Deficiency in Cardiac Dystrophin Affects the Abundance of the $\alpha$ -/ $\beta$ -Dystroglycan Complex

Although Duchenne muscular dystrophy is primarilycategorised as a skeletal muscle disease, deficiency in themembrane cytoskeletal protein dystrophin also affects the heart.The central transsarcolemmal linker between the actin membranecytoskeleton and the extracellular matrix is represented by thedystrophin-associated dystroglycans. Chemical cross-linkinganalysis revealed no significant differences in the dimericstatus of the α-/β-dystroglycan subcomplex in thedystrophic mdx heart as compared to normalcardiac tissue. In analogy to skeletal muscle fibres, heartmuscle also exhibited a greatly reduced abundance of bothdystroglycans in dystrophin-deficient cells. Immunoblottingdemonstrated that the degree of reduction inα-dystroglycan is more pronounced in matured mdxskeletal muscle as contrasted to the mdx heart. The factthat the deficiency in dystrophin triggers a similarpathobiochemical response in both types of muscle suggests thatthe cardiomyopathic complications observed inx-linked muscular dystrophy might be initiated bythe loss of the dystrophin-associated surface glycoproteincomplex.     

Novel cardiovascular findings in association with a POMT2 mutation: three siblings with α-dystroglycanopathy

Dystroglycanopathies are a genetically heterogeneous subset of congenital muscular dystrophies that exhibit autosomal recessive inheritance and are characterized by abnormal glycosylation of α-dystroglycan. In particular, POMT2 (protein O-mannosyltransferase-2) mutations have been identified in congenital muscular dystrophy patients with a wide range of clinical involvement, ranging from the severe muscle-eye-brain disease and Walker–Warburg syndrome to limb girdle muscular dystrophy without structural brain or ocular involvement. Cardiovascular disease is thought to be uncommon in congenital muscular dystrophy, with rare reports of cardiac involvement. We describe three brothers aged 21, 19, and 17 years with an apparently homozygous POMT2 mutation who all presented with congenital muscular dystrophy, intellectual disabilities, and distinct cardiac abnormalities. All three brothers were homozygous for a p.Tyr666Cys missense mutation in exon 19 of the POMT2 gene. On screening echocardiograms, all siblings demonstrated significant dilatation of the aortic root and depressed left ventricular systolic function and/or left ventricular wall motion abnormalities. Our report is the first to document an association between POMT2 mutations and aortopathy with concomitant depressed left ventricular systolic function. On the basis of our findings, we suggest patients with POMT2 gene mutations be screened not only for myocardial dysfunction but also for aortopathy. In addition, given the potential for progression of myocardial dysfunction and/or aortic dilatation, longitudinal surveillance imaging is recommended both for patients with disease as well as those that have normal baseline imaging.     

Novel bi-allelic variants expand the SPTBN4-related genetic and phenotypic spectrum

Neurodevelopmental disorder with hypotonia, neuropathy, and deafness (NEDHND, OMIM #617519) is an autosomal recessive disease caused by homozygous or compound heterozygous variants in SPTBN4 coding for type 4 βIV-spectrin, a non-erythrocytic member of the β-spectrin family. Variants in SPTBN4 disrupt the cytoskeletal machinery that controls proper localization of ion channels and the function of axonal domains, thereby generating severe neurological dysfunction. We set out to analyze the genetic causes and describe the clinical spectrum of suspected cases of NEDHND. Variant screening was done by whole exome sequencing; clinical phenotypes were described according to the human phenotype ontology, and histochemical analysis was performed with disease-specific antibodies. We report four families with five patients harboring novel homozygous and compound heterozygous SPTBN4 variants, amongst them a multi-exon deletion of SPTBN4. All patients presented with the key features of NEDHND; severe muscular hypotonia, dysphagia, absent speech, gross motor, and mental retardation. Additional symptoms comprised horizontal nystagmus, epileptiform discharges in EEG without manifest seizures, and choreoathetosis. Muscle histology revealed both characteristics of myopathy and of neuropathy. This report expands the SPTBN4 variant spectrum, highlights the spectrum of morphological phenotypes of NEDHND-patients, and reveals clinical similarities between the NEDHND, non-5q SMA, and congenital myopathies.Subject terms: Genetics research, Disease genetics     

