Content uploaded by Christoph Neuwirth
Author content
All content in this area was uploaded by Christoph Neuwirth on Jul 11, 2017
Content may be subject to copyright.
Motor Unit Number Index (MUNIX) detects motor neuron loss
in pre-symptomatic muscles in Amyotrophic Lateral Sclerosis
Christoph Neuwirth
a,
⇑
, Paul E. Barkhaus
b
, Christian Burkhardt
a
, José Castro
c
, David Czell
d
,
Mamede de Carvalho
c
, Sanjeev Nandedkar
e
, Erik Stålberg
f
, Markus Weber
a,g
a
Neuromuscular Diseases Unit/ALS Clinic, Kantonsspital St. Gallen, St. Gallen, Switzerland
b
Medical College of Wisconsin, Milwaukee, WI, USA
c
Department of Neurosciences, Hospital de Santa Maria, Instituto de Medicina Molecular, Faculty of Medicine, University of Lisbon, Lisbon, Portugal
d
Kantonsspital Winterthur, Winterthur, Switzerland
e
Natus Medical Inc, Middleton, WI, USA
f
Institute of Neurosciences, Uppsala University, Department of Clinical Neurophysiology, University Hospital, Uppsala, Sweden
g
Department of Neurology, University Hospital Basel, Basel, Switzerland
article info
Article history:
Accepted 20 November 2016
Available online xxxx
Keywords:
MUNIX
Pre-symptomatic ALS
Biomarker
Multicentre
ALSFRS-R
highlights
In pre-symptomatic muscles MUNIX can detect motor unit loss.
MUNIX is more sensitive to change compared to CMAP and ALSFRS-R.
This makes MUNIX a biomarker candidate for disease progression.
abstract
Objective: Motor Unit Number Index (MUNIX) is a quantitative neurophysiological measure that provides
an index of the number of lower motor neurons supplying a muscle. It reflects the loss of motor neurons
in patients with Amyotrophic Lateral Sclerosis (ALS). However, it is unclear whether MUNIX also detects
motor unit loss in strong, non-wasted muscles.
Methods: Three centres measured MUNIX in 49 ALS patients every three months in six different muscles
(abductor pollicis brevis, abductor digiti minimi, biceps brachii, tibialis anterior, extensor digitorum bre-
vis, abductor hallucis) on the less affected side. The decline of MUNIX in initially non-wasted, clinically
strong muscles (manual muscle testing, MMT grade 5) was analysed before and after onset of weakness.
Results: In 49 subjects, 151 clinically strong muscles developed weakness and were included for analysis.
The average monthly relative loss of MUNIX was 5.0% before and 5.6% after onset of weakness. This rate of
change was significantly higher compared to ALS functional rating scale (ALSFRS-R) and compound mus-
cle action potential (CMAP) change over 12 months prior to the onset of muscle weakness (p= 0.024).
Conclusion: MUNIX is an electrophysiological marker that detects lower motor neuron loss in ALS, before
clinical weakness becomes apparent by manual muscle testing.
Significance: This makes MUNIX a good biomarker candidate for disease progression and possibly phar-
macodynamics responds.
Ó2016 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights
reserved.
1. Introduction
Motor Unit Number Index (MUNIX) is a quantitative electro-
physiological technique that provides an index of the number of
functional lower motor neurons (LMN) supplying a muscle. Recent
studies have demonstrated a good test-retest reliability in healthy
subjects and ALS patients and its capability to track loss of func-
tional LMNs over time (Ahn et al., 2010; Nandedkar et al., 2010,
http://dx.doi.org/10.1016/j.clinph.2016.11.026
1388-2457/Ó2016 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
Abbreviations: ADM, abductor digiti minimi muscle; AH, abductor hallucis muscle;
ALS, Amyotrophic Lateral Sclerosis; ALSFRS-R, revised amyotrophic lateral sclerosis
functional rating scale; APB, abductor pollicis brevis muscle; BB, biceps brachii
muscle; CI, Confidence Intervals; CMAP, compound muscle action potential; EDB,
extensor digitorum brevis muscle; FDI, first dorsal interosseus muscle; LMN, lower
motor neuron; MMT, manual muscle testing; MUNE, motor unit number estima-
tion; MUNIX, motor unit number index; SD, standard deviation; TA, tibialis anterior
muscle; UMN, upper motor neuron.
⇑
Corresponding author at: Neuromuscular Diseases Unit/ALS Clinic, Kantonsspi-
tal St. Gallen, Rorschacherstrasse 95, CH-9007 St. Gallen, Switzerland. Fax: +41
714946389.
