Distinct Disease Phases in Muscles of
Facioscapulohumeral Dystrophy Patients Identified by
MR Detected Fat Infiltration
Barbara H. Janssen1*, Nicoline B. M. Voet2, Christine I. Nabuurs1, Hermien E. Kan1,3, Jacky W. J. de Rooy1,
Alexander C. Geurts2, George W. Padberg4, Baziel G. M. van Engelen4, Arend Heerschap1
1Department of Radiology, Radboud University Medical Center, Nijmegen, The Netherlands, 2Department of Rehabilitation, Radboud University Medical Center,
Nijmegen, The Netherlands, 3Department of Radiology, Leiden University Medical Centre, Leiden, The Netherlands, 4Department of Neurology, Radboud University
Medical Center, Nijmegen, The Netherlands
Facioscapulohumeral muscular dystrophy (FSHD) is an untreatable disease, characterized by asymmetric progressive
weakness of skeletal muscle with fatty infiltration. Although the main genetic defect has been uncovered, the downstream
mechanisms causing FSHD are not understood. The objective of this study was to determine natural disease state and
progression in muscles of FSHD patients and to establish diagnostic biomarkers by quantitative MRI of fat infiltration and
phosphorylated metabolites. MRI was performed at 3T with dedicated coils on legs of 41 patients (28 men/13 women, age
34–76 years), of which eleven were re-examined after four months of usual care. Muscular fat fraction was determined with
multi spin-echo and T1 weighted MRI, edema by TIRM and phosphorylated metabolites by 3D
imaging. Fat fractions were compared to clinical severity, muscle force, age, edema and phosphocreatine (PCr)/ATP.
Longitudinal intramuscular fat fraction variation was analyzed by linear regression. Increased intramuscular fat correlated
with age (p,0.05), FSHD severity score (p,0.0001), inversely with muscle strength (p,0.0001), and also occurred sub-
clinically. Muscles were nearly dichotomously divided in those with high and with low fat fraction, with only 13% having an
intermediate fat fraction. The intramuscular fat fraction along the muscle’s length, increased from proximal to distal. This fat
gradient was the steepest for intermediate fat infiltrated muscles (0.0760.01/cm, p,0.001). Leg muscles in this intermediate
phase showed a decreased PCr/ATP (p,0.05) and the fastest increase in fatty infiltration over time (0.1860.15/year,
p,0.001), which correlated with initial edema (p,0.01), if present. Thus, in the MR assessment of fat infiltration as
biomarker for diseased muscles, the intramuscular fat distribution needs to be taken into account. Our results indicate that
healthy individual leg muscles become diseased by entering a progressive phase with distal fat infiltration and altered
energy metabolite levels. Fat replacement then relatively rapidly spreads over the whole muscle.
31P MR spectroscopic
Citation: Janssen BH, Voet NBM, Nabuurs CI, Kan HE, de Rooy JWJ, et al. (2014) Distinct Disease Phases in Muscles of Facioscapulohumeral Dystrophy Patients
Identified by MR Detected Fat Infiltration. PLoS ONE 9(1): e85416. doi:10.1371/journal.pone.0085416
Editor: Jan Kassubek, University of Ulm, Germany
Received August 6, 2013; Accepted November 26, 2013; Published January 14, 2014
Copyright: ? 2014 janssen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Prinses Beatrix Spierfonds [WAR08-15] (https://www.prinsesbeatrixspierfonds.nl), ZonMW [89000003 (http://www.
zonmw.nl/en/), the FSHD Global Research Foundation (http://www.fshdglobal.org/), and the FSH Society (http://www.fshsociety.org/). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Barbara.Janssen@radboudumc.nl
Facioscapulohumeral muscular dystrophy (FSHD) is the third
most common hereditary muscular disorder . The disease is
characterized by progressive asymmetric weakness and fatty
infiltration of skeletal muscles. In recent years it was demonstrated
that FSHD is associated with a contraction of D4Z4 repeats on
chromosome 4q35 , leading to lost repression of DUX4, a
protein that exerts toxic effects on muscle cells .
