Clinical and morphological phenotype of the filamin myopathy: a study of 31 German patients.

Mutations in the filamin C gene (FLNC) cause a myofibrillar myopathy (MFM), morphologically characterized by focal myofibrillar destruction and abnormal accumulation of several proteins within skeletal muscle fibres. We studied 31 patients from four German families to evaluate the phenotype of filaminopathy. All patients harboured the same p.W2710X mutation in FLNC. Haplotype analysis suggested a founder mutation in these German filaminopathy families. The mean age at onset of clinical symptoms was 44 +/- 6 years (range, 24-57 years). Slowly progressive muscle weakness was mostly pronounced proximally, initially affecting the lower extremities and involving the upper extremities in the course of disease progression, similar to the distribution of weakness seen in limb-girdle muscular dystrophies (LGMD). Patients frequently developed respiratory muscle weakness. About one-third of the patients showed cardiac abnormalities comprising conduction blocks, tachycardia, diastolic dysfunction and left ventricular hypertrophy indicating a cardiac involvement in filaminopathy. Serum creatine kinase levels varied from normal up to 10-fold of the upper limit. Magnetic resonance imaging studies showed a rather homogenous pattern of muscle involvement in the lower extremities differing from that in other types of MFM. Myopathological features included perturbation of myofibrillar alignment, accumulation of granulofilamentous material similar to that seen in primary desminopathies and abnormal intracellular protein deposits typical of MFM. Decreased activities of oxidative enzymes and fibre hypertrophy seem to be early features, whereas dystrophic changes were present in advanced stages of filaminopathy. Rimmed vacuoles were detected in only a few cases. The intracellular aggregates were composed of a variety of proteins including filamin C, desmin, myotilin, Xin, dystrophin and sarcoglycans. Therapy is so far limited to symptomatic treatment. The German filaminopathy cohort, the largest group of patients studied so far, shares phenotypic features with LGMD and presents with characteristic histopathological findings of MFM.

Peripheral nerve demyelination caused by a mutant Rho GTPase guanine nucleotide exchange factor, frabin/FGD4.

GTPases of the Rho subfamily are widely involved in the myelination of the vertebrate nervous system. Rho GTPase activity is temporally and spatially regulated by a set of specific guanine nucleotide exchange factors (GEFs). Here, we report that disruption of frabin/FGD4, a GEF for the Rho GTPase cell-division cycle 42 (Cdc42), causes peripheral nerve demyelination in patients with autosomal recessive Charcot-Marie-Tooth (CMT) neuropathy. These data, together with the ability of frabin to induce Cdc42-mediated cell-shape changes in transfected Schwann cells, suggest that Rho GTPase signaling is essential for proper myelination of the peripheral nervous system.

The 6-maleimidocaproyl hydrazone derivative of doxorubicin (DOXO-EMCH) is superior to free doxorubicin with respect to cardiotoxicity and mitochondrial damage.

Doxorubicin causes a chronic cardiomyopathy in which genetic and functional lesions of mitochondria accumulate in the long-term and explain in part the delayed onset of heart dysfunction. DOXO-EMCH a 6-maleimidocaproyl hydrazone derivative of doxorubicin, is an albumin binding prodrug which has entered clinical trials because of its superior antitumor and toxicological profile. In the present work, we examined the chronic cardiotoxicity of DOXO-EMCH in direct comparison with doxorubicin. Rats (11 weeks of age) were treated with intravenous doxorubicin (0.8 mg/kg weekly for 7 weeks), an equimolar dose of DOXO-EMCH (1.1 mg/kg), or with 3.3 mg/kg of DOXO-EMCH. Controls received saline. Animals were euthanized at 48th week. Rats exposed to doxorubicin had a severe clinical, and histopathological cardiomyopathy with depressed myocardial activity of cytochrome c-oxidase (COX, 26% of controls), reduced expression of the mtDNA-encoded COX II subunit, decreased mtDNA copy numbers (46% of controls), and high levels of malondialdehyde and superoxide (787% of controls). All parameters were highly correlated with myocardial damage. Both DOXO-EMCH groups did not differ from controls with regard to clinical symptomatology, mortality and mitochondrial enzymes, although the myocardia of the high-dose group had slightly increased histopathological abnormalities, depressed mtDNA copies (74% of controls) and elevated superoxide levels (347% of controls). Doxorubicin-exposed hearts and to a lesser extent the myocardia of both DOXO-EMCH groups contained mtDNA-deletions. In summary both DOXO-EMCH doses were superior over doxorubicin with respect to clinical and histopathological evidence of cardiomyopathy, myocardial COX-activity, COX II expression, mtDNA-content, mtDNA mutation loads and superoxide production in rats.

Myogenin (Myf4) upregulation in trans-differentiating fibroblasts from a congenital myopathy with arrest of myogenesis and defects of myotube formation.