E-mail address: christoph.neuwirth@kssg.ch (C. Neuwirth).
Clinical Neurophysiology xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Clinical Neurophysiology
journal homepage: www.elsevier.com/locate/clinph
Please cite this article in press as: Neuwirth C et al. Motor Unit Number Index (MUNIX) detects motor neuron loss in pre-symptomatic muscles in Amy-
otrophic Lateral Sclerosis. Clin Neurophysiol (2016), http://dx.doi.org/10.1016/j.clinph.2016.11.026
2011; Neuwirth et al., 2011, 2015; Boekestein et al., 2012; Fathi
et al., 2016).
MUNIX applies a mathematical model, using the area and
power of the compound muscle action potential (CMAP) after
supramaximal electrical stimulation of a mixed peripheral nerve
and area and power of the surface electromyography (EMG) at dif-
ferent levels of voluntary isometric contraction. These values are
used to compute the ‘‘ideal case motor unit count” to estimate
the amount of functioning motor neurons. MUNIX is fast, non-
invasive, and can be applied to any distal or proximal muscle in
which a CMAP can be elicited by supramaximal electrical nerve
stimulation. The method has been described in detail previously
(Nandedkar et al., 2004, 2010). Measurements are usually per-
formed in less than five minutes per muscle (Neuwirth et al.,
2015, 2016).
Onset of ALS usually starts focally in the cervical/lumbar regions
(limbs), bulbar region, or thoracic region, then spreads to contigu-
ous regions (Ravits et al., 2007).
Consequently, measurements from several arm and leg muscles
can provide information on the pattern of disease spread as
opposed to measurements in only a single muscle (Neuwirth
et al., 2015).
In a previous study, MUNIX measurements in 6 different mus-
cles revealed a significant higher decline rate than the revised
ALS functional rating score (ALSFRS-R) and was similar in different
types of ALS onset (bulbar, arm, leg onset) (Neuwirth et al., 2015).
However, it is not known whether MUNIX is able to detect LMN
loss in strong, non-wasted (here denoted pre-symptomatic)
muscles.
The aim of this study was to determine the rate of MUNIX
decline prior and after the onset of weakness in initially clinical
strong muscles (modified MRC manual muscle testing grade 5)
and to compare MUNIX decline rates with CMAP amplitude decline
rates and the ALSFRS-R, a well-established functional measure of
disease progression (Kaufmann et al., 2007).
2. Methods
2.1. Subjects
ALS patients were recruited in specialised ALS centres in St. Gal-
len, Lisbon, and Milwaukee. The study protocol was approved by
the local ethics committees. All subjects gave written informed
consent.
ALS patients fulfilled the categories for possible, probable-
laboratory supported, probable, or definite ALS according to the
revised El Escorial criteria (Brooks et al., 2000). Patients were
excluded, if they had other diseases that could influence coopera-
tion or measurements (e.g. polyneuropathy, radiculopathy, periph-
eral nerve lesion, carpal tunnel syndrome, major stroke,
frontotemporal dementia). Time from symptom onset (weakness,
dysarthria, dysphagia, dyspnoea, gait impairment) to first mea-
surement had to be less than 24 months. This criterion was added
to avoid bias towards patients with slow progression or subjects
with advanced stages and already numerous wasted muscles at
study entry. Assessments and measurements were performed
every three months ± two weeks scheduled at the regular clinic
visits.
2.2. MUNIX procedure
To reduce systematic variability caused by electrode size and
type, all centres used the same self-adhesive disposable surface
ground and disc recording electrodes with 15 mm diameter
(Ref 019-415200, Natus, Middleton, WI, USA) and arrangement of
stimulation/recording electrodes (Barkhaus et al., 2006).
Keypoint
Ò
-Classic-, Keypoint
Ò
.net- and Synergy
Ò
-electromyogra
phies were used for measurements. Stimulation electrodes were
not standardised across centres. The protocol for MUNIX measure-
ments, model and computation has been reported in detail previ-
ously (Nandedkar et al., 2004, 2010; Neuwirth et al., 2010, 2011).
MUNIX was performed in the abductor pollicis brevis (APB),
abductor digiti minimi (ADM), biceps brachii (BB), tibialis anterior
(TA), extensor digitorum brevis (EDB), and abductor hallucis (AH)
muscles.