Even though the most important genetic event for the disease
seems to be identified, a causative treatment is not yet available
. Progress is hampered because the trigger for DUX4
expression and the further unfolding of disease processes leading
to fatty infiltration and muscle weakness are not known. Thus
clarification of the underlying mechanisms is expected to offer
clues for a more targeted approach in the search for treatment .
Understanding these mechanisms first requires that some key
questions concerning the process of fatty infiltration are addressed.
What is the natural distribution of fatty infiltration? How is this
related to clinical severity, to muscle weakness and to energy
metabolism? Is there prevalence for specific muscles to be affected
and does fatty infiltration vary within muscles? What is the natural
progression over time and what are predictive signs of progression?
To answer these questions and to evaluate treatment effective-
ness, the use of a non-invasive quantitative imaging method, such
as MRI, is essential. Unlike biopsies, MRI is not limited to a single
location, and longitudinal data can be collected without risk for the
patient. MR of fatty infiltration in muscles has been used to study
muscular disorders like Duchenne muscular dystrophy [6,7]. We
have introduced a quantitative MRI measure of fatty infiltration in
muscles based on T2 relaxation time analysis and demonstrated its
value in a preliminary study of FSHD patients . Phosphorus
MR spectroscopy has been used extensively to investigate the
PLOS ONE | www.plosone.org1 January 2014 | Volume 9 | Issue 1 | e85416
energy status of diseased muscles [9–15]. Recently it was also
introduced in a pilot study with FSHD patients .
Until now quantitative MR imaging studies were performed in
limited numbers of patients. However, because of the variability in
age of onset and in degree of disease severity , a study of its
pathophysiology requires the participation of a relatively large
number of well described patients. The main aim of this study was
to determine natural disease state and progression by quantitative
MRI of skeletal muscles in the legs of a large, well-characterized
cohort of genetically confirmed FSHD patients. In particular we
wanted to address the aforementioned pathophysiological ques-
tions to ultimately uncover clues on disease mechanism and to
establish MRI biomarkers with prognostic and predictive value for
Materials and Methods
Patients and Study Design
We recruited 41 FSHD patients from the local neurology
department (28 men/13 women, age 21–81 years, see Table 1 for
patients demographics). Of 36 patients the upper leg (‘thigh’) was
examined, they were selected from a group of patients that were
entering a clinical trial to assess the effects of rehabilitation
intervention . In addition we included the MR exams of the
lower leg of five patients from a previous study , which were re-
analyzed in the exact same way as the MR exams of the
aforementioned patients (vide infra).
Eleven patients (8 men/3 women, age 34–76 years) were
randomly selected, from the group that underwent an MR
examination of the thigh, for a follow-up measurement after a
period of four months. During this period these patients were
instructed to maintain a normal level of activity (‘usual care’).
All patients were clinically and genetically diagnosed with
FSHD and able to walk independently (ankle-foot orthoses and
canes were accepted). Patients were all unrelated except for one
mother and son (patients #7 and #37). Disease severity was
assessed with the Ricci score  and maximum voluntary
isometric extension (quadriceps) and flexion (hamstrings) of the
knee were measured with a quantitative fixed myometry testing
system . Ethical approval was obtained from the Radboud
university medical center review board, and written informed
consent was obtained from all subjects.
MR measurements were performed on a 3T MR system (TIM
Trio, Siemens, Erlangen, Germany). Subjects were positioned feet
first supine inside the magnet bore. Images were acquired with a
home-built proton birdcage radiofrequency coil (inner diameter
In 36 patients, the least affected thigh, according to the subject’s
own experience was examined, unless there were contraindications
(e.g. a previous fracture or recent injury). A fish oil capsule was
positioned at one third of the distance between the spina iliaca
anterior superior and the patella and served as a landmark for
exact matching of the imaging slices between the baseline and
follow-up measurements. For the MR examinations of leg, the
upper end of the proton coil was positioned at the center of the
Scout images were made in three orthogonal
directions to position MRI slices for subsequent scans. The
transmit frequency was set on the water resonance and the
transmitter voltage was adjusted to the load.
