Development of MRC Centre MRI calf muscle fat fraction protocol as a sensitive outcome measure in Hereditary Sensory Neuropathy Type 1.

OBJECTIVES: Hereditary sensory neuropathy type 1 (HSN1) is a rare, slowly progressive neuropathy causing profound sensory deficits and often severe motor loss. L-serine supplementation is a possible candidate therapy but the lack of responsive outcome measures is a barrier for undertaking clinical trials in HSN1. We performed a 12-month natural history study to characterise the phenotype of HSN1 and to identify responsive outcome measures. METHODS: Assessments included Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNSv2), CMTNSv2-Rasch modified, nerve conduction studies, quantitative sensory testing, intraepidermal nerve fibre density (thigh), computerised myometry (lower limbs), plasma 1-deoxysphingolipid levels, calf-level intramuscular fat accumulation by MRI and patient-based questionnaires (Neuropathic Pain Symptom Inventory and 36-Short Form Health Survey version 2 [SF-36v2]). RESULTS: 35 patients with HSN1 were recruited. There was marked heterogeneity in the phenotype mainly due to differences between the sexes: males generally more severely affected. The outcome measures that significantly changed over 1 year and correlated with CMTNSv2, SF-36v2-physical component and disease duration were MRI determined calf intramuscular fat accumulation (mean change in overall calf fat fraction 2.36%, 95% CI 1.16 to 3.55, p=0.0004), pressure pain threshold on the hand (mean change 40 kPa, 95% CI 0.7 to 80, p=0.046) and myometric measurements of ankle plantar flexion (median change -0.5 Nm, IQR -9.5 to 0, p=0.0007), ankle inversion (mean change -0.89 Nm, 95% CI -1.66 to -0.12, p=0.03) and eversion (mean change -1.61 Nm, 95% CI -2.72 to -0.51, p=0.006). Intramuscular calf fat fraction was the most responsive outcome measure. CONCLUSION: MRI determined calf muscle fat fraction shows validity and high responsiveness over 12 months and will be useful in HSN1 clinical trials.

Stability and sensitivity of water T2 obtained with IDEAL-CPMG in healthy and fat-infiltrated skeletal muscle.

Quantifying muscle water T2 (T2 -water) independently of intramuscular fat content is essential in establishing T2 -water as an outcome measure for imminent new therapy trials in neuromuscular diseases. IDEAL-CPMG combines chemical shift fat-water separation with T2 relaxometry to obtain such a measure. Here we evaluate the reproducibility and B1 sensitivity of IDEAL-CPMG T2 -water and fat fraction (f.f.) values in healthy subjects, and demonstrate the potential of the method to quantify T2 -water variation in diseased muscle displaying varying degrees of fatty infiltration. The calf muscles of 11 healthy individuals (40.5 ± 10.2 years) were scanned twice at 3 T with an inter-scan interval of 4 weeks using IDEAL-CPMG, and 12 patients with hypokalemic periodic paralysis (HypoPP) (42.3 ± 11.5 years) were also imaged. An exponential was fitted to the signal decay of the separated water and fat components to determine T2 -water and the fat signal amplitude muscle regions manually segmented. Overall mean calf-level muscle T2 -water in healthy subjects was 31.2 ± 2.0 ms, without significant inter-muscle differences (p = 0.37). Inter-subject and inter-scan coefficients of variation were 5.7% and 3.2% respectively for T2 -water and 41.1% and 15.4% for f.f. Bland-Altman mean bias and ±95% coefficients of repeatability were for T2 -water (0.15, -2.65, 2.95) ms and f.f. (-0.02, -1.99, 2.03)%. There was no relationship between T2 -water (ρ = 0.16, p = 0.07) or f.f. (ρ = 0.03, p = 0.7761) and B1 error or any correlation between T2 -water and f.f. in the healthy subjects (ρ = 0.07, p = 0.40). In HypoPP there was a measurable relationship between T2 -water and f.f. (ρ = 0.59, p < 0.001). IDEAL-CPMG provides a feasible way to quantify T2 -water in muscle that is reproducible and sensitive to meaningful physiological changes without post hoc modeling of the fat contribution. In patients, IDEAL-CPMG measured elevations in T2 -water and f.f. while showing a weak relationship between these parameters, thus showing promise as a practical means of quantifying muscle water in patient populations.

