ARTICLE OPEN ACCESS Three-dimensional imaging in myotonic dystrophy type 1 Linking molecular alterations with disease phenotype Alfonsina Ballester-Lopez, MSc, Judit Núñez-Manchón, MSc, Emma Koehorst, MSc, Ian Linares-Pardo, Correspondence Dr. Nogales-Gadea Miriam Almendrote, MD, Giuseppe Lucente, MD, Nicolau Guanyabens, MD, Marta Lopez-Osias, BSc, gnogales@igtp.cat Adrián Suárez-Mesa, MSc, Shaliza Ann Hanick, MSc, Jakub Chojnacki, PhD, Alejandro Lucia, MD, PhD, Guillem Pintos-Morell, MD, PhD, Jaume Coll-Cantı́, MD, PhD, Alicia Martı́nez-Piñeiro, MD,* Alba Ramos-Fransi, MD, PhD,* and Gisela Nogales-Gadea, PhD* Neurol Genet 2020;6:e484. doi:10.1212/NXG.0000000000000484 Abstract MORE ONLINE Video Objective We aimed to determine whether 3D imaging reconstruction allows identifying molecular: clinical associations in myotonic dystrophy type 1 (DM1). Methods We obtained myoblasts from 6 patients with DM1 and 6 controls. We measured cytosine- thymine-guanine (CTG) expansion and detected RNA foci and muscleblind like 1 (MBNL1) through 3D reconstruction. We studied dystrophia myotonica protein kinase (DMPK) ex- pression and splicing alterations of MBNL1, insulin receptor, and sarcoplasmic reticulum Ca(2+)-ATPase 1. Results Three-dimensional analysis showed that RNA foci (nuclear and/or cytoplasmic) were present in 45%–100% of DM1-derived myoblasts we studied (range: 0–6 foci per cell). RNA foci represented <0.6% of the total myoblast nuclear volume. CTG expansion size was associated with the number of RNA foci per myoblast (r = 0.876 [95% confidence interval 0.222–0.986]) as well as with the number of cytoplasmic RNA foci (r = 0.943 [0.559–0.994]). Although MBNL1 colocalized with RNA foci in all DM1 myoblast cell lines, colocalization only accounted for 1% of total MBNL1 expression, with the absence of DM1 alternative splicing patterns. The number of RNA foci was associated with DMPK expression (r = 0.967 [0.079–0.999]). On the other hand, the number of cytoplasmic RNA foci was correlated with the age at disease onset (r = −0.818 [−0.979 to 0.019]). Conclusions CTG expansion size modulates RNA foci number in myoblasts derived from patients with DM1. MBNL1 sequestration plays only a minor role in the pathobiology of the disease in these cells. Higher number of cytoplasmic RNA foci is related to an early onset of the disease, a finding that should be corroborated in future studies. *These authors contributed equally to this work. From the Neuromuscular and Neuropediatric Research Group (A.B.-L., J.N.-M., E.K., I.L.-P., M.A., G.L., M.L.-O., A.S.-M., S.A.H., G.P.-M., J.C.-C., A.M.-P., A.R.-F., G.N.-G.), Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Campus Can Ruti, Universitat Autònoma de Barcelona, Badalona; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) (A.B.-L., G.P.-M., J.C.-C., G.N.-G.), Instituto de Salud Carlos III, Madrid; Neuromuscular Pathology Unit. Neurology Service. Neuroscience department (M.A., G.L., N.G., J.C.-C., A.M.-P., A.R.-F.), Hospital Universitari Germans Trias i Pujol, Badalona, Barcelona; IrsiCaixa AIDS Research Institute (J.C.), Badalona, Spain; Faculty of Sport Sciences (A.L.), Universidad Europea de Madrid; Instituto de Investigación Hospital 12 de Octubre (i+12) (A.L.), Madrid; and Division of Rare Diseases. University Hospital Vall d’Hebron (G.P.-M.), Barcelona, Spain. Go to Neurology.org/NG for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by the authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CI = confidence interval; DM1 = myotonic dystrophy type 1; DMPK = dystrophia myotonica protein kinase; IR = insulin receptor; MBNL1 = muscleblind like 1; mRS = modified Rankin Scale; qPCR = quantitative PCR; RT-PCR = reverse transcription PCR. Myotonic dystrophy type I (DM1) is a multisystemic disorder A291, for more information on the participants and clinical with autosomal dominant inheritance caused by a cytosine- data collection). thymine-guanine (CTG) repeat expansion in the 39UTR of the dystrophia