All imaging was performed in the transversal plane centered on
the middle femur for the thigh, or the largest circumference for the
T1 weighted spin echo (SE) MR images were acquired first (field
of view (FOV) 1756175 mm; base resolution 384; repetition time
Table 1. Patient demographics.
Patient nr. Ricci-scoreSex Age (years) FSHD duration (years)
10f 21 15
2 1.5f 252
3 1.5m 315
6 1.5m 38 17
103.5m 44 32
13 3.5m 5014
14 1.5f 501
15 1.5f 516
23 3.5m 57 17
373f 68 12
393m 69 19
40* 3.5m 764
*Underwent two MR exams four months apart.
MR Detected Disease Phases in FSHD Muscles
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(TR) 530 ms; echo time (TE) 16 ms; slices 23; slice thickness
4 mm; gap 0.4 mm).
Turbo Inversion Recovery Magnitude (TIRM) images were
collected with an inversion time (IT) to null the fat signals (FOV
1756175 mm; base resolution 256; TR 4000 ms; TE 41 ms; IT
22 ms; slices 23; slice thickness 4 mm; gap 0.4 mm) to visualize
edema [21–23]. To avoid inflow artifacts from venous and arterial
blood, saturation bands were placed above the upper and below
the lower slice .
Subsequently, multi SE MR images were acquired (FOV
1756175 mm; base resolution 256; TR 3000 ms; 16 equally
spaced TE’s 7.7–123.2 ms; slices 5–8, limited by specific
absorption rate; slice thickness 6 mm; gap 9 mm).
Phosphorus MR spectroscopic imaging (31P MRSI).
31P quadrature insert surface coil covered the quadriceps muscles
of the thigh, and for the leg measurements a circularly polarized
half volume31P coil covered the calf musculature. A 3D31P MRSI
dataset was acquired after imaging (FOV 15061506200 mm;
matrix-size 1461468 quadriceps/1061068 calf, TR 1000 ms;
BIR45 adiabatic pulse for excitation; 12 averages; weighted k-
space acquisition; nominal voxel volume 8.6 ml quadriceps/
16.6 ml calf). Datasets were interpolated to a matrix size of
was analyzed separately. T1weighted images were scored for fatty
infiltration using the four grade scale of Lamminen , by one
experienced musculoskeletal radiologist (J.W.J.R). When a differ-
ent score was awarded to the proximal and distal images the
average score was used.
Muscle area was assessed by drawing regions of interest (ROI’s)
for every muscle on the center slice of the T2weighted MRI. Fat
and muscle fractions were quantified from the multi SE MR
images as described earlier . Note that normal fat fraction for
healthy muscle does not exceed 10% . This method is not
suitable when edema is present, as it will affect the tissues
transverse relaxation properties. In those cases, T1 signal intensity
(SI) and TIRM SI of the individual muscles were quantified by
carefully drawing ROI’s in ImageJ (http://rsb.info.nih.gov/ij/)
and normalized to bone marrow SI.
To assess natural progression fat fraction differences were
normalized to a period of one year for every patient (for every
muscle) by dividing these fractions by the exact number of days
between the baseline and follow up measurement, multiplied by
From the middle slice of the 3D-MRSI dataset
with the largest circumference, representative voxels were assigned
to a specific muscle according to the corresponding T1weighted
image overlaid with the MRSI grid. Only spectra with a sufficient
signal-to-noise-ratio (SNR) (Cramer Rao Lower Bound (cATP)
,30%) were included for further analysis.
Free induction decays were zero-filled to double the number of
points and apodized by 8 Hz with a Lorentzian line shape and
manually phased using jMRUI 4.0 . Peak areas were obtained
from inorganic phosphate (Pi), PCr (fitted to a Lorentzian line
shape), and ATP (fitted to a Gaussian line shape), using the
AMARES algorithm  with prior knowledge on the relative line
width, frequency and amplitude.