Muscle magnetic resonance imaging in congenital myasthenic syndromes.

INTRODUCTION: In this study we investigated muscle magnetic resonance imaging in congenital myasthenic syndromes (CMS). METHODS: Twenty-six patients with 9 CMS subtypes and 10 controls were imaged. T1-weighted (T1w) and short-tau inversion recovery (STIR) 3-Tesla MRI images obtained at thigh and calf levels were scored for severity. RESULTS: Overall mean the T1w score was increased in GFPT1 and DPAGT1 CMS. T1w scans of the AChR-deficiency, COLQ, and CHAT subjects were indistinguishable from controls. STIR images from CMS patients did not differ significantly from those of controls. Mean T1w score correlated with age in the CMS cohort. CONCLUSIONS: MRI appearances ranged from normal to marked abnormality. T1w images seem to be especially abnormal in some CMS caused by mutations of proteins involved in the glycosylation pathway. A non-selective pattern of fat infiltration or a normal-appearing scan in the setting of significant clinical weakness should suggest CMS as a potential diagnosis. Muscle MRI could play a role in differentiating CMS subtypes. Muscle Nerve 54: 211-219, 2016.

Extra-ocular muscle MRI in genetically-defined mitochondrial disease.

OBJECTIVES: Conventional and quantitative MRI was performed in patients with chronic progressive external ophthalmoplegia (CPEO), a common manifestation of mitochondrial disease, to characterise MRI findings in the extra-ocular muscles (EOMs) and investigate whether quantitative MRI provides clinically relevant measures of disease. METHODS: Patients with CPEO due to single mitochondrial DNA deletions were compared with controls. Range of eye movement (ROEM) measurements, peri-orbital 3 T MRI T1-weighted (T1w) and short-tau-inversion-recovery (STIR) images, and T2 relaxation time maps were obtained. Blinded observers graded muscle atrophy and T1w/STIR hyperintensity. Cross-sectional areas and EOM mean T2s were recorded and correlated with clinical parameters. RESULTS: Nine patients and nine healthy controls were examined. Patients had reduced ROEM (patients 13.3°, controls 49.3°, p < 0.001), greater mean atrophy score and increased T1w hyperintensities. EOM mean cross-sectional area was 43 % of controls and mean T2s were prolonged (patients 75.6 ± 7.0 ms, controls 55.2 ± 4.1 ms, p < 0.001). ROEM correlated negatively with EOM T2 (rho = -0.89, p < 0.01), whilst cross-sectional area failed to correlate with any clinical measures. CONCLUSIONS: MRI demonstrates EOM atrophy, characteristic signal changes and prolonged T2 in CPEO. Correlation between elevated EOM T2 and ROEM impairment represents a potential measure of disease severity that warrants further evaluation. KEY POINTS: Chronic progressive external ophthalmoplegia is a common clinical manifestation of mitochondrial disease. • Existing extra-ocular muscle MRI data in CPEO reports variable radiological findings. MRI confirmed EOM atrophy and characteristic signal changes in CPEO. EOM T2 was significantly elevated in CPEO and correlated negatively with ocular movements. EOM T2 represents a potential quantitative measure of disease severity in CPEO.

Improved anatomical reproducibility in quantitative lower-limb muscle MRI.

PURPOSE: To compare the influence of two limb positions and slice prescription using scout-image-based and surface-anatomy-based methods on the reproducibility of quantitative MRI of lower-limb muscles. MATERIALS AND METHODS: Ten healthy subjects were scanned at 3 Tesla with a two-dimensional turbo spin-echo T1-weighted acquisition. Imaging was performed at thigh and calf level in two subject limb positions and independently repeated by a second operator. Regions-of-interest (ROI) were drawn on three muscles at thigh and calf levels on axial slices at fixed distance from the knee joint and at a level determined by surface anatomy. RESULTS: Test-retest reliability of muscle cross-sectional area and ROI area overlap were similar for both limb positioning methods. Changing limb position between scans reduced ROI overlap (P < 0.01). Scout-image-based slice prescription resulted in narrower limits of agreement and higher intraclass correlation coefficients compared with surface-anatomy-based slice prescription. CONCLUSION: Slice prescription based on fixed distance from the knee joint provided superior reproducibility of slice location than a surface anatomy-based method and should be used for longitudinal quantitative MRI studies. Exact subject positioning will depend on scanner and coil configuration, but should be consistent through a longitudinal study.

