H.B. Ginjaar

Serum matrix metalloproteinase-9 (MMP-9) as a biomarker for monitoring disease progression in Duchenne muscular dystrophy (DMD)

To identify serum biomarkers that allow monitoring of disease progression and treatment effects in Duchenne muscular dystrophy (DMD) patients, levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase-1 (TIMP-1) and osteopontin (OPN) were determined in 63 DMD patients on corticosteroid therapy. These proteins were selected for their role in the pathogenesis of muscular dystrophy. Levels of MMP-9 and TIMP-1 were significantly higher in sera of DMD patients compared to healthy controls, whereas the OPN levels showed no significant difference. MMP-9 levels were also observed to be significantly higher in older, nonambulant patients, compared to ambulant patients. Longitudinal data from a smaller cohort of DMD patients followed up for over 4years showed that MMP-9, but not TIMP-1 increased significantly with age. Hence, MMP-9 is a potential DMD biomarker for disease progression. Future studies have to confirm whether serum MMP-9 levels can be used to monitor therapeutic response.

Sarcoglycanopathies in Dutch patients with autosomal recessive limb girdle muscular dystrophy

Abstract Within a group of 76 sporadic/autosomal recessive limb girdle muscular dystrophy (LGMD) patients we tried to identify those with LGMD type 2C-E. Muscle biopsy specimens of 40 index patients, who had 22 affected sibs, were analyzed immuno-histochemically for the presence of three subunits: α-, β-, and γ,-sarcoglycans. Abnormal sarcoglycan expression was established in eight patients, with six affected sibs. In one patient γ-sarcoglycan was absent, and both α- and β-sarcoglycans were reduced. In the remaining seven patients γ-sarcoglycan was (slightly) reduced, and α- and β-sarcoglycans were absent or reduced. By DNA sequencing mutations were detected in one of the three sarcoglycan genes in all eight cases. Three patients had mutations in the α-, three in the β-, and two in the γ-sarcoglycan gene. The patients with sarcoglycanopathy comprised the more severely affected cases (P=0.04). In conclusion, sarcoglycanopathy was identified in 23% (14/62) of the autosomal recessive LGMD patients.

Application of in vitro myo-differentiation of non-muscle cells to enhance gene expression and facilitate analysis of muscle proteins

Introduction of the myogenic-determination gene MyoD forces non-muscle cell cultures into myogenesis, thereby inducing expression of muscle-specific proteins and facilitating their analysis. In several MyoD-transfected fibroblasts, immunohistochemical detection showed expression of desmin after three days, of titin after five days and of dystrophin after seven days. Cell fusion (myotube formation) could be observed after five days. After nine days a fraction of the cells showed a striated titin pattern, indicating an advanced state of muscle differentiation. Dystrophin (the protein absent in Duchenne Muscular Dystrophy patients) can be detected in MyoD-transfected and differentiated fibroblasts from healthy individuals, and is absent in those of patients. MyoD-transfection increases transcription of the dystrophin gene, facilitating RNA-based mutation detection. Using RNA from MyoD-transfected, differentiated fibroblasts of a deceased patient with an unknown, non-deletion mutation, we were able to identify a CGA-->TGA nonsense mutation in the rod domain at basepair 6492 and to establish a rapid mutation specific test for future diagnosis of the mutation in his relatives.

A novel gamma-sarcoglycan mutation causing childhood onset, slowly progressive limb girdle muscular dystrophy.

Limb girdle muscular dystrophy is a heterogeneous group of disorders. One autosomal recessive subtype, LGMD2C, has been linked to chromosome 13, and is caused by gamma-sarcoglycan deficiency in muscle. This report describes a novel missense mutation identified in a large consanguineous Dutch family with LGMD. This mutation leads to reduction of gamma-sarcoglycan, and gives rise to a childhood-onset, slowly-progressive dystrophy.

Health status in non-dystrophic myotonias: close relation with pain and fatigue

Abstract To determine self-reported health status in non-dystrophic myotonias (NDM) and its relationship to painful myotonia and fatigue. In a cross-sectional study, 32 NDM patients with chloride and 30 with sodium channelopathies, all off treatment, completed a standardised interview, the fatigue assessment scale (FAS), and the 36-item Short-Form Health Survey (SF-36). Beside formal assessment of pain, assessment of painful or painless myotonia was determined. The domain scores of the SF-36 were compared with Dutch community scores. Apart from the relationship among SF-36 scores and (1) painful myotonia and (2) fatigue, regression analyses in both NDM groups were conducted to determine the strongest determinants of the SF-36 domains general health perception, physical component (PCS) and mental component summary (MCS). All physically oriented SF-36 domains in both NDM groups (Pxa0≤xa00.01) and social functioning in the patients with sodium channelopathies (Pxa0=xa00.048) were substantially lower relative to the Dutch community scores. The patients with painful myotonia (41.9%) scored substantially (Pxa0<xa00.05) lower on most SF-36 domains than the patients without painful myotonia (58.1%). Fatigued patients (53.2%) scored substantially lower (Pxa0≤xa00.01) on all SF-36 domains than their non-fatigued counterparts (46.8%). The regression analysis showed that fatigue was the strongest predictor for the general-health perception and painful myotonia for the physical-component summary. None of the patients showed below-norm scores on the domain mental-component summary. The impact of NDM on the physical domains of patients’ health status is substantial, and particularly painful myotonia and fatigue tend to impede their physical functioning.