Metabolite ratios: PCr/ATP and Pi/ATP were evaluated to
avoid coil profile variations. The pH was calculated from the Pi-
PCr frequency shift . The value of each parameter was
averaged for all analyzed voxels in one muscle and this value was
used for further analysis.
Each of the investigated muscles (see Fig. 1)
Statistical analyses were performed with Prism 5.0 (GraphPad
Software, San Diego, California, USA). Non-parametric one-way-
ANOVA (Kruskal-Wallis test) was used to investigate differences
in the average fat fraction between muscles, with Dunn’s Multiple
Comparison Test as post-hoc test. One-tailed correlation analyses
were performed between fat fraction and patients’ age, duration of
disease, radiological score, Ricci-scores, maximum voluntary
force, PCr/ATP, Pi/ATP, and pH. Linear regression analysis
was used to assess the distribution of fatty infiltration over the
length of the muscle. Outcome parameters in this analyses are the
slope of the line, indicating the direction of fatty infiltration over
the length of the muscle, and the coefficient of determination (R2),
indicating to what extent fat fraction increases or decreases linearly
over the length of the muscle. One-way ANOVA was used to
investigate dependence of fat fraction progression on initial muscle
fraction. T1 SI difference was compared between muscles normal
and hyperintense TIRM images with a one-tailed t-test, and
correlation was investigated with linear regression.
Muscular Fat Infiltration, Edema, Clinical Grading and
Muscle Strength in FSHD
Fat infiltration in skeletal muscles is visible as hyperintense areas
on T1 weighted MR images (Fig. 1A–C). This may be
accompanied by edema, which can be identified independently
from fatty infiltration by TIRM images (Fig. 1D). In 41 FSHD
patients we investigated 446 leg muscles, of which 4.3% showed
edema, which was mostly present in the quadriceps muscles.
The quantitative assessment of muscular fat fraction revealed
that 262 of the remaining 427 muscles were normal or mildly fat
infiltrated (,0.25 fat fraction), 54 were intermediately fat
infiltrated (between 0.25 and 0.75 fat fraction) and 111 muscles
were severely infiltrated (.0.75 fat fraction). A fat fraction
distribution plot resulted in a typical hourglass shape (Fig. 2A).
Significant differences were observed in average fat fractions of
thigh muscles (p,0.01), in particular the semimembranosus had a
significant higher fat fraction than the vastus lateralis and vastus
medialis (Fig. 2B).
Average fat fraction correlated positively with patients’ age
(p,0.05, R2=0.15) (Fig. 3A) and FSHD duration (p,0.0001,
R2=0.54), (Fig. 3B). Slopes of the correlations were not
significantly different between muscles (Fig. S1 and S2). The
average yearly increase in fatty infiltration was 0.860.4% for age
and 1.960.3% for FSHD duration. The average fat fraction
showed a strong correlation with radiological scores (p,0.0001,
R2=0.70) (Fig. 3C) and with overall clinical Ricci score for FSHD
severity (p,0.0001, R2=0.90) (Fig. 3D). The fat fraction deviated
from normal at Ricci score 2 (a subclinical event as this score
excludes leg muscle involvement) and further increased at higher
Ricci scores. Muscle fraction multiplied by the muscle area
significantly correlated with muscle strength for the quadriceps
and hamstring (p,0.0001, R2=0.57) (Fig. 3E).
Intramuscular Fat Distribution
Visual inspection of MR images revealed that the fat fraction
was often not evenly distributed over the length of the muscle
(Fig. 4). Muscle with an intermediate fat fraction showed the
steepest fatty infiltration gradient over the length of the muscle
(761% cm21, mean6SEM). This value was significantly higher
compared to muscles that were normal or mildly fat infiltrated
(1.360.3% cm21, p,0.0001) and those that were heavily
MR Detected Disease Phases in FSHD Muscles
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infiltrated by fat (1.160.1% cm21, p,0.0001). Overall fat fraction
rose from proximal to distal.