Cardiomyopathy in patients with POMT1-related congenital and limb-girdle muscular dystrophy

Protein-o-mannosyl transferase 1 (POMT1) is a glycosyltransferase involved in α-dystroglycan (α-DG) glycosylation. Clinical phenotype in POMT1-mutated patients ranges from congenital muscular dystrophy (CMD) with structural brain abnormalities, to limb-girdle muscular dystrophy (LGMD) with microcephaly and mental retardation, to mild LGMD. No cardiac involvement has until now been reported in POMT1-mutated patients. We report three patients who harbored compound heterozygous POMT1 mutations and showed left ventricular (LV) dilation and/or decrease in myocardial contractile force: two had a LGMD phenotype with a normal or close-to-normal cognitive profile and one had CMD with mental retardation and normal brain MRI. Reduced or absent α-DG immunolabeling in muscle biopsies were identified in all three patients. Bioinformatic tools were used to study the potential effect of POMT1-detected mutations. All the detected POMT1 mutations were predicted in silico to interfere with protein folding and/or glycosyltransferase function. The report on the patients described here has widened the clinical spectrum associated with POMT1 mutations to include cardiomyopathy. The functional impact of known and novel POMT1 mutations was predicted with a bioinformatics approach, and results were compared with previous in vitro studies of protein-o-mannosylase function.     

Laminin-111 Restores Regenerative Capacity in a Mouse Model for α7 Integrin Congenital Myopathy

Mutations in the α7 integrin gene cause congenital myopathy characterized by delayed developmental milestones and impaired mobility. Previous studies in dystrophic mice suggest the α7β1 integrin may be critical for muscle repair. To investigate the role that α7β1 integrin plays in muscle regeneration, cardiotoxin was used to induce damage in the tibialis anterior muscle of α7 integrin-null mice. Unlike wild-type muscle, which responded rapidly to repair damaged myofibers, α7 integrin-deficient muscle exhibited defective regeneration. Analysis of Pax7 and MyoD expression revealed a profound delay in satellite cell activation after cardiotoxin treatment in α7 integrin-null animals when compared with wild type. We have recently demonstrated that the muscle of α7 integrin-null mice exhibits reduced laminin-α2 expression. To test the hypothesis that loss of laminin contributes to the defective muscle regeneration phenotype observed in α7 integrin-null mice, mouse laminin-111 (α1, β1, γ1) protein was injected into the tibialis anterior muscle 3 days before cardiotoxin-induced injury. The injected laminin-111 protein infiltrated the entire muscle and restored myogenic repair and muscle regeneration in α7 integrin-null muscle to wild-type levels. Our data demonstrate a critical role for a laminin-rich microenvironment in muscle repair and suggest laminin- 111 protein may serve as an unexpected and novel therapeutic agent for patients with congenital myopathies.     

Mutations and polymorphisms of the skeletal muscle α‐actin gene (ACTA1)

The ACTA1 gene encodes skeletal muscle α‐actin, which is the predominant actin isoform in the sarcomeric thin filaments of adult skeletal muscle, and essential, along with myosin, for muscle contraction. ACTA1 disease‐causing mutations were first described in 1999, when a total of 15 mutations were known. In this article we describe 177 different disease‐causing ACTA1 mutations, including 85 that have not been described before. ACTA1 mutations result in five overlapping congenital myopathies: nemaline myopathy; intranuclear rod myopathy; actin filament aggregate myopathy; congenital fiber type disproportion; and myopathy with core‐like areas. Mixtures of these histopathological phenotypes may be seen in a single biopsy from one patient. Irrespective of the histopathology, the disease is frequently clinically severe, with many patients dying within the first year of life. Most mutations are dominant and most patients have de novo mutations not present in the peripheral blood DNA of either parent. Only 10% of mutations are recessive and they are genetic or functional null mutations. To aid molecular diagnosis and establishing genotype–phenotype correlations, we have developed a locus‐specific database for ACTA1 variations ( http://waimr.uwa.edu.au ). Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Muscle hypertrophy as the presenting sign in a patient with a complete FHL1 deletion

