Mutations of tropomyosin 3 (TPM3) are common and associated with type 1 myofiber hypotrophy in congenital fiber type disproportion

Congenital fiber type disproportion (CFTD) is a rare congenital myopathy characterized by hypotonia and generalized muscle weakness. Pathologic diagnosis of CFTD is based on the presence of type 1 fiber hypotrophy of at least 12% in the absence of other notable pathological findings. Mutations of the ACTA1 and SEPN1 genes have been identified in a small percentage of CFTD cases. The muscle tropomyosin 3 gene, TPM3, is mutated in rare cases of nemaline myopathy that typically exhibit type 1 fiber hypotrophy with nemaline rods, and recently mutations in the TPM3 gene were also found to cause CFTD. We screened the TPM3 gene in patients with a clinical diagnosis of CFTD, nemaline myopathy, and with undefined congenital myopathies. Mutations in TPM3 were identified in 6 out of 13 patients with CFTD, as well as in one case of nemaline myopathy. Review of muscle biopsies from patients with diagnoses of CFTD revealed that patients with a TPM3 mutation all displayed marked disproportion of fiber size, without type 1 fiber predominance. Several mutation‐negative cases exhibited other abnormalities, such as central nuclei and central cores. These results support the utility of the CFTD diagnosis in directing the course of genetic testing. Hum Mutat 30:1–8, 2009. © 2009 Wiley‐Liss, Inc.     

Recessive ACTA1 variant causes congenital muscular dystrophy with rigid spine

Variants in ACTA1, which encodes α-skeletal actin, cause several congenital myopathies, most commonly nemaline myopathy. Autosomal recessive variants comprise approximately 10% of ACTA1 myopathy. All recessive variants reported to date have resulted in loss of skeletal α-actin expression from muscle and severe weakness from birth. Targeted next-generation sequencing in two brothers with congenital muscular dystrophy with rigid spine revealed homozygous missense variants in ACTA1. Skeletal α-actin expression was preserved in these patients. This report expands the clinical and histological phenotype of ACTA1 disease to include congenital muscular dystrophy with rigid spine and dystrophic features on muscle biopsy. This represents a new class of recessive ACTA1 variants, which do not abolish protein expression.     

Thin filament proteins mutations associated with skeletal myopathies: Defective regulation of muscle contraction

In humans, more than 140 different mutations within seven genes (ACTA1, TPM2, TPM3, TNNI2, TNNT1, TNNT3, and NEB) that encode thin filament proteins (skeletal α-actin, β-tropomyosin, γ-tropomyosin, fast skeletal muscle troponin I, slow skeletal muscle troponin T, fast skeletal muscle troponin T, and nebulin, respectively) have been identified. These mutations have been linked to muscle weakness and various congenital skeletal myopathies including nemaline myopathy, distal arthrogryposis, cap disease, actin myopathy, congenital fiber type disproportion, rod-core myopathy, intranuclear rod myopathy, and distal myopathy, with a dramatic negative impact on the quality of life. In this review, we discuss studies that use various approaches such as patient biopsy specimen samples, tissue culture systems or transgenic animal models, and that demonstrate how thin filament proteins mutations alter muscle structure and contractile function. With an enhanced understanding of the cellular and molecular mechanisms underlying muscle weakness in patients carrying such mutations, better therapy strategies can be developed to improve the quality of life.     

The new neuromuscular disease related with defects in the ASC‐1 complex: report of a second case confirms ASCC1 involvement

Next‐generation sequencing technology aided the identification of the underlying genetic cause in a female newborn with a severe neuromuscular disorder. The patient presented generalized hypotonia, congenital bone fractures, lack of spontaneous movements and poor respiratory effort. She died within the first days of life. Karyotyping and screening for several genes related with neuromuscular diseases all tested negative. A male sibling was subsequently born with the same clinical presentation. Whole‐exome sequencing was performed with variant filtering assuming a recessive disease model. Analysis focused on genes known to be related firstly with congenital myopathies, extended to muscle diseases and finally to other neuromuscular disorders. No disease‐causing variants were identified. A similar disorder was described in patients with recessive variants in two genes: TRIP4 (three families) and ASCC1 (one family), both encoding subunits of the nuclear activating signal cointegrator 1 (ASC‐1) complex. Our patient was also found to have a homozygous frameshift variant (c.157dupG, p.Glu53Glyfs*19) in ASCC1 , thereby representing the second known case. This confirms ASCC1 involvement in a severe neuromuscular disease lying within the spinal muscular atrophy or primary muscle disease spectra.     