Reproducibility, and age, body-weight and gender dependency of candidate skeletal muscle MRI outcome measures in healthy volunteers.

OBJECTIVES: Quantitative magnetic resonance imaging (MRI) can potentially meet the pressing need for objective, sensitive, reproducible outcome measures in neuromuscular disease trials. We tested, in healthy volunteers, the consistency, reliability and sensitivity to normal inter-subject variation of MRI methods targeted to lower limb muscle pathology to inform the design of practical but comprehensive MRI outcome measure protocols for use in imminent patient studies. METHODS: Forty-seven healthy volunteers, age 21-81 years, were subject at 3T to three-point Dixon fat-fraction measurement, T1-relaxometry, T2-relaxometry and magnetisation transfer ratio (MTR) imaging at mid-thigh and mid-calf level bilaterally. Fifteen subjects underwent repeat imaging at 2 weeks. RESULTS: Mean between-muscle fat fraction and T2 differences were small, but significant (p < 0.001). Fat fraction and T 2 correlated positively, and MTR negatively with subject age in both the thigh and calf, with similar significant correlations with weight at thigh level only (p < 0.001 to p < 0.05). Scan-rescan and inter-observer intra-class correlation coefficients ranged between 0.62-0.84 and 0.79-0.99 respectively. CONCLUSIONS: Quantitative lower-limb muscle MRI using readily implementable methods was sensitive enough to demonstrate inter-muscle differences (small in health), and correlations with subject age and weight. In combination with high reliability, this strongly supports the suitability of these methods to provide longitudinal outcome measures in neuromuscular disease treatment trials. KEY POINTS: • Quantitative lower limb muscle MRI provides potential outcome measures in neuromuscular diseases • Bilateral thigh/calf coverage using sequences sensitive to acute and chronic pathology • Measurements have excellent scan-rescan and interobserver reliability • Measurements show small but significant inter-subject age and weight dependency • Readily implementable sequences suitable for further assessment in patient studies.

Longitudinal Changes in MRI Muscle Morphometry and Composition in People With Inclusion Body Myositis.

BACKGROUND AND OBJECTIVES: Limited data suggest that quantitative MRI (qMRI) measures have potential to be used as trial outcome measures in sporadic inclusion body myositis (sIBM) and as a noninvasive assessment tool to study sIBM muscle pathologic processes. Our aim was to evaluate changes in muscle structure and composition using a comprehensive multiparameter set of qMRI measures and to assess construct validity and responsiveness of qMRI measures in people with sIBM. METHODS: This was a prospective observational cohort study with assessments at baseline (n = 30) and 1 year (n = 26). qMRI assessments include thigh muscle volume (TMV), inter/intramuscular adipose tissue (IMAT), muscle fat fraction (FF), muscle inflammation (T2 relaxation time), IMAT from T2* relaxation (T2*-IMAT), intermuscular connective tissue from T2* relaxation (T2*-IMCT), and muscle macromolecular structure from the magnetization transfer ratio (MTR). Physical performance assessments include sIBM Physical Functioning Assessment (sIFA), 6-minute walk distance, and quantitative muscle testing of the quadriceps. Correlations were assessed using the Spearman correlation coefficient. Responsiveness was assessed using the standardized response mean (SRM). RESULTS: After 1 year, we observed a reduction in TMV (6.8%, p < 0.001) and muscle T2 (6.7%, p = 0.035), an increase in IMAT (9.7%, p < 0.001), FF (11.2%, p = 0.030), connective tissue (22%, p = 0.995), and T2*-IMAT (24%, p < 0.001), and alteration in muscle macromolecular structure (ΔMTR = -26%, p = 0.002). A decrease in muscle T2 correlated with an increase in T2*-IMAT (r = -0.47, p = 0.008). Deposition of connective tissue and IMAT correlated with deterioration in sIFA (r = 0.38, p = 0.032; r = 0.34, p = 0.048; respectively), whereas a decrease in TMV correlated with a decrease in quantitative muscle testing (r = 0.36, p = 0.035). The most responsive qMRI measures were T2*-IMAT (SRM = 1.50), TMV (SRM = -1.23), IMAT (SRM = 1.20), MTR (SRM = -0.83), and T2 relaxation time (SRM = -0.65). DISCUSSION: Progressive deterioration in muscle quality measured by qMRI is associated with a decline in physical performance. Inflammation may play a role in triggering fat infiltration into muscle. qMRI provides valid and responsive measures that might prove valuable in sIBM experimental trials and assessment of muscle pathologic processes. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that qMRI outcome measures are associated with physical performance measures in patients with sIBM.