Four and a half LIM protein 1 (FHL1/SLIM1) has recently been identified as the causative gene mutated in four distinct diseases affecting skeletal muscle that have overlapping features, including reducing body myopathy, X‐linked myopathy, X‐linked dominant scapuloperoneal myopathy and Emery–Dreifuss muscular dystrophy. FHL1 localises to the sarcomere and the sarcolemma and is believed to participate in muscle growth and differentiation as well as in sarcomere assembly. We describe in this case report a boy with a deletion of the entire FHL1 gene who is now 15 years of age and presented with muscle hypertrophy, reduced subcutaneous fat, rigid spine and short stature. This case is the first, to our knowledge, with a complete loss of the FHL1 protein and MAP7D3 in combination. It supports the theory that dominant negative effects (accumulation of cytotoxic‐mutated FHL1 protein) worsen the pathogenesis. It extends the phenotype of FHL1‐related myopathies and should prompt future testing in undiagnosed patients who present with unexplained muscle hypertrophy, contractures and rigid spine, particularly if male.     

Amyloid-β Deposition in Skeletal Muscle of Transgenic Mice : Possible Model of Inclusion Body Myopathy

Inclusion body myopathy is a progressive muscle disorder characterized by nuclear and cytoplasmic inclusions and vacuolation of muscle fibers. Affected muscle fibers contain deposits of congophilic amyloid, amyloid-β immunoreactive filaments, and paired helical filaments, all of which are pathological hallmarks of Alzheimer’s disease in brain. Accumulations of amyloid-β and its precursor are thought to play important roles in the pathogenesis of both inclusion body myopathy and Alzheimer’s disease. Overexpression of mutant forms of β protein precursor in transgenic mice by neuron-specific promoters has been reported to cause amyloid deposits in the brain. Here we report that overexpression in transgenic mice of the signal plus 99-amino acid carboxyl-terminal sequences of β protein precursor, under the control of a cytomegalovirus enhancer/β-actin promoter, resulted in vacuolation and increasing accumulation of the 4-kd amyloid-β and the carboxyl-terminus in skeletal muscle fibers during aging. These deposits in transgenic muscle only rarely showed Congo red birefringence. Thus, overexpression of part of β protein precursor in transgenic mice led to development of some of the characteristic features of inclusion body myopathy. These mice may be a useful model of inclusion body myopathy, which shares a number of pathological markers with Alzheimer’s disease.     

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.     

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.     

Identification of two novel SMCHD1 sequence variants in families with FSHD-like muscular dystrophy

Facioscapulohumeral muscular dystrophy 1 (FSHD1) is caused by a contraction in the number of D4Z4 repeats on chromosome 4, resulting in relaxation of D4Z4 chromatin causing inappropriate expression of DUX4 in skeletal muscle. Clinical severity is inversely related to the number of repeats. In contrast, FSHD2 patients also have inappropriate expression of DUX4 in skeletal muscle, but due to constitutional mutations in SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1), which cause global hypomethylation and hence general relaxation of chromatin. Thirty patients originally referred for FSHD testing were screened for SMCHD1 mutations. Twenty-nine had >11 D4Z4 repeats. SMCHD1 c.1040+1G>A, a pathogenic splice-site variant, was identified in a FSHD1 family with a borderline number of D4Z4 repeats (10) and a variable phenotype (in which a LMNA1 sequence variant was previously described), and SMCHD1 c.2606 G>T, a putative missense variant (p.Gly869Val) with strong in vitro indications of pathogenicity, was identified in a family with an unusual muscular dystrophy with some FSHD-like features. The two families described here emphasise the genetic complexity of muscular dystrophies. As SMCHD1 has a wider role in global genomic methylation, the possibility exists that it could be involved in other complex undiagnosed muscle disorders. Thus far, only 15 constitutional mutations have been identified in SMCHD1, and these two sequence variants add to the molecular and phenotypic spectrum associated with FSHD.     

