Human muscular dystrophy: elevation of urinary dimethylarginines.

The amounts of the dimethylarginines NG,NG-dimethylarginine (DMA) and NG,N'G-dimethylarginine (DM'A) excreted in the urine of muscular dystrophic patients were examined and compared with the amounts excreted by normal controls, patients with other types of neuromuscular diseases, and patients with disuse muscle atrophy resulting from traumatic paralysis. The patients with muscular dystrophy excreted high concentrations of DMA and this urine showed high ratios of DMA to DM'A. This finding indicates a relation between protein methylation processes and muscular dystrophy.

Titin in muscular dystrophy and cardiomyopathy: Urinary titin as a novel marker.

Titin, encoded by the gene TTN, is the largest human protein, and plays central roles in sarcomeric structures and functions in skeletal and cardiac muscles. Mutations of TTN are causally related to specific types of muscular dystrophies and cardiomyopathies. A developed methodology of next generation sequencing has recently led to the identification of novel TTN mutations in such diseases. The clinical significance of titin is now emerging as a target for genetic strategies. Titin-related muscular dystrophies include tibial muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, hereditary myopathy with early respiratory failure, central core myopathy, centronuclear myopathies, and Salih myopathy. Truncation mutations of TTN have been identified as the most frequent genetic cause of dilated cardiomyopathy. In this review article, we highlight the role of titin and impact of TTN mutations in the pathogenesis of muscular dystrophies and cardiomyopathies. Recently, a novel sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of the urinary titin N-terminal fragments (U-TN) has been established. We discuss the clinical significance of U-TN in the diagnosis of muscular dystrophies and differential diagnosis of cardiomyopathies, as well as risk stratification in dilated cardiomyopathy.

Urinary N-terminal fragment of titin is a marker to diagnose muscular dystrophy in patients with cardiomyopathy.

The differential diagnosis of cardiomyopathy is important. It has been recently reported that urinary titin N (U-TN) is increased in patients with muscular dystrophy (MD), and is associated with muscular damage. We aimed to clarify whether U-TN is useful as a diagnostic tool for distinguishing MD from various cardiomyopathies [e.g. dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM)]. We measured and compared the U-TN/creatinine ratio (U-TN/Cr; pmol/mg/dl) in 278 control subjects and 331 patients with various cardiomyopathies (DCM, n = 199; sarcoidosis, n = 18; HCM, n = 86; amyloidosis, n = 15; Fabry disease, n = 6; MD, n = 7). The U-TN/Cr was significantly higher in MD patients than in patients with various cardiomyopathies and the control subjects (P < 0.001). From the ROC analysis, the U-TN/Cr (with a cut-off value of 8.7) identified MD with 100% sensitivity, 82% specificity, and an area under the curve (AUC) of 0.92 (95% CI 0.87-0.98, P < 0.001). The AUC of the U-TN/Cr that was able to predict MD was superior to those of U-TN, creatinine kinase, B-type natriuretic peptide, and troponin I. Urinary Titin-N is a novel marker to diagnose MD.

Analysis of extracellular mRNA in human urine reveals splice variant biomarkers of muscular dystrophies.

Urine contains extracellular RNA (exRNA) markers of urogenital cancers. However, the capacity of genetic material in urine to identify systemic diseases is unknown. Here we describe exRNA splice products in human urine as a source of biomarkers for the two most common forms of muscular dystrophies, myotonic dystrophy (DM) and Duchenne muscular dystrophy (DMD). Using a training set, RT-PCR, droplet digital PCR, and principal component regression, we identify ten transcripts that are spliced differently in urine exRNA from patients with DM type 1 (DM1) as compared to unaffected or disease controls, form a composite biomarker, and develop a predictive model that is 100% accurate in our independent validation set. Urine also contains mutation-specific DMD mRNAs that confirm exon-skipping activity of the antisense oligonucleotide drug eteplirsen. Our results establish that urine mRNA splice variants can be used to monitor systemic diseases with minimal or no clinical effect on the urinary tract.

Random urine calcium/osmolality in the assessment of calciuria in children with decreased muscle mass.