A mandatory step in the procedure was to move the active
recording electrode several times until the maximum CMAP ampli-
tude with a clear negative take-off (first negative deflection) was
obtained. The clinically less affected side was selected for measure-
ments, which was generally the opposite side of symptom onset in
order to prevent measurements in severely affected muscles and to
reduce the risk of an early ‘‘floor-effect” (Neuwirth et al., 2015). In
case of no detectable weakness in limb muscles, the right side was
chosen.
2.3. Measures
At each visit, ALSFRS-R score was determined by the same ALS
research nurse or neurologist. MUNIX raters were blinded to the
results of the ALSFRS-R score and previously obtained CMAP and
MUNIX measurements. Results of these longitudinal MUNIX mea-
surements have partially been published (Neuwirth et al., 2015).
This study aimed to analyse only pre-symptomatic muscles for
which additional longitudinal data sets were available.
Manual muscle testing (MMT), using a modified 8-grade scale,
was performed in all muscles prior to MUNIX measurements by
an experienced ALS neurologist. The 8-grade scale was selected
to distinguish different force levels (Table 1). Numeric values for
statistical analysis for MMT4 + was 4.3 and consequently 3.7 for
MMT4-.
For analysis of pre-symptomatic muscles, only clinically strong
(MMT 5) and non-wasted muscles were considered for which at
least one measurement before onset of weakness on follow up
was available.
To evaluate change of MUNIX and CMAP in pre-symptomatic
muscles, the last point in time with MMT grade 5 was arbitrarily
set to ‘‘month 0” (M0). In case the rater documented slight clinical
weakness in a muscle which was not detectable on the next follow-
up visit with MMT grade 5, the last measurement with MMT grade
5 was set to M0. The relative change of MUNIX, ALSFRS-R, CMAP,
and MMT in percent change over time before and after the last
measurement with MMT grade 5 (M0) was analysed.
2.4. Statistics
Linear mixed-effect models with the dependent variable ‘‘per-
centage change from baseline”, fixed factor time (month), variables
(MUNIX, CMAP, ALS-FRS-R, MMT) and random factor subject were
Table 1
Manual muscle testing (MMT) grading scale.
Grade
5 Normal strength
4+ Inability to resist against maximal pressure
4 Ability to resist against moderate pressure
4Ability to resist against minimal pressure
3 Ability to move through full ROM AG
2 Ability to move with GE
1 Visible muscle contraction
0 No movement/contraction at all
ROM = range of movement; AG = against gravity; GE = gravity eliminated.
2C. Neuwirth et al. / Clinical Neurophysiology xxx (2016) xxx–xxx
Please cite this article in press as: Neuwirth C et al. Motor Unit Number Index (MUNIX) detects motor neuron loss in pre-symptomatic muscles in Amy-
otrophic Lateral Sclerosis. Clin Neurophysiol (2016), http://dx.doi.org/10.1016/j.clinph.2016.11.026
performed. In order to calculate the relative change per month for
each muscle, a nested model design was used (months nested in
muscles). To compare the relative change for each variable
(MUNIX, ALSFRS-R, CMAP, MMT) at each time point, another
nested model design was used where the variables were nested
in months. Decline rates and p-values with 95% Confidence Inter-
vals (CI) of relative change values are presented. A p-value <0.05
was considered significant. For an appropriate comparison of the
time series data, the Standard Error (SE) was applied. All analyses
were performed using the statistical program R version 3.1.2
(https://www.r-project.org)(R Core Team, 2012).
3. Results
Sixty-seven subjects were recruited from September 2010 until
May 2015. Each centre recruited 15 to 28 ALS patients. Three
patients did not fulfil inclusion criteria and were excluded from
analysis because of long disease duration (27–58 months) with
very slow disease progression. Forty-nine out of 67 patients
(36 men, 13 women) presented with at least one muscle with full
force (MMT grade 5) and weakness on follow up. Seventeen sub-
jects had bulbar onset (35%) and 33 spinal onset (65%), with almost
equal distribution of lower and upper limb onset. The mean age of
subjects was 59.3 years ± 11.3 (SD) and the mean disease duration
at the time of first measurement was 13.4 ± 5.8 months.
MUNIX measurements were well tolerated in all subjects
except in one subject who declined longitudinal measurement of
the biceps.
