ORIGINAL RESEARCH published: 16 March 2021 doi: 10.3389/fneur.2021.634766 Magnetization Transfer Ratio in Lower Limbs of Late Onset Pompe Patients Correlates With Intramuscular Fat Fraction and Muscle Function Tests Claudia Nuñez-Peralta 1,2*, Paula Montesinos 3 , Alicia Alonso-Jiménez 4 , Jorge Alonso-Pérez 5 , David Reyes-Leiva 5 , Javier Sánchez-González 3 , Jaume Llauger-Roselló 1 , Sonia Segovia 5,6 , Izaskun Belmonte 7 , Irene Pedrosa 7 , Edited by: Antonio Martínez-Noguera 1 , Briano Matellini-Mosca 1 , Glenn Walter 8 and Edoardo Malfatti, Jordi Díaz-Manera 5,6,9* INSERM U1179 Handicap 1 Neuromusculaire: Physiopathologie, Radiology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 2 Departament de Medicina, Universidad Biothérapie et Pharmacologie Autónoma de Barcelona, Barcelona, Spain, 3 Philips Healthcare Iberia, Madrid, Spain, 4 Neuromuscular Reference Center, Appliquées (END-ICAP), France Neurology Department, University Hospital of Antwerp, Edegem, Belgium, 5 Neuromuscular Disorders Unit, Neurology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 6 Centro de Investigación Biomédica en Red de Reviewed by: Enfermedades Raras (CIBERER), Madrid, Spain, 7 Rehabilitation Department, Hospital de la Santa Creu i Sant Pau, Marianne De Visser, Barcelona, Spain, 8 Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL, United States, University of Amsterdam, Netherlands 9 John Walton Muscular Dystrophy Research Center, Newcastle University, Newcastle upon Tyne, United Kingdom Carmelo Rodolico, University of Messina, Italy *Correspondence: Objectives: Magnetization transfer (MT) imaging exploits the interaction between bulk Claudia Nuñez-Peralta water protons and protons contained in macromolecules to induce signal changes cnunez@santpau.cat Jordi Díaz-Manera through a special radiofrequency pulse. MT detects muscle damage in patients with jordi.diaz-manera@newcastle.ac.uk neuromuscular conditions, such as limb-girdle muscular dystrophies or Charcot-Marie- Tooth disease, which are characterized by progressive fiber loss and replacement by fatty Specialty section: This article was submitted to tissue. In Pompe disease, in which there is, in addition, an accumulation of glycogen Neuromuscular Disorders and inside the muscle fibers, MT has not been tested yet. Our aim is to estimate MT ratio Peripheral Neuropathies, a section of the journal (MTR) in the skeletal muscle of these patients and correlate it with intramuscular fat Frontiers in Neurology fraction (FF) and results of muscle function tests. Received: 28 November 2020 Methods: We obtained two-point axial Dixon and Dixon-MT sequences of the right thigh Accepted: 01 February 2021 Published: 16 March 2021 on a 1.5 Teslas MRI scanner in 60 individuals, including 29 late onset Pompe disease Citation: patients, 2 patients with McArdle disease, and 29 age and sex matched healthy controls. Nuñez-Peralta C, Montesinos P, FF and MTR were estimated. Muscle function using several muscle function tests, Alonso-Jiménez A, Alonso-Pérez J, including quantification of muscle strength, timed test quality of life scales, conventional Reyes-Leiva D, Sánchez-González J, Llauger-Roselló J, Segovia S, spirometry obtaining forced vital capacity while sitting and in the supine position, were Belmonte I, Pedrosa I, assessed in all patients. Martínez-Noguera A, Matellini-Mosca B, Walter G and Results: MTR was significantly lower in Pompe patients compared with controls (45.5 ± Díaz-Manera J (2021) Magnetization 8.5 vs. 51.7 ± 2.3, Student T-test, p < 0.05). There was a negative correlation between Transfer Ratio in Lower Limbs of Late Onset Pompe Patients Correlates the MTR and FF muscles studied (correlation coefficient: −0.65, Spearman test: p < With Intramuscular Fat Fraction and 0.05). MTR correlated with most of the muscle function test results. We analyzed if there Muscle Function Tests. Front. Neurol. 