Different Approaches to Analyze Muscle Fat Replacement With Dixon MRI in Pompe Disease.

Quantitative MRI is an increasingly used method to monitor disease progression in muscular disorders due to its ability to measure changes in muscle fat content (reported as fat fraction) over a short period. Being able to objectively measure such changes is crucial for the development of new treatments in clinical trials. However, the analysis of the images involved continues to be a daunting task because of the time needed. Whether a more specific analysis selecting individual muscles or a global one analyzing the whole thigh or compartments could be a suitable alternative has only been marginally studied. In our study we compare three methods of analysis of 2-point-dixon images in a cohort of 34 patients with late onset Pompe disease followed over a period of one year. We measured fat fraction on MRIs obtained at baseline and at year 1, and we calculated the increment of fat fraction. We correlated the results obtained with the results of muscle function tests to investigate whether the three methods of analysis were equivalent or not. We observed significant differences between the three methods in the estimation of the fat fraction at both baseline and year 1, but no difference was found in the increment in fat fraction between baseline and year 1. When we correlated the fat fraction obtained with each method and the muscle function tests, we found a significant correlation with most tests in all three methods, although in most comparisons the highest correlation coefficient was found with the analysis of individual muscles. We conclude that the fastest strategy of analysis assessing compartments or the whole thigh could be reliable for certain cohorts of patients where the variable to study is the fat increment. In other sorts of studies, an individual muscle approach seems the most reliable technique.

Corrigendum: Magnetization Transfer Ratio in Lower Limbs of Late Onset Pompe Patients Correlates With Intramuscular Fat Fraction and Muscle Function Tests.

[This corrects the article DOI: 10.3389/fneur.2021.634766.].

Platelet Derived Growth Factor-AA Correlates With Muscle Function Tests and Quantitative Muscle Magnetic Resonance in Dystrophinopathies.

Introduction: Duchenne (DMD) and Becker (BMD) muscular dystrophy are X-linked muscular disorders produced by mutations in the DMD gene which encodes the protein dystrophin. Both diseases are characterized by progressive involvement of skeletal, cardiac, and respiratory muscles. As new treatment strategies become available, reliable biomarkers and outcome measures that can monitor disease progression are needed for clinical trials. Methods: We collected clinical and functional data and blood samples from 19 DMD patients, 13 BMD patients, and 66 healthy controls (8 pediatric and 58 adult controls), and blood samples from 15 patients with dysferlinopathy (DYSF) and studied the serum concentration of 4 growth factors involved in the process of muscle fibrosis. We correlated the serum concentration of these growth factors with several muscle function tests, spirometry results and fat fraction identified by quantitative Dixon muscle MRI. Results: We found significant differences in the serum concentration of Platelet Derived Growth Factor-AA (PDGF-AA) between DMD patients and pediatric controls, in Connective Tissue Growth Factor (CTGF) between BMD patients and adult controls, and in and Transforming Growth Factor- ß1 (TGF-ß1) between BMD and DYSF patients. PDGF-AA showed a good correlation with several muscle function tests for both DMD and BMD patients and with thigh fat fraction in BMD patients. Moreover, PDGF-AA levels were increased in muscle biopsies of patients with DMD and BMD as was demonstrated by immunohistochemistry and Real-Time PCR studies. Conclusion: Our study suggests that PDGF-AA should be further investigated in a larger cohort of DMD and BMD patients because it might be a good biomarker candidate to monitor the progression of these diseases.

Magnetization Transfer Ratio in Lower Limbs of Late Onset Pompe Patients Correlates With Intramuscular Fat Fraction and Muscle Function Tests.

