Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1.

Myotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we decided to study the presence of DM1 antisense (DM1-AS) transcripts (the origin of the polyglutamine (polyGln) RAN protein) using RT-PCR and FISH, and that of RAN translation via immunoblotting and immunofluorescence in distinct DM1 primary cell cultures, e.g., myoblasts, skin fibroblasts and lymphoblastoids, from ten patients. DM1-AS transcripts were found in all DM1 cells, with a lower expression in patients compared to controls. Antisense RNA foci were found in the nuclei and cytoplasm of a subset of DM1 cells. The polyGln RAN protein was undetectable in all three cell types with both approaches. Immunoblots revealed a 42 kD polyGln containing protein, which was most likely the TATA-box-binding protein. Immunofluorescence revealed a cytoplasmic aggregate, which co-localized with the Golgi apparatus. Taken together, DM1-AS transcript levels were lower in patients compared to controls and a small portion of the transcripts included the expanded repeat. However, RAN translation was not present in patient-derived DM1 cells, or was in undetectable quantities for the available methods.

A New Methodology for Intraoperative Monitoring of the Functional Integrity of the Phrenic Nerve During Cardiothoracic Surgery.

INTRODUCTION: The phrenic nerve could be easily injured during cardiothoracic surgeries because of its anatomical relationships. The aim of this study is to describe a new, feasible, and reproducible methodology to achieve a continuous intraoperative neuromonitoring of the phrenic nerve. METHODS: Consecutive patients who underwent open-chest surgery were included. The recording active electrode was placed 5 cm superior to the tip of the xiphoid process, and a hook wire inserted at the motor point of the ipsilateral hemidiaphragm was used as the reference electrode. RESULTS: We studied 45 patients (92% men, mean age 67 years). Mean height and weight were 167 ± 6.9 cm and 75.6 ± 12.3 kg, respectively. A reproducible compound motor action potential was recorded in 38 (85%) subjects. The mean latency and amplitude values were 9.68 ± 2.40 ms and 1.36 ± 3.83 mV, respectively. No intraoperative events were recorded. CONCLUSIONS: We reported a new methodology which allows the assessment of phrenic nerve functional integrity during surgical procedures.

Preliminary Findings on CTG Expansion Determination in Different Tissues from Patients with Myotonic Dystrophy Type 1.

Myotonic Dystrophy type 1 (DM1) is characterized by a high genetic and clinical variability. Determination of the genetic variability in DM1 might help to determine whether there is an association between CTG (Cytosine-Thymine-Guanine) expansion and the clinical manifestations of this condition. We studied the variability of the CTG expansion (progenitor, mode, and longest allele, respectively, and genetic instability) in three tissues (blood, muscle, and tissue) from eight patients with DM1. We also studied the association of genetic data with the patients' clinical characteristics. Although genetic instability was confirmed in all the tissues that we studied, our results suggest that CTG expansion is larger in muscle and skin cells compared with peripheral blood leukocytes. While keeping in mind that more research is needed in larger cohorts, we have provided preliminary evidence suggesting that the estimated progenitor CTG size in muscle could be potentially used as an indicator of age of disease onset and muscle function impairment.

Three-dimensional imaging in myotonic dystrophy type 1: Linking molecular alterations with disease phenotype.

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) expression 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.

The Need for Establishing a Universal CTG Sizing Method in Myotonic Dystrophy Type 1.

The number of cytosine-thymine-guanine (CTG) repeats ('CTG expansion size') in the 3'untranslated region (UTR) region of the dystrophia myotonica-protein kinase (DMPK) gene is a hallmark of myotonic dystrophy type 1 (DM1), which has been related to age of disease onset and clinical severity. However, accurate determination of CTG expansion size is challenging due to its characteristic instability. We compared five different approaches (heat pulse extension polymerase chain reaction [PCR], long PCR-Southern blot [with three different primers sets-1, 2 and 3] and small pool [SP]-PCR) to estimate CTG expansion size in the progenitor allele as well as the most abundant CTG expansion size, in 15 patients with DM1. Our results indicated variability between the methods (although we found no overall differences between long PCR 1 and 2 and SP-PCR, respectively). While keeping in mind the limited sample size of our patient cohort, SP-PCR appeared as the most suitable technique, with an inverse significant correlation found between CTG expansion size of the progenitor allele, as determined by this method, and age of disease onset (r = -0.734, p = 0.016). Yet, in light of the variability of the results obtained with the different methods, we propose that an international agreement is needed to determine which is the most suitable method for assessing CTG expansion size in DM1.