12:634766. was any difference in MTR values between Pompe patients and healthy controls in those doi: 10.3389/fneur.2021.634766 muscles that did not have an increase in fat, a measure that could be related to the Frontiers in Neurology | www.frontiersin.org 1 March 2021 | Volume 12 | Article 634766 Nuñez-Peralta et al. Magnetization Transfer Imaging in Pompe 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. Keywords: late onset Pompe disease, lower limb muscle, magnetic transfer ratio, intramuscular fat fraction, muscle function tests INTRODUCTION patients do not always show an increase in fat accumulation even if they develop muscle weakness, suggesting that the Magnetization transfer (MT) imaging is a magnetic resonance identification of changes related to glycogen accumulation could (MRI) technique that exploits the magnetization exchange be useful to monitor progression of the disease, especially in between water and tissue protons present in different these presymptomatic cases. Our hypothesis is that MT could environments. In normal tissue, hydrogen protons are present identify early structural changes related to the accumulation of in two compartments: the so-called “free pool” with mobile glycogen before degeneration of the muscle fibers to substitution protons in free water and a second compartment called the by fatty tissue takes place in the muscles. To confirm our “bound pool,” consisting of protons bound to proteins and other hypothesis, we studied a cohort of patients with late-onset Pompe macromolecules, such as glycogen (1). Protons within the free disease (LOPD) using the MT technique and compared the pool are responsible for the conventional MRI signal because results with those obtained in healthy controls. Additionally, they have a long and easily detectable T2 signal; on the other we correlated MTR values with intramuscular FF and muscle hand, protons bound to macromolecules have short T2 values function tests. due to their highly restricted motion and are not detected in routine MRI. The interaction between these two compartments can be METHODS probed by measuring the exchange of energy from the bound Study Design and Participants to the free pool of protons. This is achieved by applying a This is a transversal cross-sectional study involving 60 individuals special radiofrequency (RF) pulse called MT pulse (a low- who were studied at the Hospital de la Santa Creu i power RF saturation pulse) that gives origin to the MT Sant Pau (HSCSP) in Barcelona. The study is registered in effect (2). The MT effect can be used to create additional ClinicalTrials.gov with the identifier NCT01914536. The HSCSP contrast (MTC) in different tissues, and it is widely used ethics committee approved the study, and all participants signed in MR angiography, enhancing the representation of smaller an informed consent form. All study procedures were performed peripheral branches of the vessels (3), or in multiple sclerosis in accordance with Spanish regulations. in which the background suppression improves detection of The inclusion criteria for the study were (1) genetically acute lesions (4). The MT effect can also be quantified confirmed diagnosis of Pompe disease or McArdle disease; (2) obtaining the magnetization transfer ratio (MTR), which no contraindications to MRI; and (3) willingness to complete provides insight into relaxation and exchange rates of free water all muscle function tests, respiratory assessment, and patient- and macromolecules (5). reported outcomes measures. As control, we included a group Musculoskeletal tissue displays a pronounced MT effect, and of healthy volunteers age- and sex-matched with the Pompe it is already demonstrated to be a sensitive measure of muscle patients. We ruled out other neuromuscular conditions in all damage in patients with neuromuscular conditions, such as limb- participants in the study based on clinical data and results of girdle muscular dystrophies (LGMD) or Charcot-Marie-Tooth complementary tests, such as blood analysis, spinal MRI, or EMG disease, which are disorders characterized by progressive muscle when needed. fiber loss and replacement by fatty tissue (6, 7). Nevertheless, All patients were studied by three physiotherapists with the utility of MT has not been tested in Pompe disease yet, a long experience in neuromuscular disorders at HSCSP. The disease in which there is an accumulation of glycogen within physiotherapists evaluated muscle function using the following muscle fibers leading to cell death and replacement by fat. tests: the 6-min walking test, time to walk 10 m, timed up- Estimation of the