myotonica protein (DMPK) gene.1 The main Myoblast cultures pathogenic process underlying DM1 is a toxic RNA gain-of- We extracted muscle cells from the biopsy explants and placed function effect of expanded DMPK transcripts forming hairpin- them on culture plates between 2015 and 2016 in the cell culture structured aggregates, called “RNA foci.”2,3 These aggregates room of our institution (see supplemental data, links.lww.com/ are able to sequester splicing factors, thereby affecting the al- NXG/A291, for more information) and analyzed data from ternative splicing of some pre-mRNAs such as insulin receptor these myoblasts during 2018 and 2019 as described below. (IR), sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 1 (ATP2A1),4,5 or muscleblind like 1 (MBNL1).6 Expansion repeat sizing in myoblasts We determined CTG expansion size using long PCR- Most studies on DM1 have relied on 2D imaging at a single southern blotting (see supplemental data, links.lww.com/ cell level. Yet, this classic approach does not allow detection of NXG/A291, for a detailed description of the protocol). all RNA foci aggregates nor identification of their location Three-dimensional imaging of RNA foci inside cells. In addition, with this technique, it is not possible and MBNL1 to identify the distribution and sequestration of MBNL1 To detect RNA foci and MBNL1, we performed fluorescence protein. These limitations have hindered recognition of the in situ hybridization and immunostaining. The protocol we full cascade of molecular events associated with the pheno- used and the details of 3D imaging can be found in supple- type manifestation of DM1. mental data, links.lww.com/NXG/A291. Here, we used 3D imaging in whole myoblasts derived from Alternative splicing analysis and patients with DM1 with different degrees of affectation in an DMPK expression attempt to gain insight into the link between DM1 molecular We analyzed alternative splicing and DMPK expression with players and disease phenotype. Thus, we assessed the relationship reverse transcription (RT) PCR and quantitative PCR (qPCR), between CTG repeat expansion size, RNA foci, MBNL1 coloc- respectively (see supplemental data, links.lww.com/NXG/ alization, and splicing defects in DM1 patient-derived myoblasts, A291, for more information on the protocol and primers). on the one hand, and the clinical characteristics of the patients, on the other hand. We have obtained new pathophysiology data Statistical analysis related to how DM1 might affect patient-derived myoblasts. Details of the statistical analysis can be found in supplemental data, links.lww.com/NXG/A291. Methods Data availability The data that support the findings of this study are available Standard protocol approvals, registrations, from the corresponding author on reasonable request. and patient consents This study was approved (reference # PI-15-009) by the Ethic Committee of the University Hospital Germans Trias i Pujol (Badalona, Spain) and was performed in agreement with the Results Declaration of Helsinki for Human Research. All participants Patients’ phenotype signed a written informed consent to participate in the study. We studied 6 patients with DM1—4 women and 2 men— with age at disease onset ranging from 15 to 48 years (table 1). Participants Patients were unrelated except P3 and P4 (who were sisters). Six patients were recruited for this study, with DM1 di- The majority of patients had cardiac problems, with 3 showing agnosis confirmed in our laboratory by bidirectional triplet mild ECG alterations, one (P2) a structural cardiopathy PCR, as previously described.7 Patient muscle biopsies were (valvulopathy), and another one (P5) using a pacemaker. Half obtained from the left biceps brachialis (n = 5) and vastus of the patients needed nocturnal mechanical ventilation, and lateralis muscle (n = 1). The control biopsies were obtained the other 3 had mild changes in respiratory functional tests from the hand muscle abductor pollicis longus of