High-throughput data-driven analysis of myofiber composition reveals muscle-specific disease and age-associated patterns.

Contractile properties of myofibers are dictated by the abundance of myosin heavy chain (MyHC) isoforms. MyHC composition designates muscle function, and its alterations could unravel differential muscle involvement in muscular dystrophies and aging. Current analyses are limited to visual assessments in which myofibers expressing multiple MyHC isoforms are prone to misclassification. As a result, complex patterns and subtle alterations are unidentified. We developed a high-throughput, data-driven myofiber analysis to quantitatively describe the variations in myofibers across the muscle. We investigated alterations in myofiber composition between genotypes, 2 muscles, and 2 age groups. We show that this analysis facilitates the discovery of complex myofiber compositions and its dependency on age, muscle type, and genetic conditions.-Raz, V., Raz, Y., van de Vijver, D., Bindellini, D., van Putten, M., van den Akker, E. B. High-throughput data-driven analysis of myofiber composition reveals muscle-specific disease and age-associated patterns.

Identification of a novel SGCA missense mutation in a case of limb-girdle muscular dystrophy 2D with the absence of four sarcoglycan proteins.

Limb-girdle muscular dystrophy 2D (LGMD2D) is caused by mutations in the α-sarcoglycan gene (SGCA). Due to lack of specificity, it is impossible to identify LGMD2D only by clinical symptoms and conventional immunohistochemical staining. The loss of any protein (α-, ß-, γ-, δ-sarcoglycan) that represent sarcoglycanopathy may cause reduction or absence of the other three proteins. Here, we report a patient with a complete loss of all the four proteins. Next generation sequencing (NGS) results showed a missense mutation (C.218 C > T) and a partial heterozygous deletion containing exons 7 and 8 of SGCA, which led to the final diagnosis of the patient. The discovery of this new mutation could broaden the spectrum of SGCA mutations, which may be associated with putative LGMD2D, especially when all the four proteins are completely missing.

LGMD2E is the most common type of sarcoglycanopathies in the Iranian population.

Sarcoglycanopathies (SGCs) which are caused by mutations in SGCA, SGCB, SGCG or SGCD genes are a subgroup of autosomal-recessive limb-girdle-muscular-dystrophies (LGMD2). Although frequencies of mutations in these genes are different among populations, mutations in SGCA and SGCD, respectively, have the highest and lowest frequencies in most populations. Here, we report the proportion of mutations in SGC genes among a group of Iranian SGCs patients. Clinical features and results of SGC genes screening of 25 SGCs probands are presented. Large deletion mutations are confirmed with MLPA assays. In total, 15 candidate disease causing mutations were observed in the SGCA, SGCB, SGCG and SGCD genes; ten were novel. Fourteen (56%), seven (28%), three (12%) and one (4%) patient, respectively, carried mutations in SGCB, SGCG, SGCD and SGCA. The findings suggest that LGMD2E is the most common form of SGCs in the Iranian population and that LGMD2D is the rarest. Twelve LGMD2E cases carried the same mutation. To the best of knowledge, the mutation spectrum in SGCs is being reported for the first time in Iranian population. The finding will be beneficial for screening and genetic-counseling of SGCs patients in Iran.

Identification of Genetic Variants Associated with Severe Myocardial Bridging through Whole-Exome Sequencing.

Myocardial bridging (MB) is a congenital coronary artery anomaly and an important cause of angina. The genetic basis of MB is currently unknown. This study used a whole-exome sequencing technique and analyzed genotypic differences. Eight coronary angiography-confirmed cases of severe MB and eight age- and sex-matched control patients were investigated. In total, 139 rare variants that are potentially pathogenic for severe MB were identified in 132 genes. Genes with multiple rare variants or co-predicted by ClinVar and CADD/REVEL for severe MB were collected, from which heart-specific genes were selected under the guidance of tissue expression levels. Functional annotation indicated significant genetic associations with abnormal skeletal muscle mass, cardiomyopathies, and transmembrane ion channels. Candidate genes were reviewed regarding the functions and locations of each individual gene product. Among the gene candidates for severe MB, rare variants in DMD, SGCA, and TTN were determined to be the most crucial. The results suggest that altered anchoring proteins on the cell membrane and intracellular sarcomere unit of cardiomyocytes play a role in the development of the missed trajectory of coronary vessels. Additional studies are required to support the diagnostic application of cardiac sarcoglycan and dystroglycan complexes in patients with severe MB.