POMT2 mutations cause alpha-dystroglycan hypoglycosylation and Walker-Warburg syndrome

Background: Walker-Warburg syndrome (WWS) is an autosomal recessive condition characterised by congenital muscular dystrophy, structural brain defects, and eye malformations. Typical brain abnormalities are hydrocephalus, lissencephaly, agenesis of the corpus callosum, fusion of the hemispheres, cerebellar hypoplasia, and neuronal overmigration, which causes a cobblestone cortex. Ocular abnormalities include cataract, microphthalmia, buphthalmos, and Peters anomaly. WWS patients show defective O-glycosylation of α-dystroglycan (α-DG), which plays a key role in bridging the cytoskeleton of muscle and CNS cells with extracellular matrix proteins, important for muscle integrity and neuronal migration. In 20% of the WWS patients, hypoglycosylation results from mutations in either the protein O-mannosyltransferase 1 (POMT1), fukutin, or fukutin related protein (FKRP) genes. The other genes for this highly heterogeneous disorder remain to be identified. Objective: To look for mutations in POMT2 as a cause of WWS, as both POMT1 and POMT2 are required to achieve protein O-mannosyltransferase activity. Methods: A candidate gene approach combined with homozygosity mapping. Results: Homozygosity was found for the POMT2 locus at 14q24.3 in four of 11 consanguineous WWS families. Homozygous POMT2 mutations were present in two of these families as well as in one patient from another cohort of six WWS families. Immunohistochemistry in muscle showed severely reduced levels of glycosylated α-DG, which is consistent with the postulated role for POMT2 in the O-mannosylation pathway. Conclusions: A fourth causative gene for WWS was uncovered. These genes account for approximately one third of the WWS cases. Several more genes are anticipated, which are likely to play a role in glycosylation of α-DG.     

Localizations of γ-Actins in Skin,Hair, Vibrissa,Arrector Pili Muscle and Other Hair Appendages of Developing Rats

Six isoforms of actins encoded by different genes have been identified in mammals including α-cardiac, α-skeletal, α-smooth muscle (α-SMA), β-cytoplasmic, γ-smooth muscle (γ-SMA), and γ-cytoplasmic actins (γ-CYA). In a previous study we showed the localization of α-SMA and other cytoskeletal proteins in the hairs and their appendages of developing rats (Morioka K., et al. (2011) Acta Histochem. Cytochem. 44, 141–153), and herein we determined the localization of γ type actins in the same tissues and organs by immunohistochemical staining. Our results indicate that the expression of γ-SMA and γ-CYA is suggested to be poor in actively proliferating tissues such as the basal layer of the epidermis and the hair matrix in the hair bulb, and as well as in highly keratinized tissues such as the hair cortex and hair cuticle. In contrast, the expression of γ-actins were high in the spinous layer, granular layer, hair shaft, and inner root sheath, during their active differentiations. In particular, the localization of γ-SMA was very similar to that of α-SMA. It was located not only in the arrector pili muscles and muscles in the dermis, but also in the dermal sheath and in a limited area of the outer root sheath in both the hair and vibrissal follicles. The γ-CYA was suggested to be co-localized with γ-SMA in the dermal sheath, outer root sheath, and arrector pili muscles. Sparsely distributed dermal cells expressed both types of γ-actin. The expression of γ-actins is suggested to undergo dynamic changes according to the proliferation and differentiation of the skin and hair-related cells.     

