Expanding the histopathological spectrum of CFL2-related myopathies.

Congenital myopathies (CMs) caused by mutation in cofilin-2 gene (CFL2) show phenotypic heterogeneity ranging from early-onset and rapid progressive forms to milder myopathy. Muscle histology is also heterogeneous showing rods and/or myofibrillar changes. Here, we report on three new cases, from two unrelated families, of severe CM related to novel homozygous or compound heterozygous loss-of-function mutations in CFL2. Peculiar histopathological changes showed nemaline bodies and thin filaments accumulations together to myofibrillar changes, which were evocative of the muscle findings observed in Cfl2-/- knockout mouse model.

MYH7-related myopathies: clinical, histopathological and imaging findings in a cohort of Italian patients.

BACKGROUND: Myosin heavy chain 7 (MYH7)-related myopathies are emerging as an important group of muscle diseases of childhood and adulthood, with variable clinical and histopathological expression depending on the type and location of the mutation. Mutations in the head and neck domains are a well-established cause of hypertrophic cardiomyopathy whereas mutation in the distal regions have been associated with a range of skeletal myopathies with or without cardiac involvement, including Laing distal myopathy and Myosin storage myopathy. Recently the spectrum of clinical phenotypes associated with mutations in MYH7 has increased, blurring this scheme and adding further phenotypes to the list. A broader disease spectrum could lead to misdiagnosis of different congenital myopathies, neurogenic atrophy and other neuromuscular conditions. RESULTS: As a result of a multicenter Italian study we collected clinical, histopathological and imaging data from a population of 21 cases from 15 families, carrying reported or novel mutations in MYH7. Patients displayed a variable phenotype including atypical pictures, as dropped head and bent spine, which cannot be classified in previously described groups. Half of the patients showed congenital or early infantile weakness with predominant distal weakness. Conversely, patients with later onset present prevalent proximal weakness. Seven patients were also affected by cardiomyopathy mostly in the form of non-compacted left ventricle. Muscle biopsy was consistent with minicores myopathy in numerous cases. Muscle MRI was meaningful in delineating a shared pattern of selective involvement of tibialis anterior muscles, with relative sparing of quadriceps. CONCLUSION: This work adds to the genotype-phenotype correlation of MYH7-relatedmyopathies confirming the complexity of the disorder.

Phenotypic heterogeneity in two unrelated Danon patients associated with the same LAMP-2 gene mutation.

Danon disease, an X-linked cardioskeletal myopathy caused by primary deficiency of lysosome-associated membrane protein-2 (LAMP-2), is clinically characterized by cardiomyopathy, myopathy, and variable mental retardation. The pathological hallmark of the disease is the absence of LAMP-2 immunohistochemical staining in muscle. The LAMP-2 gene mutations reported thus far are generally private mutations. We describe two cases of Danon disease with different clinical presentation, in whom we identified the same exon skipping mutation c.928G>A in the LAMP-2 gene. The first patient was affected by an early onset myopathy and hypertrophic cardiomyopathy (HCM) that partially improved with drug treatment. A first muscle biopsy at age 4 months showed markedly increased glycogen, and acid maltase deficiency was ruled out biochemically. A second muscle biopsy, performed at age 3(1/2) years, showed very mild abnormalities. The second child at age 15 years had mild, diffuse muscle weakness and wasting, moderate mental deficiency, and HCM. Two serial biopsies performed at age 8 and 15 years showed similar findings of multiple esterase-positive vacuoles in type I myofibers. In both patients the immunohistochemical study demonstrated the absence of LAMP-2 in skeletal muscle.

Mutation in the CAV3 gene causes partial caveolin-3 deficiency and hyperCKemia.

Mutations in the caveolin-3 (CAV3) gene are associated with autosomal dominant limb-girdle muscular dystrophy (LGMD1C). The authors report a novel sporadic mutation in the CAV3 gene in two unrelated children with persistent elevated levels of serum creatine kinase (hyperCKemia) without muscle weakness. Immunohistochemistry and quantitative immunoblot analysis of caveolin-3 showed reduced expression of the protein in muscle fibers. Our data indicate that a partial caveolin-3 deficiency should be considered in the differential diagnosis of idiopathic hyperCKemia.

Increased number of caveolae and caveolin-3 overexpression in Duchenne muscular dystrophy.

Caveolae are small pockets or invaginations localized at the plasma membrane. Caveolins are the principal protein components of caveolae and play an important structural role in the formation of caveolae membranes. Here, we studied by freeze fracture and immunological techniques the spatial organization of caveolae at the muscle cell plasma membrane and the expression of caveolin-3 in Duchenne muscular dystrophy (DMD) muscle fibers. In DMD muscle, we found an increased number of caveolae at the sarcolemma that corresponds to an overexpression of caveolin-3 by immunohistochemistry and by Western blot analysis. These findings suggest a possible role for caveolae and caveolin-3 in the pathogenesis of DMD.