How a paternal uniparental isodisomy of chromosome 17 leads to autosomal recessive limb-girdle muscular dystrophy R3.

Uniparental isodisomy is a condition where both chromosomes of a pair are inherited from one parental homologue. If a deleterious variant is present on the duplicated chromosome, its homozygosity can reveal an autosomal recessive disorder in the offspring of a heterozygous carrier. Limb-girdle muscular dystrophy (LGMD) R3 is an autosomal recessive inherited disease that is associated with alpha-sarcoglycan gene (SGCA) variants. We report the first published case of LGMDR3 due to a homozygous variant in SGCA unmasked by uniparental isodisomy. The patient is an 8-year-old who experienced delayed motor milestones but normal cognitive development. He presented with muscle pain and elevated plasma creatine kinase. Sequencing of the SGCA gene showed a homozygous pathogenic variant. Parents were not related and only the father was heterozygous for the pathogenic variant. A chromosomal microarray revealed a complete chromosome 17 copy number neutral loss of heterozygosity encompassing SGCA, indicating paternal uniparental isodisomy.

Novel mutations in the SGCA gene in unrelated Vietnamese patients with limb-girdle muscular dystrophies disease.

Background: Limb-girdle muscular dystrophy (LGMD) is a group of inherited neuromuscular disorders characterized by atrophy and weakness in the shoulders and hips. Over 30 subtypes have been described in five dominant (LGMD type 1 or LGMDD) and 27 recessive (LGMD type 2 or LGMDR). Each subtype involves a mutation in a single gene and has high heterogeneity in age of onset, expression, progression, and prognosis. In addition, the lack of understanding of the disease and the vague, nonspecific symptoms of LGMD subtypes make diagnosis difficult. Even as next-generation sequencing (NGS) genetic testing has become commonplace, some patients remain undiagnosed for many years. Methods: To identify LGMD-associated mutations, Targeted sequencing was performed in the patients and Sanger sequencing was performed in patients and family members. The in silico analysis tools such as Fathmm, M-CAP, Mutation Taster, PolyPhen 2, PROVEAN, REVEL, SIFT, MaxEntScan, Spliceailookup, Human Splicing Finder, NetGene2, and Fruitfly were used to predict the influence of the novel mutations. The pathogenicity of the mutation was interpreted according to the ACMG guidelines. Results: In this study, six patients from four different Vietnamese families were collected for genetic analysis at The Center for Gene and Protein Research and The Department of Molecular Pathology Faculty of Medical Technology, Hanoi Medical University, Hanoi, Vietnam. Based on clinical symptoms and serum creatine kinase (CK) levels, the patients were diagnosed with limb-girdle muscular dystrophies. Five mutations, including four (c.229C>T, p.Arg77Cys; exon one to three deletion; c.983 + 5G>C; and c.257_258insTGGCT, p.Phe88Leufs*125) in the SGCA gene and one (c.946-4_946-1delACAG) in the CAPN3 gene, were detected in six LGMD patients from four unrelated Vietnamese families. Two homozygous mutations (c.983 + 5G>C and c.257_258insTGGCT) in the SGCA gene were novel. These mutations were identified as the cause of the disease in the patients. Conclusion: Our results contribute to the general understanding of the etiology of the disease and provide the basis for definitive diagnosis and support genetic counseling and prenatal screening.

Nintedanib Reduces Muscle Fibrosis and Improves Muscle Function of the Alpha-Sarcoglycan-Deficient Mice.