A DM1 family with interruptions associated with atypical symptoms and late onset but not with a milder phenotype.

Carriage of interruptions in CTG repeats of the myotonic dystrophy protein kinase gene has been associated with a broad spectrum of myotonic dystrophy type 1 (DM1) phenotypes, mostly mild. However, the data available on interrupted DM1 patients and their phenotype are scarce. We studied 49 Spanish DM1 patients, whose clinical phenotype was evaluated in depth. Blood DNA was obtained and analyzed through triplet-primed polymerase chain reaction (PCR), long PCR-Southern blot, small pool PCR, AciI digestion, and sequencing. Five patients of our registry (10%), belonging to the same family, carried CCG interruptions at the 3'-end of the CTG expansion. Some of them presented atypical traits such as very late onset of symptoms ( > 50 years) and a severe axial and proximal weakness requiring walking assistance. They also showed classic DM1 symptoms including cardiac and respiratory dysfunction, which were severe in some of them. Sizes and interrupted allele patterns were determined, and we found a contraction and an expansion in two intergenerational transmissions. Our study contributes to the observation that DM1 patients carrying interruptions present with atypical clinical features that can make DM1 diagnosis difficult, with a later than expected age of onset and a previously unreported aging-related severe disease manifestation.

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.

Missense mutations have unexpected consequences: The McArdle disease paradigm.

McArdle disease is a disorder of muscle glycogen metabolism caused by mutations in the PYGM gene, encoding for the muscle-specific isoform of glycogen phosphorylase (M-GP). The activity of this enzyme is completely lost in patients' muscle biopsies, when measured with a standard biochemical test which, does not allow to determine M-GP protein levels. We aimed to determine M-GP protein levels in the muscle of McArdle patients, by studying biopsies of 40 patients harboring a broad spectrum of PYGM mutations and 22 controls. Lack of M-GP protein was found in muscle in the vast majority (95%) of patients, irrespective of the PYGM genotype, including those carrying missense mutations, with few exceptions. M-GP protein biosynthesis is not being produced by PYGM mutations inducing premature termination codons (PTC), neither by most PYGM missense mutations. These findings explain the lack of PYGM genotype-phenotype correlation and have important implications for the design of molecular-based therapeutic approaches.

Correction to: Manifesting heterozygotes in McArdle disease: a myth or a reality-role of statins.

Unfortunately the name of one of the authors was spelled incorrectly in the published original article. The correct name is Alejandro Santos-Lozano. The original article got updated.

Manifesting heterozygotes in McArdle disease: a myth or a reality-role of statins.

McArdle disease is an autosomal recessive condition caused by deficiency of the PYGM gene-encoded muscle isoform of glycogen phosphorylase. Some cases of "manifesting" heterozygotes or carriers (i.e., patients who show some McArdle-like symptoms or signs despite being carriers of only one mutated PYGM allele) have been reported in the literature but there is controversy, with misdiagnosis being a possibility. The purpose of our study was to determine if there are actually "manifesting" heterozygotes of McArdle disease and, if existing, whether statin treatment can trigger such condition. Eighty-one relatives of McArdle patients (among a total of 16 different families) were studied. We determined whether they were carriers of PYGM mutations and also collected information on exercise tests (second wind and modified Wingate anaerobic test) and statin intake. We found 50 carriers and 31 non-carriers of PYGM mutations. Although we found existence of heterozygotes manifesting some exercise-related muscle problems such as exacerbated myalgia or weakness, they only accounted for 14% of the carriers and muscle symptoms were milder than those commonly reported in patients. Further, no carrier (whether reporting symptoms or not) showed the second wind phenomenon or a flat blood lactate response to maximal-intensity exercise, both of which are hallmarks of McArdle disease. On the other hand, statin myotoxicity was not associated with muscle symptom onset.