healthy without actual clinical impairment. The muscular impairment individuals (see supplemental data, links.lww.com/NXG/ status according to the Muscular Impairment Rating Scale was 2 Neurology: Genetics | Volume 6, Number 4 | August 2020 Neurology.org/NG Table 1 Clinical data of patients with DM1 Patient with Biceps DM1 Sex Age at onset (y) MRC Myotonia (s) Cataracts Cardiopathy Spirometry 6MWD (m) MIRS mRS P1 F 15a 4 0.52 No LAFB Altered PFT 348 4 2 P2 M 48 5 0.67 Yes Valvulopathy NMV 251 3 2 P3 F 36 5 0.73 Yes None NMV 368 2 1 P4 F 42 5 0.98 Yes 1st-degree AV NMV 338 3 1 block P5 F 27 4 NP Yes Pacemaker Altered PFT NP 4 4 P6 M 36 5 0.96 No LAFB Altered PFT 519 3 2 Abbreviations: 6MWD = 6-minute walking distance; AV = atrioventricular; NP = not performed; LAFB = left anterior fascicular block; mRS = modified Rankin Scale; MIRS = Muscular Impairment Rating Scale; MRC = Medical Research Council Grade; NMV = nocturnal mechanical ventilation; PFT = pulmonary function tests. a It was not possible to determine the actual age at onset of patient P1, but since at the first visit (age 36 years), she had obvious signs that commonly appear in the early patient’s life—including oval pallor and temporal atrophy—we considered that the disease onset occurred during adolescence. variable, with P3 showing minimal signs of muscular impair- foci (cytoplasmic and/or nuclear) per cell (range 0–6). On ment on exploration, P2, P4, and P6 presenting with distal the other hand, there were no RNA foci in the control myo- weakness, and P1 and P5 with mild-to-moderate proximal blasts (supplemental figure 1, links.lww.com/NXG/A298). weakness. Most patients were independent in activities of daily living (modified Rankin Scale [mRS] score 0–2), yet one Heterogeneity was also found for the percentage of cells (P5) had severely limitations (mRS score 4). P5 did not to do carrying RNA foci. Thus, 4 patients had RNA foci in 100% of the 6-minute walking distance test or the myotonic evaluation their myoblasts, whereas P2 and P6 had RNA foci in 80% and because she died unexpectedly due to respiratory insufficiency 45% of myoblasts, respectively. Of interest, the cell lines from at age 40 years. P2 and P6 were those carrying the lowest CTG repeat number (195 and 230, respectively). We found a strong positive CTG expansion correlates with RNA foci correlation between CTG repeat number and (1) the average We determine the CTG expansion size and the RNA foci and number of total (cytoplasmic + nuclear) RNA foci per myo- MBNL1 staining analyses in the same cells by dividing the cell blast (figure 1A), as well as with (2) the average number of lines in different pools. The CTG expansion size in DM1 nuclear RNA foci per myoblast (r = 0.830; 95% confidence myoblast cell lines ranged from 195 to 1568 CTG repeats interval [CI] 0.056–0.981). (table 2). RNA foci in DM1 myoblasts are mostly, but not For the staining of RNA foci and MBNL1, we studied 71 and only, intranuclear 84 myoblasts from patients and controls, respectively. There RNA foci were mainly present in the nuclei of DM1 cells. was a high heterogeneity among DM1 myoblasts with regard Nevertheless, all 6 DM1 myoblast lines also showed RNA foci to number of RNA foci. Thus, there was an average of 3 RNA outside this organelle. The prevalence of cytoplasmic RNA Table 2 CTG expansion size and 3D analysis data in DM1 myoblasts RNA foci Derived No. of Minimum Maximum RNA area % of cells % RNA foci % of cells containing DM1 cell CTG cells no. of RNA no. of RNA foci/ average containing in the both nuclear and line repeats analyzed foci/cell foci/cell cell (μm2) RNA foci cytoplasm cytoplasmic RNA foci P1 758 11 1 6 2.7 1.6 100 23 36 P2 195 10 0 5 1.8 0.7 80 6 10 P3 899 16 2 6 3.5 0.5 100 14 38 P4 1,568 11 2 6 4.4 0.3 100 8 45 P5 292 12 1 6 3.2 0.9 100 11 25 P6 230 11 0 5 2.3 0.2 45 4 9 Abbreviation: DM1 = myotonic dystrophy type 1. Neurology.org/NG Neurology: Genetics | Volume 6, Number 4 | August 2020 3 Figure 1 Significant correlations