A total of 151 muscles from these 49 patients fulfilled the crite-
ria for analysis (clinically not affected at the first assessment and
weakness during the follow-up period). Total time for measuring
all six muscles ranged between 20 and 40 min. The individual
number of follow-up measurements varied from muscle to muscle
depending on the total number of follow-up visits and the time of
onset of muscle weakness. The number of analysed muscles prior
and after the onset of weakness is shown in Fig. 1. Fifty-five out
of 151 muscles could be measured 3-monthly up to 1 year after
onset of weakness and 44 muscles were measured 3-monthly up
to 1 year prior to onset of weakness.
The average relative monthly decline was 5.0% for MUNIX (all
muscles) before and 5.6% after onset of clinical weakness (Fig. 2,
Table 2). This rate of change was significantly higher compared
to ALSFRS-R prior to and after the onset of weakness (Fig. 2,
Table 2). Relative CMAP change was not significantly different from
ALSFRS-R over 12 months prior to clinical weakness, but after
onset of weakness (Table 3,Fig. 2A and B).
The decline rates of MUNIX were significantly higher compared
to the CMAP decline between month-12 and -3 prior to onset of
weakness (Fig. 2,Table 2). CMAP and MUNIX decline rates varied
muscle-specific (Table 2).
MUNIX decline rates were generally higher than CMAP decline
before and after onset of weakness, with the exception of the AH,
which revealed higher CMAP decline rates. ADM MUNIX had the
lowest decline rate prior to onset of clinical weakness compared
to other muscles and accelerated after onset of weakness. APB
revealed the highest pre-symptomatic MUNIX decline rate of all
muscles.
However, these differences were not statistically significant.
Decline rates were separately analysed in 6-month periods
before and after onset of muscle weakness (Table 4). CMAP and
MUNIX decline was lowest between month-12 and month-6 and
then almost doubled in the period -6 to M0 (non-significant).
MUNIX decline was most prominent in the 6-months period prior
to M0. Analysis of MMT revealed a slight increase of 0.18% per
month before onset of weakness, which was caused by prior
measurements with slight weakness which were revised to
MMT5 at follow up visits.
4. Discussion
It has been recognised in previous studies that neurophysiolog-
ical abnormalities occur (e.g. MUNE and EMG) before this is
reflected in force measurements, e.g. MMT (Swash and Ingram,
1988; Bromberg and Brownell, 2008; de Carvalho and Swash,
2013; de Carvalho et al., 2014).
In this study we could demonstrate that this is also the case for
MUNIX. A marked decline of MUNIX – expressed by relative
change of means – was already detectable12 months before
clinical muscle weakness was detected by MMT. These results in
pre-symptomatic muscles are similar to those obtained in asymp-
tomatic carriers of SOD1 mutations (Aggarwal and Nicholson,
2002). In this study in single cases of asymptomatic SOD1 muta-
tions carriers, the statistical motor unit number estimation
(MUNE) technique was applied every 6 months demonstrating
motor neuron loss prior to symptom onset. Ongoing studies utilise
MUNE to estimate the number of LMN in asymptomatic familial
ALS gene carriers (Benatar and Wuu, 2012). However, in contrast
to MUNE, where only single or few muscles are studied (usually
ADM or APB), the fast performance of MUNIX in individual muscles
yields an examination over several regions in multiple muscles in
an appropriate amount of time (Neuwirth et al., 2016).
Our results also reveal that prior to onset of weakness the
MUNIX decline is more prominent than the CMAP amplitude
Fig. 1. Number of MUNIX measurements per individual muscle. (A) Arm muscles:
APB = abductor pollicis brevis, ADM = abductor digiti minimi, BB = biceps brachii.
(B) Leg muscles: TA = tibialis anterior, EDB = extensor digitorum brevis, AH abduc-
tor hallucis. X-axis: month 0 = last point in time with MMT was grade 5.
C. Neuwirth et al. / Clinical Neurophysiology xxx (2016) xxx–xxx 3
Please cite this article in press as: Neuwirth C et al. Motor Unit Number Index (MUNIX) detects motor neuron loss in pre-symptomatic muscles in Amy-
otrophic Lateral Sclerosis. Clin Neurophysiol (2016), http://dx.doi.org/10.1016/j.clinph.2016.11.026
decline (Tables 3 and 4). This can be explained by compensatory
reinnervation (distal axonal sprouting), which can at least tem-
porarily preserve motor unit function and in turn, CMAP amplitude
(Swash and Ingram, 1988). It has been estimated that approxi-
mately 50% of LMNs supplying a muscle may be lost (i.e. motor
units) in a chronic process before clinical symptoms such as muscle
wasting or weakness become evident (Wohlfart, 1957; Aggarwal
and Nicholson, 2002; Bromberg and Brownell, 2008).