Simultaneous T2 and lipid quantitation using IDEAL-CPMG.

Muscle damage, edema, and fat infiltration are hallmarks of a range of neuromuscular diseases. The T(2) of water, T(2,w) , in muscle lengthens with both myocellular damage and inflammation and is typically measured using multiple spin-echo or Carr-Purcell-Meiboom-Gill acquisitions. However, microscopic fat infiltration in neuromuscular diseases prevents accurate T(2,w) quantitation as the longer T(2) of fat, T(2,f) , masks underlying changes in the water component. Fat saturation can be inconsistent across the imaging volume and removes valuable physiological fat information. A new method is presented that combines iterative decomposition of water and fat with echo asymmetry and least squares estimation with a Carr-Purcell-Meiboom-Gill-sequence. The sequence results in water and fat separated images at each echo time for use in T(2,w) and T(2,f) quantification. With knowledge of the T(2,w) and T(2,f) , a T(2) -corrected fat fraction map can also be calculated. Monte-Carlo simulations and measurements in phantoms, volunteers, and a patient with inclusion body myositis are demonstrated. In healthy volunteers, uniform T(2,w) and T(2) -corrected fat fraction maps are present within all muscle groups. However, muscle-specific patterns of fat infiltration and edema are evident in inclusion body myositis, which demonstrates the power of separating and quantifying the fat and water components.

Quantitative magnetization transfer in in vivo healthy human skeletal muscle at 3 T.

The value of quantitative MR methods as potential biomarkers in neuromuscular disease is being increasingly recognized. Previous studies of the magnetization transfer ratio have demonstrated sensitivity to muscle disease. The aim of this work was to investigate quantitative magnetization transfer imaging of skeletal muscle in healthy subjects at 3 T to evaluate its potential use in pathological muscle. The lower limb of 10 subjects was imaged using a 3D fast low-angle shot acquisition with variable magnetization transfer saturation pulse frequencies and amplitudes. The data were analyzed with an established quantitative two-pool model of magnetization transfer. T(1) and B(1) amplitude of excitation radiofrequency field maps were acquired and used as inputs to the quantitative magnetization transfer model, allowing properties of the free and restricted proton pools in muscle to be evaluated in seven different muscles in a region of interest analysis. The average restricted pool T(2) relaxation time was found to be 5.9 ± 0.2 µs in the soleus muscle and the restricted proton pool fraction was 8 ± 1%. Quantitative magnetization transfer imaging of muscle offers potential new biomarkers in muscle disease within a clinically feasible scan time.

MRI quantifies lumbosacral nerve root and sciatic nerve hypertrophy in chronic inflammatory demyelinating polyradiculoneuropathy.

PURPOSE: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a treatable, immune-mediated condition characterised by progressive or relapsing motor and sensory neurological deficits. The diagnosis is based on a combination of clinical, neurophysiological and supportive criteria, but can be challenging. In this study, we quantified the diameter and cross-sectional area of the lumbosacral nerve roots, and explored the imaging characteristics of the sciatic nerves, in patients with CIDP versus healthy controls using MRI. METHODS: MRI of the lumbosacral plexus and both thighs was performed at 3 T. Orthogonal diameter and cross-sectional area of the lumbosacral nerve roots were measured, along with sciatic nerve cross-sectional area at the mid-thigh level. The MRI appearance of the sciatic nerves was also evaluated qualitatively. All measurements were performed by an observer blinded to the diagnosis. RESULTS: 10 patients with CIDP and 10 healthy controls (age and sex-matched) were studied. Lumbosacral nerve root diameter and cross-sectional area were significantly increased in patients with CIDP compared to controls (mean diameter 6.0 ±â€¯1.1 mm vs 4.8 ±â€¯0.3 mm; p = 0.006), with a high sensitivity (89 %) and specificity (90 %) on ROC analysis. Sciatic nerve cross sectional area was also significantly increased in the CIDP group, and was accompanied by qualitative MRI changes. CONCLUSIONS: Quantitative MRI reveals significant hypertrophy of the lumbosacral nerve roots and sciatic nerves in patients with CIDP compared to controls. This study provides further evidence for the inclusion of lumbosacral nerve root and sciatic nerve hypertrophy on MRI as a supportive feature in the diagnostic criteria for CIDP.