Objectives: Magnetization transfer (MT) imaging exploits the interaction between bulk water protons and protons contained in macromolecules to induce signal changes through a special radiofrequency pulse. MT detects muscle damage in patients with neuromuscular conditions, such as limb-girdle muscular dystrophies or Charcot-Marie-Tooth disease, which are characterized by progressive fiber loss and replacement by fatty tissue. In Pompe disease, in which there is, in addition, an accumulation of glycogen inside the muscle fibers, MT has not been tested yet. Our aim is to estimate MT ratio (MTR) in the skeletal muscle of these patients and correlate it with intramuscular fat fraction (FF) and results of muscle function tests. Methods: We obtained two-point axial Dixon and Dixon-MT sequences of the right thigh on a 1.5 Teslas MRI scanner in 60 individuals, including 29 late onset Pompe disease patients, 2 patients with McArdle disease, and 29 age and sex matched healthy controls. FF and MTR were estimated. Muscle function using several muscle function tests, including quantification of muscle strength, timed test quality of life scales, conventional spirometry obtaining forced vital capacity while sitting and in the supine position, were assessed in all patients. Results: MTR was significantly lower in Pompe patients compared with controls (45.5 ± 8.5 vs. 51.7 ± 2.3, Student T-test, p < 0.05). There was a negative correlation between the MTR and FF muscles studied (correlation coefficient: -0.65, Spearman test: p < 0.05). MTR correlated with most of the muscle function test results. We analyzed if there was any difference in MTR values between Pompe patients and healthy controls in those muscles that did not have an increase in fat, a measure that could be related to the presence of glycogen in skeletal muscles, but we did not identify significant differences except in the adductor magnus muscle (48.4 ± 3.6 in Pompe vs. 51 ± 1.3 in healthy controls, Student T-test = 0.023). Conclusions: MTR is a sensitive tool to identify muscle loss in patients with Pompe disease and shows a good correlation with muscle function tests. Therefore, the MT technique can be useful in monitoring muscle degeneration in Pompe disease in clinical trials or natural history studies.

Correlation Between Respiratory Accessory Muscles and Diaphragm Pillars MRI and Pulmonary Function Test in Late-Onset Pompe Disease Patients.

Objectives: Pompe disease is a rare genetic disease produced by mutations in the GAA gene leading to progressive skeletal and respiratory muscle weakness. T1-weighted magnetic resonance imaging is useful to identify fatty replacement in skeletal muscles of late-onset Pompe disease (LOPD) patients. Previous studies have shown that replacement by fat correlates with worse results of muscle function tests. Our aim was to investigate if fat replacement of muscles involved in the ventilation process correlated with results of the spirometry and predicted respiratory muscle impairment in LOPD patients over time. Materials and Methods: We studied a cohort of 36 LOPD patients followed up annually in our center for a period of 4 years. We quantified muscle fat replacement using Mercuri score of the thoracic paraspinal and abdominal muscles and the pillars of the diaphragm. We correlated the combined Mercuri scores of these areas with spirometry results and the need of respiratory support. Results: We found a statistically significant correlation (Spearman test, p < 0.05; coefficient of correlation > 0.6) between forced vital capacity seated and lying and fat fraction score of all muscle groups studied. The group of patients who needed respiratory support had higher fat fraction scores than patients not requiring ventilatory support. Higher fat replacement in these areas correlated with worse progression in spirometry values over time. Conclusions: Fat replacement of paraspinal, abdominal, and trunk muscles correlates with results of spirometry and is able to predict worsening in respiratory muscle function tests that could lead to an emerging ventilatory dysfunction. Therefore, the identification of fat replacement in these muscle groups should lead to a closer monitorization of patients. Radiologic evaluation of diaphragm pillars in T1-weighted imaging axial sequences could also be helpful to predict respiratory insufficiency.

Follow-up of late-onset Pompe disease patients with muscle magnetic resonance imaging reveals increase in fat replacement in skeletal muscles.