Sarcoglycanopathies are a group of recessive limb-girdle muscular dystrophies, characterized by progressive muscle weakness. Sarcoglycan deficiency produces instability of the sarcolemma during muscle contraction, leading to continuous muscle fiber injury eventually producing fiber loss and replacement by fibro-adipose tissue. Therapeutic strategies aiming to reduce fibro-adipose expansion could be effective in muscular dystrophies. We report the positive effect of nintedanib in a murine model of alpha-sarcoglycanopathy. We treated 14 Sgca-/- mice, six weeks old, with nintedanib 50 mg/kg every 12 h for 10 weeks and compared muscle function and histology with 14 Sgca-/- mice treated with vehicle and six wild-type littermate mice. Muscle function was assessed using a treadmill and grip strength. A cardiac evaluation was performed by echocardiography and histological study. Structural analysis of the muscles, including a detailed study of the fibrotic and inflammatory processes, was performed using conventional staining and immunofluorescence. In addition, proteomics and transcriptomics studies were carried out. Nintedanib was well tolerated by the animals treated, although we observed weight loss. Sgca-/- mice treated with nintedanib covered a longer distance on the treadmill, compared with non-treated Sgca-/- mice, and showed higher strength in the grip test. Moreover, nintedanib improved the muscle architecture of treated mice, reducing the degenerative area and the fibrotic reaction that was associated with a reversion of the cytokine expression profile. Nintedanib improved muscle function and muscle architecture by reducing muscle fibrosis and degeneration and reverting the chronic inflammatory environment suggesting that it could be a useful therapy for patients with alpha-sarcoglycanopathy.

Alpha-sarcoglycanopathy presenting as myalgia and hyperCKemia in two adults with a long-term follow-up. Case reports.

Two patients with a paucisymptomatic hyperckemia underwent a skeletal muscle biopsy and massive gene panel to investigate mutations associated with inherited muscle disorders. In the SGCA gene, sequence analyses revealed a homozygous c.850C > T/p.Arg284Cys in patient 1 and two heterozygous variants (c.739G > A/p.Val247Met and c.850C > T/p.Arg284Cys) in patient 2. Combination of histology and immunofluorence studies showed minimal changes for muscular proteins including the α-sarcoglycan. These two cases highlight the advantages of next-generation sequencing in the differential diagnosis of mild myopathic conditions before considering the more invasive muscle biopsy in sarcoglycanopathies.

A new mutation of the SCGA gene is the cause of a late onset mild phenotype limb girdle muscular dystrophy type 2D with axial involvement.

Mutations in the SGCA gene cause limb girdle muscular dystrophy type 2D (LGMD2D). We report a family with three affected siblings with a mild phenotype consisting of late onset glutei and axial muscle weakness produced by a new mutation in the SGCA gene leading to a partial expression of the alpha-sarcoglycan protein. The MRI showed muscle atrophy involving paraspinal, pelvic and thigh muscles and a dystrophic pattern was observed in the muscle biopsy. Exome sequencing revealed a homozygous intronic deletion of SGCA and mRNA analysis showed the presence of three different transcripts. The presence, though in a lower proportion, of wild type transcript leads to a milder presentation of the disease. Although clinical symptoms did not entirely correspond with a sarcoglycanopathy, a compatible muscle MRI drove us to look for changes in the sarcoglycan genes. These cases are an example of how clinical, radiological and pathological data enriches the interpretation of exome analysis.

Milder forms of α-sarcoglicanopathies diagnosed in adulthood by NGS analysis.

INTRODUCTION: Sarcoglycanopathies (LGMD 2C2F) are a subgroup of limb-girdle muscular dystrophies (LGMD), caused by mutations in sarcoglycan genes. They usually have a childhood onset and rapidly progressive course with loss of ability to walk over 12-16 years. METHODS: Next generation sequencing (NGS) targeted gene panel was performed in three adult patients with progressive muscle weakness in which routine muscle histology and immunohistochemistry were not diagnostic. RESULTS: Genetic analysis revealed homozygous or compound heterozygous mutations in SGCA gene and Western Blot demonstrated protein reduction confirming the diagnosis of α-sarcoglicanopathy. DISCUSSION: Our cases evidence that the diagnosis of mild forms of alfa sarcoglicanopathy could be a challenge and suggest the possibility that they could be underdiagnosed. The use of Next generation Sequencing targeted gene panels is very helpful in the diagnosis of these patients.

