Pankaj B. Agrawal

Nemaline myopathy with minicores caused by mutation of the CFL2 gene encoding the skeletal muscle actin-binding protein, cofilin-2.

Nemaline myopathy (NM) is a congenital myopathy characterized by muscle weakness and nemaline bodies in affected myofibers. Five NM genes, all encoding components of the sarcomeric thin filament, are known. We report identification of a sixth gene, CFL2, encoding the actin-binding protein muscle cofilin-2, which is mutated in two siblings with congenital myopathy. The probands muscle contained characteristic nemaline bodies, as well as occasional fibers with minicores, concentric laminated bodies, and areas of F-actin accumulation. Her affected sisters muscle was reported to exhibit nonspecific myopathic changes. Cofilin-2 levels were significantly lower in the probands muscle, and the mutant protein was less soluble when expressed in Escherichia coli, suggesting that deficiency of cofilin-2 may result in reduced depolymerization of actin filaments, causing their accumulation in nemaline bodies, minicores, and, possibly, concentric laminated bodies.

Heterogeneity of nemaline myopathy cases with skeletal muscle α-actin gene mutations†

Nemaline myopathy (NM) is the most common of several congenital myopathies that present with skeletal muscle weakness and hypotonia. It is clinically heterogeneous and the diagnosis is confirmed by identification of nemaline bodies in affected muscles. The skeletal muscle α‐actin gene (ACTA1) is one of five genes for thin filament proteins identified so far as responsible for different forms of NM. We have screened the ACTA1 gene in a cohort of 109 unrelated patients with NM. Here, we describe clinical and pathological features associated with 29 ACTA1 mutations found in 38 individuals from 28 families. Although ACTA1 mutations cause a remarkably heterogeneous range of phenotypes, they were preferentially associated with severe clinical presentations (p < 0.0001). Most pathogenic ACTA1 mutations were missense changes with two instances of single base pair deletions. Most patients with ACTA1 mutations had no prior family history of neuromuscular disease (24/28). One severe case, caused by compound heterozygous recessive ACTA1 mutations, demonstrated increased α‐cardiac actin expression, suggesting that cardiac actin might partially compensate for ACTA1 abnormalities in the fetal/neonatal period. This cohort also includes the first instance of an ACTA1 mutation manifesting with adult‐onset disease and two pedigrees exhibiting potential incomplete penetrance. Overall, ACTA1 mutations are a common cause of NM, accounting for more than half of severe cases and 26% of all NM cases in this series. Ann Neurol 2004;56:86–96

Recessive truncating titin gene, TTN, mutations presenting as centronuclear myopathy.

Objective: To identify causative genes for centronuclear myopathies (CNM), a heterogeneous group of rare inherited muscle disorders that often present in infancy or early life with weakness and hypotonia, using next-generation sequencing of whole exomes and genomes. Methods: Whole-exome or -genome sequencing was performed in a cohort of 29 unrelated patients with clinicopathologic diagnoses of CNM or related myopathy depleted for cases with mutations of MTM1, DNM2, and BIN1. Immunofluorescence analyses on muscle biopsies, splicing assays, and gel electrophoresis of patient muscle proteins were performed to determine the molecular consequences of mutations of interest. Results: Autosomal recessive compound heterozygous truncating mutations of the titin gene, TTN, were identified in 5 individuals. Biochemical analyses demonstrated increased titin degradation and truncated titin proteins in patient muscles, establishing the impact of the mutations. Conclusions: Our study identifies truncating TTN mutations as a cause of congenital myopathy that is reported as CNM. Unlike the classic CNM genes that are all involved in excitation-contraction coupling at the triad, TTN encodes the giant sarcomeric protein titin, which forms a myofibrillar backbone for the components of the contractile machinery. This study expands the phenotypic spectrum associated with TTN mutations and indicates that TTN mutation analysis should be considered in cases of possible CNM without mutations in the classic CNM genes.

X-linked myotubular and centronuclear myopathies.

Recent work has significantly enhanced our understanding of the centronuclear myopathies and, in particular, myotubular myopathy. These myopathies share similar morphologic appearances with other diseases, namely the presence of hypotrophic myofibers with prominent internalized or centrally placed nuclei. Early workers suggested that this alteration represented an arrest in myofiber maturation, while other hypotheses implicated either failure in myofiber maturation or neurogenic causes. Despite similarities in morphology, distinct patterns of inheritance and some differences in clinical features have been recognized among cases. A severe form, known as X-linked myotubular myopathy (XLMTM), presents at or near birth. Affected males have profound global hypotonia and weakness, accompanied by respiratory difficulties that often require ventilation. Most of these patients die in infancy or early childhood, but some survive into later childhood or even adulthood. The responsible gene (MTM1) has been cloned; it encodes a phosphoinositide lipid phosphatase known as myotubularin that appears to be important in muscle maintenance. In autosomal recessive centronuclear myopathy (AR CNM), the onset of weakness typically occurs in infancy or early childhood. Some investigators have divided AR CNM into 3 subgroups: 1) an early-onset form with ophthalmoparesis, 2) an early-onset form without ophthalmoparesis, and 3) a late-onset form without ophthalmoparesis. Clinically, autosomal dominant CNM (AD CNM) is relatively mild and usually presents in adults with a diffuse weakness that is slowly progressive and may be accompanied by muscle hypertrophy. Overall, the autosomal disorders are not as clinically uniform as XLMTM, which has made their genetic characterization more difficult. Currently the responsible gene(s) remain unknown. This review will explore the historical evolution in understanding of these myopathies and give an update on their histopathologic features, genetics and pathogenesis.