Congenital myopathies: diseases of the actin cytoskeleton

Congenital myopathies are clinical and genetic heterogeneous disorders characterized by skeletal muscle weakness ranging in severity. Three major forms have been identified: actin myopathy, intranuclear rod myopathy, and nemaline myopathy. Nemaline myopathy is the most common of these myopathies and is further subdivided into seven groups according to severity, progressiveness, and age of onset. At present, five genes have been linked to congenital myopathies. These include alpha-actin (ACTA1), alpha- and beta-tropomyosin (TPM3 and TPM2), troponin T (TNNT1), and nebulin (NEB). Their protein products are all components of the thin filament of the sarcomere. The mutations identified within these genes have varying impacts on protein structure and give rise to different forms of congenital myopathies. Greater understanding of muscle formation and cause of disease can be established through the study of the effect of mutations on the functional proteins. However, a major limitation in the understanding of congenital myopathies is the lack of correlation between the degree of sarcomeric disruption and disease severity. Consequently, great difficulty may be encountered when diagnosing patients and predicting the progression of the disorders. There are no existing cures for congenital myopathies, although improvements can be made to both the standard of living and the life expectancy of the patient through various therapies.     

An updated and upgraded L1CAM mutation database

The L1 syndrome is an X‐linked recessive disease caused by mutations in the L1CAM gene. To date more than 200 different mutations have been reported, scattered over the entire gene, about 35% being missense mutations. Although it is tempting to consider these missense mutations as being disease‐causing, one should be careful in drawing any firm conclusions, unless there is additional supporting information. This is in contrast to truncating mutations, which are always considered to be disease‐causing, unless they involve truncations close to the gene stop codon. In order to allow conclusions to be drawn on the disease‐causing nature of L1CAM (missense) mutations, we have updated and upgraded our LICAM mutation database with more pathogenicity data and clinical information collected from the literature or generated by our own research. As a result, the renewed database offers condensed scientific information, allowing conclusions to be drawn on the pathogenicity and severity of LICAM mutations based on multiple factors. The L1CAMMutation Database is at: www.l1cammutationdatabase.info . © 2009 Wiley‐Liss, Inc.     

Congenital posterior pole cataract and adult onset dilating cardiomyopathy: expanding the phenotype of αB‐crystallinopathies

Mutations in the αB‐crystallin gene (CRYAB) have been reported in desmin‐related myopathies, with or without cardiac involvement. Mutations in this gene have also been documented in large multi‐generation families with autosomal dominant congenital posterior pole cataract (CPPC). In these congenital cataract families no cardiac or muscular phenotype was reported. This report describes a family with an unusual read‐through mutation in CRYAB, leading to the elongation of the normal αB‐crystallin protein with 19 amino acid residues. Affected family members combine a CPPC with an adult onset dilated cardiomyopathy (DCM), thereby expanding the αB‐crystallinopathy phenotype. Repolarisation abnormalities preceded the onset of cardiomyopathy and were already present in childhood. No skeletal myopathy was observed. This report illustrates that congenital cataract can be a prelude to more severe disease even outside the context of inborn errors of metabolism. The identification of a CRYAB mutation in this family supports the notion that mutations in this gene are a rare cause of genetically determined DCM. The combined congenital cataract/cardiomyopathy phenotype adds to our understanding of the complex phenotypic spectrum of αB‐crystallinopathies.     