Natural Progression of Fatty Infiltration
muscles of eleven patients. An average increase in fat fraction of
0.05460.12 per year was observed. In intermediately affected
muscles (n=12) the progression of fatty infiltration was much faster
(0.1860.15 per year) as compared to heavily fat infiltrated muscles
(0.0060.10 per year, n=20) and to normal to mildly infiltrated
muscles (0.04360.10 per year, n=53). Natural progression in fat
infiltration depended on the initial muscle fraction (p,0.01) and
appeared to increasefrom distal to proximal(Fig. 4).
Six muscles, in two patients, showed hyperintensity on the
baseline TIRM images, indicating edema. The T1 SI difference
between baseline and follow up exam, representing fat infiltration,
was significantly different in muscles with hyperintense signal on
baseline TIRM images compared to TIRM normal muscles
(n=14) of the same patients (p,0.01) (Fig. 5). Linear regression
analysis showed a trend between the TIRM SI and the difference
in T1 SI (p,0.1, R2=0.1).
Figure 1. Typical transversal T1 weigthed and TIRM MR images of FSHD patients. (A) Transverse T1 weigthed image of the thigh of a male
FSHD patient (age 38), showing fatty infiltration (hyperintense signal) in the semimembranosus and semitendinosus muscles. (B) Transverse T1
weighted image of the leg of a male FSHD patient (age 66 year). Fatty infiltration of the soleus muscles is clearly visible. (C) Transverse T1 weighted
image of the thigh of a 39-year-old male FSHD patient. (D) Corresponding TIRM image. The semi-membranosus is clearly fat infiltrated (grey striped
arrow), this results in a nulled signal on the corresponding TIRM image. In contrast, the vastus lateralis and vastus intermedius show hyperintense
signal in the TIRM images (white arrows) reflecting edema or inflammation. The different muscles in the thigh (Fig. 1.A) and calf (Fig. 1.B) are indicated
by the following abbreviations: rectus femoris (RF), vastus lateralis (VL), vastus intermedius (VI), vastus medialis (VM), sartorius (S), adductor longus
(AL), adductor magnus (AM), gracillis (G), semi membranosus (SM), semi tendinosus (ST), biceps femoris long head (BFL) and biceps femoris short
head (BFS), tibialis anterior (TA), extensor digitorum, longus (EDL), peroneus brevis (PB), tibialis posterior (TP), soleus medialis (SOM), soleus lateralis
(SL), gastrocnemius medialis (GM) and gastrocnemius lateralis (GL).
MR Detected Disease Phases in FSHD Muscles
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High-energy Phosphate Metabolites
Analysis of phosphate metabolites by31P MRS revealed that the
PCr/ATP ratio correlated with the fat fraction of the specific
muscle (quadriceps p,0.05, R2=0.06, calf p,0.01, R2=0.33).
This PCr/ATP ratio is significantly decreased in the intermedi-
ately fat infiltrated muscles compared to muscles with a normal fat
fraction (p,0.05), but was not further decreased in muscles with a
high fat fraction (Fig. 6A). The PCr/ATP ratio also correlated with
muscle force (p,0.001, Fig. 6B).
In this study we identified three distinct phases of fat infiltration
in lower limb muscles of FSHD patients by quantitative MR. An
analysis of the average fat fraction for all individual muscles
uncovered an hourglass pattern of many muscles with either very
Figure 2. Distribution of naturally occuring fat fraction of the thigh muscles of a large cohort of FSHD patients. (A) Fat fraction
distribution over all muscles. Fat fraction of 0 signifies 100% muscle, 1 indicates 100% fat. Muscles with an intermediary fat fraction (.0.25 and ,0.75)
are observed, in ,13% of the investigated muscles. (B) Involvement of individual thigh muscles in FSHD. Average fat fraction of 36 patients. Error
bars (SEM) reflect the high variability in this fraction between patients. The SM appears to be the most affected muscle of the upper leg, having a
significantly higher average fat fraction (0.5460.41) compared to the VL or VI (0.2060.29, 0.2060.27, respectively). Note that fat fractions are not
Gaussian distributed therefore reporting only mean6error values is not a good representation of the data.