BACKGROUND: Random urine Ca/creatinine (UCa/Cr) is used to estimate 24-hour Ca excretion. However, due to decreased urine creatinine excretion in children with decreased muscle mass (DMM), UCa/Cr overestimates their Ca excretion. OBJECTIVE: To evaluate whether in children with DMM random urine Ca/osmolality (UCa/Osm) can accurately predict hypercalciuria (24-hour urine Ca > 4.0 mg/kg) and at which "cutoff" value. METHODS: 19 children with DMM and 29 with normal muscle mass (NMM), ages 6 - 17 years, were studied. DMM was diagnosed based on clinical findings and decreased serum creatinine, and confirmed by low urine creatinine excretion. Over 24 hours, subjects collected each void separately. After each sample was analyzed, samples of each participant were combined to form a 24-hour specimen from which an aliquot (AL) was obtained; 24-hour urine Ca was first correlated with the corresponding AL Ca/Cr and Ca/Osm. As an internal control, a similar assessment ofproteinuria was conducted. In the next step, AL data were compared with individual urine samples to identify the time of day when a random sample best correlates with AL. RESULTS: The correlation coefficient between 24-hour Ca and AL Ca/Cr in all children was 0.61, in NMM 0.96, and in DMM 0.69 (in all p < 0.001). The correlation coefficient between 24-hour urine Ca and AL Ca/Osm in all children was 0.90, in NMM 0.90, and in DMM 0.91 (in all p < 0.001). In children with DMM, the correlation coefficient of 24-hour protein with AL protein/Cr was 0.75, and with protein/Osm 0.98 (both p < 0.001). Receiver operating characteristic curves showed UCa/Cr as a better predictor of 24-hour Ca > 4.0 mg/kg in NMM, whereas UCa/Osm was a better predictor of hypercalciuria in DMM patients. In NMM, UCa/Cr ratio > 0.20 had sensitivity of 88% and specificity of 96% in detecting 24-hour Ca > 4.0 mg/kg, whereas in those with DMM UCa/Osm (x 10) ratio of > 0.25 had sensitivity of 100% and specificity of 93% in detecting hypercalciuria. It was further found that random urine specimens collected between 9:00 a.m. and 2:00 p.m. best represented 24-hour urine data. CONCLUSION: In children with DMM, UCa/Osm can successfully replace UCa/Cr as a screening tool for hypercalciuria.

Generation of induced pluripotent stem cells from muscular dystrophy patients: efficient integration-free reprogramming of urine derived cells.

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy(1). Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2-3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

Urinary sodium, potassium and aldosterone in Duchenne muscular dystrophy.

Four adolescent boys with Duchenne (progressive) muscular dystrophy (DMD) of 10-11 years duration and six normal boys of similar age were studied on a metabolism ward for 22 days. Sodium and potassium intake was as follows: Period I, Na 60 mEq, K 60 mEq; Period II, Na 10, K 60; Period III, Na 10, K 95-150; Period IV, Na 60, K 60. The differences between the DMD group and the group of normal boys for sodium and potassium in serum and urine and for urinary aldosterone were not significant. These findings show that the pathologically elevated sodium-potassium ratio in skeletal muscle of patients with DMD is not due to increased aldosterone or other causes of renal wastage of potassium.

Enhanced urinary spontaneous luminescence in Duchenne muscular dystrophy.

Urinary spontaneous visible luminescence is enhanced in children with Duchenne Muscular Dystrophy (DMD). This result is indicative of systemic oxidative stress in DMD patients. It is proposed that measurement of the urinary luminescence could be employed to follow the progress of the disease, as well as the response of the patients to antioxidant therapy.

Response of serum creatine phosphokinase to steroid hormone.

Serum creatine phosphokinase activity increased significantly four or six hours after the administration of prednisolone in patients with muscular dystrophy of various types, whereas it did not increase in other muscular disorders. The extent of the response correlated inversely with the grade of disability. The prednisolone test may help to differentiate muscular dystrophy from polymyositis.

Urinary excretion of carnitine in Duchenne muscular dystrophy.

The urinary excretion of carnitine by boys with Duchenne dystrophy did not differ significantly from age-matched controls. Since serum and muscle concentrations of carnitine are also normal in Duchenne dystrophy, it cannot be proved that this small molecule leaks from muscle in Duchenne dystrophy. If the muscle surface membrane is abnormally leaky in Duchenne dystrophy, different molecules seem to be affected in different ways.