intramuscular fat fraction (FF) using the Dixon and-go test, time to climb up and down four steps, and the technique in Pompe patients is a useful biomarker for the follow- Motor Function Measure 20-item scale (MFM-20). All timed up of patients as we demonstrate that there is a progressive tests were performed asking the patient to not use aids for accumulation of fat in the skeletal muscles that precedes changes walking. Muscle strength was studied using both the Medical in muscle function tests in patients that have already started Research Council (MRC) scale and hand-held myometry. Daily enzyme replacement therapy (8). However, presymptomatic life activities were studied using the activity limitations scale Frontiers in Neurology | www.frontiersin.org 2 March 2021 | Volume 12 | Article 634766 Nuñez-Peralta et al. Magnetization Transfer Imaging in Pompe FIGURE 1 | An example of Dixon and MT images. This figure shows an axial image of the right medial thigh of a Pompe patient including the muscles studied in this paper with the ROIs drawn and the values obtained for FF and MTR. (A) 3-D Dixon imaging of the thigh. (B) ROIs drawn in (A) showing the results of FF observed. (C) Dixon-MTC imaging of the thigh. (D) ROIs drawn in (C) showing the results of MTR. for patients with upper and/or lower limb impairments, and MRI quality of life was analyzed using both the Individualized All patients were examined in a 1.5 MR system (1.5 Achieva Neuromuscular Quality of Life Questionnaire and the Short dStream; Philips, Best, NL) at HSCSP in the supine position with Form 36 questionnaire. We obtained forced vital capacity stretched legs using a 32-channel body coil. Axial 3-D Dixon (FVC), both seated and lying down, using the Carefusion FFE on the middle third of both thighs was performed with the Microlab ML 3500 MK8 spirometer (Carefusion, Yorba Linda, following parameters: TR/TE1/TE2 = 5.78/1.8/4 ms, flip angle = CA, USA). These last two tests were added due their being 15, FOV = 520 × 340 × 300, acquired voxel size = 1 × 1 × 3 commonly used in Pompe disease patients to measure their mm3 ; acquisition time was 2 min 33 s. Also, another 3-D Dixon clinical status (9). Finally, Creatine kinase levels in serum were FFE sequence was acquired with and without an off-resonance quantified in the HSCSP biochemistry laboratory following magnetization transfer contrast prepulse active, TR/TE1/TE2 = standard protocols. 32/1.8/4 ms, flip angle = 15, FOV = 400 × 200 × 114 mm, Frontiers in Neurology | www.frontiersin.org 3 March 2021 | Volume 12 | Article 634766 Nuñez-Peralta et al. Magnetization Transfer Imaging in Pompe FIGURE 2 | Dixon water and fat maps and MTC-Dixon images obtained. Examples of fat and water maps generated from the data obtained with the Dixon sequence and of the images obtained with the MTC-Dixon sequence and the map generated. Axial sections of the thighs of a control (A) and two patients with Pompe disease, one with moderate involvement (B) and the other with severe muscle involvement (C). TABLE 1 | Mean demographic and clinical data of the patients included in the MTR values were expressed in percentage. Figure 1 shows an study. example of MT imaging, and the ROIs drawn and Figure 2 shows Pompe McArdle Controls an example of the images obtained in this study. Individuals (n) 29 2 29   Mo − M Sat Age at MRI 41.2 ± 21.3 y.o. 44 and 38 y.o 45.2 ± 22.7 y.o. MTR = (1) Mo Use of ERT (n) 21 Aids for walking (n) 12 – – Statistics Need of ventilation (n) 11 – – We confirmed that the variables to be analyzed were normally MRI, magnetic resonance imaging; ERT, enzymatic replacement therapy; N, number distributed using a Kolmogorov–Smirnov test, and therefore, of patients. parametric statistical studies were used. We used the Student T-test to analyze if the differences observed in FF and MTR between Pompe and controls were statistically significant. We acquired voxel size = 1 × 1 × 3 mm3 ; total acquisition time for used the Pearson test to study if correlations between variables images with and without MTC module active was 7 min 58 s. were significant. We consider that a correlation was good if the correlation coefficient was higher than 0.6. The significance Analysis level for all statistical studies was set at p < 