1例肝豆状核变性合并杜兴型肌营养不良症及其家系报道

YAN Jian-guo;CHEN Da-wei;DONG Yi;XU Zhi-qiang;WANG Li-min;GAN Yu;WANG Fu-chuan;ZHANG Min(Pediatric Liver Disease Treatment and Research Center,the Fifth Medical Center of Chinese PLA General Hospital,Beijing 100039,China)     

Unusual respiratory manifestations in two young adults with Duchenne muscular dystrophy

Adult respirologists are often involved in the evaluation and treatment of young adult patients with Duchenne muscular dystrophy. In this context, the most frequent respiratory complication is nocturnal and daytime hypoventilation related to respiratory muscle weakness. The present article describes cases of Duchenne muscular dystrophy involving two brothers, 17 and 19 years of age, respectively, who presented with less frequently reported respiratory complications of their disease: obstructive sleep apnea and Cheyne-Stokes respiration with central apnea, which were believed to be partially or completely related to congestive cardiomyopathy.     

Spirometry is affected by intelligence and behavior in Duchenne muscular dystrophy

Children with Duchenne muscular dystrophy (DMD) have progressive respiratory muscle weakness. Spirometry monitors progress, but is effort-dependent. Intelligence quotients (IQ) average one standard deviation below normal, and behavioral disturbance is common. Our aim was to assess if impaired intelligence or behavior influences spirometry in children with DMD, and if computerized visual incentives (CVI) are beneficial. Forty-seven boys with DMD, of mean age 12.6 years (range, 6-19), were recruited. Full-scale, performance, and verbal IQ, and parent-and-teacher-reported oppositional behavior scores, were recorded. Each was divided into moderate, mild, and no impairment groups. A randomized crossover design was applied to performing spirometry with or without CVI first. A Jaeger Masterscope (version 4.60) was used. Linear regression defined the relationship between spirometry and both IQ and behavior scores. The paired Student's t- test compared spirometry performed with and without CVI for the overall group and subgroups. Boys with DMD can adequately perform spirometry. There is an association between %FEV1 and %FVC with full-scale (r = 0.50, P = 0.002; r = 0.49, P = 0.003, respectively), performance (r = 0.68, P < 0.0005; r = 0.68, P < 0.0005, respectively) and verbal (r = 0.39, P = 0.043; r = 0.36, P = 0.037, respectively) IQ, but not with parent (P = 0.77, P = 0.70, respectively) or teacher (P = 0.90, P = 0.90, respectively)-reported oppositional behavior scores. The effect of CVI was significant in those with moderate full-scale (P = 0.03), performance (P = 0.002), and verbal (P = 0.02) intellectual impairment, and moderately severe teacher-reported oppositional behavior (P = 0.02). In conclusion, spirometry results are related to intelligence in DMD. Using CVI improves the FVC obtained in those with moderate intellectual or behavioral impairment.     

中国一进行性肌营养不良家系相关致病基因Dystrophin突变检测结果分析

目的 探讨进行性肌营养不良（DMD）家系相关致病基因Dystrophin基因突变情况.方法 收集一个DMD家系的临床资料,采用聚合酶链反应及直接测序法对此家系成员进行Dystrophin基因突变检测,同时对140例家系外健康对照者的该基因位点进行限制性核酸内切酶分析（PELP）.结果 在DMD家系中先证者（DMD患者1例）发现了一个纯合变异基因,即Dystrophin基因59号外显子上发现一个错义变异（G9017A）,导致代表精氨酸的2937位密码子转变为为谷氨酰胺（R2937Q）.在健康对照者中未发现此位点的变异.结论 在一个中国DMD家系先证者的Dystrophin基因上发现了一个尚未报道的基因突变位点.     