Probable high prevalence of limb-girdle muscular dystrophy type 2D in Taiwan.

Limb-girdle muscular dystrophy type 2D (LGMD2D), an autosomal-recessive inherited LGMD, is caused by the mutations in SGCA. SGCA encodes alpha-sarcoglycan (SG) that forms a heterotetramer with other SGs in the sarcolemma, and comprises part of the dystrophin-glycoprotein complex. The frequency of LGMD2D is variable among different ethnic backgrounds, and so far only a few patients have been reported in Asia. We identified five patients with a novel homozygous mutation of c.101G>T (p.Arg34Leu) in SGCA from a big aboriginal family ethnically consisting of two tribes in Taiwan. Patient 3 is the maternal uncle of patients 1 and 2. All their parents, heterozygous for c.101G>T, denied consanguineous marriages although they were from the same tribe. The heterozygous parents of patients 4 and 5 were from two different tribes, originally residing in different geographic regions in Taiwan. Haplotype analysis showed that all five patients shared the same mutation-associated haplotype, indicating the probability of a founder effect and consanguinity. The results suggest that the carrier rate of c.101G>T in SGCA may be high in Taiwan, especially in the aboriginal population regardless of the tribes. It is important to investigate the prevalence of LGMD2D in Taiwan for early diagnosis and treatment.

Alpha-sarcoglycanopathy presenting as exercise intolerance and rhabdomyolysis in two adults.

Two patients with exercise-induced myalgias and rhabdomyolysis with myoglobinuria were evaluated with muscle biopsy and comprehensive myopathy next generation sequencing (NGS) gene panels. Genetic analysis revealed homozygosity for two known pathogenic SGCA mutations (R284C in Patient 1 and V247M in Patient 2). Muscle biopsy showed minimal changes with normal immunohistochemistry for α-sarcoglycan. Western blotting showed 27% and 35% of normal α-sarcoglycan immunoreactivity when compared to age matched controls, confirming the diagnosis of α-sarcoglycanopathy in both patients. The sarcoglycan genes should be added to the differential diagnosis for cases that present with rhabdomyolysis, exercise intolerance, and hyperCKemia, even in the absence of muscle weakness or normal α-sarcoglycan immunohistochemistry. Work-up of patients with these types of non-specific presentation may be best facilitated through the use of non-specific NGS myopathy panels.

Clinical, radiological, and pathological features in 43 cases of intracranial subependymoma.

OBJECT: Intracranial subependymomas are rarely reported due to their extremely low incidence. Knowledge about subependymomas is therefore poor. This study aimed to analyze the incidence and clinical, radiological, and pathological features of intracranial subependymomas. METHODS: Approximately 60,000 intracranial tumors were surgically treated at Beijing Tiantan Hospital between 2003 and 2013. The authors identified all cases in which patients underwent resection of an intracranial tumor that was found to be pathological examination demonstrated to be subependymoma and analyzed the data from these cases. RESULTS: Forty-three cases of pathologically confirmed, surgically treated intracranial subependymoma were identified. Thus in this patient population, subependymomas accounted for approximately 0.07% of intracranial tumors (43 of an estimated 60,000). Radiologically, 79.1% (34/43) of intracranial subependymomas were misdiagnosed as other diseases. Pathologically, 34 were confirmed as pure subependymomas, 8 were mixed with ependymoma, and 1 was mixed with astrocytoma. Thirty-five patients were followed up for 3.0 to 120 months after surgery. Three of these patients experienced tumor recurrence, and one died of tumor recurrence. Univariate analysis revealed that shorter progression-free survival (PFS) was significantly associated with poorly defined borders. The association between shorter PFS and age < 14 years was almost significant (p = 0.51), and this variable was also included in the multivariate analysis. However, multivariate analysis showed showed only poorly defined borders to be an independent prognostic factor for shorter PFS (RR 18.655, 95% CI 1.141-304.884, p = 0.040). In patients 14 years of age or older, the lesions tended to be pure subependymomas located in the unilateral supratentorial area, total removal tended to be easier, and PFS tended to be longer. In comparison, in younger patients subependymomas tended to be mixed tumors involving the bilateral infratentorial area, with a lower total removal rate and shorter PFS. CONCLUSIONS: Intracranial subependymoma is a rare benign intracranial tumor with definite radiological features. Long-term survival can be expected, although poorly defined borders are an independent predictor of shorter PFS. All the features that differ between tumors in younger and older patients suggest that they might have different origins, biological behaviors, and prognoses.

