Supplemental data Pedigrees_20170209_Revision Neurology

This file contains the pedigrees of the families described in: A family-based study into penetrance in facioscapulohumeral muscular dystrophy type 1. The file numbers refer to the family numbers in the text and supplemental data 2. All participants have a patient number that is designated in the file. Patients without number were below 18 years

Distinct Disease Phases in Muscles of Facioscapulohumeral Dystrophy Patients Identified by MR Detected Fat Infiltration

<div><p>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 ³¹P MR spectroscopic 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 (0.07±0.01/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 (0.18±0.15/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.</p></div

Intramuscular distribution and progression of fatty infiltration.

<p>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.</p

High-energy phosphates in the different stages of fatty infiltration and correlation with muscle force.

<p>(<b>A</b>) Representative phosphorous MR spectra of VL muscle of FSHD patients, upper with a normal fat fraction, lower with a high fat fraction. (<b>B</b>) PCr/ATP decreases with fat fraction (mean±SD). In intermediately fat infiltrated muscles the PCr/ATP is already decreased significantly from 4.15±1.00 to 3.57±0.88. Completely fat infiltrated muscles do not show a further decrease of this ratio. (<b>C</b>) Significant correlation between PCr/ATP and muscle strength (p<0.001, R² = 0.29). Pi = inorganic phosphate; PCr = phosphocreatine; ATP = adenosine triphosphate.</p

Correlation of fat or muscle fraction, determined by quantitative MRI, with clinical scores.

<p>(<b>A</b>) Correlation between age of the patient and average fat fraction of the thigh (p<0.05, R² = 0.15). (<b>B</b>) Average fat fraction of the thigh and FSHD duration are highly correlated (p<0.0001, R² = 0.54). (<b>C</b>) Fat fraction highly correlates with the radiological Lamminen score of the corresponding muscle (p<0.0001, R² = 0.70). (<b>D</b>) 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. (<b>E</b>) Correlation between muscle fraction (1-fat fraction) and force of quadriceps and hamstring muscle groups (p<0.0001 and R² = 0.76).</p

Patient demographics.

<p>Underwent two MR exams four months apart.</p><p>na = not available.</p

Distribution of naturally occuring fat fraction of the thigh muscles of a large cohort of FSHD patients.

<p>(<b>A</b>) 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>B</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 (0.54±0.41) compared to the VL or VI (0.20±0.29, 0.20±0.27, respectively). Note that fat fractions are not Gaussian distributed therefore reporting only mean±error values is not a good representation of the data.</p

Typical transversal T1 weigthed and TIRM MR images of FSHD patients.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) Transverse T1 weighted image of the thigh of a 39-year-old male FSHD patient. (<b>D</b>) 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).</p

The presence of edema, as identified by TIRM imaging, correlates with increased fatty infiltration, as reflected in changes in T1 weighted images.

<p>(<b>A</b>) TIRM and T1 weighted images of a 76 year-old male FSHD patient. (<b>B</b>) TIRM and T1 weighted images of a 39 year-old male FSHD patient. (<b>A–B.1</b>) VL(*) and VM(**) muscles of two FSHD patients show hyperintensity on TIRM images, indicating edema. (<b>A–B.2</b>) Baseline T1 weighted images. (<b>A–B.3</b>) Follow-up T1 weighted images showing an increase of fatty infiltration after about 4 months in the VL(*) and VM(**) muscles. (<b>C)</b> 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).</p

Analysis of transcriptional activity of DUX4 in a panel of tissues of D4Z4-2.5 and D4Z4-12.5 mice.

<p>DUX4 transcripts measured in 7 weeks old D4Z4-2.5 and D4Z4-12.5 mice (n = 3) in A) muscle tissue: Hea = Heart, Dia = Diaphragm, Pec = Pectoralis Mas = Masseter, Orb = Orbicularis oris, Qua = Quadriceps, TA = Tibialis anterior, Gas = Gastrocnemius, Ton = Tongue; and B) somatic non-muscle and germline tissue: Tes = Testis, Ute = Uterus, Ova = Ovarium, Eye, Cer = Cerebellum, Spl = Spleen, Kid = Kidney, Liv = Liver C) DUX4 transcripts measured in satellite-cell-derived myoblasts, myotubes and interstitial fibroblast extracted from EDL muscle of D4Z4-12.5 and D4Z4-2.5 transgenic mice. D) Quantitative RT-PCR data of DUX4 expression in D4Z4-2.5 myoblasts (n = 2) and myotubes (n = 2) 48 hours after induction of differentiation. Errors indicate SEM of the plotted mean.</p