Ventilatory parameters and maximal respiratory pressure changes with age in Duchenne muscular dystrophy patients

The aim of this longitudinal study was to precise, in children with Duchenne muscular dystrophy, the respective functional interest of ventilatory parameters (Vital capacity, total lung capacity and forced expiratory volume in one second [FEV₁]) in comparison to maximal inspiratory pressure (Pimax) during growth. In ten boys the mean age of 9.1 ± 1 years) to mean age of 16 ± 1.4 years followed over a period of 7 years, we found that: (1) ventilatory parameters expressed in percentage of predicted value, after a normal ascending phase, start to decrease between 11 and 12 years, (2) Pimax presented only a decreasing phase since the beginning of the study and thus was already at 67% of predicted value at 12 years while ventilatory parameters was still normal, (3) after 12 years the mean slopes of decrease per year of vital capacity and FEV1 were higher (10.7 and 10.4%) than that of Pimax (6.9%), (4) at 15 years mean values of vital capacity and FEV1 (53.3 and 49.5% of predicted values) was simlar to that of Pimax (48.3%). In conclusion, if at early stages of the disease, Pimax is a more reliable index of respiratory impaiment than ventilatory parameters, the follow‐up of ventilatory parameters, when they start to decrease, is a better indicator of disease progression and, at advanced stages they provided same information about the functional impact of disease. Pediatr Pulmonol. 2010; 45:552–559. © 2010 Wiley‐Liss, Inc.     

Relationship between peak cough flow and spirometry in Duchenne muscular dystrophy

Spirometry is used to monitor respiratory progress in children with Duchenne muscular dystrophy (DMD). Mucociliary clearance depends on cough strength, which can be measured by peak cough flow (PCF). It is not routinely measured in most centers. When the PCF falls below 270 l/min, mucociliary clearance is likely to be impaired during viral illnesses, and techniques to assist mucociliary clearance should be taught. There is no known association between spirometry and PCF. Our aim was to assess if PCF relates to spirometry measures, and if spirometry can be used to predict when the PCF <270 l/min. Children with DMD aged 6-19 years were recruited. Spirometry was performed with a Jaeger Masterscope with version 4.60 software. PCF was performed with a Wright peak flow meter. Data were collected into an Access '97 database, and statistics were performed with Stata 7.0. The association between PCF and spirometry was defined with linear regression. Logistic regression was used to predict the probability that the PCF would be <270 l/min for any given forced vital capacity (FVC) or forced expired volume in 1 sec (FEV1). The risk ratios for PCF <270 l/min were calculated for the spirometry parameters. PCF is associated with FVC (R2, 0.72) and FEV1 (R2, 0.69). The likelihood of PCF <270 l/min rises when FVC <2.l and FEV1 <2.l/sec. The risk ratio for PCF <270 l/min when FVC <2.1 l is 4.80 (1.72-13.40) and when FEV1 <2.1 l/sec is 3.94 (1.43-10.85). In children with DMD, PCF should be measured when FVC <2.1 l or FEV1 <2.1 l/sec, so that techniques to assist with mucociliary clearance can be effectively used.     

Understanding adherence to noninvasive ventilation in youth with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X‐linked, progressive neuromuscular disorder that results in chronic respiratory insufficiency and subsequently failure requiring noninvasive ventilation (NIV). Adherence to NIV in neuromuscular disorders and related barriers are poorly described. The aim of the current study was to assess NIV adherence, adherence barriers, and identify psychosocial predictors of adherence in young boys with early DMD‐related sleep disordered breathing and recommended nocturnal NIV. This cross‐sectional study included 42 youth with DMD with prescribed nocturnal NIV, and their caregivers. Caregivers and youth completed questionnaires assessing adherence barriers, psychosocial symptoms (eg, anxiety and depressive symptoms), and stress. Medical information pertinent to cardiopulmonary health and neurologic status at both enrollment and initiation of NIV was reviewed. Adherence to NIV, defined as percent days used and days used ≥4 hours/day was 56.1 ± 38.7% and 46.2 ± 40.6%, respectively. Average duration of use on days worn was 5.61 ± 4.23 hours. NIV usage was correlated with the severity of obstructive sleep apnea but not cardiopulmonary variables. Mask discomfort was the most commonly reported adherence barrier followed by behavioral barriers (eg, refusing to use). Multiple regression analyses revealed that internalizing behaviors (eg, anxiety and depressive symptoms) and total adherence barriers significantly predicted NIV adherence. Adherence to NIV in DMD is poor and similar to other pediatric chronic diseases. Our data suggest interventions targeting adherence barriers and patient internalizing symptoms may improve adherence to NIV in DMD.     