0.05, and post-hoc We analyzed both sequences in the middle right thigh of Bonferroni corrections were used when needed. Statistical studies all patients. Regions of interest (ROIs) were manually drawn were performed with SPSS for Mac computers (Version 21, SPSS by three investigators on one slice of the following muscles: Inc., Chicago, IL). vastus lateralis (VL), sartorius (Sar), the long head of the biceps femoris (BLH), and adductor magnus (AM), keeping a RESULTS reasonable distance from the fascia and subcutaneous fat tissue. FF was obtained using a Philips Research Image Development Patients Included Environment (PRIDE) tool developed for this purpose. MTR was We included in the study 29 LOPD patients of which 23 patients calculated using the following formula in which Mo and MSat were symptomatic and treated with enzymatic replacement refer to the images without and with the saturation prepulse. therapy (ERT) and 6 patients were presymptomatic and not Frontiers in Neurology | www.frontiersin.org 4 March 2021 | Volume 12 | Article 634766 Nuñez-Peralta et al. Magnetization Transfer Imaging in Pompe FIGURE 3 | MTR and FF in controls and Pompe patients. (A) Mean thigh MTR value in controls (red) and Pompe patients (blue). (B) MTR value of VL, BLH, AM, and Sar in controls (red) and Pompe patients (blue). (C) Mean thigh FF value in controls (red) and Pompe patients (blue). (D) FF of VL, BLH, AM, and Sar in controls (red) and Pompe patients (blue). (E) Mean thigh MTR value in individuals with mean thigh FF lower than 20% in controls (red) and Pompe patients (blue). (F) Mean thigh MTR value in individuals with mean thigh FF higher than 20% in controls (red) and Pompe patients (blue). Mean value and standard deviation are shown. treated with ERT. Clinical features of LOPD patients included in MTR average value was lower in symptomatic compared with this study have been reported before (10). We also included in the presymptomatic (41.9 ± 9.8 vs. 51 ± 4, respectively, Student study 2 patients with McArdle disease without muscle weakness T-test p < 0.001). There were no differences in MTR between at clinical examination and 29 healthy age- and sex-matched controls and presymptomatic patients. controls. Main demographic and clinical data of the individuals We found higher mean thigh FF in LOPD patients’ muscles included in the study are summarized in Table 1. (44.9 ± 32.8%) compared with controls (17.86 ± 6.8%) (Student T-test, p <0.001; Figure 3C). When analyzed separately, we MRI Results identified higher values in AM, BLH, and VL but not in Sar We found lower MTR average value in LOPD patients’ muscles in LOPD patients compared with controls (Figure 3D). Mean (45.5 ± 8.5) compared with controls (51.7 ± 2.3) (Student thigh FF was significantly higher in symptomatic compared with T-test, p < 0.001; Figure 3A). When analyzed separately, we presymptomatic LOPD patients (32.6 ± 7.7 vs. 21.24 ± 7.3, identified significant lower values in AM and BLH but not in VL respectively, Student T-test p < 0.001). There were no differences nor Sar in LOPD patients compared with controls (Figure 3B). in mean thigh FF between controls and presymptomatic patients. Frontiers in Neurology | www.frontiersin.org 5 March 2021 | Volume 12 | Article 634766 Nuñez-Peralta et al. Magnetization Transfer Imaging in Pompe FIGURE 4 | Correlation between MTR value and FF. The figure shows the correlation between MTR value and FF in each muscle analyzed from controls (red) and patients with Pompe disease (blue). We did not identify significant differences in MTR in those TABLE 2 | Correlation between MTC values and results of the muscle function muscles with FF lower than 20% between LOPD and healthy tests. controls when the four individual muscles were analyzed together Mean MTC value (Figures 3E,F). However, when analyzed separately, we observed significant differences in MTR in the AM (48.4 ± 3.6 in Pompe Correlation coefficient Significance vs. 51 ± 1.3 in controls, Student T-test = 0.023). We observed Time to walk 10 m −0.72 0.0001 a negative correlation between MTR value and FF in muscles 6 MWT 0.813 0.0001 studied (correlation coefficient: −0.65, Spearman test p < 0.0001; Time up go −0.78 0.0001 Figure 4). Time climb up 4 Steps −0.79 0.0001 