Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.

Early-Onset Myopathies: Clinical Findings, Prevalence of Subgroups and Diagnostic Approach in a Single Neuromuscular Referral Center in Germany.

BACKGROUND: Early-onset myopathies are a heterogeneous group of neuromuscular diseases with broad clinical, genetic and histopathological overlap. The diagnostic approach has considerably changed since high throughput genetic methods (next generation sequencing, NGS) became available. OBJECTIVE: We present diagnostic subgroups in a single neuromuscular referral center and describe an algorithm for the diagnostic work-up. METHODS: The diagnostic approach of 98 index patients was retrospectively analysed. In 56 cases targeted sequencing of a known gene was performed, in 44 patients NGS was performed using large muscle specific panels, and in 12 individuals whole exome sequencing (WES) was undertaken. One patient was diagnosed via array CGH. Clinical features of all patients are provided. RESULTS: The final diagnosis could be found in 63 out of 98 patients (64%) with molecular genetic analysis. In 55% targeted gene sequencing could establish the genetic diagnosis. However, this rate largely depended on the presence of distinct histological or clinical features. NGS (large myopathy-related panels and WES) revealed genetic diagnosis in 58.5% (52% and 67%, respectively). The genes detected by WES in our cohort of patients were all covered by the panels. Based on our findings we propose an algorithm for a practical diagnostic approach.Prevalences:MTM1- and LAMA2-patients are the two biggest subgroups, followed by SEPN1-, RYR1- and Collagen VI-related diseases. 31% of genetically confirmed cases represents a group with overlap between "congenital myopathies (CM)" and "congenital muscular dystrophies (CMD)". In 36% of the patients a specific genetic diagnosis could not be assigned. CONCLUSIONS: A final diagnosis can be confirmed by high throughput genetic analysis in 58.5% of the cases, which is a higher rate than reported in the literature for muscle biopsy and should in many cases be considered as a first diagnostic tool. NGS cannot replace neuromuscular expertise and a close discussion with the geneticists on NGS is mandatory. Targeted candidate gene sequencing still plays a role in selected cases with highly suspicious clinical or histological features. There is a relevant clinical and genetic overlap between the entities CM and CMD.

No muscle involvement in myoclonus-dystonia caused by epsilon-sarcoglycan gene mutations.

Mutations in the epsilon-sarcoglycan gene (SGCE) can cause autosomal dominant inherited myoclonus-dystonia (M-D). Defects in other sarcoglycans; alpha-, beta-, gamma-, and delta can cause autosomal recessive inherited limb girdle muscular dystrophies. epsilon- and alpha-sarcoglycans are very homologous and may substitute for one-another in different tissues. We therefore investigated whether mutations in SGCE also cause abnormalities of skeletal and myocardial muscle. Six patients with clinically and genetically verified M-D and no signs of limb-girdle muscular dystrophy were included. Skeletal muscle biopsies were obtained from all patients, and endomyocardial muscle biopsy from one of the patients. Morphological and immunohistological investigations were performed and compared with controls. Histological and immunohistological investigations of muscle and clinical assessment of muscle strength and mass showed no difference between M-D patients and controls. Our findings indicate that patients with M-D have no signs or symptoms of muscle disease. This suggests a different role of the sarcoglycan complex epsilonbetagammadelta versus alphabetagammadelta complex in humans, as earlier suggested in rodents.

Brain imaging and neuropsychology in late-onset dementia due to a novel mutation (R93C) of valosin-containing protein.

Inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD, MIM 167320) is a recently identified autosomal dominant disorder due to mutations in the valosin-containing protein (VCP) that affects muscle, bone and brain. Brain involvement and neuropsychological findings of IBMPFD have not been described in detail. A patient carried a novel heterozygous base pair change, 47832C>T, in the VCP gene that resulted in substitution of an arginine residue by cysteine at position 93 (R93C). He presented first with myopathy while bone involvement remained subclinical. The patient developed behavioral abnormalities in his 60s and showed frank personality change with fluent empty speech at the age of 74 years. This syndrome was best classified as semantic dementia. Magnetic resonance imaging disclosed slight but progressive cerebral atrophy with prominent callosal and frontal white matter loss. Positron emission tomography demonstrated glucose hypometabolism of the frontal and temporal lobes disproportionate to their structural involvement. This first comprehensive clinical and neuroimaging study in IBMPFD may raise the awareness among clinicians as well as basic scientists for this exemplary genetic model of dementia.

Scapuloperoneal syndrome type Kaeser and a wide phenotypic spectrum of adult-onset, dominant myopathies are associated with the desmin mutation R350P.