of 3D analysis data, CTG expansion size, and phenotype in DM1 myoblasts (A) Correlation between CTG repeat expansion and RNA foci number. The 95% CI for the Pearson correlation coefficient was 0.222–0.986. (B) Correlation between CTG repeat expansion and percentage of cells presenting cytoplasmic RNA foci. The 95% CI for the Pearson correlation coefficient was 0.559–0.994. (C) Correlation between RNA foci number and % of cells presenting cytoplasmic RNA foci. The 95% CI for the Pearson correlation coefficient was 0.263–0.987. (D) Correlation between % of cells presenting RNA foci and % of cells presenting cytoplasmic RNA foci. The 95% CI for the Pearson correlation coefficient was 0.105–0.983. (E) Correlation between the RNA foci area (μm2) and the presence of cytoplasmic RNA foci. The 95% CI for the Pearson correlation coefficient was 0.085–0.982. (F) Correlation between age at onset of the disease and the presence of cytoplasmic RNA foci. The 95% CI for the Pearson correlation coefficient was −0.979 to −0.019. rep exp = repeat expansion; n = number; cyto = cytoplasm. foci was, however, heterogeneous—ranging from 4% to 23% RNA foci only represent a small fraction of the of all RNA foci found (mean = 12%) (table 2). The per- total nucleus volume centage of myoblasts showing cytoplasmic RNA foci also The portion of the total nuclear dimension occupied by RNA varied among the different DM1 patient lines, ranging from foci was analyzed by determining the volume of all the RNA 10% to 45% of cells. On the other hand, there were no cells foci inside the nucleus and by comparing this variable with the showing cytoplasmic-only RNA foci. total nuclear volume. RNA foci occupied 0.34%–0.53% of the total nuclear volume in DM1 myoblasts. We found a strong, positive correlation between the number of CTG repeats and the percentage of cells with cytoplasmic Trapped MBNL1 in RNA foci represent a RNA foci (figure 1B). We also found a positive correlation negligible fraction of total MBNL1 expression between the average total number (nuclear + cytoplasmic) of Given the main role that MBNL1 plays in DM1 disease, we RNA foci per myoblast and the percentage of cells presenting aimed to analyze the trapping of this protein in the RNA foci. RNA foci in the cytoplasm (figure 1C) and also between the MBNL1 protein was present in control and DM1 myoblasts. percentage of DM1 myoblasts presenting RNA foci (nuclear In all analyzed myoblasts, MBNL1 was located both in the and/or cytoplasmic) and the percentage of RNA foci in the nucleus and in the cytoplasm (figure 2, B and E). Colocali- cytoplasm (figure 1D). We further analyzed the area occupied zation of RNA foci together with MBNL1 was found in all by RNA foci (table 2) and found a positive correlation be- DM1 patients’ lines. The trapped MBNL1 was not only found tween this variable and the presence of cytoplasmic foci in the nucleus (figure 2, A–C), but also in the cytoplasm (figure 1E). Thus, those cell lines that contained RNA foci (figure 2, D–F), representing 9% of the total cellular coloc- with larger areas had more foci in the cytoplasm. alization (Video 1). Trapped MBNL1 represented less than 1% of the total MBNL1 expression in DM1 myoblasts. Higher number of cytoplasmic RNA foci is associated with an earlier disease onset RNA foci are associated with DMPK transcripts When analyzing the relationship between the 3D molecular As RNA foci are the product of DMPK gene transcription, we findings and the phenotype of patients with DM1, we found aimed to analyze DMPK transcripts in our myoblast cell lines. that the number of cytoplasmic RNA foci was inversely re- To this end, we chose qPCR as it is a more sensitive technique lated to the age at disease onset (figure 1F). than RT-PCR and used a probe that was located outside the 4 Neurology: Genetics | Volume 6, Number 4 | August 2020 Neurology.org/NG Figure 2 Confocal microscopy of DM1 myoblasts MBNL1 protein (green), RNA foci (red), nucleus (blue). Scale bars: 2.5 