Expression of Cellular Prion Protein in Activated Hepatic Stellate Cells

Suppression subtractive hybridization was used to clone genes associated with the activation of hepatic stellate cells and 13 genes were found to be dominantly expressed in activated stellate cells. Among them, one was identical to the 421-837th base pairs of cDNA sequence reported for rat prion-related protein (PrP). In cultured stellate cells, PrP mRNA expression increased in a time-dependent manner in parallel with smooth muscle (SM) α-actin mRNA expression. In situ hybridization demonstrated that PrP mRNA was localized in and around the fibrous septa of carbon tetrachloride (CCl₄)-treated liver. Cellular PrP (PrP^c) was produced by culture-activated stellate cells, and immunohistochemically detected in the fibrous septa of CCl₄-damaged liver and sinusoidal linings of common bile duct-ligated liver, consistent with the localization of SM α-actin. Immunoelectron microscopy revealed that PrP^c resided on the plasma membrane of stellate cells. These results indicate that PrP expression is closely related to stellate cell activation associated with fibrogenic stimuli.     

Erythropoietin reduces the expression of myostatin in mdx dystrophic mice

Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO=0.60±0.11, control=1.07±0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO=0.95±0.14, control=1.05±0.16) and TNF-α (rhEPO=0.73±0.20, control=1.01±0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle.     

Myotendinous Junction Defects and Reduced Force Transmission in Mice that Lack α7 Integrin and Utrophin

The α7β1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of α7β1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and α7 integrin (mdx/α7^−/−), or dystrophin and utrophin (mdx/utr^−/−), exhibit severe muscle pathology and die prematurely. To explore the contribution of the α7β1 integrin and utrophin to muscle integrity and function, we generated mice lacking both α7 integrin and utrophin. Surprisingly, mice that lack both α7 integrin and utrophin (α7/utr^−/−) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to α7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in α7/utr^−/− skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The α7/utr^−/− mouse, therefore, demonstrates the critical roles of α7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the α7β1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle.Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease that affects 1 in every 3500 live male births. Patients with DMD have impaired mobility, are restricted to a wheelchair by their teens, and die from cardiopulmonary failure in their early twenties.^1,2 Currently, there is no cure or effective treatment for this devastating disease. Mutations in the dystrophin gene resulting in loss of the dystrophin protein are the cause of disease in DMD patients and the mdx mouse model.^3,4,5,6,7The dystrophin glycoprotein complex links laminin in the extracellular matrix to the actin cytoskeleton. The N-terminal region of dystrophin interacts with cytoskeletal F-actin⁸ and the C-terminal region associates with the dystrophin-associated protein complex, which include α- and β-dystroglycan, α- and β-syntrophin, the sarcoglycans, and sarcospan.⁹ In DMD, the absence of dystrophin leads to disruption of the dystrophin glycoprotein complex, resulting in increased muscle fragility and altered cell signaling.⁹ Loss of this critical transmembrane linkage complex in DMD patients and mdx mice results in progressive muscle damage and weakness, inflammation, necrosis, and fibrosis. Lack of dystrophin also leads to abnormalities at myotendinous and neuromuscular junctions (MTJ and NMJ), which further contribute to skeletal muscle damage.^{10,11,12,13,14,15,16,17} In addition, defective muscle repair in DMD patients eventually results in muscle degeneration exceeding the rate of regeneration.¹⁸ Overall, dystrophin is critical for muscle function, structure, and stability, and its absence results in progressive muscle wasting and severe muscular dystrophy. In the absence of dystrophin two additional laminin-binding receptors, the α7β1 integrin and utrophin, are up-regulated in the skeletal muscle of DMD patients and mdx mice, which may compensate for the loss of the dystrophin glycoprotein complex.^19,20,21The α7β1 integrin is a heterodimeric laminin receptor involved in bidirectional cell signaling and is localized at junctional and extrajunctional sites in skeletal muscle.^22,23 At least six α7 integrin isoforms produced by developmentally regulated RNA splicing are expressed in skeletal muscle.²⁴ Mutations in the α7 integrin gene (ITGA7) cause myopathy in humans.²⁵ Mice lacking the α7 integrin develop myopathy, exhibit vascular smooth muscle defects and have altered extracellular matrix deposition.^{26,27,28,29,30} The observation that the α7β1 integrin is elevated in the muscle of DMD patients and mdx mice led to the hypothesis that the α7β1 integrin may compensate for the loss of dystrophin.¹⁹ Enhanced expression of the α7 integrin in the skeletal muscle of severely dystrophic mice reduced muscle pathology and increased lifespan by threefold.^10,11 In contrast, loss of both dystrophin and α7 integrin in mice results in severe muscular dystrophy and premature death by 4 weeks of age.^28,31 The α7β1 integrin is therefore a major modifier of disease progression in DMD.The utrophin glycoprotein complex is a third major laminin receptor in skeletal muscle. Utrophin has significant sequence homology to dystrophin.^32,33 In normal adult muscle utrophin is restricted to neuromuscular and myotendinous junctions.³⁴ During development or in damaged or diseased muscle, utrophin expression is increased and becomes localized at extrajunctional sites.^35,36 Utrophin interacts with the same proteins as dystrophin, but binds to actin filaments at different sites.³⁷ In mice, loss of utrophin results in a mild form of myasthenia with reduced sarcolemmal folding at the postsynaptic membrane of the neuromuscular junction.^12,15 Transgenic overexpression of utrophin has been shown to rescue mdx mice.³⁸ Mice that lack both dystrophin and utrophin exhibit severe muscular dystrophy and die by 14 weeks of age.^13,14 Thus, utrophin is also a major laminin receptor that modifies disease progression in DMD.To understand the functional overlap between the α7β1 integrin and utrophin in skeletal muscle, we produced mice that lack both α7 integrin and utrophin (α7/utr^−/−). Since both complexes are highly enriched at the MTJ and NMJ, we hypothesized that α7/utr^−/− mice may have severe abnormalities at these critical junctional sites. Our study demonstrates α7/utr^−/− mice exhibit partial embryonic lethality comparable with that observed in α7^−/− mice. Dystrophin is increased in these animals and enriched at the NMJ but not the MTJ. α7/utr^−/− mice display ultrastructural defects in their MTJ and compromised force transmission. Together, these results indicate that the α7β1 integrin, dystrophin and utrophin laminin binding complexes provide continuity between laminin in the extracellular matrix and the cell cytoskeleton, which are necessary for the normal structural and functional properties of skeletal muscle.     