SPEG interacts with myotubularin, and its deficiency causes centronuclear myopathy with dilated cardiomyopathy.

Centronuclear myopathies (CNMs) are characterized by muscle weakness and increased numbers of central nuclei within myofibers. X-linked myotubular myopathy, the most common severe form of CNM, is caused by mutations in MTM1, encoding myotubularin (MTM1), a lipid phosphatase. To increase our understanding of MTM1 function, we conducted a yeast two-hybrid screen to identify MTM1-interacting proteins. Striated muscle preferentially expressed protein kinase (SPEG), the product of SPEG complex locus (SPEG), was identified as an MTM1-interacting protein, confirmed by immunoprecipitation and immunofluorescence studies. SPEG knockout has been previously associated with severe dilated cardiomyopathy in a mouse model. Using whole-exome sequencing, we identified three unrelated CNM-affected probands, including two with documented dilated cardiomyopathy, carrying homozygous or compound-heterozygous SPEG mutations. SPEG was markedly reduced or absent in two individuals whose muscle was available for immunofluorescence and immunoblot studies. Examination of muscle samples from Speg-knockout mice revealed an increased frequency of central nuclei, as seen in human subjects. SPEG localizes in a double line, flanking desmin over the Z lines, and is apparently in alignment with the terminal cisternae of the sarcoplasmic reticulum. Examination of human and murine MTM1-deficient muscles revealed similar abnormalities in staining patterns for both desmin and SPEG. Our results suggest that mutations in SPEG, encoding SPEG, cause a CNM phenotype as a result of its interaction with MTM1. SPEG is present in cardiac muscle, where it plays a critical role; therefore, individuals with SPEG mutations additionally present with dilated cardiomyopathy.

Normal myofibrillar development followed by progressive sarcomeric disruption with actin accumulations in a mouse Cfl2 knockout demonstrates requirement of cofilin-2 for muscle maintenance

Cofilin-2, a small actin-binding protein and member of the AC protein family that includes cofilin-1 and destrin, is predominantly expressed at sarcomeres in skeletal and cardiac muscles. The role of cofilin-2 in muscle development and function is unclear. In humans, recessive cofilin-2 mutations have been associated with nemaline myopathy with minicores. To investigate the functional role of cofilin-2 in vivo, we generated constitutive and muscle-specific cofilin-2-deficient mice using a cre-loxP strategy. Cofilin-2-deficient mice were similar to their wild-type (WT) littermates at birth, but died by day 8. They were significantly smaller, severely weak and had very little milk in their stomachs. The sarcomeric structure was intact at birth, but by Day 7, skeletal muscles showed severe sarcomeric disruptions starting at the Z-line, along with filamentous actin accumulations consistent with a lack of actin depolymerization activity. Cofilin-2-deficient muscles contained elevated numbers of slow fibers and exhibited upregulation of slow fiber-specific genes. Increased amounts of other sarcomeric proteins including α-actinin-2, α-sarcomeric actin and tropomyosin were also present. While destrin was not expressed in either WT or cofilin-2-deficient muscles, cofilin-1 was similarly expressed in developing myofibers of both genotypes. There was no evidence for compensatory changes in expression of either family member in cofilin-2-deficient tissues. The onset of pathology and weakness in cofilin-2-deficient muscles correlated with normal developmental loss of cofilin-1 expression within myofibers, suggesting that cofilin-1 serves as an early developmental sarcomeric isoform. Overall, cofilin-2, although not critical for muscle development, is essential for muscle maintenance.