MUNIX seems to overcome this ‘‘blind spot” of pre-
symptomatic LMN loss. This could be especially relevant for early
phase II clinical ALS trials and proof-of-concept studies. Such stud-
ies are usually underpowered for functional measures, such as the
Table 2
Relative monthly decline of MUNIX, MMT and CMAP in individual muscles 12 months before and after last measurement with MMT.
Muscle Monthly decline rates [%]
Pre-symptomatic 12 months Symptomatic 12 months
MUNIX CMAP MMT MUNIX CMAP MMT
APB 6.8 2.2 0.18 5.1 3.9 2.7
ADM 2.2 2.3 0.29 5.2 4.4 3.0
BB 4.6 3.4 0.06 4.2 4.3 2.4
TA 6.3 4.9 0.24 7.3 6.0 4.1
EDB 6.1 2.7 0.06 6.2 5.9 4.3
AH 3.7 4.6 0.25 5.6 6.7 4.8
All muscles 5.0 3.4 0.18 5.6 5.2 3.6
ALSFRS-R 3.5 3.0
Table 3
Difference of means (95% CI) and p-values for differences of relative decline rates comparing MUNIX, CMAP and ALSFRS-R prior and after month 0 (corresponding to Fig. 2).
Month MUNIX vs ALSFRS CMAP vs ALSFRS MUNIX vs CMAP
12 21.8 (7.15, 36.51); 0.0036 5.7 (8.88, 20.28); 0.44 16.1 (2.47, 29.79); 0.0207
921.8 (7.90, 35.76); 0.0022 6.4 (7.46, 20.30); 0.36 15.4 (2.01, 28.82); 0.0243
620.6 (9.35, 31.87); 0.0003 5.8 (5.36, 16.98); 0.31 14.8 (4.02, 25.58); 0.0071
314.7 (4.24, 25.09); 0.0059 7.1 (3.20, 17.36); 0.18 7.6 (2.35, 17.53); 0.13
0 1.2 (7.47, 9.87); 0.79 0.1 (8.30, 8.16); 0.99 1.3 (6.88, 9.42); 0.76
36.8 (12.06, 1.59); 0.0106 7.1 (12.32, 1.93); 0.0072 0.3 (4.83, 5.44); 0.91
613.0 (18.82, 7.24);<0.0001 13.4 (19.14, 7.59);<0.0001 0.3 (5.40, 6.08); 0.91
920.9 (27.42, 14.39);<0.0001 16.4 (22.90, 9.98);<0.0001 4.5 (10.94, 2.01); 0.18
12 24.4 (31.94, 16.80);<0.0001 19.8 (27.28, 12.25);<0.0001 4.6 (12.10, 2.88); 0.23
Significant differences (p< 0.05) are presented in bold numerics.
Table 4
Decline rates in 6-months periods before and after onset of weakness for all muscles for ALSFRS-R, MUNIX, CMAP and MMT.
Month Monthly decline rates [%], all muscles
ALSFRS-R MUNIX CMAP MMT
12 to 63.2 3.5 2.3 0.1
6to0 3.9 6.6 4.5 0.3
0to6 3.1 5.7 5.5 4.1
6to12 2.9 5.0 3.9 2.9
Fig. 2. Relative decline/increase of MMT, MUNIX, ALSFRS-R (ALS), and CMAP 12 months prior and after onset of weakness in muscles. Month 0 = last measurement with
MMT5. (A) Comparison of MMT, MUNIX and ALSFRS-R. (B) Comparison of MMT, CMAP and ALSFRS-R. Corresponding p-values and confidence intervals are shown separately
in Table 3. Error bars = standard error.
4C. Neuwirth et al. / Clinical Neurophysiology xxx (2016) xxx–xxx
Please cite this article in press as: Neuwirth C et al. Motor Unit Number Index (MUNIX) detects motor neuron loss in pre-symptomatic muscles in Amy-
otrophic Lateral Sclerosis. Clin Neurophysiol (2016), http://dx.doi.org/10.1016/j.clinph.2016.11.026
ALSFRS-R and MMT. However, a biological signal (e.g. potentially
effective medications or treatments slow down or stop motor neu-
ron loss) would be detected by MUNIX, thus paving the way for
longer phase III trials (Benatar and Wuu, 2012; Turner and
Benatar, 2015; Benatar et al., 2016).
