan–Meier method.

(DOCX)

pone.0313307.s005.docx^{(74.8KB, docx)}

S6 Fig

(DOCX)

pone.0313307.s006.docx^{(131.8KB, docx)}

S7 Fig. The survival curves for ALS patients with neutrophils < 3499/μL vs. neutrophils > 3500/μL using Kaplan–Meier method.

(DOCX)

pone.0313307.s007.docx^{(74.4KB, docx)}

Attachment

Submitted filename: Reviewer-Comments.pdf

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Submitted filename: Response_to_Reviewer.docx

pone.0313307.s010.docx^{(15.7KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Fasciculation potentials are related to the prognosis of amyotrophic lateral sclerosis

Keiko Ohnari

Kosuke Mafune

Hiroaki Adachi

Roles

Abstract

Introduction

Methods

Patients

Electrophysiological studies

Statistical analysis

Results

Table 1. Clinical findings in patients with ALS classified according to clinical course.

Table 2. EMG findings in patients with ALS classified according to clinical course.

Fig 1.

Table 3. Nerve conduction studies of patients with ALS classified according to the clinical course.

Table 4. Laboratory data of patients with ALS classified according to clinical course.

Table 5. Factors related to the frequency of fasciculation potential.

Fig 2. Correlation between the percentage of muscles with fasciculation potentials and uric acid level (grand mean centered uric acid level).

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Aditya Kumar Padhi

Roles

Author response to Decision Letter 0

Decision Letter 1

Aditya Kumar Padhi

Roles

Author response to Decision Letter 1

Decision Letter 2

Aditya Kumar Padhi

Roles

Acceptance letter

Aditya Kumar Padhi

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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