Effects of high‐intensity intermittent swimming on PGC‐1α protein expression in rat skeletal muscle

Aim: The aim of the present investigation was to elucidate the effects of exercise intensity on exercise‐induced expression of peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α) protein in rat skeletal muscle. Methods: We measured PGC‐1α content in the skeletal muscles of male Sprague–Dawley rats (age: 5–6 weeks old; body weight: 150–170 g) after a single session of high‐intensity intermittent exercise (HIE) or low‐intensity prolonged swimming exercise (LIE). During HIE, the rats swam for fourteen 20‐s periods carrying a weight (14% of body weight), and the periods of swimming were separated by a 10‐s pause. LIE rats swam with no load for 6 h in two 3‐h sessions, separated by 45 min of rest. Results: After HIE, the PGC‐1α protein content in rat epitrochlearis muscle had increased by 126, 140 and 126% at 2, 6 and 18 h, respectively, compared with that of the age‐matched sedentary control rats’ muscle. Immediately, 6 and 18‐h after LIE, the PGC‐1α protein content in the muscle was significantly elevated by 84, 95 and 67% respectively. The PGC‐1α protein content observed 6 h after HIE tended to be higher than that observed after LIE. However, there was no statistically significant difference between the two values (P = 0.12). Conclusion: The present investigation suggests that irrespective of the intensity of the exercise, PGC‐1α protein content in rat skeletal muscle increases to a comparable level when stimuli induced by different protocols are saturated. Further, HIE is a potent stimulus for enhancing the expression of PGC‐1αprotein, which may induce mitochondrial biogenesis in exercise‐activated skeletal muscle.     

De novo RYR1 heterozygous mutation (I4898T) causing lethal core-rod myopathy in twins

"Core-rod myopathy" is a rare congenital myopathy characterized by the presence of "cores" and "rods" in distinct locations in the same or different muscle fibres. This association is linked currently to mutations in RYR1, NEB and ACTA1 genes. We report identical twins who presented with polyhydramnios and loss of fetal motility during pregnancy; hypotonia, arthrogryposis and swallowing impairment at birth; need of immediate respiratory support and death at 27 and 50 days of life. Muscle biopsies, performed at 27 days of life in twin 1 and at 49 days in twin 2, showed the presence of separate cores and rods in the muscle fibres, both at light and electron microscopy. The molecular analysis showed a heterozygous de novo mutation (Ile4898Thr) of the RYR1 gene. The molecular study of ACTA1, TMP2 and TMP3 genes did not show abnormalities. This is the first report of a lethal form of congenital "core-rod myopathy". The mutation Ile4898Thr has been previously described in central core disease but not in core-rod myopathy. The report enlarges the phenotypic spectrum of "core-rod myopathy" and highlights the morphological variability associated to special RYR1 mutations.     

Novel Mutations Widen the Phenotypic Spectrum of Slow Skeletal/β‐Cardiac Myosin (MYH7) Distal Myopathy

Laing early onset distal myopathy and myosin storage myopathy are caused by mutations of slow skeletal/β‐cardiac myosin heavy chain encoded by the gene MYH7, as is a common form of familial hypertrophic/dilated cardiomyopathy. The mechanisms by which different phenotypes are produced by mutations in MYH7, even in the same region of the gene, are not known. To explore the clinical spectrum and pathobiology, we screened the MYH7 gene in 88 patients from 21 previously unpublished families presenting with distal or generalized skeletal muscle weakness, with or without cardiac involvement. Twelve novel mutations have been identified in thirteen families. In one of these families, the father of the proband was found to be a mosaic for the MYH7 mutation. In eight cases, de novo mutation appeared to have occurred, which was proven in four. The presenting complaint was footdrop, sometimes leading to delayed walking or tripping, in members of 17 families (81%), with other presentations including cardiomyopathy in infancy, generalized floppiness, and scoliosis. Cardiac involvement as well as skeletal muscle weakness was identified in nine of 21 families. Spinal involvement such as scoliosis or rigidity was identified in 12 (57%). This report widens the clinical and pathological phenotypes, and the genetics of MYH7 mutations leading to skeletal muscle diseases.     