This concept is supported by our recent study which showed
that the time to detect a 25% relative difference of decline with
80% power between two groups (each n= 50) would take
11.6 months for MUNIX (using 4 of the 6 muscle groups), but
26.5 months for ALSFRS-R (Neuwirth et al., 2015).
Twelve to 6 months prior to onset of weakness, MUNIX and
CMAP decline rates were smaller and then increased in the time
period 6 months before and 12 months after onset of weakness
in the observed muscles (Table 4). This difference was not statisti-
cally significant, yet might reflect acceleration of motor neuron loss
prior to onset of weakness. Alternatively, the different decline rates
might be also caused by bias: muscles in slowly progressing
patients are more likely to be trackable longer backward and for-
ward than fast progressing muscles, resulting in higher decline
rates around month 0, before clinical onset of weakness.
The ADM showed smaller change rates and deterioration of
MUNIX and CMAP than the APB 12 months prior to onset of weak-
ness and then increased. These different change rates may be the
electrophysiological correlate of the clinical ‘‘split-hand” phe-
nomenon (Weber et al., 2000; Eisen and Kuwabara, 2012). The
electrophysiological ‘‘split-hand-index”, which uses the CMAP
amplitudes of the APB, ADM, and first dorsal interosseus (FDI)
muscle for calculation, has been recently adapted for MUNIX mea-
surements (Weber et al., 2000; Eisen and Kuwabara, 2012; Menon
et al., 2013; Kim et al., 2016). The initially slower decline rates in
the ADM might reflect the relative resistance to LMN degeneration
compared to the APB and FDI until the onset of weakness and mus-
cle wasting and further progression of LMN degeneration. The
cause of the spilt hand phenomenon is still incompletely under-
stood, but is most likely of cortical origin (Weber et al., 2000;
Eisen and Kuwabara, 2012).
There are some limitations of this study.
MUNIX and manual muscle testing need active cooperation of
the patients. Manual muscle testing is subjective and depends on
the general physical condition and strength of the rater and the
tested subject. This might be prone to error, especially when the
raters – which were specialized neurologists in the 3 centres –
have to distinguish between normal force and slight weakness. In
our cohort, some MMT grade 4 + measurements were revised on
follow up visit again to MMT grade 5, resulting in a slight increase
of MMT in the pre-symptomatic period (Tables 2 and 4).
In addition, MMT was not standardised across the centres,
which might have been another source of variability. It should also
be noted that the transformation of the MMT rating scale into a
numeric value was arbitrary, and therefore the calculation of rela-
tive decline may not mirror decline in force appropriately. How-
ever, for the purpose of this study not the grading system was
relevant, but the definitive onset of weakness (‘‘point of no
return”). Nevertheless for future studies, quantitative measure-
ments such as hand-held-dynamometry may be advantageous.
It seems possible that upper motor neuron (UMN) involvement
had an effect on force measurements. However, it has been shown
that LMN loss is the primary cause of weakness in ALS, while UMN
impairment leads to slowing of contraction speed (Kent-Braun
et al., 1998). In addition, MMT has been suggested as an accurate
tool to observe global strength in ALS patients (Great Lakes ALS
Support Group, 2003; Visser et al., 2003).
Like in our previous study, the relative change of MUNIX was
significantly higher compared to the ALSFRS-R (Neuwirth et al.,
2015). This comparison has its limitations, as different features
are compared: loss of functioning LMN versus a global functional
score. The latter is also influenced by upper motor neuron (UMN)
involvement. It should therefore be emphasised, that MUNIX is
not intended to replace the ALSFRS-R or other functional measures
in phase III clinical trials, but may provide a biological signal in
terms of a pharmacodynamic response in early phase II studies.
Finally the absolute number of individual muscles amongst the
set of 6 muscles was relatively small, invalidating statistical anal-
ysis and interpretation of muscle-specific data.
5. Conclusion
MUNIX is an electrophysiological method that detects loss of
functional lower motor neurons in pre-symptomatic muscles in
ALS, before clinical weakness becomes apparent by manual muscle
testing. This suggests MUNIX as a marker of disease progression
and potential pharmacodynamics response in early phase II clinical
ALS trials (Turner and Benatar, 2015).
Disclosures
Dr. Neuwirth and Dr. Weber have received honoraria from
Hänseler AG, Switzerland and Biogen, USA, as advisory board
members. Dr. Weber has received advisory board honoraria from
Merz Pharma, Switzerland. Dr. Nandedkar is an employee of Natus
Medical Inc, and also a consultant to Biogen, USA. Dr. Burkhardt
receives honoraria for services from Biogen, USA. The other authors
declared they have no competing interests.