Skeletal muscle MRI differentiates SBMA and ALS and correlates with disease severity.

OBJECTIVE: To investigate the use of muscle MRI for the differential diagnosis and as a disease progression biomarker for 2 major forms of motor neuron disorders: spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS). METHODS: We applied quantitative 3-point Dixon and semiquantitative T1-weighted and short tau inversion recovery (STIR) imaging to bulbar and lower limb muscles and performed clinical and functional assessments in ALS (n = 21) and SBMA (n = 21), alongside healthy controls (n = 16). Acquired images were analyzed for the presence of fat infiltration or edema as well as specific patterns of muscle involvement. Quantitative MRI measurements were correlated with clinical measures of disease severity in ALS and SBMA. RESULTS: Quantitative imaging revealed significant fat infiltration in bulbar (p < 0.001) and limb muscles in SBMA compared to controls (thigh: p < 0.001; calf: p = 0.001), identifying a characteristic pattern of muscle involvement. In ALS, semiquantitative STIR imaging detected marked hyperintensities in lower limb muscles, distinguishing ALS from SBMA and controls. Finally, MRI measurements correlated significantly with clinical scales of disease severity in both ALS and SBMA. CONCLUSIONS: Our findings show that muscle MRI differentiates between SBMA and ALS and correlates with disease severity, supporting its use as a diagnostic tool and biomarker for disease progression. This highlights the clinical utility of muscle MRI in motor neuron disorders and contributes to establish objective outcome measures, which is crucial for the development of new drugs.

Natural history of limb girdle muscular dystrophy R9 over 6 years: searching for trial endpoints.

OBJECTIVE: Limb girdle muscular dystrophy type R9 (LGMD R9) is an autosomal recessive muscle disease for which there is currently no causative treatment. The development of putative therapies requires sensitive outcome measures for clinical trials in this slowly progressing condition. This study extends functional assessments and MRI muscle fat fraction measurements in an LGMD R9 cohort across 6 years. METHODS: Twenty-three participants with LGMD R9, previously assessed over a 1-year period, were re-enrolled at 6 years. Standardized functional assessments were performed including: myometry, timed tests, and spirometry testing. Quantitative MRI was used to measure fat fraction in lower limb skeletal muscle groups. RESULTS: At 6 years, all 14 muscle groups assessed demonstrated significant increases in fat fraction, compared to eight groups in the 1-year follow-up study. In direct contrast to the 1-year follow-up, the 6-min walk test, 10-m walk or run, timed up and go, stair ascend, stair descend and chair rise demonstrated significant decline. Among the functional tests, only FVC significantly declined over both the 1- and 6-year studies. INTERPRETATION: These results further support fat fraction measurements as a primary outcome measure alongside functional assessments. The most appropriate individual muscles are the vastus lateralis, gracilis, sartorius, and gastrocnemii. Using composite groups of lower leg muscles, thigh muscles, or triceps surae, yielded high standardized response means (SRMs). Over 6 years, quantitative fat fraction assessment demonstrated higher SRM values than seen in functional tests suggesting greater responsiveness to disease progression.

Exploration of New Contrasts, Targets, and MR Imaging and Spectroscopy Techniques for Neuromuscular Disease - A Workshop Report of Working Group 3 of the Biomedicine and Molecular Biosciences COST Action BM1304 MYO-MRI.