A reporter system for analysis of regulatable promoter functions in the basidiomycete fungus Phanerochaete chrysosporium.

To establish a system for functional analysis of regulatable gene promoters involved in lignocellulose degradation by Phanerochaete chrysosporium, a DNA construct was made in which a minimal promoter region, 410 bp of the 5' untranslated region (UTR) of the cbhI.1 gene of P. chrysosporium, was fused to the phlR sequence of Streptoallotiechus hindustanus. This construct was used to transform P. chrysosporium to phleomycin resistance. Southern blot analysis revealed that the incoming DNA was maintained extrachromosomally in the transformants. The donor DNA was methylated by the fungus; inhibition of such methylation allowed transformants to be distinguished from 'false-positive' phlR colonies. Reverse transcriptase-polymerase chain reaction analysis of gene expression revealed that the cbhI.1 5' UTR/phlR sequence construct was transcribed, but that an intron within the cbhI.1 promoter was not excised from transcripts of the transforming DNA construct. Moreover, catabolite repression of cbhI.1 expression is relaxed in the construct. These observations suggest that normal function of the cbhI.1 promoter requires more than 410 bp.

Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophy (LGMD) is a clinically and genetically heterogeneous group of myopathies, including autosomal dominant and recessive forms. To date, two autosomal dominant forms have been recognized: LGMD1A, linked to chromosome 5q, and LGMD1B, associated with cardiac defects and linked to chromosome 1q11-21. Here we describe eight patients from two different families with a new form of autosomal dominant LGMD, which we propose to call LGMD1C, associated with a severe deficiency of caveolin-3 in muscle fibres. Caveolin-3 (or M-caveolin) is the muscle-specific form of the caveolin protein family, which also includes caveolin-1 and -2. Caveolins are the principal protein components of caveolae (50-100 nm invaginations found in most cell types) which represent appendages or sub-compartments of plasma membranes. We localized the human caveolin-3 gene (CAV3) to chromosome 3p25 and identified two mutations in the gene: a missense mutation in the membrane-spanning region and a micro-deletion in the scaffolding domain. These mutations may interfere with caveolin-3 oligomerization and disrupt caveolae formation at the muscle cell plasma membrane.

Lignocellulose degradation by Phanerochaete chrysosporium: gene families and gene expression for a complex process.

Phanerochaete chrysosporium completely degrades lignocellulose. The most recalcitrant component, lignin, is oxidized by the radical products of lignin and manganese peroxidases, whereas cellulose and hemicellulose are hydrolysed. Both peroxidases and cellulases exist as complex families at both the DNA and protein levels. The lignin peroxidases may function principally when mycelium-bound and, therefore, undetectable in culture supernatants. Moreover, methods for the study of P. chrysosporium must be applicable to solid substrate as well as liquid-culture conditions. For these reasons, detailed studies of gene expression, made possible by the reverse transcriptase-polymerase chain reaction method, are essential. Such studies reveal that gene families are subject to differential expression. The cellulase system has some differences from that of Trichoderma reesei; the distinction made between the activities of exocellobiohydrolases and endoglucanases needs to be re-appraised in both species. Current studies also seek to reconstruct the systems of degradation of lignocellulose and its individual components by heterologous expression of individual proteins in recombinant systems, and their use in mechanistic studies singly and in combinations.

Informed strain improvement for lignin degradation by Phanerochaete chrysosporium.

The effect of breeding from the white rot fungus Phanerochaete chrysosporium ME446 on performance for lignin mineralization was examined. This model for informed strain improvement without mutagenesis is based on abundant restriction fragment length polymorphisms (RFLPs). Under optimized conditions for lignin mineralization, extracellular manganese peroxidase (MnP) but not lignin peroxidase (LiP) could be detected, so measurement of LiP activity is not a valid assay for lignin degradation. Mineralization of 14C-labelled synthetic lignin (14C-DHP) was used to compare the performance of the wild-type strain ME446 with those of sets of progeny strains. Meiotic progeny from strain ME446, heterokaryotic progeny of crosses between such strains, and meiotic progeny of one heterokaryotic strain were examined. In each case, a minority of strains performed more efficiently than the parental strain ME446. The greatest range of lignin-mineralization performance (70-fold) was found within the set of initial progeny of ME446 and the narrowest was within the set of secondary homokaryotic strains. This is consistent with the view that a moderate number of determinants contribute to lignin mineralization performance. However, performance did not correlate with the possession of any single allele of those for 38 previously defined RFLP markers. The results show that lignin mineralization performance can be improved by cycles of crosses and fruiting, without mutagenesis.