Acknowledgements
This work was supported by the Swiss ALS Foundation, the
Swiss NeRAB Foundation and the EU Joint Programme Neurode-
generative Disease Research (JPND) project. The project is sup-
ported through the following funding organisations under the
aegis of JPND – www.jpnd.eu: France, Agence Nationale de la
Recherche; Germany, Bundesministerium für Bildung und For-
schung; Ireland, Health Research Board; Italy, Ministero della
Salute; The Netherlands, The Netherlands Organisation for Health
Research and Development; Poland, Narodowe Centrum Badan
´i
Rozwoju; Portugal, Fundação a Ciência e a Tecnologia; Spain, Min-
isterio de Ciencia e Innovación; Switzerland, Schweizerischer
Nationalfonds zur Förderung der Wissenschaftlichen Forschung;
Turkey, Tübitak; United Kingdom, Medical Research Council (grant
number SNF 31ND30_141622). We would like to thank all partici-
pating patients and their caregivers. We are thankful to Urs (Sim-
men Statistical Consulting, Basel, Switzerland) for performing the
statistical analysis. We also like to thank Dr Michael Benatar,
Miami, USA, for fruitful discussions. Dr Nandedkar thanks Natus
Medical Inc for support. Dr Barkhaus acknowledges funding sup-
port from the Dolores and Carroll Fund from the Milwaukee Med-
ical Foundation.
References
Aggarwal A, Nicholson G. Detection of preclinical motor neurone loss in SOD1
mutation carriers using motor unit number estimation. J Neurol Neurosurg
Psychiatry 2002;73:199–201.
Ahn SW, Kim SH, Kim JE, Kim SM, Kim SH, Park KS, et al. Reproducibility of the
motor unit number index (MUNIX) in normal controls and amyotrophic lateral
sclerosis patients. Muscle Nerve 2010;42:808–13.
Barkhaus PE, Periquet MI, Nandedkar SD. Influence of the surface EMG electrode on
the compound muscle action potential. Electromyogr Clin Neurophysiol
2006;46:235–9.
Benatar M, Boylan K, Jeromin A, Rutkove SB, Berry J, Atassi N, et al. ALS biomarkers
for therapy development: state of the field and future directions. Muscle Nerve
2016;53:169–82.
Benatar M, Wuu J. Presymptomatic studies in ALS: rationale, challenges, and
approach. Neurology 2012;79:1732–9.
C. Neuwirth et al. / Clinical Neurophysiology xxx (2016) xxx–xxx 5
Please cite this article in press as: Neuwirth C et al. Motor Unit Number Index (MUNIX) detects motor neuron loss in pre-symptomatic muscles in Amy-
otrophic Lateral Sclerosis. Clin Neurophysiol (2016), http://dx.doi.org/10.1016/j.clinph.2016.11.026
Boekestein WA, Schelhaas HJ, van Putten MJ, Stegeman DF, Zwarts MJ, van Dijk JP.
Motor unit number index (MUNIX) versus motor unit number estimation
(MUNE): a direct comparison in a longitudinal study of ALS patients. Clin
Neurophysiol 2012;123:1644–9.
Bromberg MB, Brownell AA. Motor unit number estimation in the assessment of
performance and function in motor neuron disease. Phys Med Rehabil Clin N
Am 2008;19:509–32. ix.
Brooks BR, Miller RG, Swash M, Munsat TL. World Federation of Neurology Research
Group on Motor Neuron D. El Escorial revisited: revised criteria for the
diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor
Neuron Disord 2000;1:293–9.
de Carvalho M, Swash M. Fasciculation potentials and earliest changes in motor unit
physiology in ALS. J Neurol Neurosurg Psychiatry 2013;84:963–8.
de Carvalho M, Turkman A, Swash M. Sensitivity of MUP parameters in detecting
change in early ALS. Clin Neurophysiol 2014;125:166–9.
Eisen A, Kuwabara S. The split hand syndrome in amyotrophic lateral sclerosis. J
Neurol Neurosurg Psychiatry 2012;83:399–403.
Fathi D, Mohammadi B, Dengler R, Boselt S, Petri S, Kollewe K. Lower motor neuron
involvement in ALS assessed by motor unit number index (MUNIX):long-term
changes and reproducibility. Clin Neurophysiol 2016;127:1984–8.