μm. (A) XY and XZ volume slices of individual channels corresponding to nucleus (blue), MBNL1 protein (green), and RNA foci (red). DM1 myoblast showing the nuclear colocalization between the RNA foci and the MBNL1 protein. (B) Merged image of XY and XZ volume slices of representative nuclear colocali- zation between RNA foci and MBNL1 protein in DM1 myoblast. White arrow indicates a single RNA foci aggregate colocalizing with MBNL1 protein. (C) Isosurface rendering of nuclear colocalization between RNA foci and MBNL1 protein in DM1 myoblasts. (D) XY and XZ volume slices of in- dividual channels corresponding to nucleus (blue), MBNL1 protein (green), and RNA foci (red). DM1 myoblast showing the cytoplasmic colocalization between the RNA foci and the MBNL1 protein. (E) Merged image of XY and XZ volume slices of rep- resentative cytoplasmic colocalization between RNA foci and MBNL1 protein in DM1 myoblast. White arrow indicates a single RNA foci aggregate colocalizing with MBNL1 protein. (F) Isosurface rendering of cytoplasmic colocalization between RNA foci and MBNL1 protein in DM1 myoblasts. MBNL1 = muscleblind like 1. CTG repeat for detecting expression of both wild-type and altered in DM1.4,8,9 No differences were found between expanded DMPK alleles. We did not find significant differ- controls and patients in the expression of MBNL1 or ATP2A1 ences between the DMPK expression of patients and controls isoforms. However, the expression levels of the INSR exon 11 myoblasts (figure 3A). In turn, we found a positive correlation exclusion isoform—the most expressed isoform in some between DMPK expression and (1) total (nuclear and/or DM1 models—showed a tendency of higher expression in cytoplasmic) RNA foci number (r = 0.967 [95% CI controls than in patients (figure 3B). 0.079–0.999], p = 0.033), as well as with the RNA foci that were present in the nucleus only (r = 0.993 [95% CI 0.682–0.999], p = 0.008). Discussion We studied the quantity and distribution of RNA foci and Absence of DM1 alternative splicing patterns in MBNL1 protein in DM1 patient–derived myoblasts using a DM1 myoblasts novel 3D imaging approach. In addition, in the same myoblast To study the extent to which was alternative splicing affected cultures, we measured CTG expansion, DMPK expression, in DM1 myoblasts, we analyzed the expression of inclusion and some alternative splicing events. All the molecular data and exclusion isoforms—MBNL1 exon 7, INSR exon 11, and were correlated with the clinical characteristics of the patients ATP2A1 exon 22—that have been previously reported to be with DM1. Thus, although more research is needed, the 3D Neurology.org/NG Neurology: Genetics | Volume 6, Number 4 | August 2020 5 Figure 3 qDMPK expression and splicing analysis Data are mean ± SD. (A) qDMPK expression in patients with DM1 vs controls. (B) Percentage of the DM1 spliced-related isoform in patients with DM1 compared with controls for MBNL1, IR, and ATP2A1 transcripts measured with RT-PCR. MBNL1 and ATP2A1 showed no significant differences, whereas IR showed a tendency of higher expression in controls compared with patients (p = 0.057; effect size [Cohen d] = 1.83). DMPK = dystrophia myotonica protein; IR = insulin receptor; MNBL1 = muscleblind like 1. method we used appears as a potential tool to identify po- DM1 cell lines that we studied, no myoblast presented tential associations between molecular alterations and clinical cytoplasmic-only RNA foci. CTG expansion size appears to manifestations in patients with DM1. regulate the formation of cytoplasmic RNA foci. Thus, larger CTG expansions will produce a higher number of nuclear foci, Our results indicate that the number of RNA foci per DM1 and therefore, more cells will contain cytoplasmic foci because myoblast is regulated by the CTG expansion length. A pre- the latter only appeared in association with nuclear foci. The vious study showed that RNA foci number differed between presence and origin of cytoplasmic foci have not yet