Clinical and molecular description of a 17q21.33 microduplication in a girl with severe kyphoscoliosis and developmental delay.

High proportion of disease-associated copy number variant maps to chromosome 17. Genomic studies have provided an insight into its complex genomic structure such as relative abundance of segmental duplication and intercepted repetitive elements. 17q21.31, 17q11.2 and 17q12 loci are well known on this chromosome and are associated with microdeletion and microduplication syndrome. No syndrome associated with 17q21.33 locus have been described. We report clinical, cytogenetic and molecular investigations of a 13 years-old girl admitted for evaluation of microcephaly, scoliosis, skeletal defects and learning difficulties. We carried out detailed analysis of the clinical phenotype of this patient and investigated the genetic basis using Agilent 180K Array Comparative Genomic Hybridization. We identified a ∼0.9 Mb de novo microduplication on chromosome 17q21.33. Four genes, COL1A1, SGCA, PPP1R9B and CHAD located within the duplicated region are possible candidates for clinical features present in our patients. Gene expression studies by real-time RT-PCR assay only showed an overexpression of SGCA (P < 0.01), a component of the dystrophin glycoprotein complex. Defect of SGCA was previously shown to lead to severe childhood autosomal recessive muscular dystrophy (LGMD2D) which result in progressive muscle weakness and can also be associated with hyperlordosis or scoliosis. Further cases with similar duplications are expected to be diagnosed. This will contribute to the delineation of this potential new microduplication syndrome and to improve genetic counseling.

Cardiac Characterization of sgca-Null Mice Using High Resolution Echocardiography.

Limb-girdle muscular dystrophy 2D (LGMD2D) is an inherited myogenic disorder belonging to the group of muscular dystrophies. Sgca-null mouse is a knock-out model of LGMD2D. Little is known about cardiac phenotype characterization in this model at different ages. We conducted a prospective study to characterize cardiac sgca-null mice phenotype using high resolution Doppler echocardiography at different ages. Conventional echocardiography was performed on anesthetised mice using a Vevo 770 (Visualsonics) with 30 MHz cardiac probe. Wild Type (WT) and sgca-null mice were scanned at 13, 15 and 17 months. From M-mode, we measured interventricular septal (IVS) wall thickness, posterior wall (PW) thickness, and end-left ventricular diameter in systolic and diastolic. From the above parameters, we calculated left ventricular (LV) shortening fraction (SF), LV ejection fraction (EF) and LV mass. At age 13 months, PW diastolic thickness was increased in sgca-null mice (0.89±0.14 mm vs 0.73±0.2 mm; P=0.020) and LV mass was higher in sgca-null mice (LV mass 205.2 mg vs 143 mg; P=0.001). We found also dilation of the LV (LVEDD: 4.84 mm vs 4.29 mm; P=0.019) in sgca-null mice. At age 15 months, dilation of the LV (LVEDD: 4.86 mm vs 4 mm; P=0.05) with an increase of the LV mass (165.7 mg vs 127.12; P=0.03) are found in sgca-null mice. At age 17 months, we found a decrease of the PW thickening (17% vs 30%; P=0.036). This work provides echocardiographic insights for the assessment of pharmaceutical therapies in sgca-null mice.