Effects of BMSCs interactions with adventitial fibroblasts in transdifferentiation and ultrastructure processes

In this study an in vitro model of simulated blood vessel injury was used to study the effects of bone marrow-derived mesenchymal stem cells (BMSCs) morphology and to detect vascular smooth muscle actin (SM α-actin) expression in the presence of adventitial fibroblasts. BMSCs from rats with DAPI-labeled nuclei were co-cultured with adventitial fibroblasts for 7 days, while BMSCs cultured alone served as controls. Cell morphology of BMSCs was assessed by laser confocal microscopy and SM α-actin or calponin expression in BMSCs was detected by immunofluorescence staining. The expression of SM α-actin mRNA was identified using RT-PCR. Cell ultrastructure was assessed by electron microscopy. The results demonstrate that BMSCs with DAPI-labeled nuclei were smaller compared with fibroblasts, and their nuclei emitted a blue fluorescence. Most BMSCs displayed a polygonal shape changing from their original long fusiform shape. BMSCs with blue nuclei and red cytoplasm (SM α-actin positive or calponin positive) were observed, and a substantial number of filaments were present in the cytoplasm as observed under electron microscopy. The number of these cells increased as a function of culture duration. However, SM α-actin expression was weak and calponin expression was not detected in the control group. This study provides important new information on the characterization of artherosclerosis pathogenesis and vascular restenosis after blood vessel injury. Our findings demonstrate that direct interactions with adventitial fibroblasts can induce vascular smooth muscle-like cell differentiation in BMSCs.     