been patients with DM1 carrying <500 or >1000 CTG repeats, studied in depth. Some authors have suggested that cyto- respectively.10 In this regard, we have analyzed patients car- plasmic RNA foci are a product of the cell cycle as well as of rying CTG expansions within the 500–1,000 range, with RNA the breakdown of the nuclear membrane and as such are more foci related to CTG repeat number. On the other hand, our likely to appear in dividing cells.13 However, a study in an data indicate that there are a small number of RNA foci per animal model of DM1 expressing CTG expansions in the DM1 myoblast, averaging only 3 per cell (range 0–6), which is heart tissue found RNA foci exclusively in the cytoplasm of consistent with the findings of previous research in muscle cardiomyocytes, which are highly differentiated, nondividing tissue.3,10–12 cells.15 On the other hand, our results show that cytoplasmic RNA foci colocalize with MBNL1. The role of cytoplasmic Of interest, not all the DM1 myoblasts derived from the same foci and whether they have a toxic effect should be studied in patient had RNA foci. Two lines of patient-derived myoblasts future research. who carried the shorter expansions showed RNA foci only in 40% and 80% of the cells. Thus, our results suggest that CTG RNA foci represent less than 0.6% of the total nuclear volume expansion not only regulates the amount of RNA foci per in DM1 myoblasts. Although researchers in the DM1 area are myoblast but also the number of myoblasts presenting RNA foci. aware of the small size of RNA foci and of the difficulty to In this regard, previous research has suggested that RNA foci can identify them under the microscope by naked eye, the data undergo dynamic changes during the cell cycle13 and travel from presented here add some information on the impact that RNA one cell to another.14 Thus, although we cannot discard a certain foci have on the nuclear volume. In addition, when analyzing effect of the cell cycle phase on our results, we tried to minimize the area of RNA foci in relation to other molecular findings, this potential confounder by seeding the different DM1 myo- we observed a correlation with the number of cytoplasmic blast cell lines following a well-defined protocol and using foci. The fact that scarce data are currently available on cy- consistently the same treatment method. Based on the expan- toplasmic RNA foci makes it difficult to interpret our results. sion sizes that we studied, DM1 myoblasts carrying expansions However, as previously described by other authors,16 RNA close to—or larger than—300 CTG repeats would carry RNA foci can fuse or divide randomly with no apparent or known foci in all their cells. To study whether 300 CTG repeats is an purpose. actual threshold for all patients with DM1 will require a chal- lenging 3D study in muscle tissue of patients with DM1. Our analysis of MBNL1 revealed that the sequestration of this protein inside RNA foci represents less than 1% of the total We found both nuclear and cytoplasmic RNA foci. Although MBNL1 myoblast expression. Although there are studies we found myoblasts with cytoplasmic RNA foci in all the showing the pathologic role of MBNL1 in DM1,17–21 our 6 Neurology: Genetics | Volume 6, Number 4 | August 2020 Neurology.org/NG results are consistent with a previous study by Coleman Our 3D imaging study allowed us to analyze the role of et al.22 reporting that only a small proportion of MBNL1 potential DM1 players using a novel approach. Thus, our protein (0.2%) was sequestered by RNA foci in DM1 lens results would indicate that CTG expansion determines the epithelial cells. The study by Coleman and coworkers also number of RNA foci per myoblast and of myoblasts that showed that MBNL1 can freely travel from the nucleus to the contain RNA foci, as well as the appearance of cytoplasmic cytoplasm or from the cytoplasm to the nucleus depending on foci. An additional finding is that RNA foci only represent a the transcriptional