Prednisolone rescues Duchenne muscular dystrophy phenotypes in human pluripotent stem cell–derived skeletal muscle in vitro

Duchenne muscular dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced pluripotent stem cells (iPSC) to a late myogenic stage. This allows us to recapitulate classical DMD phenotypes (mislocalization of proteins of the dystrophin-associated glycoprotein complex, increased fusion, myofiber branching, force contraction defects, and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion, and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are caused by antiinflammatory properties. Our work introduces a human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.

Duchenne muscular dystrophy (DMD) is an X-linked muscular dystrophy (affecting 1 in 5,000 boys) caused by mutations in the dystrophin gene (DMD) (1). There is currently no cure for the disease and the only available treatment is glucocorticoids, which can prolong the ambulatory phase (2). The dystrophin protein plays a key role in organizing a molecular complex (dystrophin-associated glycoprotein complex [DGC]) spanning the sarcolemma at the level of costameres and linking the actin cytoskeleton to laminin and extracellular matrix. In DMD patients fibers are more sensitive to mechanical stress and experience formation of membrane tears upon muscle contraction (3). DMD mutant myofibers exhibit abnormal calcium homeostasis, displaying higher resting calcium levels (4). The DGC also acts as an important scaffold necessary for the function of several signaling proteins such as nitric oxide synthase (nNOS) (5). In the early stages of the disease, the degeneration of muscle fibers stimulates regeneration of new fibers from satellite cells, a physiological response that counterbalances fiber loss and maintains a normal muscle function. This increased generation of fibers is accompanied by structural defects such as branching of the newly generated fibers, possibly resulting from fusion defects of the regenerating cells (6, 7). As the disease progresses, satellite cell regeneration capacity decreases, leading to tissue fibrosis. This myofiber degeneration and fibrosis are considered to be largely responsible for the decrease in muscle strength observed in patients. There is also evidence suggesting intrinsic contractile dysfunction in zebrafish, mice, and dogs lacking dystrophin (8–10). Due to difficulties in accessing patient muscle fibers, evidence for such contractile defects and their cause and significance in the progression of the human diseased phenotypes has remained very limited (11).Much of the research on the etiology of DMD as well as preclinical tests for the validation of DMD therapeutic strategies have been carried out in the mdx mouse, a spontaneous dystrophin mutant (12). In the mdx mouse myofibers, defects such as branching and misalignment are detected as early as embryonic day 13.5 at the beginning of the fetal period (13). A significant limitation of the mdx model is that the dystrophy is much less severe and only partly phenocopies the human disease (12). There is therefore a critical need for a better preclinical model in which the disease can be recapitulated with human myofibers. The recent development of protocols to differentiate human pluripotent stem cells such as induced pluripotent stem cells (iPSCs) to skeletal myofibers in vitro (14) now offers the possibility to generate DMD models better reflecting the physiology of human cells. Several studies describing the establishment of DMD patient iPSC lines and their differentiation to skeletal muscles have been reported (15). However, only a limited set of relevant phenotypes have been analyzed and the impact on skeletal muscle contractility has not been investigated.Here we describe an optimized myogenic differentiation protocol resulting in significantly improved myofiber maturation from human pluripotent cells in vitro, as shown by the expression of all fast myosin isoforms. Using this optimized protocol, we show that muscle fibers derived from two human isogenic iPSC cell lines carrying different DMD mutations engineered in a healthy iPSC line recapitulate most hallmarks of the DMD phenotype compared with the parental line. These include mislocalization of DGC proteins such as nNOS, branching/fusion defects, and calcium signaling hyperactivation. We also demonstrate that skeletal myofibers derived from the DMD mutant lines exhibit defective contractions, strongly supporting the notion that an intrinsic contractility defect also contributes to the muscle weakness phenotype in DMD patients. Remarkably, this contraction defect can be largely rescued by prednisolone treatment, indicating that the drug directly acts on mutant fibers. Finally, these DMD phenotypes are also observed in an iPSC line derived from a DMD patient differentiated using the optimized protocol and they are rescued when restoring the DMD coding frame using CRISPR-Cas9, demonstrating their specificity. Thus, our work provides an in vitro platform to study the etiology of DMD in human myofibers. Our human DMD in vitro model will allow for exploration of the early contraction and branching defects caused by absence of dystrophin at the origin of the pathology and offers a platform for preclinical testing of candidate therapies for this devastating disease.     