Neuromuscular diseases are characterized by progressive muscle degeneration and muscle weakness resulting in functional disabilities. While each of these diseases is individually rare, they are common as a group, and a large majority lacks effective treatment with fully market approved drugs. Magnetic resonance imaging and spectroscopy techniques (MRI and MRS) are showing increasing promise as an outcome measure in clinical trials for these diseases. In 2013, the European Union funded the COST (co-operation in science and technology) action BM1304 called MYO-MRI (www.myo-mri.eu), with the overall aim to advance novel MRI and MRS techniques for both diagnosis and quantitative monitoring of neuromuscular diseases through sharing of expertise and data, joint development of protocols, opportunities for young researchers and creation of an online atlas of muscle MRI and MRS. In this report, the topics that were discussed in the framework of working group 3, which had the objective to: Explore new contrasts, new targets and new imaging techniques for NMD are described. The report is written by the scientists who attended the meetings and presented their data. An overview is given on the different contrasts that MRI can generate and their application, clinical needs and desired readouts, and emerging methods.

Validation of MRC Centre MRI calf muscle fat fraction protocol as an outcome measure in CMT1A.

OBJECTIVE: To translate the quantitative MRC Centre MRI protocol in Charcot-Marie-Tooth disease type 1A (CMT1A) to a second site; validate its responsiveness in an independent cohort; and test the benefit of participant stratification to increase outcome measure responsiveness. METHODS: Three healthy volunteers were scanned for intersite standardization. For the longitudinal patient study, 11 patients with CMT1A were recruited with 10 patients rescanned at a 12-month interval. Three-point Dixon MRI of leg muscles was performed to generate fat fraction (FF) maps, transferred to a central site for quality control and analysis. Clinical data collected included CMT Neuropathy Score. RESULTS: Test-retest reliability of FF within individual healthy calf muscles at the remote site was excellent: intraclass correlation coefficient 0.79, limits of agreement -0.67 to +0.85 %FF. In patients, mean calf muscle FF was 21.0% and correlated strongly with disease severity and age. Calf muscle FF significantly increased over 12 months (+1.8 ± 1.7 %FF, p = 0.009). Patients with baseline FF >10% showed a 12-month FF increase of 2.9% ± 1.3% (standardized response mean = 2.19). CONCLUSIONS: We have validated calf muscle FF as an outcome measure in an independent cohort of patients with CMT1A. Responsiveness is significantly improved by enrolling a stratified patient cohort with baseline calf FF >10%.

Semi-Automated Analysis of Diaphragmatic Motion with Dynamic Magnetic Resonance Imaging in Healthy Controls and Non-Ambulant Subjects with Duchenne Muscular Dystrophy.

Subjects with Duchenne Muscular Dystrophy (DMD) suffer from progressive muscle damage leading to diaphragmatic weakness that ultimately requires ventilation. Emerging treatments have generated interest in better characterizing the natural history of respiratory impairment in DMD and responses to therapy. Dynamic (cine) Magnetic Resonance Imaging (MRI) may provide a more sensitive measure of diaphragm function in DMD than the commonly used spirometry. This study presents an analysis pipeline for measuring parameters of diaphragmatic motion from dynamic MRI and its application to investigate MRI measures of respiratory function in both healthy controls and non-ambulant DMD boys. We scanned 13 non-ambulant DMD boys and 10 age-matched healthy male volunteers at baseline, with a subset (n = 10, 10, 8) of the DMD subjects also assessed 3, 6, and 12 months later. Spirometry-derived metrics including forced vital capacity were recorded. The MRI-derived measures included the lung cross-sectional area (CSA), the anterior, central, and posterior lung lengths in the sagittal imaging plane, and the diaphragm length over the time-course of the dynamic MRI. Regression analyses demonstrated strong linear correlations between lung CSA and the length measures over the respiratory cycle, with a reduction of these correlations in DMD, and diaphragmatic motions that contribute less efficiently to changing lung capacity in DMD. MRI measures of pulmonary function were reduced in DMD, controlling for height differences between the groups: at maximal inhalation, the maximum CSA and the total distance of motion of the diaphragm were 45% and 37% smaller. MRI measures of pulmonary function were correlated with spirometry data and showed relationships with disease progression surrogates of age and months non-ambulatory, suggesting that they provide clinically meaningful information. Changes in the MRI measures over 12 months were consistent with weakening of diaphragmatic and inter-costal muscles and progressive diaphragm dysfunction. In contrast, longitudinal changes were not seen in conventional spirometry measures during the same period. Dynamic MRI measures of thoracic muscle and pulmonary function are, therefore, believed to detect meaningful differences between healthy controls and DMD and may be sensitive to changes in function over relatively short periods of follow-up in non-ambulant boys with DMD.