Altered polyamine excretion in Duchenne muscular dystrophy.

Increased urinary levels of the polyamines, putrescine, spermidine, and spermine are present in Duchenne muscular dystrophy (DMD) patients. Alterations in excretion are detectable when based on nanomoles per milligram creatinine, micromoles per 24-hour urine collection, or nanomoles per kilogram body weight per 24-hour urine collection. Evaluation of creatinine levels in spot collections of urine versus 24-hour collections suggests a more consistent urinary creatinine in spot urines from DMD patients and normals, with wider variability in 24-hour specimens. We postulate that polyamines may be useful markers to evaluate disease activity and to screen for drug efficacy, as they have proved useful in cancer patients in assessing alterations in tumor kinetic parameters.

3-methylhistidine excretion in myotonic dystrophy.

3-Methylhistidine (3-MH) excretion reflects the rate of muscle protein catabolism, since 3-MH occurs almost exclusively in muscle actin and myosin and is not reutilized or catabolized. We studied 3-MH excretion in 9 patients with myotonic dystrophy, 8 normals, and 10 disease controls with Duchenne dystrophy and other disorders. 3-MH excretion was expressed relative to muscle mass as determined by both urinary creatinine and total body potassium (40K method). Absolute 3-MH excretion was decreased in myotonic dystrophy patients but was normal when related to muscle mass. The finding of normal 3-MH excretion in myotonic dystrophy suggests that the muscle wasting in this disorder results from impaired anabolic processes rather than accelerated muscle destruction.

Primary beta-sarcoglycanopathy manifesting as recurrent exercise-induced myoglobinuria.

We report an unusual presentation of a primary beta-sarcoglycanopathy (LGMD type 2E). A 12- year-old boy came to our attention after six episodes of exercise-induced myoglobinuria. Electromyogram showed mild myopathic features of the proximal lower limb muscles. Electrocardiogram was normal. Neurological examination revealed normal muscle strength and reduced deep tendon reflexes. A muscle biopsy showed rare regenerating fibers; the immunohistochemistry was normal for dystrophin, while all the sarcoglycans were diffusely decreased. Western blot analysis showed a relevant decrease of all sarcoglycan proteins and a mild dystrophin reduction. beta-Sarcoglycan gene analysis demonstrated a compound heterozygous status for these mutations: a novel A-T base pair substitution at nucleotide 85 in exon 2, changing the codon Arg to a stop codon; a C-T base pair substitution at nucleotide 272 in exon 3 changing a Arg to a Cys residue. We consider that exercise-induced myoglobinuria may be the presenting sign of primary beta-sarcoglycanopathy.

Decrease of 3-methylhistidine and increase of NG,NG-dimethylarginine in the urine of patients with muscular dystrophy.

The amounts of 3-methylhistidine, N epsilon,N epsilon-dimethyllysine, N epsilon, N epsilon, N epsilon-trimethyllysine, NG,NG-dimethylarginine, and NG,N'G-dimethylarginine were determined in the urine specimens of healthy subjects and patients of corresponding ages with Duchenne, limb-girdle, and congenital types of muscular dystrophy, and motor neuron diseases. The amount of excretion of 3-methylhistidine decreased and that of NG,NG-dimethylarginine increased significantly in Duchenne and limb-girdle types of muscular dystrophy, but not in diseases with neurogenic muscular atrophy. The decrease of 3-methylhistidine was observed consistently throughout the course of the Duchenne type of muscular dystrophy. The amounts of the other methylamino acids both in myogenic and neurogenic myopathies were not different from those in healthy subjects.

Decrease in urinary excretion of 3-methylhistidine by patients with Duchenne muscular dystrophy during glucocorticoid treatment.