Substrate-dependent differential splicing of introns in the regions encoding the cellulose binding domains of two exocellobiohydrolase I-like genes in Phanerochaete chrysosporium.

Recently, we have shown differential splicing of an intron in the cbhI.2 gene of Phanerochaete chrysosporium ME446; this intron lies within the region of the gene encoding the cellulose binding domain (P.F.G. Sims, M. S. Soares-Felipe, Q. Wang, M.E. Gent, C. Tempelaars, and P. Broda, Mol. Microbiol. 12:209-216, 1994). Here, we show that such differential splicing occurs in the cbhI.1 gene of this fungus as well as in the cbhI.2 gene and that this phenomenon is substrate dependent. Avicel elicits the synthesis of both classes of mRNA transcripts from both of these genes. In contrast, carboxymethyl cellulose predominantly elicits the synthesis of fully spliced transcripts from both genes. Such differential splicing might allow this fungus to regulate the specificities of substrate binding for these cellulases.

PCR-mediated analysis of lignocellulolytic gene transcription by Phanerochaete chrysosporium: substrate-dependent differential expression within gene families.

We compare the kinetics of appearance of supernatant enzyme activities (lignin peroxidase, manganese peroxidase, and cellulase) and gene expression (LIG, mnp, and cbhI gene families and the unique cbhII gene) in Phanerochaete chrysosporium ME446 when grown on four different carbon sources: ball-milled straw, representing the natural substrate lignocellulose; Avicel as a crystalline cellulose; and high and low concentrations of glucose, in all cases with limiting nitrogen. PCR-based technology utilizing pairs of primers specific for particular genes showed that there is differential expression between and within the families. There were a number of instances of mRNA species being present only on a single day, implying tight regulation of lignocellulose degradation at the mRNA level. The patterns of extracellular enzyme activities and mnp and cbh gene expression are similar whereas LIG gene expression can be detected when no corresponding enzyme activity is observed in the extracellular supernatant. The enzyme produced under these conditions is presumably sequestered by the mycelium and is likely to be functionally significant. Another striking result is that cellulose, in the form of Avicel, elicits the expression of three LIG gene for which there is no expression under the same conditions with the other carbon sources.

Isolation, characterization, and analysis of the expression of the cbhII gene of Phanerochaete chrysosporium.

Two cDNA sequences representing putative allelic variants of the Phanerochaete chrysosporium cbhII gene were isolated by hybridization to the Trichoderma reesei cbhII gene. Both of the equivalent genomic sequences were subsequently isolated by the inverse PCR technique. DNA sequencing showed that the cbhII open reading frame of 1,380 bp codes for a putative polypeptide of 460 amino acids which is interrupted by six introns. The domain structure found in T. reesei cbhII is conserved in the equivalent P. chrysosporium protein. The overall similarity between the two gene products is 54%, with the region of highest conservation being found in the cellulose-binding domain (65%). Unlike the cbhI gene of P. chrysosporium, cbhII does not appear to be a member of a class of closely related genes. CBHII is a new member of family B of the beta-1, 4-glucanases. Alignment of the P. chrysosporium and T. reesei CBHII protein sequences showed that all of the residues important for the formation of the extended loops of the catalytic domain and those residues that are involved in the catalytic action of the T. reesei enzyme are also present in the P. chrysosporium equivalent. The profiles of cbh gene expression in P. chrysosporium reveal that while cbhI.1 and cbhI.2 could be coregulated, cbhII can be independently controlled. The latter is so far the only cellulase gene found to be expressed when the fungus is grown on oat spelt arabinoxylan, suggesting that it may play an active role in the xylanolytic as well as the cellulolytic systems.

[Muscle phosphorylase deficiency in childhood. A case report]. / Il deficit di fosforilasi muscolare nell' età pediatrica. Contributo clinico.

Myophosphorylase deficiency or McArdle's disease is rarely recognized in childhood. The onset is generally in adolescence or in adult age with exercise intolerance, muscle cramps and myoglobinuria. Two siblings of 6 and 2 years of age are described. The first patient showed early fatigue and both had elevated CK levels. Morphological and biochemical studies of muscle biopsies revealed a defect of myophosphorylase.

Differential expression of multiple exo-cellobiohydrolase I-like genes in the lignin-degrading fungus Phanerochaete chrysosporium.

The genome of Phanerochaete chrysosporium strain ME446 contains multiple, non-allelic, cellobiohydrolase I (CBHI)-like sequences, at least two of which are expressed in a cellulose-dependent manner. Each of the expressed genes contains two identically positioned introns within its coding region. The lengths and sequences of these introns are different and one is not excised from all transcripts, raising the possibility that subtly different protein products may be expressed from a common gene. Introns are also present upstream of both genes but these differ in number and position, as well as sequence and length. Endoglucanase-like sequences could not be identified and it is suggested that variant CBHI-like proteins may provide endoglucanase activity in this fungus.