Figure 3. Correlation of fat or muscle fraction, determined by quantitative MRI, with clinical scores. (A) Correlation between age of the
patient and average fat fraction of the thigh (p,0.05, R2=0.15). (B) Average fat fraction of the thigh and FSHD duration are highly correlated
(p,0.0001, R2=0.54). (C) Fat fraction highly correlates with the radiological Lamminen score of the corresponding muscle (p,0.0001, R2=0.70). (D)
Quantitative fat fraction of lower limb correlates with patients Ricci score (p,0.0001, R2=0.90). Fat fraction starts to increase above normal levels at
Ricci score 2. The high standard deviation depicted in the error bars signifies the large variation in fat fraction determined in the limb and the
appointed Ricci score. (E) Correlation between muscle fraction (1-fat fraction) and force of quadriceps and hamstring muscle groups (p,0.0001 and
MR Detected Disease Phases in FSHD Muscles
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low or high fat, and few muscles with an intermediate fat fraction.
This quasi-binary distribution has not been reported for other
muscular dystrophies  and may be FSHD specific. The
intermediate phase is most characteristic, showing a relative steep
fat gradient over the length of the muscles, an altered energy
metabolism and rapid progression of fatty infiltration.
For other dystrophies often average values of all subjects or
muscles are presented, which obscures the presence of a specific
distribution. The average fat fraction as calculated over all
investigated muscles in this study (0.360.1), actually only was
present in 36 out of 427 muscles. Fat fractions were highest in the
semi membranosus, semi tendinosus and adductor muscles as has
been previously described by Wattjes et al. . The vastus
muscles were largely preserved.
We found that in leg muscles the intramuscular fat fraction
increased linearly from proximal to distal, as was also observed in
a pilot study of only the lower leg . The steepest fat gradient
occurred in the intermediate affected muscles indicating that these
muscles are progressing towards a complete fat infiltrated state.
This interpretation is supported by the follow-up measurements,
which revealed that intermediate affected muscles were most
prone to increase their fat-muscle ratio. In these muscles the
average increase of this ratio was about 10% in four months. This
may seem fast for a disease that is characterized by slow
progression, but we observed it in only a relative small fraction
of muscles. The quasi-binary fat distribution of muscles in FSHD
patients mentioned above also indicates that relative rapid
transitions occur. Moreover, a sudden disease progression within
individual muscles is in accordance with the often reported
observation in FSHD patients of periods of rapid deterioration of
single muscles or muscle groups, interrupting long stable periods
[30,31]. In some cases the lower performance of a single muscle
Figure 4. Intramuscular distribution and progression of fatty infiltration. Transveral T1 weighted images at different positions of the thigh
of a FSHD patient. Baseline measurement (left panels) reveals an uneven distruibution over the length of the muscle with an increasing fat infiltration
from proximal (top) to distal (bottom), especially prominent in the VM, VI, AM. This fatty gradient was largest in intermediate fat infiltrated muscles, as
was shown by the linear regression analyses. These intermediately fat infiltrated muscles also showed the largest increase in fatty infiltration over
time. From the follow-up measurement (right panels) it is clear to see that fat is increasing distally. AM=adductor magnus; BFL=biceps femoris long
head; VI=vastus intermedius; VL=vastus lateris; VM=vastus medialis.
MR Detected Disease Phases in FSHD Muscles
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Figure 5. The presence of edema, as identified by TIRM imaging, correlates with increased fatty infiltration, as reflected in changes
in T1 weighted images. (A) TIRM and T1 weighted images of a 76 year-old male FSHD patient. (B) TIRM and T1 weighted images of a 39 year-old
male FSHD patient. (A–B.1) VL(*) and VM(**) muscles of two FSHD patients show hyperintensity on TIRM images, indicating edema. (A–B.2) Baseline
T1 weighted images. (A–B.3) Follow-up T1 weighted images showing an increase of fatty infiltration after about 4 months in the VL(*) and VM(**)
muscles. (C) SI difference between baseline and follow-up T1 weighted images is significantly different in TIRM hyperintense FSHD muscles (N=6)
compared to TIRM normal FSHD muscles (n=14) (p,0.01).