Seven patients, aged 10-17 years, with Duchenne muscular dystrophy were treated orally with prednisolone (PSL) at a dose of 0.8-1.0 mg/kg per day for 8 weeks. During the treatment their muscle strength, serum creatine kinase (CK) activity, serum levels of myoglobin (Mb), and urinary excretion of 3-methylhistidine (3-MeH) and glycine (Gly) were measured serially. In all the patients, the motor function or muscle strength improved, and the serum CK activity and Mb level decreased during PSL treatment. Urinary excretion of 3-MeH, a unique constituent of muscle contractile proteins, decreased to 51-63% of the baseline value in weeks 6-9 after the start of PSL administration, and returned to the baseline level in week 12. The ratios of 3-MeH to creatinine and to Gly also decreased during the treatment. Urinary excretion of Gly, which is ubiquitous in all tissues including muscle, did not decrease during the treatment. These findings suggest that PSL inhibits proteolysis of muscle contractile protein.

Urinary excretion of 3-methylhistidine and creatine in carriers of Duchenne muscular dystrophy.

The mean molar ratio (0.0171) of 3-methylhistidine to creatinine in 24-h urine samples from carriers of Duchenne muscular dystrophy did not differ significantly from that of controls. The creatine/creatinine ratio was also normal.

Spontaneous urinary visible luminescence: characteristics and modification by oxidative stress-related clinical conditions.

Urinary visible luminescence is the result of the excretion of oxidized biomolecules and, as such, could provide a valuable index of systemic oxidative stress. The characteristics of the urinary luminescence that support this proposal are reviewed and the data obtained for patients with hyperthyroidism and children with Duchenne muscular dystrophy are also discussed. Enhanced urinary chemiluminescence was observed in both pathologies. A similar enhancement was obtained when the urinary luminescence of smokers was compared to that of non-smokers. The possibilities and limitations of this noninvasive methodology for the evaluation of systemic oxidative stress is critically evaluated.

[Effect of retabolyl on the dynamics of urinary catecholamine and DOPA excretion and electroneuromyographic indices in patients with Duchenne's myodystrophy]. / Vliianie retabolila na dinamiku urinarnoi ékskretsii katekholaminov, DOFA i élektroneiromiograficheskikh pokazatelei u bol'nykh s miodistrofiei Diushenna.

The monitoring of the use of anabolic hormones in patients with progressive Duchenne's muscular dystrophy was shown to be possible with the help of the clinical analysis of the patients' motor possibilities, electroneuromyographic examination and determination of the time-course of the urinary excretion of catecholamines and DOPA.

[Circadian rhythm of catecholamine excretion, effect of L-DOPA and Nacom in various diseases of the nervous system]. / Sutochnyi ritm ékskretsii katekholaminov, vliianie L-DOFA i nakoma pri nekotorykh zabolevaniiakh nervnoi sistemy.

The activity of the sympathoadrenal system (SAS) was studied in 132 patients with central nervous system diseases such as parkinsonism, deforming muscular dystonia (DMD), Huntington's chorea, myopathy and asthenic neurosis. The estimation was based on determinations of urine catecholamine (CA) excretion with the help of the fluorometric method developed by E. Sh. Matlina et. al. (1965). The control group included 50 healthy subjects. The findings obtained confirmed the reported data concerning the role of CA in the pathogenesis of the studied forms of nervous pathology. The study showed a decrease in dopamine excretion (DA) in parkinsonism, its increase in Huntington's chorea and DMD, and insufficiency of SAS activity in myopathy. Furthermore, additional criteria pointing to alterations in the diurnal SAS activity in the patients were revealed. These changes manifested themselves in the disruption of the diurnal rhythm of CA excretion as well as in the deficiency of DOPA and DA synthesis and deposition following a single dose of L-DOPA and nacome.

[Status of connective tissue in progressive muscular dystrophies]. / Sostoianie soedinitel'noi tkani pri progressiruiushchikh myshechnykh distrofiiakh.

60 patients with different forms of neuromuscular disorders were examined. Morphological studies of skeletal muscles and of diurnal excretion with urine of acetic GAG were carried out. It was established that the changes in the stromal connective tissue in the progressive muscular dystrophia appear in the early stages of the disease and affect both the essential substance and the fibrillar structures. The excretion with the urine of acetic GAG was increased. In denervative amyotrophy the changes became apparent against the background of marked clinical symptoms. The data obtained may be important for a differential diagnosis of progressive muscular dystrophy and denervative amyotrophy, and for the development of differential drug therapy.