Time go down 4 Steps −0.75 0.0001 Correlation With Results of the Muscle MRC lower limbs 0.71 0.0001 Activlim 0.59 0.001 Function Tests CK levels 0.12 0.71 We observed a significant correlation between mean thigh MTR MTC adductor magnus value and the results of muscle function tests, including timed tests, such as the 6MWT or the time to walk 10 m, and measures Hip adduction 0.63 0.0001 of muscle strength, such as the composite MT value of the MTC biceps long head lower limbs. Moreover, we identified a good correlation between Knee flexion 0.54 0.003 individual muscle MTR values and specific muscle assessments as MTC vastus lateralis is shown in Table 2. Knee extension 0.38 0.52 6MWT, 6 min walking Test. Pearson test was performed, and p-values are shown. DISCUSSION We observed that MT is an indirect measurement of muscle Quantitative muscle MRI is progressively being implemented loss in LOPD patients, and it correlates with results of different in the follow-up of patients with neuromuscular diseases, muscle function tests commonly used in clinical trials and natural including Pompe disease, in clinical trials and natural history history studies. We also identified a good correlation between studies (11–13). Dixon imaging allows calculating the FF, which muscle FF and muscle MTR value, suggesting that MTR values is the amount of skeletal muscle replaced by fatty tissue. FF is decrease in relation to progressive loss of muscle fibers that are shown to correlate with the results of muscle function tests in replaced by fatty tissue. different diseases, including but not limited to Pompe disease. Frontiers in Neurology | www.frontiersin.org 6 March 2021 | Volume 12 | Article 634766 Nuñez-Peralta et al. Magnetization Transfer Imaging in Pompe FIGURE 5 | Example of Dixon and MTR images in a presymptomatic Pompe patient. (A) AM muscle is not macroscopically infiltrated by fat, and the estimation of FF using the Dixon technique is 12%. (B) MTR value in AM is 45. Moreover, Dixon has proved to be useful to follow up the disease, or spinal muscle atrophy (6, 7). We observed lower MTR progression of fat replacement in LOPD patients in longitudinal values in symptomatic patients compared with non-symptomatic studies. Previous studies have identified risk factors associated patients and controls. To fully understand the biophysical origins with FF, such as early age of onset of symptoms and disease of MT in Pompe muscle, a more detailed quantitative MT study duration (8). is warranted that takes into account pool sizes, T1, lipids, and Based on these results, Dixon sequences are being multiple transfer magnetization transfer mechanisms (5, 19). implemented in the new clinical trials that are being designed in Our initial hypothesis was that MT could be useful as an this disease. MT imaging is based on the magnetization exchange indirect measurement of glycogen. Glycogen is a highly hydrated between protons present in the free-water compartment molecule, and therefore, it was tempting to hypothesize that, in and protons bound to macromolecules. The more hydrated patients with Pompe disease who accumulate glycogen in their the macromolecules are, the more magnetization transfer is muscles, MTR could increase because of the accumulation of obtained. MT from fat is low as it is a hydrophobic tissue. water bound to the glycogen. In this study, we observed that Hydrophobic properties of lipids cause them to experience fat replacement is a confounding factor because it reduces the limited MT; for that reason, MT is generally low in tissues MTR signal considerably and makes difficult the identification with high fat content (14–16). However, it is known that fat of the effect of other tissue components on MTR. However, generates chemical shift artifacts that could compromise the MT values in muscles with low levels of fat replacement characterization and analysis of tissue. The use of traditional (<20% FF) are not statistically different between healthy controls fat-suppression techniques in MT studies can affect the MTR and LOPD patients except for the AM. Additionally, we have measurements, but the use of water images obtained from Dixon not seen differences between presymptomatic non-treated and sequences avoids possible