Novel mutations in CRB1 and ABCA4 genes cause Leber congenital amaurosis and Stargardt disease in a Swedish family

This study aimed to identify genetic mechanisms underlying severe retinal degeneration in one large family from northern Sweden, members of which presented with early-onset autosomal recessive retinitis pigmentosa and juvenile macular dystrophy. The clinical records of affected family members were analysed retrospectively and ophthalmological and electrophysiological examinations were performed in selected cases. Mutation screening was initially performed with microarrays, interrogating known mutations in the genes associated with recessive retinitis pigmentosa, Leber congenital amaurosis and Stargardt disease. Searching for homozygous regions with putative causative disease genes was done by high-density SNP-array genotyping, followed by segregation analysis of the family members. Two distinct phenotypes of retinal dystrophy, Leber congenital amaurosis and Stargardt disease were present in the family. In the family, four patients with Leber congenital amaurosis were homozygous for a novel c.2557C>T (p.Q853X) mutation in the CRB1 gene, while of two cases with Stargardt disease, one was homozygous for c.5461-10T>C in the ABCA4 gene and another was carrier of the same mutation and a novel ABCA4 mutation c.4773+3A>G. Sequence analysis of the entire ABCA4 gene in patients with Stargardt disease revealed complex alleles with additional sequence variants, which were evaluated by bioinformatics tools. In conclusion, presence of different genetic mechanisms resulting in variable phenotype within the family is not rare and can challenge molecular geneticists, ophthalmologists and genetic counsellors.     

Characterisation of the human GFRalpha-3 locus and investigation of the gene in Hirschsprung disease

BACKGROUND—The GDNF family receptor alpha (GFRα) proteins are extracellular cell surface bound molecules that act as adapters in binding of the GDNF family of soluble neurotrophic factors to the RET receptor. These molecules are essential for development of many neural crest derived cell types and the kidney. Mutations in RET and in two members of the GDNF ligand family are associated with Hirschsprung disease (HSCR), a congenital absence of the enteric ganglia. Members of the GFRα family are also candidates for HSCR mutations. One such gene is GFRα-3, which is expressed in the peripheral nervous system and developing nerves.
OBJECTIVE—We have characterised the structure of the human GFRα-3 locus and investigated the gene for sequence variants in a panel of HSCR patients.
METHODS—Long range PCR or subcloning of PAC clones was used to investigate GFRα-3 intron-exon boundaries. A combination of single strand conformation polymorphism (SSCP) analysis and direct sequencing was used to investigate GFRα-3 sequence variants.
RESULTS—GFRα-3 spans eight coding exons and has a gene structure and organisation similar to that of GFRα-1. We identified three polymorphic variants in GFRα-3 in a normal control population, a subset of which also occurred in HSCR patients. We did not detect any sequence variants within the coding sequence of GFRα-3. We found a base substitution in the 5' UTR of GFRα-3, 15 base pairs upstream of the translation start site. A second substitution was identified in intron 4 (IVS4-30G>A) between the splice branch site and the splice acceptor site. The final variant was a 2 base pair insertion within the splice donor consensus sequence of exon 7 (IVS7+4ins GG).
CONCLUSIONS—We did not detect any correlation between variants of GFRα-3 and the HSCR phenotype. Our data suggest that mutations of this gene are not a cause of HSCR.


Keywords: GFRα-3; Hirschsprung disease; RET     

Emery-Dreifuss syndrome: Genetic and clinical varieties

Two familial and 2 sporadic cases of Emery-Dreifuss syndrome are reported. One family presented a rare autosomal dominant variant of Emery-Dreifuss muscular dystrophy, another with X-linked recessive inheritance showed unusual intrafamilial variability. One of sporadic cases closely resembled rigid spine syndrome, the other was clinically intermediate between Emery-Dreifuss muscular dystrophy and rigid spine syndrome, showing that they are not distinct disorders. © 1994 Wiley-Liss, Inc.