Large-scale serum protein biomarker discovery in Duchenne muscular dystrophy

Serum biomarkers in Duchenne muscular dystrophy (DMD) may provide deeper insights into disease pathogenesis, suggest new therapeutic approaches, serve as acute read-outs of drug effects, and be useful as surrogate outcome measures to predict later clinical benefit. In this study a large-scale biomarker discovery was performed on serum samples from patients with DMD and age-matched healthy volunteers using a modified aptamer-based proteomics technology. Levels of 1,125 proteins were quantified in serum samples from two independent DMD cohorts: cohort 1 (The Parent Project Muscular Dystrophy–Cincinnati Children’s Hospital Medical Center), 42 patients with DMD and 28 age-matched normal volunteers; and cohort 2 (The Cooperative International Neuromuscular Research Group, Duchenne Natural History Study), 51 patients with DMD and 17 age-matched normal volunteers. Forty-four proteins showed significant differences that were consistent in both cohorts when comparing DMD patients and healthy volunteers at a 1% false-discovery rate, a large number of significant protein changes for such a small study. These biomarkers can be classified by known cellular processes and by age-dependent changes in protein concentration. Our findings demonstrate both the utility of this unbiased biomarker discovery approach and suggest potential new diagnostic and therapeutic avenues for ameliorating the burden of DMD and, we hope, other rare and devastating diseases.There is an urgent need for a reliable surrogate biomarker or set of biomarkers for Duchenne muscular dystrophy (DMD), ideally based on readily accessible and measurable molecules (1). DMD is a severe form of myopathy with an incidence of about 1 in 3,600–9,337 boys worldwide (2, 3), and is a result of different types of mutations in the X-linked DMD gene that abolish the expression and biological activity of dystrophin, an essential protein for muscle-fiber plasma membrane integrity and myofiber function (4, 5). Clinically, the disease is characterized by progressive muscle wasting, leading to loss of ambulation by 8–15 y of age and early death from complications from respiratory, orthopedic, and cardiac problems (2, 6).Several current drug-development programs are focused on slowing or preventing the progressive muscle loss in DMD either in conjunction with the standard of care treatment or as stand-alone therapies. Standard of care is currently chronic high-dose glucocorticoids, which are able to prolong ambulation by 3–4 y (7, 8) and slow disease progression, but are associated with a significant array of side effects (2, 6, 9, 10). Promising therapeutic approaches for DMD include restoring expression of the dystrophin gene via exon-skipping strategies (11–13), viral-based gene therapies (14, 15), and nonsense suppression/read-through strategies (16). Other genetic approaches include delivering minidystrophins, up-regulation of utrophin to compensate for the missing dystrophin, and many others (17). Pharmacological strategies in development include dissociative steroid drugs, which offer the potential of greater efficacy and lesser side effects (18), other anti-inflammatory therapies, and effectors of signaling pathways (19). The current primary clinical endpoint used for determining efficacy in the majority of these therapeutic approaches for ambulatory boys with DMD is the “six-minute walk test” (20, 21), although it is not ideal (22).Blood provides a circulating protein representation of all body tissue in both normal and pathological conditions, and serum proteins are emerging as useful biomarkers for diagnosis and prognosis of a growing number of diseases (23, 24). Mass spectrometry (MS)-based proteomic screens recently have proved successful at de novo biomarker identification in DMD (25). However, verification and validation of MS-discovered serum biomarkers remain challenging (24). Other approaches, such as multiplexed antibody or aptamer-based assays, are being considered for proteome screens because of their potential for higher throughput and better sensitivity, which may help overcome the validation challenges of identified