confounding effects derived from the symptomatic treated patients in the MTR values in muscles fat signal for MTR calculation (17, 18). For these reasons, in this with FF lower than 20% (Figure 5). There are two potential study, we decided to use a two-point Dixon sequence with an explanations for our findings. On one hand, it is possible off-resonance prepulse. However, the signal obtained in muscles that the amount of glycogen accumulated in these muscles replaced by fat could also be influenced by other concomitant was too low to induce any change in MTR. It is well-known components, such as fibrotic tissue or inflammation. Therefore, that LOPD patients accumulate less glycogen than infantile MTR is not directly measuring only fat replacement, but in our onset patients (IOPD). Muscle biopsies of LODP patients opinion, is an indirect measurement of loss of muscle fibers can show mild accumulation of glycogen or even be normal, that reduce the values obtained. In this sense, our results are and muscle biopsies in IOPD patients are characterized by similar to the ones reported in other neuromuscular diseases, a massive accumulation of glycogen. On the other hand and such as LGMD, inclusion body myositis, Charcot-Marie-Tooth based on the physiopathology of the disease, it is probable that Frontiers in Neurology | www.frontiersin.org 7 March 2021 | Volume 12 | Article 634766 Nuñez-Peralta et al. Magnetization Transfer Imaging in Pompe the accumulation of glycogen was more prominent in those CONCLUSION muscles undergoing fatty replacement, but if that was the case, MTR could be influenced by the loss of muscle fibers and The MTR estimation constitutes a sensitive tool for the expansion of fat, producing, as a result, a decrease in MTR identification of cellular damage in patients with Pompe disease. values (20–22). We have also tested MT in 2 patients with It is correlated with muscle loss and muscle function tests. glucogenosis type V, usually known as McArdle disease, who Therefore, MT should be further explored as a tool for had mean thigh FF lower than 10%, and we have not seen monitoring muscle degeneration in Pompe disease in clinical any change in MTR values compared with controls. In our trials or natural history studies. opinion, to completely rule out MTR as an indirect measure of glycogen in patients, we should explore it in pretreated IOPD DATA AVAILABILITY STATEMENT patients because they accumulate larger amounts of glycogen in their muscles. The original contributions generated for the study are included Interestingly, we have seen a statistically significant decrease in the article/supplementary material, further inquiries can be in MTR values in the AM muscle in presymptomatic patients directed to the corresponding author/s. without an increase in FF. We previously identified that this muscle is one of the muscles earlier replaced by fat ETHICS STATEMENT in the progression of the disease (8). Two of these patients developed mild hip adduction weakness soon after being The studies involving human participants were reviewed and scanned in this study. In our opinion, this result probably approved by the Ethics Committee of the Hospital de la Santa reflects the loss of muscle fibers that could be the cause of Creu i Sant Pau, Barcelona. Written informed consent to the weakness rather than accumulation of glycogen as we participate in this study was provided by the participants’ legal would expect an increase in the MTR values. It is possible guardian/next of kin. that MT has high sensitivity to detect mild muscle fiber loss even when these fibers have not been replaced by fat yet, AUTHOR CONTRIBUTIONS and Dixon studies do not detect an increase in fatty tissue. Accordingly, McDaniel et al. reported a decrease in MTR value CN-P and PM: concept and design of the study, acquisition in muscles of patients with muscular dystrophies that were and analysis of data, and drafting the paper. AA-J and JA-P: not replaced by fat, which could be related, in our opinion, acquisition and analysis of data, and drafting the paper. DR-L, to the existence of early muscle damage not yet leading to JL-R, IB, IP, and BM-M: concept and design of the study, fat replacement. acquisition of data, and drafting the paper. JS-G, AM-N