Quantifying fat replacement of muscle by quantitative MRI in muscular dystrophy.

The muscular dystrophies are rare orphan diseases, characterized by progressive muscle weakness: the most common and well known is Duchenne muscular dystrophy which affects young boys and progresses quickly during childhood. However, over 70 distinct variants have been identified to date, with different rates of progression, implications for morbidity, mortality, and quality of life. There are presently no curative therapies for these diseases, but a range of potential therapies are presently reaching the stage of multi-centre, multi-national first-in-man clinical trials. There is a need for sensitive, objective end-points to assess the efficacy of the proposed therapies. Present clinical measurements are often too dependent on patient effort or motivation, and lack sensitivity to small changes, or are invasive. Quantitative MRI to measure the fat replacement of skeletal muscle by either chemical shift imaging methods (Dixon or IDEAL) or spectroscopy has been demonstrated to provide such a sensitive, objective end-point in a number of studies. This review considers the importance of the outcome measures, discusses the considerations required to make robust measurements and appropriate quality assurance measures, and draws together the existing literature for cross-sectional and longitudinal cohort studies using these methods in muscular dystrophy.

MRI biomarker assessment of neuromuscular disease progression: a prospective observational cohort study.

BACKGROUND: A substantial impediment to progress in trials of new therapies in neuromuscular disorders is the absence of responsive outcome measures that correlate with patient functional deficits and are sensitive to early disease processes. Irrespective of the primary molecular defect, neuromuscular disorder pathological processes include disturbance of intramuscular water distribution followed by intramuscular fat accumulation, both quantifiable by MRI. In pathologically distinct neuromuscular disorders, we aimed to determine the comparative responsiveness of MRI outcome measures over 1 year, the validity of MRI outcome measures by cross-sectional correlation against functionally relevant clinical measures, and the sensitivity of specific MRI indices to early muscle water changes before intramuscular fat accumulation beyond the healthy control range. METHODS: We did a prospective observational cohort study of patients with either Charcot-Marie-Tooth disease 1A or inclusion body myositis who were attending the inherited neuropathy or muscle clinics at the Medical Research Council (MRC) Centre for Neuromuscular Diseases, National Hospital for Neurology and Neurosurgery, London, UK. Genetic confirmation of the chromosome 17p11.2 duplication was required for Charcot-Marie-Tooth disease 1A, and classification as pathologically or clinically definite by MRC criteria was required for inclusion body myositis. Exclusion criteria were concomitant diseases and safety-related MRI contraindications. Healthy age-matched and sex-matched controls were also recruited. Assessments were done at baseline and 1 year. The MRI outcomes-fat fraction, transverse relaxation time (T2), and magnetisation transfer ratio (MTR)-were analysed during the 12-month follow-up, by measuring correlation with functionally relevant clinical measures, and for T2 and MTR, sensitivity in muscles with fat fraction less than the 95th percentile of the control group. FINDINGS: Between Jan 19, 2010, and July 7, 2011, we recruited 20 patients with Charcot-Marie-Tooth disease 1A, 20 patients with inclusion body myositis, and 29 healthy controls (allocated to one or both of the 20-participant matched-control subgroups). Whole muscle fat fraction increased significantly during the 12-month follow-up at calf level (mean absolute change 1.2%, 95% CI 0.5-1.9, p=0.002) but not thigh level (0.2%, -0.2 to 0.6, p=0.38) in patients with Charcot-Marie-Tooth disease 1A, and at calf level (2.6%, 1.3-4.0, p=0.002) and thigh level (3.3%, 1.8-4.9, p=0.0007) in patients with inclusion body myositis. Fat fraction correlated with the lower limb components of the inclusion body myositis functional rating score (ρ=-0.64, p=0.002) and the Charcot-Marie-Tooth examination score (ρ=0.63, p=0.003). Longitudinal T2 and MTR changed consistently with fat fraction but more variably. In muscles with a fat fraction lower than the control group 95th percentile, T2 was increased in patients compared with controls (regression coefficients: inclusion body myositis thigh 4.0 ms [SE 0.5], calf 3.5 ms [0.6]; Charcot-Marie-Tooth 1A thigh 1.0 ms [0.3], calf 2.0 ms [0.3]) and MTR reduced compared with controls (inclusion body myositis thigh -1.5 percentage units [pu; 0.2], calf -1.1 pu [0.2]; Charcot-Marie-Tooth 1A thigh -0.3 pu [0.1], calf -0.7 pu [0.1]). INTERPRETATION: MRI outcome measures can monitor intramuscular fat accumulation with high responsiveness, show validity by correlation with conventional functional measures, and detect muscle water changes preceding marked intramuscular fat accumulation. Confirmation of our results in further cohorts with these and other muscle-wasting disorders would suggest that MRI biomarkers might prove valuable in experimental trials. FUNDING: Medical Research Council UK.