levels and cell requirements. In this con- small—and in fact negligible—part of the total nucleus text, the low values of sequestration of MBNL1 would suggest volume and the sequestration of MBNL1 is an infrequent that this phenomenon plays only a minor role in the patho- event. Thus, no DM1 splicing alterations were found. The physiology of the disease, as 99% of total MBNL1 expression DMPK expression levels are related to the number of RNA is free and DM1 myoblasts showed a distribution pattern foci found per myoblast. Last, we found that cytoplasmic similar to that of control myoblasts. RNA foci are inversely related to the age at onset of the patients with DM1 studied. Cytoplasmic RNA foci should be The levels of DMPK expression were related to the number of considered in future studies because the role of these mol- RNA foci per cell. This was an expected finding: indeed, if ecules in the pathobiology of DM1 needs to be clearly there are more RNA foci inside a cell, theoretically there elucidated. should also be more DMPK, as for both RNA foci and DMPK determination, we used a technique, qPCR, which allows Acknowledgment detecting expanded as well as wild-type alleles. How DMPK The authors gratefully acknowledge the participants in this expression is affected in DM1 remains to be determined. research and other researchers in the myotonic dystrophy DMPK expression levels have been analyzed in several studies type I field for their insightful advice regarding data presented with inconclusive or contradictory results. Some studies in the present research work. found that DMPK transcripts were increased in myotubes derived from patients with DM1,23 but other authors found Study funding the opposite result.10,24 In this regard, heterogeneity among The research of G. Nogales-Gadea, A. Ramos-Fransi, and A. studies with regard to assessment techniques and patient Lucia is funded by Instituto de Salud Carlos III (grant num- characteristics limits the ability to gain insight into the alter- bers PI15/01756, PI15/00558, and PI18/00713) and cofi- ation of DMPK expression in DM1. nanced by Fondos FEDER. G. Nogales-Gadea is supported by a Miguel Servet research contract (ISCIII CD14/00032, We did not find the expected altered DM1 splicing when ISCIII CPII19/00021, and FEDER) and by a Trampoline analyzing IR, ATP2A1, and MBNL1 transcripts. As men- Grant #21108 from AFM Telethon. A. Ballester-Lopez is tioned above, the trapped MBNL1 in RNA foci was a minor funded by an FI Agaur fellowship ref. FI_B 01090 and SGR event in DM1 myoblasts, and thus, the cascade of molecular 1520 (GRC) Generalitat de Catalunya. E. Koehorst is funded pathomechanisms in which the splicing alteration occurs due by the La Caixa Foundation (ID 100010434), fellowship code to MBNL1 protein sequestration does not seem to play a LCF/BQ/IN18/11660019, cofunded by the European relevant role. These results could also be explained by the fact Union’s Horizon 2020 research and innovation program un- that the expression of the aforementioned aberrant isoforms is der the Marie Skłodowska-Curie grant agreement no. 713673. related to developmental stages,4,9 and because we used un- I. Linares-Pardo is funded by CP14/00032 and SGR 1520 differentiated cells (i.e., myoblasts), the expression pattern (GRC) Generalitat de Catalunya. J. Núñez-Manchón was should differ from the pattern of fully differentiated myotubes, funded by AFM Telethon Trampoline Grant #21108. G. and especially of skeletal muscle fibers. Lucente was supported by a Rio Hortega contract (ISCIII CM16/00016 and FEDER). J. Chojnacki is supported by Our results indicate that an earlier age at disease onset is asso- European Union’s Horizon 2020 research and innovation ciated with a higher number of cytoplasmic RNA foci. By con- program under the Marie Skłodowska-Curie grant agreement trast, the age at onset was not correlated with the number of no. 793830. The funding bodies had no role in the design of CTG repeats or RNA foci per myoblast or to DMPK expression the study and collection, analysis, and interpretation of data. levels. Several studies25–28 have analyzed the possible association between CTG expansion size and the different DM1-related Disclosure symptoms, but CTG instability adds heterogeneity to the dis- A. Ballester-Lopez is funded by an FI Agaur fellowship FI_B ease, which further complicates to establish these associations. 