Great Lakes ALS Support Group. A comparison of muscle strength testing techniques
in amyotrophic lateral sclerosis. Neurology 2003;61:1503–7.
Kaufmann P, Levy G, Montes J, Buchsbaum R, Barsdorf AI, Battista V, et al. Excellent
inter-rater, intra-rater, and telephone-administered reliability of the ALSFRS-R
in a multicenter clinical trial. Amyotroph Lateral Scler 2007;8:42–6.
Kent-Braun JA, Walker CH, Weiner MW, Miller RG. Functional significance of upper
and lower motor neuron impairment in amyotrophic lateral sclerosis. Muscle
Nerve 1998;21:762–8.
Kim DG, Hong YH, Shin JY, Park KH, Sohn SY, Lee KW, et al. Split-hand phenomenon
in amyotrophic lateral sclerosis: a motor unit number index study. Muscle
Nerve 2016;53:885–8.
Menon P, Kiernan MC, Yiannikas C, Stroud J, Vucic S. Split-hand index for the
diagnosis of amyotrophic lateral sclerosis. Clin Neurophysiol 2013;124:410–6.
Nandedkar SD, Barkhaus PE, Stalberg EV. Motor unit number index (MUNIX):
principle, method, and findings in healthy subjects and in patients with motor
neuron disease. Muscle Nerve 2010;42:798–807.
Nandedkar SD, Barkhaus PE, Stalberg EV. Reproducibility of MUNIX in patients with
amyotrophic lateral sclerosis. Muscle Nerve 2011;44:919–22.
Nandedkar SD, Nandedkar DS, Barkhaus PE, Stalberg EV. Motor unit number index
(MUNIX). IEEE Trans Biomed Eng 2004;51:2209–11.
Neuwirth C, Barkhaus PE, Burkhardt C, Castro J, Czell D, de CM, et al. Tracking motor
neuron loss in a set of six muscles in amyotrophic lateral sclerosis using the
Motor Unit Number Index (MUNIX): a 15-months longitudinal multicentre trial.
J Neurol Neurosurg Psychiatry 2015;86:1172–9.
Neuwirth C, Burkhardt C, Alix J, Castro J, de Carvalho M, Gawel M, et al. Quality
control of Motor Unit Number Index (MUNIX) measurements in 6 muscles in a
single-subject ‘‘Round-Robin” setup. PLoS One 2016;11:e0153948.
Neuwirth C, Nandedkar S, Stalberg E, Barkhaus PE, Carvalho M, Furtula J, et al. Motor
Unit Number Index (MUNIX): a novel neurophysiological marker for
neuromuscular disorders; test-retest reliability in healthy volunteers. Clin
Neurophysiol 2011;122:1867–72.
Neuwirth C, Nandedkar S, Stalberg E, Weber M. Motor unit number index (MUNIX):
a novel neurophysiological technique to follow disease progression in
amyotrophic lateral sclerosis. Muscle Nerve 2010;42:379–84.
R Core Team. A language and environment for statistical computing. R Foundation
for Statistical Computing; 2012.
Ravits J, Paul P, Jorg C. Focality of upper and lower motor neuron degeneration at
the clinical onset of ALS. Neurology 2007;68:1571–5.
Swash M, Ingram D. Preclinical and subclinical events in motor neuron disease. J
Neurol Neurosurg Psychiatry 1988;51:165–8.
Turner MR, Benatar M. Ensuring continued progress in biomarkers for amyotrophic
lateral sclerosis. Muscle Nerve 2015;51:14–8.
Visser J, Mans E, de Visser M, van den Berg-Vos RM, Franssen H, de Jong JM, et al.
Comparison of maximal voluntary isometric contraction and hand-held
dynamometry in measuring muscle strength of patients with progressive
lower motor neuron syndrome. Neuromuscul Disord 2003;13:744–50.
Weber M, Eisen A, Stewart H, Hirota N. The split hand in ALS has a cortical basis. J
Neurol Sci 2000;180:66–70.
Wohlfart G. Collateral regeneration from residual motor nerve fibers in
amyotrophic lateral sclerosis. Neurology 1957;7:124–34.
6C. Neuwirth et al. / Clinical Neurophysiology xxx (2016) xxx–xxx
Please cite this article in press as: Neuwirth C et al. Motor Unit Number Index (MUNIX) detects motor neuron loss in pre-symptomatic muscles in Amy-
otrophic Lateral Sclerosis. Clin Neurophysiol (2016), http://dx.doi.org/10.1016/j.clinph.2016.11.026