CSGALNACT1‐congenital disorder of glycosylation: A mild skeletal dysplasia with advanced bone age

Congenital disorders of glycosylation (CDGs) comprise a large number of inherited metabolic defects that affect the biosynthesis and attachment of glycans. CDGs manifest as a broad spectrum of disease, most often including neurodevelopmental and skeletal abnormalities and skin laxity. Two patients with biallelic CSGALNACT1 variants and a mild skeletal dysplasia have been described previously. We investigated two unrelated patients presenting with short stature with advanced bone age, facial dysmorphism, and mild language delay, in whom trio‐exome sequencing identified novel biallelic CSGALNACT1 variants: compound heterozygosity for c.1294G>T (p.Asp432Tyr) and the deletion of exon 4 that includes the start codon in one patient, and homozygosity for c.791A>G (p.Asn264Ser) in the other patient. CSGALNACT1 encodes CSGalNAcT‐1, a key enzyme in the biosynthesis of sulfated glycosaminoglycans chondroitin and dermatan sulfate. Biochemical studies demonstrated significantly reduced CSGalNAcT‐1 activity of the novel missense variants, as reported previously for the p.Pro384Arg variant. Altered levels of chondroitin, dermatan, and heparan sulfate moieties were observed in patients’ fibroblasts compared to controls. Our data indicate that biallelic loss‐of‐function mutations in CSGALNACT1 disturb glycosaminoglycan synthesis and cause a mild skeletal dysplasia with advanced bone age, CSGALNACT1‐CDG.     

OBSL1 mutations in 3‐M syndrome are associated with a modulation of IGFBP2 and IGFBP5 expression levels

3‐M syndrome is an autosomal recessive disorder characterized by severe pre‐ and postnatal growth retardation and minor skeletal changes. We have previously identified CUL7 as a disease‐causing gene but we have also provided evidence of genetic heterogeneity in the 3‐M syndrome. By homozygosity mapping in two inbred families, we found a second disease locus on chromosome 2q35–36.1 in a 5.2‐Mb interval that encompasses 60 genes. To select candidate genes, we performed microarray analysis of cultured skin fibroblast RNA from one patient, looking for genes with altered expression; we found decreased expression of IGFBP2 and increased expression of IGFBP5. However, direct sequencing of these two genes failed to detect any anomaly. We then considered other candidate genes by their function/location and found nine distinct mutations in the OBSL1 gene in 13 families including eight nonsense and one missense mutations. To further understand the links between OBSL1, CUL7, and insulin‐like growth factor binding proteins (IGFBPs), we performed real‐time quantitative PCR (RT‐PCR) analysis for OBSL1, CUL7, IGFBP2, and IGFBP5, using cultured fibroblast RNAs from two patients with distinct OBSL1 mutations (p.F697G; p.H814RfsX15). We found normal CUL7 mRNA levels but abnormal IGFBP2 and IGFBP5 mRNA levels in the two patients, suggesting that OBSL1 modulates the expression of IGFBP proteins. Hum Mutat 30:1–7, 2009. © 2009 Wiley‐Liss, Inc.     

Spectrum and detection rate of L1CAM mutations in isolated and familial cases with clinically suspected L1‐disease

Mutations in L1CAM, the gene encoding the L1 neuronal cell adhesion molecule, lead to an X‐linked trait characterized by one or more of the symptoms of hydrocephalus, adducted thumbs, agenesis or hypoplasia of corpus callosum, spastic paraplegia, and mental retardation (L1‐disease). We screened 153 cases with prenatally or clinically suspected X‐chromosomal hydrocephalus for L1CAM mutations by SSCP analysis of the 28 coding exons and regulatory elements in the 5′‐untranslated region of the gene. Forty‐six pathogenic mutations were found (30.1% detection rate), the majority consisting of nonsense, frameshift, and splice site mutations. In eight cases, segregation analysis disclosed recent de novo mutations. Statistical analysis of the data indicates a significant effect on mutation detection rate of (i) family history, (ii) number of L1‐disease typical clinical findings, and (iii) presence or absence of signs not typically associated with L1CAM‐disease. Whereas mutation detection rate was 74.2% for patients with at least two additional cases in the family, only 16 mutations were found in the 102 cases with negative family history (15.7% detection rate). Our data suggest a higher than previously assumed contribution of L1CAM mutations in the pathogenesis of the heterogeneous group of congenital hydrocephalus. Am. J. Med. Genet. 92:40–46, 2000. © 2000 Wiley‐Liss, Inc.     