Genotypic and phenotypic features of all Spanish patients with McArdle disease: a 2016 update.

BACKGROUND: We recently described the genotype/phenotype features of all Spanish patients diagnosed with McArdle disease as of January 2011 (n = 239, prevalence of ~1/167,000) (J Neurol Neurosurg Psychiatry 2012;83:322-8). Several caveats were however identified suggesting that the prevalence of the disease is actually higher. METHODS: We have now updated main genotype/phenotype data, as well as potential associations within/between them, of all Spanish individuals currently diagnosed with McArdle disease (December 2016). RESULTS: Ninety-four new patients (all Caucasian) have been diagnosed, yielding a prevalence of ~1/139,543 individuals. Around 55% of the mutated alleles have the commonest PYGM pathogenic mutation p.R50X, whereas p.W798R and p.G205S account for 10 and 9% of the allelic variants, respectively. Seven new mutations were identified: p.H35R, p.R70C, p.R94Q, p.L132WfsX163, p.Q176P, p.R576Q, and c.244-3_244-2CA. Almost all patients show exercise intolerance, the second wind phenomenon and high serum creatine kinase activity. There is, however, heterogeneity in clinical severity, with 8% of patients being asymptomatic during normal daily life, and 21% showing limitations during daily activities and fixed muscle weakness. A major remaining challenge is one of diagnosis, which is often delayed until the third decade of life in 72% of new patients despite the vast majority (86%) reporting symptoms before 20 years. An important development is the growing proportion of those reporting a 4-year improvement in disease severity (now 34%) and following an active lifestyle (50%). Physically active patients are more likely to report an improvement after a 4-year period in the clinical course of the disease than their inactive peers (odds ratio: 13.98; 95% confidence interval: 5.6, 34.9; p < 0.001). Peak oxygen uptake is also higher in the former (20.7 ± 6.0 vs. 16.8 ± 5.3 mL/kg/min, p = 0.0013). Finally, there is no association between PYGM genotype and phenotype manifestation of the disease. CONCLUSIONS: The reported prevalence of McArdle disease grows exponentially despite frequent, long delays in genetic diagnosis, suggesting that many patients remain undiagnosed. Until a genetic cure is available (which is not predicted in the near future), current epidemiologic data support that adoption of an active lifestyle is the best medicine for these patients.

Taking advantage of an old concept, "illegitimate transcription", for a proposed novel method of genetic diagnosis of McArdle disease.

PURPOSE: McArdle disease is a metabolic disorder caused by pathogenic mutations in the PYGM gene. Timely diagnosis can sometimes be difficult with direct genomic analysis, which requires additional studies of cDNA from muscle transcripts. Although the "nonsense-mediated mRNA decay" (NMD) eliminates tissue-specific aberrant transcripts, there is some residual transcription of tissue-specific genes in virtually all cells, such as peripheral blood mononuclear cells (PBMCs). METHODS: We studied a subset of the main types of PYGM mutations (deletions, missense, nonsense, silent, or splicing mutations) in cDNA from easily accessible cells (PBMCs) in 12 McArdle patients. RESULTS: Analysis of cDNA from PBMCs allowed detection of all mutations. Importantly, the effects of mutations with unknown pathogenicity (silent and splicing mutations) were characterized in PBMCs. Because the NMD mechanism does not seem to operate in nonspecific cells, PBMCs were more suitable than muscle biopsies for detecting the pathogenicity of some PYGM mutations, notably the silent mutation c.645G>A (p.K215=), whose effect in the splicing of intron 6 was unnoticed in previous muscle transcriptomic studies. CONCLUSION: We propose considering the use of PBMCs for detecting mutations that are thought to cause McArdle disease, particularly for studying their actual pathogenicity.Genet Med 18 11, 1128-1135.