Connective tissue growth factor causes EMT-like cell fate changes in vivo and in vitro

Summary Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of chronic fibrotic diseases. However, the mechanism by which paracrine effects of CTGF control the cell fate of neighboring epithelial cells is not known. In this study, we investigated the paracrine effects of CTGF overexpressed in fibroblasts of Col1a2-CTGF transgenic mice on epithelial cells of skin and lung. The skin and lungs of Col1a2-CTGF transgenic mice were examined for phenotypic markers of epithelial activation and differentiation and stimulation of signal transduction pathways. In addition to an expansion of the dermal compartment in Col1a2-CTGF transgenic mice, the epidermis was characterized by focal hyperplasia, and basal cells stained positive for &agr;SMA, Snail, S100A4 and Sox9, indicating that these cells had undergone a change in their genetic program. Activation of phosphorylated p38 and phosphorylated Erk1/2 was observed in the granular and cornified layers of the skin. Lung fibrosis was associated with a marked increase in cells co-expressing epithelial and mesenchymal markers in the lesional and unaffected lung tissue of Col1a2-CTGF mice. In epithelial cells treated with TGF&bgr;, CTGF-specific siRNA-mediated knockdown suppressed Snail, Sox9, S100A4 protein levels and restored E-cadherin levels. Both adenoviral expression of CTGF in epithelial cells and treatment with recombinant CTGF induced EMT-like morphological changes and expression of &agr;-SMA. Our in vivo and in vitro data supports the notion that CTGF expression in mesenchymal cells in the skin and lungs can cause changes in the differentiation program of adjacent epithelial cells. We speculate that these changes might contribute to fibrogenesis.

Whole genome sequencing identifies SCN2A mutation in monozygotic twins with Ohtahara syndrome and unique neuropathologic findings

Mutations in SCN2A gene cause a variety of epilepsy syndromes. We report a novel SCN2A‐associated epilepsy phenotype in monozygotic twins with tonic seizures soon after birth and a suppression‐burst electroencephalography (EEG) pattern. We reviewed the medical records, EEG tracings, magnetic resonance imaging (MRI), and neuropathologic findings, and performed whole genome sequencing (WGS) on Twin Bs DNA and Sanger sequencing (SS) on candidate gene mutations. Extensive neurometabolic evaluation and early neuroimaging studies were normal. Twin A died of an iatrogenic cause at 2 weeks of life. His neuropathologic examination was remarkable for dentate‐olivary dysplasia and granule cell dispersion of the dentate gyrus. Twin B became seizure free at 8 months and was off antiepileptic drugs by 2 years. His brain MRI, normal at 2 months, revealed evolving brainstem and basal ganglia abnormalities at 8 and 15 months that resolved by 20 months. At 2.5 years, Twin B demonstrated significant developmental delay. Twin Bs WGS revealed a heterozygous variant c.788C>T predicted to cause p.Ala263Val change in SCN2A and confirmed to be de novo in both twins by SS. In conclusion, we have identified a de novo SCN2A mutation as the etiology for Ohtahara syndrome in monozygotic twins associated with a unique dentate‐olivary dysplasia in the deceased twin.

Congenital myopathy caused by a novel missense mutation in the CFL2 gene

Nemaline myopathy and myofibrillar myopathy are heterogeneous myopathies that both comprise early-onset forms. We present two sisters from a consanguineous Iraqi Kurdish family with predominant axial and limb girdle weakness. Muscle biopsies showed features of both nemaline myopathy and myofibrillar myopathy. We performed homozygosity mapping in both siblings using an Affymetrix 250K Nspl SNP array. One of the overlapping homozygous regions harbored the gene CFL2. Because a mutation in CFL2 was identified in a family with nemaline myopathy, we performed sequence analysis of the gene and a novel homozygous missense mutation in exon 2 (c.19G>A, p.Val7Met) of CFL2 was identified in both siblings. CFL2 encodes the protein cofilin-2, which plays an important role in regulation of sarcomeric actin filaments. To our knowledge, this is the second family in which a mutation in CFL2 causes an autosomal recessive form of congenital myopathy with features of both nemaline and myofibrillar myopathy. Given the clinical variability and the multitude of histological features of congenital myopathies, CFL2 sequence analysis should be considered in patients presenting with an autosomal recessive form of congenital myopathy.

Epidemiology of Systemic Candidiasis in a Tertiary Care Neonatal Unit

143 neonates were diagnosed to have acquired systemic candidiasis out of a total 4530 admissions (3.2 per cent) to the neonatal intensive care unit (NICU) during a period of 6 1/2 years from January 1990 to June 1996. Mean age at onset was 10.4 days, mean birth weight 1454 g, and mean gestation was 31.7 weeks. Ninety-four per cent were premature, 95 per cent low birth weight (LBW), and all had undergone peripheral vein catheterization and had received broad spectrum antibiotics, except one, prior to the diagnosis. Fifty-eight per cent were ventilated and 15 per cent received parenteral nutrition. Persistent/recurrent pneumonia, apnoea, lethargy, high gastric aspirates, and abdominal distension were the common clinical manifestations. Candida tropicalis, C. albicans, and C. guillermondii were the most common isolates. Blood and urine were the predominant sites for isolation of Candida. Fluconazole was the most used antifungal agent, with 24 per cent resistance against it. Fifty per cent of babies died due to all causes. Of all the deaths, two-thirds were Candida related. Candida-attributable deaths occurred in 24 cases (17 per cent).