The novel αB‐crystallin (CRYAB) mutation p.D109G causes restrictive cardiomyopathy

Restrictive cardiomyopathy (RCM) is a rare heart disease characterized by diastolic dysfunction and atrial enlargement. The genetic etiology of RCM is not completely known. We identified by a next‐generation sequencing panel the novel CRYAB missense mutation c.326A>G, p.D109G in a small family with RCM in combination with skeletal myopathy with an early onset of the disease. CRYAB encodes αB‐crystallin, a member of the small heat shock protein family, which is highly expressed in cardiac and skeletal muscle. In addition to in silico prediction analysis, our structural analysis of explanted myocardial tissue of a mutation carrier as well as in vitro cell transfection experiments revealed abnormal protein aggregation of mutant αB‐crystallin and desmin, supporting the deleterious effect of this novel mutation. In conclusion, CRYAB appears to be a novel RCM gene, which might have relevance for the molecular diagnosis and the genetic counseling of further affected families in the future.     

Recessive RYR1 mutations in a patient with severe congenital nemaline myopathy with ophthalomoplegia identified through massively parallel sequencing

Nemaline myopathy (NM) is a group of congenital myopathies, characterized by the presence of distinct rod-like inclusions "nemaline bodies" in the sarcoplasm of skeletal muscle fibers. To date, ACTA1, NEB, TPM3, TPM2, TNNT1, and CFL2 have been found to cause NM. We have identified recessive RYR1 mutations in a patient with severe congenital NM, through high-throughput screening of congenital myopathy/muscular dystrophy-related genes using massively parallel sequencing with target gene capture. The patient manifested fetal akinesia, neonatal severe hypotonia with muscle weakness, respiratory insufficiency, swallowing disturbance, and ophthalomoplegia. Skeletal muscle histology demonstrated nemaline bodies and small type 1 fibers, but without central cores or minicores. Congenital myopathies, a molecularly, histopathologically, and clinically heterogeneous group of disorders are considered to be a good candidate for massively parallel sequencing.     

Characterization of mutations in fifty North American patients with X‐linked myotubular myopathy

X‐linked myotubular myopathy (MTM1) is a rare developmental disorder of skeletal muscle that is characterized by the presence of abnormal central nuclei in biopsy specimens taken from affected individuals. To date 133 different mutations have been identified in the MTM1 gene worldwide. We report here mutations detected in 50 additional U.S. families with biopsy‐proven MTM1. Forty‐one of the patients have not been described previously, including 18 with novel mutations. Eighty‐eight percent of the mothers of sporadic cases that were studied were identified as carriers, extending the previously reported high‐carrier frequency for this disorder. Clinical information collected on the majority of patients helps to further correlate genotype with phenotype, and implications of these data for genetic counseling in families are discussed. Hum Mutat 19:114–121, 2002. © 2002 Wiley‐Liss, Inc.     

Clustering of mutations associated with mild Marfan‐like phenotypes in the 3′ region of FBN1 suggests a potential genotype–phenotype correlation

Mutations in the gene for fibrillin‐1 (FBN1) cause Marfan syndrome, a dominantly inherited disorder of connective tissue that primarily involves the cardiovascular, ocular, and skeletal systems. There is a remarkable degree of variability both within and between families with Marfan syndrome, and FBN1 mutations have also been found in a range of other related connective tissue disorders collectively termed type‐1 fibrillinopathies. FBN1 mutations have been found in almost all of the 65 exons of the FBN1 gene and for the most part have been unique to one affected patient or family. Aside from the “hot spots” for the neonatal Marfan syndrome in exons 24–27 and 31–32, genotype–phenotype correlations have been slow to emerge. Here we present the results of temperature‐gradient gel electrophoresis analysis of FBN1 exons 59–65. Six mutations were identified, only one of which had been previously reported. Two of the six mutations were found in patients with mild phenotypes. Taken together with other published reports, our results suggest that a sizable subset (ca. 40%) of mutations in this region is associated with mild phenotypes characterized by the lack of significant aortic pathology, compared with about 7% in the rest of the gene. In two cases, mutations affecting analogous positions within one of the 43 cbEGF modules of FBN1 are associated with mild phenotypes when found in one of the 6 C‐terminal modules (encoded by exons 59–63), but are associated with classic or severe phenotypes when found in cbEGF modules elsewhere in the gene. Am. J. Med. Genet. 91:212–221, 2000. © 2000 Wiley‐Liss, Inc.