Exercise and Preexercise Nutrition as Treatment for McArdle Disease.

McArdle disease is due to an inborn defect in the muscle isoform of glycogen phosphorylase (or "myophosphorylase"), the enzyme that catalyzes the first step of glycogenolysis. This condition is still not fully understood, and although advances in research would help patients immeasurably, these would also enhance our understanding of exercise metabolism. It has been 10 yr since the first published report demonstrating the benefits of regular aerobic exercise for these patients. However, misconceptions remain and the value of exercise prescription for patients with McArdle disease is still overlooked. Here, we review the role of exercise in McArdle disease with the aim to better inform health-care professionals and thus better serve the interests of patients. Recommendations for regular exercise together with preexercise nutrition in children and adult patients are also provided along with examples of exercise practice and its benefits.

Genes and exercise intolerance: insights from McArdle disease.

McArdle disease (glycogen storage disease type V) is caused by inherited deficiency of a key enzyme in muscle metabolism, the skeletal muscle-specific isoform of glycogen phosphorylase, "myophosphorylase," which is encoded by the PYGM gene. Here we review the main pathophysiological, genotypic, and phenotypic features of McArdle disease and their interactions. To date, moderate-intensity exercise (together with pre-exercise carbohydrate ingestion) is the only treatment option that has proven useful for these patients. Furthermore, regular physical activity attenuates the clinical severity of McArdle disease. This is quite remarkable for a monogenic disorder that consistently leads to the same metabolic defect at the muscle tissue level, that is, complete inability to use muscle glycogen stores. Further knowledge of this disorder would help patients and enhance understanding of exercise metabolism as well as exercise genomics. Indeed, McArdle disease is a paradigm of human exercise intolerance and PYGM genotyping should be included in the genetic analyses that might be applied in the coming personalized exercise medicine as well as in future research on genetics and exercise-related phenotypes.

Implementation of Intraoperative Neurophysiological Monitoring during Endovascular Procedures in the Central Nervous System.

BACKGROUND AND OBJECTIVE: Intraoperative monitoring (IOM) has been used in different surgical disciplines since the 1980s. Nonetheless, regular routine use of IOM in interventional neuroradiology units has only been reported in a few centers. The aim of this study is to report our experience, 1 year after deciding to implement standardized IOM during endovascular treatment of vascular abnormalities of the central nervous system. METHODS: Basic recordings included somatosensory-evoked potentials (SEPs) and motor-evoked potentials (MEPs). Corticobulbar motor-evoked potentials and flash-visual-evoked potentials were also recorded depending on the topography of the lesion. Intra-arterial provocative tests (PTs) with amobarbital and lidocaine were also performed. All patients except 1 were under total intravenous anesthesia. Clinical outcome was assessed prospectively and correlated with IOM events. RESULTS: Twelve patients and 15 procedures were monitored during the inclusion period. Significant IOM events were detected during 3 of the 15 procedures (20%). We observed temporary MEP changes in 2 cases which resolved after interruption of the embolization or application of corrective measures, leaving no postoperative neurological deficits. In 1 case, persistent SEP and MEP deterioration was detected secondary to a frontal hematoma, resulting in mild sensory-motor deficit in the right upper extremity after the procedure. Overall, 12 PTs (4 spinal cord and 8 brain abnormalities) were performed using lidocaine and sodium amytal injections. One positive result occurred after the injection of lidocaine. No false negatives were detected. CONCLUSIONS: IOM may provide continuous real-time data about the functional status of eloquent areas and pathways of the central nervous system in patients under general anesthesia. It therefore allows us to detect early neurological damage in time to perform specific actions that may prevent irreversible neurological deficits.

Paraplejía por mielitis tuberculosa / Tuberculous myelitis with paraplegia

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