biomarkers. For example, a recent study using an antibody-based array against 384 target proteins identified 11 protein biomarkers of disease across different muscular dystrophies from patient samples gathered from four different clinical sites (26). In addition, a modified aptamer-based technology (the SOMAscan assay) is emerging as another highly sensitive and multiplexed assay for biomarker discovery and validation (27–29). Based on novel reagents (Slow Off-rate Modified Aptamers, or SOMAmer reagents) that recognize specific conformational epitopes of native 3D proteins with high specificity and high sensitivity (30–32), the SOMAscan assay measures levels of 1,125 analytes in only 65 µL of serum over a wide dynamic range (>8 logs of concentration). Because the SOMAscan assay relies on the availability of the protein epitopes (i.e., the epitopes are not blocked by other protein binding, posttranslational modifications, and so forth), what is measured in the assay and the actual protein concentration in the sample being interrogated is frequently but not always correlated. In the same manner, ELISAs for the same proteins also are frequently but not always correlated.Because blood is the preferred diagnostic clinical material, and biomarkers in the blood can differ by several orders-of-magnitude in abundance, the SOMAscan assay may be a path forward to identify and verify key blood-based biomarkers for DMD and other diseases. We used the SOMAscan technology to screen for protein biomarkers associated with DMD using serum samples from two independent cohorts collected in different locations and run at different times (cohort information in Demographics, Characteristics, and Enrollment Criteria of the PPMD-C and CINRG Cohorts and Dataset S1). The first cohort analyzed was from The Parent Project Muscular Dystrophy–Cincinnati Children’s Hospital Medical Center (hereafter PPMD-C), which included the goal of identifying alternative treatment paths (i.e., nondystrophin-centric) for patients with DMD. The second cohort analyzed was from The Cooperative International Neuromuscular Research Group, Duchenne Natural History Study (hereafter CINRG) (33), which included the goal of identifying changes in biomarkers with age in patients with DMD. In the present study, we compared the data from these two independent studies. This process enabled us to identify 44 biomarkers in the blood associated with DMD: 24 that are significantly increased and 20 that are significantly decreased in patients with DMD.These data suggest new protein targets and biomarkers for further DMD studies. The data also may facilitate future clinical studies designed to identify new therapeutics for DMD, as well as further demonstrating the utility of the SOMAscan assay technology for identifying protein biomarkers for both rare and common diseases. We are making our data fully available to the DMD research community to enable further studies that may be suggested by these findings.     

Discrepancy between systolic and diastolic dysfunction of the left ventricle in patients with Duchenne muscular dystrophy

To assess the systolic and diastolic dysfunction of the leftventricle (LV) in relation to age and the severity of impairmentin Duchenne muscular dystrophy (DMD), we performed M-mode, two-dimensionaland pulsed-wave Doppler echocardi-ography in 45 male subjectswith DMD aged 8 to 25 years and in 40 age-matched healthy controls.Systolic dysfunction started in the first decade of life, withsome patients showing severe systolic dysfunction in their earlyteens. This dysfunction, however, did not always depend on theseverity of the skeletal muscle disease. No patients with DMDshowed an increase in peak atrial velocity and time-velocityintegrals of the atrial contraction velocity curve, findingsfrequently reported to precede the abnormalities in many cardiacdiseases; it was thought therefore that these patients had noincrease in left atrial compensation. Diastolic dysfunctionmay not routinely precede or accompany the systolic dysfunctionin DMD, in contrast with what is reported in patients with ischaemicor hypertensive heart disease. DMD patients usually show a predominantsystolic dysfunction.