Figure 6. High-energy phosphates in the different stages of fatty infiltration and correlation with muscle force. (A) Representative
phosphorous MR spectra of VL muscle of FSHD patients, upper with a normal fat fraction, lower with a high fat fraction. (B) PCr/ATP decreases with
fat fraction (mean6SD). In intermediately fat infiltrated muscles the PCr/ATP is already decreased significantly from 4.1561.00 to 3.5760.88.
Completely fat infiltrated muscles do not show a further decrease of this ratio. (C) Significant correlation between PCr/ATP and muscle strength
(p,0.001, R2=0.29). Pi=inorganic phosphate; PCr=phosphocreatine; ATP=adenosine triphosphate.
MR Detected Disease Phases in FSHD Muscles
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may be compensated by unaffected synergistic muscles, which
would clinically mask its dysfunction [32,33]. Assuming that
replacement of muscular tissue by fat occurs at a constant rate
after entering this intermediate phase, fat replacement of entire
muscles will, on the average, be completed within approximately
three and a half years. This can be relevant for prognostication
and monitoring therapy effectiveness in FSHD. There is no report
on fat gradients over the length of muscles in other neuromuscular
disorders, which may be FSHD specific. Recently, a muscular fatty
content gradient was also found in inherited poly-neuropathy, but
this was not associated with disease progression .
The low percentage of muscles involved in a rapid progression
towards complete fatty infiltration indicates that this process is
triggered by an infrequent event. The nature of this event is
agreement with findings that only 0.1% of muscle nuclei express
DUX4 in FSHD patients . A recent paper by Tassin and
colleagues  describes a model of initiation and propagation of a
transcriptional cascade, which provides an elegant explanation for
the DUX4 gene in (one or) few myonuclei yields DUX4 protein
where they activate target genes, which causes expansion into a
transcriptional cascade of deregulation. Because of the multinucle-
ated nature of myofibers this model predicts a gradient of
deregulation over the length of muscles. The amplification of
DUX4 gene activation into a transcriptional cascade may also
explain the fast progression observed in the intermediate fat
infiltrated muscles. Preferential involvement of particular muscles
the initial DUX4 gene activation trigger. Our finding that MR-
visible fat content increases more in the case of initial edema
supports the involvement of inflammation in early disease onset, as
TIRM positive muscles are associated with muscle inflammation
[21–23,37–39]. However, whether inflammation is cause or
consequence of DUX4 transcription in the initiation process
The correlation between increased fat fraction and lower
strength of skeletal muscles is coherent with the loss of muscle mass
and also explains the (weak) relation with the age of the patients.
Clinical severity scores (Lamminen  and Ricci ) strongly
correlated with fat fraction, but abnormal high fat fractions were
also present in lower limb muscles without clinical symptoms, as
was observed in patients with Ricci score 2 (excludes lower limb
involvement). Thus, imaging fatty infiltration is a potential tool to
predict clinical muscle affliction [32,33]. The extent of edema in
our study (4.3%) is somewhat lower than reported in two recent
FSHD studies, that however, included more muscles per patient
and more severely affected patients [23,40].
The lower PCr/ATP ratios observed in intermediately fatty
infiltrated muscles suggest an early change in high-energy
phosphate metabolism in disease development. Lower steady state
PCr/ATP ratios were also found in muscles of Becker and
Duchenne patients [10,41]. This may represent a lower cellular
(phospho)creatine pool due to a lower energy state. Alternatively, it
may represent a change in fiber type composition, if the fraction of
oxidative fibers, which have lower PCr/ATP ratio’s [42,43],
increase due to preferred involvement of type II fibers. This is
supported by histological findings of biopsies, showing more
dominant type I fibers among the remaining fibers in FSHD
affected muscles  and is also congruent with the correlation
between PCr/ATP and muscle force.