Limb girdle muscular dystrophy : A pathological and immunohistochemical reevaluation

Ninety‐seven muscle biopsies from 81 limb girdle muscular dystrophy (LGMD) patients [32 autosomal recessive (AR), 15 autosomal dominant (AD), 34 sporadic] were morphologically reevaluated. Sarcoglycan analysis was done in 37 available muscle biopsies of AR and sporadic patients. Chi‐square tests were used to analyze the relation between abnormalities in AR/sporadic versus AD cases. Eighty percent of the muscle biopsies showed a predominantly dystrophic pattern, 20% showed myopathic changes, and 17% of these also had neurogenic changes. Muscle histology was not significantly different between AR/sporadic and AD LGMD; however, the observed abnormalities were more pronounced in the AR/sporadic group. Collections of inflammatory cells were observed in 25% and 10% of the AR/sporadic and AD group, respectively. Sarcoglycanopathy was diagnosed in 25% of the AR and sporadic patients of the 37 families tested. We conclude that the histological picture of AR/sporadic and AD LGMD is essentially the same, and sarcoglycanopathy constitutes an important part of the AR/sporadic patients.

Warm-up phenomenon in myotonia associated with the V445M sodium channel mutation

Sirs: Myotonia is a clinical phenomenon consisting of uncontrolled temporary muscle stiffness after voluntary or evoked muscle contractions [1]. It is a cardinal symptom in non-dystrophic myotonias, including chloride and sodium channelopathies. Myotonia typically occurs after a period of rest and decreases with continuing exercise, commonly referred to as the warm-up phenomenon. This is in contrast with what occurs in paradoxical myotonia, where muscle stiffness increases as a result of continuing exercise. n nThe warm-up phenomenon is an established clinical feature in chloride channelopathies, both in recessive myotonia congenita (Becker’s disease) as well as in dominant myotonia congenita (Thomsen’s disease). It has also been shown to occur in limb muscles of patients with a sodium channelopathy [1, 4]. By contrast, paradoxical myotonia has been established by others as the characteristic feature of sodium channel myotonias [3]. n nThus, unlike the phenotypic homogeneity of chloride channelopathies, sodium channel mutations are associated with a broad spectrum of clinical phenotypes [5]. Here we report three patients with a predominant and generalised warm-up phenomenon associated with the V445M missense mutation of the SCN4A gene encoding the alpha-subunit of the voltage gated sodium channel. n nThree patients, from two families, with an autosomal dominant non-dystrophic myotonia and a predominant warm-up phenomenon were referred to our clinic. All patients had complaints of generalised muscle stiffness since birth and had earlier been diagnosed as Thomsen’s disease, but recent chloride channel gene (CLCN1) mutation screening was negative. Clinical myotonia was generalised, severe and painful. All patients noticed aggravation of myotonia at cold temperatures and only one patient reported aggravation after eating potassium rich food. They all used sodium channel blockers (mexiletine 200xa0mg three times a day, procainamide 1000xa0mg three times a day and quinine 200xa0mg three times a day respectively) with a good subjective effect. Needle EMG of all patients revealed myotonic discharges in all muscles investigated (left biceps muscle, right first interosseus muscle, right rectus femoris muscle, left tibialis anterior muscle and left orbicularis oculi muscle). We measured the warm-up phenomenon in three different muscle groups during a drug free period. The warm-up phenomenon of eyelid muscles and right hand flexor muscles was quantified by measuring the difference between two relaxation times - timed with a stopwatch - , i.e. after maximum voluntary contraction of three seconds following ten minutes of rest and after ten successive contractions. For leg muscles we compared the first and tenth trial on a ‘chair test’ in which the time required to rise from a standardised chair, to move around it and to sit down again was measured. DNA extracted from 20xa0ml of blood was screened for mutations in SCN4A by direct nucleotide sequence analysis. n nAll patients showed a marked generalised warm-up phenomenon (Tablexa01). Direct nucleotide sequence analysis of all three patients showed the same missense mutation (c.1333G>A; p.V445M) in SCN4A. n n n nTablexa01 n nClinical relaxation times (in seconds) of three patients after the first (1st) and tenth (10th) maximum voluntary contraction of eyelid muscles (eyes), right hand flexor muscles (hand) and leg muscles (legs) n n n nThe V445M sodium channel mutation was first reported by Rosenfeld et al. causing a painful myotonia congenita [4]. The warm-up phenomenon was mentioned in one patient. Here we report three patients with a predominant and generalised (eyelid muscles, right hand flexor muscles and leg muscles) warm-up phenomenon in association with the same V445M sodium channel mutation. Although quantitative computer analysis for the warm-up phenomenon of a single muscle group has already been developed, we used clinical tests in order to be able to measure this phenomenon in three various muscle groups [2]. n nFor a definite molecular diagnosis clinicians should be aware that a predominant and generalised warm-up phenomenon may be found in chloride channelopathies but also in sodium channelopathies. A definite diagnosis is important for informing patients about their disease, orienting therapy and genetic counselling [5]. Thus, in patients with severe, painful myotonia in combination with a clear warm-up phenomenon, we recommend screening of SCN4A with particular attention for the V445M mutation.