In 1965, an adult-onset, autosomal dominant disorder with a peculiar scapuloperoneal distribution of weakness and atrophy was described in a large, multi-generation kindred and named 'scapuloperoneal syndrome type Kaeser' (OMIM #181400). By genetic analysis of the original kindred, we discovered a heterozygous missense mutation of the desmin gene (R350P) cosegregating with the disorder. Moreover, we detected DES R350P in four unrelated German families allowing for genotype-phenotype correlations in a total of 15 patients carrying the same mutation. Large clinical variability was recognized, even within the same family, ranging from scapuloperoneal (n = 2, 12%), limb girdle (n = 10, 60%) and distal phenotypes (n = 3, 18%) with variable cardiac (n = 7, 41%) or respiratory involvement (n = 7, 41%). Facial weakness, dysphagia and gynaecomastia were frequent additional symptoms. Overall and within each family, affected men seemingly bear a higher risk of sudden, cardiac death as compared to affected women. Moreover, histological and immunohistochemical examination of muscle biopsy specimens revealed a wide spectrum of findings ranging from near normal or unspecific pathology to typical, myofibrillar changes with accumulation of desmin. This study reveals that the clinical and pathological variability generally observed in desminopathies may not be attributed to the nature of the DES mutation alone, but may be influenced by additional genetic and epigenetic factors such as gender. In addition, mutations of the desmin gene should be considered early in the diagnostic work-up of any adult-onset, dominant myopathy, even if specific myofibrillar pathology is absent.

Clinical consequences of bone bruise around the knee.

The aim of this study is to evaluate the relation between bone bruise and (peri-)articular derangement and to assess the impact of bone bruise on presentation and short term course of knee complaints. We recorded MR abnormalities in 664 consecutive patients with sub-acute knee complaints. Patients were divided in four groups: patients with and without intra-articular knee pathology, subdivided in patients with and without bone bruise. We assessed function and symptoms at the time of MR and 6 months thereafter. Bone bruises were diagnosed in 124 of 664 patients (18.7%). Patients with bone bruise had significantly more complete ACL, lateral meniscal, MCL and LCL tears. Both with and without intra-articular pathology patients with bone bruise had a significantly poorer function at the time of MR (Noyes score of, respectively, 313.21 versus 344.81 and 306.98 versus 341.19). Patients with bone bruise and intra-articular pathology showed significantly more decrease in activity (decrease of Tegner score from 6.28 to 2.12 versus 5.70-2.55). At 6 months there were no significant differences in clinical parameters between the four groups. We concluded that bone bruise in combination with MCL tear is an important cause of initial clinical impairment in patients with sub-acute knee complaints. Clinical improvement within 6 months is more pronounced than in patients without bone bruise.

LGMD 2I due to the common mutation 826C>A in the FKRP gene presenting as myopathy with vacuoles and paired-helical filaments.

We report on two unrelated patients clinically presenting with late-onset progressive limb girdle weakness; cardiomyopathy was seen in one patient. Muscle biopsy revealed a necrotic myopathy with numerous rimmed vacuoles, ultrastructurally typical paired-helical filaments, and reduced immunohistochemical staining for alpha-dystroglycan. Quadriceps sparing hereditary inclusion body myopathy due to mutations in GNE gene, and OPMD due to PABPN1 mutations were excluded, genetically. We detected a homozygous mutation of the FKRP gene (826C>A) in both patients. Mutations of FKRP have been reported in congenital muscular dystrophies, LGMD2I, cardiomyopathy and hyperCKemia, but not in myopathies with vacuoles and paired-helical filaments. Therefore, our findings further extend the morphological variability of muscular dystrophies due to FKRP mutations.

Effectiveness of MR imaging in selection of patients for arthroscopy of the knee.

PURPOSE: To determine the effectiveness of magnetic resonance (MR) imaging in the appropriate identification of those patients with a high clinical suspicion of internal derangements of the knee who require arthroscopic therapy. MATERIALS AND METHODS: In a prospective multicenter study, MR imaging was performed at 0.5 T in 430 consecutive patients. The sensitivity and specificity of MR imaging in the patients who underwent arthroscopy and the corrected sensitivity and specificity of MR in all the study patients were calculated. For this correction, patients with negative MR and arthroscopic results were considered representative of the patients with negative MR results who were conservatively treated, and the number of the former was doubled. The standard errors of the corrected values were adjusted with the delta method. RESULTS: At MR imaging, arthroscopy was indicated in 221 patients, 200 of whom underwent arthroscopy. Two hundred nine patients with negative MR imaging results were randomized for arthroscopic (105 patients) or for conservative treatment (104 patients). Of the 105 patients randomized for arthroscopy, 93 actually underwent arthroscopy. Arthroscopic treatment was necessary in 13 of 93 patients with a negative diagnosis at MR imaging. Arthroscopic treatment was necessary in 179 of 200 patients with a positive diagnosis at MR (sensitivity, 93.2%; specificity, 79.2%). Sensitivity and specificity corrected for randomization were 87.3% and 88.4%. Sensitivity and specificity corrected for randomization, respectively, were 84.1% and 94.2% for the diagnosis of medial meniscal tears and 69.5% and 94.5% for the diagnosis of lateral meniscal tears at MR. CONCLUSION: MR imaging is an effective tool in the selection of patients for arthroscopy from among a general population.

Acquired antral pyloric hypertrophy in the dog.