01090 and SGR 1520 (GRC) Generalitat de Catalunya. J. Núñez-Manchón is funded by AFM Telethon Trampoline Our study is limited by the low number of myoblast cell lines Grant #21108. E Koehorst is funded by the La Caixa Foun- studied and by the complexity of 3D molecular imaging at the dation (ID 100010434), fellowship code LCF/BQ/IN18/ single cell level, which is a highly time-consuming approach. 11660019, cofunded by the European Union’s Horizon While keeping this limitation in mind, we were able to eval- 2020 research and innovation program under the Marie uate the molecular pathologic cascade in 6 patients with DM1 Skłodowska-Curie grant agreement no. 713673. I. Linares- with different clinical manifestations of this disease. Pardo is funded by CP14/00032 and SGR 1520 (GRC) Neurology.org/NG Neurology: Genetics | Volume 6, Number 4 | August 2020 7 Generalitat de Catalunya. M. Almendrote reports no disclo- sures relevant to the manuscript. G. Lucente is supported by a Appendix (continued) Rio Hortega contract (ISCIII CM16/00016 and FEDER). N. Name Location Contribution Guanyabens, M. Lopez-Osias, A. Suárez-Mesa, S. Hanick, and J. Chojnack report no disclosures relevant to the manuscript. Shaliza Ann Institut d’Investigació en Data acquisition and Hanick, MSc Ciències de la Salut revision of the manuscript A. Lucia declares grants from the Spanish government Germans Trias i Pujol, granting agency Instituto de Salud Carlos III, Madrid, Spain. Badalona, Spain G. Pintos-Morell reports personal honoraria from Shire- Jakub IrsiCaixa AIDS Research Image analysis Takeda, Amicus, and Sanofi-Genzyme, outside the submitted Chojnacki, Institute, Badalona, Spain PhD work. J. Coll-Cantı́, A. Martı́nez-Piñeiro, and A. Ramos-Fransi report no disclosures relevant to the manuscript. G. Nogales- Alejandro Universidad Europea de Analysis of the data and Lucia, MD, Madrid and Instituto de revision of the manuscript Gadea declares grants from Instituto de Salud Carlos III, PhD Investigación Hospital 12 Madrid, Spain, and AFM Telethon, France. G. Nogales-Gadea de Octubre (i+12), Madrid, Spain is supported by a Miguel Servet research contract (ISCIII CD14/00032, CPII19/00021, and FEDER). Go to Neurol- Guillem Hospital Universitari Vall Revision of the ogy.org/NG for full disclosures. Pintos- d’Hebron, Barcelona, Spain manuscript Morell, MD, PhD Publication history Jaume Coll- Hospital Universitari Revision of the Received by Neurology: Genetics February 21, 2020. Accepted in final Cantı́, MD, Germans Trias i Pujol, manuscript form June 5, 2020. PhD Badalona, Barcelona, Spain Alicia Hospital Universitari Revision of the Martı́nez- Germans Trias i Pujol, manuscript Piñeiro, MD Badalona, Barcelona, Appendix Authors Spain Name Location Contribution Alba Ramos- Hospital Universitari Clinical data collection Fransi, MD, Germans Trias i Pujol, and drafting of the Alfonsina Institut d’Investigació en Data collection; PhD Badalona, Barcelona, manuscript Ballester- Ciències de la Salut acquisition and analysis; Spain Lopez, MSc Germans Trias i Pujol, and drafting of the Badalona, Spain manuscript Gisela Institut d’Investigació en Design of the study and Nogales- Ciències de la Salut drafting of the manuscript Judit Núñez- Institut d’Investigació en Data acquisition and Gadea, PhD Germans Trias i Pujol, Manchón, Ciències de la Salut drafting of the manuscript Badalona, Spain MSc Germans Trias i Pujol, Badalona, Spain Emma Institut d’Investigació en Revision of the References Koehorst, Ciències de la Salut manuscript 1. 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