Upper Limb Evaluation in Duchenne Muscular Dystrophy: Fat-Water Quantification by MRI, Muscle Force and Function Define Endpoints for Clinical Trials.

OBJECTIVE: A number of promising experimental therapies for Duchenne muscular dystrophy (DMD) are emerging. Clinical trials currently rely on invasive biopsies or motivation-dependent functional tests to assess outcome. Quantitative muscle magnetic resonance imaging (MRI) could offer a valuable alternative and permit inclusion of non-ambulant DMD subjects. The aims of our study were to explore the responsiveness of upper-limb MRI muscle-fat measurement as a non-invasive objective endpoint for clinical trials in non-ambulant DMD, and to investigate the relationship of these MRI measures to those of muscle force and function. METHODS: 15 non-ambulant DMD boys (mean age 13.3 y) and 10 age-gender matched healthy controls (mean age 14.6 y) were recruited. 3-Tesla MRI fat-water quantification was used to measure forearm muscle fat transformation in non-ambulant DMD boys compared with healthy controls. DMD boys were assessed at 4 time-points over 12 months, using 3-point Dixon MRI to measure muscle fat-fraction (f.f.). Images from ten forearm muscles were segmented and mean f.f. and cross-sectional area recorded. DMD subjects also underwent comprehensive upper limb function and force evaluation. RESULTS: Overall mean baseline forearm f.f. was higher in DMD than in healthy controls (p<0.001). A progressive f.f. increase was observed in DMD over 12 months, reaching significance from 6 months (p<0.001, n = 7), accompanied by a significant loss in pinch strength at 6 months (p<0.001, n = 9) and a loss of upper limb function and grip force observed over 12 months (p<0.001, n = 8). CONCLUSIONS: These results support the use of MRI muscle f.f. as a biomarker to monitor disease progression in the upper limb in non-ambulant DMD, with sensitivity adequate to detect group-level change over time intervals practical for use in clinical trials. Clinical validity is supported by the association of the progressive fat transformation of muscle with loss of muscle force and function.

Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5.

BACKGROUND: Protein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level. RESULTS: The specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth. CONCLUSIONS: Many characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log phase growth suggests that this enzyme is involved in cell growth or its expression is controlled by metabolic or nutritional signals.

Quantitative magnetic resonance imaging in limb-girdle muscular dystrophy 2I: a multinational cross-sectional study.

We conducted a prospective multinational study of muscle pathology using magnetic resonance imaging (MRI) in patients with limb-girdle muscular dystrophy 2I (LGMD2I). Thirty eight adult ambulant LGMD2I patients (19 male; 19 female) with genetically identical mutations (c.826C>A) in the fukutin-related protein (FKRP) gene were recruited. In each patient, T1-weighted (T1w) imaging was assessed by qualitative grading for 15 individual lower limb muscles and quantitative Dixon imaging was analysed on 14 individual lower limb muscles by region of interest analysis. We described the pattern and appearance of muscle pathology and gender differences, not previously reported for LGMD2I. Diffuse fat infiltration of the gastrocnemii muscles was demonstrated in females, whereas in males fat infiltration was more prominent in the medial than the lateral gastrocnemius (p = 0.05). In the anterior thigh of males, in contrast to females, median fat infiltration in the vastus medialis muscle (45.7%) exceeded that in the vastus lateralis muscle (11.2%) (p<0.005). MRI is non-invasive, objective and does not rely on patient effort compared to clinical and physical measures that are currently employed. We demonstrated (i) that the quantitative Dixon technique is an objective quantitative marker of disease and (ii) new observations of gender specific patterns of muscle involvement in LGMD2I.