Phanerochaete chrysosporium and its natural substrate.

We seek to define more fully how Phanerochaete chrysosporium degrades its natural substrate, lignocellulose. This contribution concerns several relevant topics. Mineralisation of [14C]DHP, as a model for lignin degradation, showed that a set of genetically defined meiotically derived products of strain ME446 differed in their degradative ability and also that, under optimum conditions for mineralisation, extracellular lignin peroxidase activity was absent. Xylanolytic and xylosidase/beta(1-->3) glucanase activities are also described. The complexity of the CBHI gene family is described and differential splicing of a CBHI gene transcript is proposed. In contrast to the multiplicity of CHBI genes there is a single CBHII gene. PCR methods were developed to analyse differential gene expression on different substrates. We have also developed a transformation system involving a reporter construct for the analysis of CBHI promoter function.

A Phanerochaete chrysosporium beta-D-glucosidase/beta-D-xylosidase with specificity for (1-->3)-beta-D-glucan linkages.

Phanerochaete chrysosporium is the best studied organism with respect to lignin degradation, but its degradation of the xylan component of lignocellulose is only now being studied. When grown on oat spelt xylan (mainly arabinoxylan), it produces an enzyme with beta-D-xylosidase and beta-D-glucosidase activity. This enzyme was purified by ultrafiltration followed by ammonium sulphate precipitation, anion-exchange chromatography using DEAE Biogel and Mono Q, and gel filtration using Superose 12. It is extracellular, with an apparent M(r) value of 44,500 as determined by SDS-PAGE; the pI is 4.67 and activity is maximal at pH 5 and 60 degrees C. The enzyme is of particular interest because its principal activity is against laminaribiose (3-O-beta-D-glucopyranosyl-D-glucopyranose and laminarin [(1-->3)-beta-D-glucan with ca. 3% of beta-(1-->6) branches] rather than cellobiose and xylobiose. It was competitively inhibited by D-glucono-1,5-lactone and deoxynojirimycin; with p-nitrophenyl beta-D-xylopyranoside as substrate, the Ki values were 32 and 87.5 microM, respectively, and with p-nitrophenyl beta-D-glucopyranoside, they were 35 and 68.7 microM, respectively. The Km values with p-nitrophenyl beta-D-xylopyranoside and p-nitrophenyl beta-D-glucopyranoside as substrates were 3.51 and 5.30 mM, respectively.

Isozyme specific polymerase chain reaction analysis of differential gene expression: a general method applied to lignin peroxidase genes of Phanerochaete chrysosporium.

Analysis of differential expression of closely related genes is a general problem. The lignin peroxidase genes of Phanerochaete chrysosporium represent a typical case. They are differentially expressed according to the conditions encountered during growth. We show that the expression of two such genes, LIG1 and LIG5, can be differentiated at the mRNA level using a highly specific form of the polymerase chain reaction. This generally applicable method allows rapid and accurate analysis of complex and closely related mRNA populations.

Polarimetry and 13C n.m.r. show that the hydrolyses of beta-D-glucopyranosyl fluoride by beta(1-->3)-glucanases from Phanerochaete chrysosporium and Sporotrichum dimorphosporum have opposite stereochemistries.

The time courses of optical rotation and fluoride ion release during hydrolysis of beta-D-glucopyranosyl fluoride by the beta(1-->3)-glucanase of Phanerochaete chrysosporium (J. L. Copa-Patiño and P. Broda, unpublished work) indicated that the initial sugar product was beta-D-glucopyranose. This was confirmed by monitoring the hydrolysis of 1-[13C]beta-D-glucopyranosyl fluoride by this enzyme with 13C n.m.r. (without proton decoupling). The same two techniques were used to confirm that hydrolysis of beta-D-glucopyranosyl fluoride by the exo beta(-->3)-glucanase of 'Basidiomycete QM 806' (identified as Sporotrichum dimorphosporum) yielded alpha-glucopyranose as first sugar product, in accordance with previous results using laminarin as substrate [Parrish and Reese (1963) Carbohydr. Res. 3, 424-429; Nelson (1970) J. Biol. Chem. 245, 869-872].

Stable defined substrate for turbidimetric assay of endoxylanases.

A stable xylan suspension was prepared and characterized. Hydrolysis of the particles converts them into soluble fragments, thereby lowering the turbidity of the suspension. The small volume of the assay mixture, the short incubation time required, and the simplicity of the procedure permit the rapid analysis of many samples. Furthermore, the procedure can be used to assay xylanase activities in the presence of other reducing materials and is also useful for monitoring low-level xylanase activities.