Taking muscle biopsies remains the gold standard to examine
muscular dystrophies, but this is invasive, painful, restricted to a
observed in the present and a previous study  fatty infiltration is
very heterogeneous, both between and within muscles, which
demonstrates the need to know in advance which (part of a) muscle
is affected, to acquire representative tissue. Our study indicates that
MRI guidance in taking muscle biopsies is needed. Other common
computer tomography, or poor signal to noise and limited
penetration depth in ultrasound. In clinical trials muscle strength is
often assessed to evaluate treatment effects, but this may show a
placebo effect . Muscular fat fraction determined by MRI does
not involvea placebo effect.
A limitation of our study was the lack of including a component
for the presence of edema in the T2 analysis. However, we
identified muscles with edema by TIRM and excluded the very
small fraction of edematous muscles from this T2 analysis.
Progression of fatty infiltration in these muscles was then derived
from T1 images. Furthermore, we chose to investigate lower
extremity muscles in these patients even though FSHD is a disease
known to first involve the facial and scapular muscles. However,
for this study we aimed for the highest image quality, which could
be achieved with a dedicated coil for the lower extremity. To
compare different disease phases we had to introduce fat fraction
cut-off values, for which we chose 25% and 75% of fatty
infiltration. Shifting these values by 65% did not change the main
results of this study.
In conclusion, this study established fat fraction as assessed by
MR imaging as an objective quantitative and sensitive biomarker
for muscular affliction in FSHD, detecting even subclinical muscle
involvement. This MR biomarker may serve to predict disease
progression, to guide biopsies and to evaluate treatments to
preserve or improve muscle performance. Importantly, in these
applications the intramuscular fat distribution may have to be
taken into account. Our data suggest a specific sequence of events
that leads towards full muscle pathology in FSHD, in which
muscles first progress from normal to being distally fat infiltrated,
with an altered metabolic profile, after which fat rapidly infiltrates
the whole muscles. This process of disease unfolding may direct
new treatment strategies.
the individual thigh muscles. Solid line gives best linear
correlation with 95% confidence interval indicated by the dotted
lines. Slopes of the lines were statistically tested to identify possible
differences between the muscles. However analyses showed no
significant differences. VL=vastus lateralis, VI=vastus interme-
dius, RF=rectus femoris, VM=vastus medialis, BFS biceps
femoris short head, BFL=biceps femoris long head, S=sartorius,
G=gracillis, ST=semit endinosus, SM=semi membranosus,
AM=adductor magnus, AL=adductor longus.
Correlation between fat fractions and age for
duration for the individual thigh muscles. Solid line gives
best linear correlation with 95% confidence interval indicated by
the dotted lines. Slopes of the lines were statistically tested to
identify possible differences between the muscles. However
analyses showed no significant differences. VL=vastus lateralis,
VI=vastus intermedius, RF=rectus femoris, VM=vastus med-
ialis, BFS biceps femoris short head, BFL=biceps femoris long
Correlation between fat fractions and disease
MR Detected Disease Phases in FSHD Muscles
PLOS ONE | www.plosone.org8 January 2014 | Volume 9 | Issue 1 | e85416
head, S=sartorius, G=gracillis, ST=semi tendinosus, SM=semi Download full-text
membranosus, AM=adductor magnus, AL=adductor longus.
We thank Rob J.W. Arts for helping with data processing and Jos IJspeert
for performing the force measurements.
Conceived and designed the experiments: AG BE GP AH. Performed the
experiments: BJ NV CN HK. Analyzed the data: BJ NV CN HK JR.
Wrote the paper: BJ NV CN HK JR AG GP BE AH.
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PLOS ONE | www.plosone.org9 January 2014 | Volume 9 | Issue 1 | e85416