P.7.15 What can we learn from an assisted bicycle training in a symptomatic girl with Duchenne muscular dystrophy? A case study

In this case study a 9-year old ambulatory symptomatic girl with Duchenne muscular dystrophy participated in a dynamic training. Since the role of exercise is far from clear in both boys and girls with DMD, a recently developed assisted bicycle training was evaluated for its feasibility and effectiveness in this girl. The girl trained at home, 15xa0min with her arms followed by her legs, 5 times a week, for 24xa0weeks. The primary outcomes were the Motor Function Measure, and the Assisted Six-Minute Cycling Test. Secondary outcomes were the Vignos and Brooke scale for lower and upper extremity functioning, timed tests (time to rise from a floor, to rise from a chair, to climb 3 stairs and to walk 10xa0m), the Medical Research Scale scale for muscle strength, and quantitative muscle ultrasound to determine the echo intensity of the biceps brachii muscle, the forearm flexors, the rectus femoris muscle and the tibialis anterior muscle. This case study showed that the assisted bicycle training was feasible and safe. Additionally, we found that no physical deterioration occurred during the training period: she remained stable on the Motor Function Measure and the Assisted Six-Minute Cycling Test. Slight improvements in quantitative muscle ultrasound intensity were found, indicating less fatty infiltration in the muscles. Since there are several indications from this case study that physical training could be beneficial in this population, we recommend further research on the effects of dynamic training in girls with DMD and its relation to the level of dystrophin.

Thought Ripples on Muscle Waves : Recognition of Rippling Muscle Disease

We report on a 16-year-old Dutch patient in whom rippling muscle disease (RMD) was diagnosed years after his mother had been falsely diagnosed with acid maltase deficiency. The autosomal dominant mode of inheritance of the neuromuscular symptoms in this family had led to a re-evaluation of the diagnosis of acid maltase deficiency. Physical examination revealed the three key features leading to the clinical diagnosis of RMD: rippling, mounding, and percussion-induced rapid muscle contraction. Mutation analysis revealed a novel heterozygous missense mutation in the caveolin-3 gene (c.79C > G; p.Arg27Gly) in both the index patient and his mother. This case report stresses the importance of adhering to the mode of inheritance in the diagnosis of neuromuscular disorders. It also indicates that typical RMD phenomena are not easily acknowledged among paediatricians or neurologists. We therefore present an overview of these clinical characteristics of rippling muscle disease RMD.

Prevalence and mutation spectrum of skeletal muscle channelopathies in the Netherlands

Few reliable data exist on the prevalence of skeletal muscle channelopathies. We determined the minimum point prevalence of genetically-defined skeletal muscle channelopathies in the Netherlands and report their mutation spectrum. Minimum point prevalence rates were calculated as number of genetically-confirmed skeletal muscle channelopathy patients (CLCN1, SCN4A, CACNA1S and KCNJ2 gene mutations) in the Netherlands (1990-2015) divided by the total number of at-risk individuals. Rates were expressed as cases/100.000 and 95% confidence intervals were calculated based on Poisson distribution. Results of standardized genetic diagnostic procedures were used to analyze mutation spectra. We identified 405 patients from 234 unrelated pedigrees, resulting in a minimum point prevalence of 2.38/100.000 (95% CI 2.16-2.63) for skeletal muscle channelopathies in the Netherlands. Minimum point prevalence rates for the disease groups, non-dystrophic myotonia and periodic paralysis, were 1.70/100.000 and 0.69/100.000 respectively. Sixty-one different CLCN1 mutations (including 12 novel mutations) were detected in myotonia congenita. Twenty-eight different SCN4A missense mutations (including three novel mutations) were identified in paramyotonia congenita/sodium channel myotonia, hypokalemic periodic paralysis and hyperkalemic periodic paralysis. Four different CACNA1S missense mutations were detected in hypokalemic periodic paralysis and five KCNJ2 missense mutations in Andersen-Tawil syndrome. The minimum point prevalence rates for genetically-defined skeletal muscle channelopathies confirm their rare disease status in the Netherlands. Rates are almost twice as high as in the UK and more in line with pre-genetic prevalence estimates in parts of Scandinavia. Future diagnostic and therapeutic studies may benefit from knowledge of the mutation spectrum of skeletal muscle channelopathies.