BACKGROUND: Late-onset Pompe disease (LOPD) is a genetic disorder characterized by progressive degeneration of the skeletal muscles produced by a deficiency of the enzyme acid alpha-glucosidase. Enzymatic replacement therapy with recombinant human alpha-glucosidase seems to reduce the progression of the disease; although at the moment, it is not completely clear to what extent. Quantitative muscle magnetic resonance imaging (qMRI) is a good biomarker for the follow-up of fat replacement in neuromuscular disorders. The aim of this study was to describe the changes observed in fat replacement in skeletal muscles using qMRI in a cohort of LOPD patients followed prospectively. METHODS: A total of 36 LOPD patients were seen once every year for 4 years. qMRI, several muscle function tests, spirometry, activities of daily living scales, and quality-of-life scales were performed on each visit. Muscle MRI consisted of two-point Dixon studies of the trunk and thigh muscles. Computer analysis of the images provided the percentage of muscle degenerated and replaced by fat in every muscle (known as fat fraction). Longitudinal analysis of the measures was performed using linear mixed models applying the Greenhouse-Geisser test. RESULTS: We detected a statistically significant and continuous increase in mean thigh fat fraction both in treated (+5.8% in 3 years) and in pre-symptomatic patients (+2.6% in 3years) (Greenhouse-Geisser p < 0.05). As an average, fat fraction increased by 1.9% per year in treated patients, compared with 0.8% in pre-symptomatic patients. Fat fraction significantly increased in every muscle of the thighs. We observed a significant correlation between changes observed in fat fraction in qMRI and changes observed in the results of the muscle function tests performed. Moreover, we identified that muscle performance and mean thigh fat fraction at baseline visit were independent parameters influencing fat fraction progression over 4 years (analysis of covariance, p < 0.05). CONCLUSIONS: Our study identifies that skeletal muscle fat fraction continues to increase in patients with LOPD despite the treatment with enzymatic replacement therapy. These results suggest that the process of muscle degeneration is not stopped by the treatment and could impact muscle function over the years. Hereby, we show that fat fraction along with muscle function tests can be considered a good outcome measures for clinical trials in LOPD patients.

Study of the effect of anti-rhGAA antibodies at low and intermediate titers in late onset Pompe patients treated with ERT.

Late onset Pompe disease (LOPD) is a genetic disorder characterized by slowly progressive skeletal and respiratory muscle weakness. Symptomatic patients are treated with enzyme replacement therapy (ERT) with alglucosidase alpha (rhGAA). Although most of ERT treated patients develop antibodies against rhGAA, their influence on clinical progression is not completely known. We studied the impact of anti-rhGAA antibodies on clinical progression of 25 ERT treated patients. We evaluated patients at visit 0 and, after 1 year, at visit 1. We performed several muscle function tests, conventional spirometry and quantitative muscle MRI (qMRI) using 3-point Dixon analysis of thigh muscles at both visits. We also obtained serum samples at both visits and anti-rhGAA antibodies were quantified using ELISA. Antibody titers higher than 1:200 were identified in 18 patients (72%) of our cohort. Seven patients (28%) did not develop antibodies (0 to <1:200), 17 patients (68%) developed low to intermediate titers (1:200 to <1:31,200) and 1 patient (4%) developed high titers (>1:31,200). We analyzed the effect of low and intermediate antibody titers in clinical and radiological progression. There were no differences between the results of muscle function tests, spirometry or fat fraction analyzed using qMRI between patients with and without antibodies groups at baseline. Moreover, antibody titers did not influence muscle function test, spirometry results or qMRI results at year 1 visit. Most of the LOPD patients developed antibodies against ERT that persisted over time at low or intermediate levels. However, antibodies at these low and intermediate titers might not influence clinical response to the drug.

Identification of serum microRNAs as potential biomarkers in Pompe disease.

OBJECTIVE: To analyze the microRNA profile in serum of patients with Adult Onset Pompe disease (AOPD). METHODS: We analyzed the expression of 185 microRNAs in serum of 15 AOPD patients and five controls using microRNA PCR Panels. The expression levels of microRNAs that were deregulated were further studied in 35 AOPD patients and 10 controls using Real-Time PCR. Additionally, the skeletal muscle expression of microRNAs which showed significant increase levels in serum samples was also studied. Correlations between microRNA serum levels and muscle function test, spirometry, and quantitative muscle MRI were performed (these data correspond to the study NCT01914536 at ClinicalTrials.gov). RESULTS: We identified 14 microRNAs that showed different expression levels in serum samples of AOPD patients compared to controls. We validated these results in a larger cohort of patients and we found increased levels of three microRNAs, the so called dystromirs: miR-1-3p, miR-133a-3p, and miR-206. These microRNAs are involved in muscle regeneration and the expression of these was increased in patients' muscle biopsies. Significant correlations between microRNA levels and muscle function test were found. INTERPRETATION: Serum expression levels of dystromirs may represent additional biomarkers for the follow-up of AOPD patients.