and The main limitation of our study is that we have not GW: concept and design of the study, and drafting the paper. used a gold standard test to study the amount of glycogen SS: concept and design of the study, coordination of the study, present in the muscles of the patients, such as a muscle and drafting the paper. JD-M: concept and design of the study, biopsy or specific imaging sequences able to identify glycogen, obtaining funding for the study, acquisition and analysis of data, such as 13C and 1H spectroscopy (23), chemical exchange and drafting the paper. All authors contributed to the article and saturation transfer imaging of glycogen (GlycoCEST) (24–26), approved the submitted version. and the recently described nuclear Overhauser enhancement of glycogen (Glyco-nOe) (27, 28). All these sequences are FUNDING able to identify glycogen in muscles and/or liver but are not available in conventional hospitals not focused on This study was sponsored by grants from Sanofi Genzyme and research. Some of the advantages of the MT technique are from the Spanish Ministry of Health, Fondos FEDER-ISCIII that it is available in many hospitals, does not require a PI18/01525 to JD-M. The company has neither reviewed the data specific coil, and the acquisition is relatively fast and easy nor suggested changes in any of the conclusions of the paper. to analyze. We have seen that MT values correlate with FF and, therefore, ACKNOWLEDGMENTS could be used to monitor disease progression over time although this should be explored in future longitudinal studies. The only We would like to thank all members of the Spanish Association longitudinal study published so far shows that MT measurements of Patients with Glycogenosis (www.glucogenosis.org) and the decreased in Charcot-Marie-Tooth and IBM patients after 1 Philips Healthcare engineer team for their collaboration. We year of follow-up in relation to the accumulation of fatty tissue also thank all patients and relatives for their collaboration in identified using Dixon (29). this study. REFERENCES 2. Henkelman RM, Stanisz GJ, Graham SJ. Magnetization transfer in MRI: a review. NMR Biomed. (2001) 14:57–64. doi: 10.1002/nbm.683 1. Sinclair CDJ, Samson RS, Thomas DL, Weiskopf N, Lutti A, Thornton JS, et al. 3. Catalano C, Pavone P, Laghi A, Faroni J, Clementi M, Di Girolamo M, Quantitative magnetization transfer in in vivo healthy human skeletal muscle et al. Magnetic resonance angiography of the intracranial circle using at 3 T. Magn Reson Med. 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(2015) 28:1716–25. doi: 10.1002/nbm.3437 The remaining authors declare that the research was conducted in the absence of 17. Smith AK, Dortch RD, Dethrage LM, Lyttle BD, Kang H, Welch EB, et al. any commercial or financial relationships that could be construed as a potential Incorporating dixon multi-echo fat water separation for novel quantitative conflict of interest. magnetization transfer of the human optic nerve in vivo. Magn Reson Med. (2017) 77:707–16. doi: 10.1002/mrm.26164 Copyright © 2021 Nuñez-Peralta, Montesinos, Alonso-Jiménez, Alonso-Pérez, Reyes- 18. Ercan E, Varma G, Dimitrov IE, Xi Y, Pinho MC, Yu FF, et al. Leiva, Sánchez-González, Llauger-Roselló, Segovia, Belmonte, Pedrosa, Martínez- Combining inhomogeneous magnetization transfer and multipoint Dixon Noguera, Matellini-Mosca, Walter and Díaz-Manera. This is an open-access article acquisition: potential utility and evaluation. Magn Reson Med. (2020) distributed under the terms of the Creative Commons Attribution License (CC BY). 85:2136–44. doi: 10.1002/mrm.28571 The use, distribution or reproduction in other forums is permitted, provided the 19. Zhou Y, van Zijl PCM, Xu J, Yadav NN. Mechanism and quantitative original author(s) and the copyright owner(s) are credited and that the original assessment of saturation transfer for water-based detection of the aliphatic publication in this journal is cited, in accordance with accepted academic practice. protons in carbohydrate polymers. Magn Reson Med. (2020) 85:1643– No use, distribution or reproduction is permitted which does not comply with these 54. doi: 10.1002/mrm.28503 terms. Frontiers in Neurology | www.frontiersin.org 9 March 2021 | Volume 12 | Article 634766