Acquired antral pyloric hypertrophy is one of the most common causes of pyloric obstruction in the small (10 kg) mature or old dog. Clinical signs include chronic intermittent vomiting after meals and occasional weight loss or abdominal distension. Definitive diagnosis is made by exploratory laparotomy and histologic examination of excised tissues. Most dogs with acquired antral pyloric hypertrophy become clinically normal after surgical correction of the gastric outlet obstruction.

Ureterocolonic anastomosis in clinically normal dogs.

Ureterocolonic anastomosis was evaluated in 13 clinically normal dogs. Urinary continence was maintained after surgery, and the procedure was completed without technique errors in all but 2 dogs. Three dogs died within 5 weeks (2 of undetermined causes and 1 of aspiration pneumonia and neurologic disease), and 1 dog was euthanatized 4 months after surgery because of neurologic signs. Two healthy dogs were euthanatized 3 months after surgery for light microscopic evaluation of their kidneys. Five dogs were euthanatized 6 months after surgery for light microscopic evaluation of their kidneys. Gastrointestinal and neurologic disturbances developed in 4 dogs at various postoperative intervals. Plasma ammonia concentration measured in 2 dogs with neurologic signs was increased. Plasma ammonia concentration measured in 5 dogs without neurologic signs was within normal limits. All 5 dogs, in which metabolic acidosis was diagnosed, had high normal or above normal serum chloride concentration. Serum urea nitrogen values were increased after surgery because of colonic absorption of urea. Serum creatinine concentration was increased in 1 dog 6 months after surgery. Individual kidney glomerular filtration rate was reduced in 38% (3/8) of the kidneys from 4 other dogs at 6 months after surgery. Of 5 dogs euthanatized at 3 to 4 months after surgery, 4 had bilateral pyelitis, and 1 had unilateral pyelonephritis. Six months after surgery, pyelonephritis was diagnosed in 40% (4/10) of the kidneys from 5 dogs. The ureterocolonic anastomosis procedure is a salvage procedure that should allow complete cystectomy. However, variable degrees of metabolic acidosis, hyperammonemia, and neurologic disease may result.

Pylorectomy and gastroduodenostomy in the dog: technique and clinical results in 28 cases.

Pylorectomy and end-to-end gastroduodenostomy are surgical procedures that allow excision of abnormal pyloric tissue and provide improved gastric outflow. These techniques were used for the treatment of benign, malignant, and ulcerative conditions that were judged to be not adequately treatable with pyloromyotomies or pyloroplasties. End-to-end gastroduodenostomy was not much more difficult than a standard intestinal anastomosis; however, a thorough knowledge of the pyloric area anatomy was required to avoid serious surgical errors. In addition, gentle tissue manipulation and precise suture placement reduced the chance of iatrogenic pancreatitis, biliary obstruction, tissue ischemia, and/or suture line leakage. The results of surgery depended on the underlying disease process. Dogs with benign lesions such as chronic hypertrophic pyloric gastropathy responded favorably to treatment. Dogs with malignant disease and perforated ulcers had low long-term survival rate. Pyloric adenocarcinoma was not adequately treated with this method alone.

Chronic hypertrophic pyloric gastropathy as a cause of pyloric obstruction in the dog.

Six small-breed, middle-age dogs with a history of chronic intermittent vomiting had benign pyloric lesions causing gastric outlet obstruction. Marked similarities were found in clinical signs, pathologic changes, and treatment results. The condition was classified as a syndrome and was named chronic hypertrophic pyloric gastropathy. The appearance of the obstructive lesions at surgery were various forms of mucosal hypertrophy. Microscopically, the syndrome was characterized by mucosal foveolar and glandular hyperplasia, cystic glandular dilatation, superficial mucosal ulcerations, and various cellular infiltrates. The affected dogs were successfully treated by surgical correction of the gastric outlet obstruction.

Use of serum beta-chain human chorionic gonadotropin in the management of testis tumor.

One-third of all testis tumors secrete HCG which can be detected in the serum by radioimmunoassay. beta-HCG levels can be used for diagnosis and follow-up of testis tumors. Seminomas with elevated serum beta-HCG levels probably have nonseminomatous metastases.

Effect of sera of patients with genitourinary tract cancers on transformation of lymphocytes stimulated with anti-thymocyte globulin.

Serum from patients with genitourinary cancer was tested for ability to support transformation of autologous or normal lymphocytes stimulated with ATG. Selected for specificity for T-lymphocytes, ATG at times gave a more sensitive indication of a serum defect than did PHA. Although poor transformability in the presence of patient serum suggests an inhibitor, this test cannot rule out the possibility of a limitation of an essential nutrient usually supplied by serum. Presence of inhibitor(s) was more strongly indicated by contrasting transformation of normal lymphocytes with patient serum against a panel of normal sera. The presence of circulating inhibitor(s) was confirmed by demonstrating depression of transformation of normal lymphocytes in complete medium by chromatographic fractions from patient serum that did not support transformation. In several instances, patient serum was more inhibitory of autologous than normal lymphocytes, which suggested an additional way of examining host immune competence.