PDGF-BB serum levels are decreased in adult onset Pompe patients.

Adult onset Pompe disease is a genetic disorder characterized by slowly progressive skeletal and respiratory muscle weakness. Symptomatic patients are treated with enzymatic replacement therapy with human recombinant alfa glucosidase. Motor functional tests and spirometry are commonly used to follow patients up. However, a serological biomarker that correlates with the progression of the disease could improve follow-up. We studied serum concentrations of TGFß, PDGF-BB, PDGF-AA and CTGF growth factors in 37 adult onset Pompe patients and 45 controls. Moreover, all patients performed several muscle function tests, conventional spirometry, and quantitative muscle MRI using 3-point Dixon. We observed a statistically significant change in the serum concentration of each growth factor in patients compared to controls. However, only PDGF-BB levels were able to differentiate between asymptomatic and symptomatic patients, suggesting its potential role in the follow-up of asymptomatic patients. Moreover, our results point to a dysregulation of muscle regeneration as an additional pathomechanism of Pompe disease.

Quantitative muscle MRI to follow up late onset Pompe patients: a prospective study.

Late onset Pompe disease (LOPD) is a slow, progressive disorder characterized by skeletal and respiratory muscle weakness. Enzyme replacement therapy (ERT) slows down the progression of muscle symptoms. Reliable biomarkers are needed to follow up ERT-treated and asymptomatic LOPD patients in clinical practice. In this study, 32 LOPD patients (22 symptomatic and 10 asymptomatic) underwent muscle MRI using 3-point Dixon and were evaluated at the time of the MRI with several motor function tests and patient-reported outcome measures, and again after one year. Muscle MRI showed a significant increase of 1.7% in the fat content of the thigh muscles in symptomatic LOPD patients. In contrast, there were no noteworthy differences between muscle function tests in the same period of time. We did not observe any significant changes either in muscle MRI or in muscle function tests in asymptomatic patients over the year. We conclude that 3-point Dixon muscle MRI is a useful tool for detecting changes in muscle structure in symptomatic LOPD patients and could become part of the current follow-up protocol in daily clinics.

Muscle MRI Findings in Childhood/Adult Onset Pompe Disease Correlate with Muscle Function.

OBJECTIVES: Enzyme replacement therapy has shown to be effective for childhood/adult onset Pompe disease (AOPD). The discovery of biomarkers useful for monitoring disease progression is one of the priority research topics in Pompe disease. Muscle MRI could be one possible test but the correlation between muscle MRI and muscle strength and function has been only partially addressed so far. METHODS: We studied 34 AOPD patients using functional scales (Manual Research Council scale, hand held myometry, 6 minutes walking test, timed to up and go test, time to climb up and down 4 steps, time to walk 10 meters and Motor Function Measure 20 Scale), respiratory tests (Forced Vital Capacity seated and lying, Maximun Inspiratory Pressure and Maximum Expiratory Pressure), daily live activities scales (Activlim) and quality of life scales (Short Form-36 and Individualized Neuromuscular Quality of Life questionnaire). We performed a whole body muscle MRI using T1w and 3-point Dixon imaging centered on thighs and lower trunk region. RESULTS: T1w whole body muscle MRI showed a homogeneous pattern of muscle involvement that could also be found in pre-symptomatic individuals. We found a strong correlation between muscle strength, muscle functional scales and the degree of muscle fatty replacement in muscle MRI analyzed using T1w and 3-point Dixon imaging studies. Moreover, muscle MRI detected mild degree of fatty replacement in paraspinal muscles in pre-symptomatic patients. CONCLUSION: Based on our findings, we consider that muscle MRI correlates with muscle function in patients with AOPD and could be useful for diagnosis and follow-up in pre-symptomatic and symptomatic patients under treatment. TAKE HOME MESSAGE: Muscle MRI correlates with muscle function in patients with AOPD and could be useful to follow-up patients in daily clinic.