Congenital myopathies often have an unclear aetiology. Here, we studied a novel case of a severe congenital myopathy with a failure of myotube formation. Polymerase chain reaction-based analysis was performed to characterize the expression patterns of the Desmin, p21, p57, and muscle regulatory factors (MRFs) MyoD, Myf4, Myf5 and Myf6 in differentiating skeletal muscle cells (SkMCs), normal human fibroblasts and patient-derived fibroblasts during trans-differentiation. The temporal and spatial pattern of MRFs was further characterized by immunocyto- and immunohistochemical stainings. In differentiating SkMCs, each MRF showed a characteristic expression pattern. Normal trans-differentiating fibroblasts formed myotubes and expressed all of the MRFs, which were detected. Interestingly, the patient's fibroblasts also showed some fusion events during trans-differentiation with a comparable expression profile for the MRFs, particularly, with increased expression of Myf4 and p21. Immunohistochemical analysis of normal and patient-derived skeletal musculature revealed that Myf4, which is downregulated during normal fetal development, was still present in patient-derived skeletal head muscle, which was also positive for Desmin and sarcomeric actin. The abnormal upregulation of Myf4 and p21 in the patient who suffered from a severe congenital myopathy suggests that the regulation of Myf4 and p21 gene expression during myogenesis might be of interest for further studies.

Tissue-specific mtDNA lesions and radical-associated mitochondrial dysfunction in human hearts exposed to doxorubicin.

Doxorubicin causes a chronic cardiomyopathy. Although the exact pathogenesis is unknown, recent animal data suggest that somatically acquired alterations of mitochondrial DNA (mtDNA) and concomitant mitochondrial dysfunction play an important role in its onset. In this study, skeletal and myocardial muscles were examined from human autopsies. Compared to controls (n = 8), doxorubicin-exposed hearts (n = 6) showed low absolute enzyme activity of mtDNA-encoded nicotinamide adenine dinucleotide hydrogen dehydrogenase (NADH DH, 79% residual activity, p = 0.03) and cytochrome c oxidase (COX, 59% residual activity, p < 0.001), but not of succinate dehydrogenase (SDH), which is encoded exclusively by nuclear DNA. NADH DH/SDH and COX/SDH ratios were 37% (p < 0.001) and 27% (p < 0.001) of controls. Expression of the mtDNA-encoded subunit II of COX was reduced (82%, p = 0.04), compared to its unchanged nucleus-encoded subunit IV. MtDNA-content was diminished (56%, p = 0.02), but the 'common' mtDNA-deletion was increased (9.2-fold, p = 0.004). Doxorubicin-exposed hearts harboured numerous additional mtDNA rearrangements lacking direct repeats. They contained elevated levels of malondialdehyde (MDA) (p = 0.006, compared to controls), which correlated inversely with the COX/SDH ratio (r = -0.45, p = 0.02) and the mtDNA-content (r = -0.75, p = 0.002), and correlated positively with the levels of the 'common' deletion (r = 0.80, p < 0.001). Doxorubicin-exposed hearts also contained the highest levels of superoxide (p < 0.001, compared to controls), which correlated negatively with the mtDNA-encoded respiratory chain activities, such as the COX/SDH ratio (r = -0.57, p = 0.02) and the NADH/SDH ratio (r = -0.52, p = 0.04), as well as with the mtDNA content (r = -0.69, p = 0.003), and correlated positively with the frequency of the 'common' deletion (r = 0.76, p < 0.001) and the MDA levels (r = 0.86, p < 0.001). Doxorubicin-exposed hearts contained electron-dense deposits within mitochondria. Hearts exposed to other anthracyclines (n = 6) or skeletal muscle (all groups) had no mitochondrial dysfunction. Doxorubicin, unlike other anthracyclines, augments lipid peroxidation, induces mtDNA mutations and decreases mtDNA content in human hearts. These lesions have an impact on mitochondrial function and could be of importance in the pathogenesis of clinical cardiomyopathy.

p.S143F mutation in lamin A/C: a new phenotype combining myopathy and progeria.

We report a young girl with a phenotype combining early-onset myopathy and a progeria. She had myopathy and marked axial weakness during the first year of life; progeroid features, including growth failure, sclerodermatous skin changes, and osteolytic lesions, developed later. We identified the underlying cause to be a hitherto unreported de novo missense mutation in the LMNA gene (S143F) encoding the nuclear envelope proteins lamins A and C. Although LMNA mutations have been known to cause Hutchinson-Gilford progeria syndrome and Emery-Dreifuss muscular dystrophy, this is the first report of a patient combining features of these two phenotypes because of a single mutation in LMNA.

Neonatal type IV glycogen storage disease associated with "null" mutations in glycogen branching enzyme 1.

The fatal neonatal form of type IV glycogen storage disease (GSD IV) was diagnosed on light and electron microscopy and by analysis of GBE1 , the gene encoding glycogen branching enzyme. We report two novel truncating mutations, as well as the first genomic mutational analysis of GBE1 using denaturing high performance liquid chromatography.

Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependymal flow and reveals a novel mechanism for hydrocephalus formation.

Motility of unicellular organisms occurred early in evolution with the emergence of cilia and flagella. In vertebrates, motile cilia are required for numerous functions such as clearance of the airways and determination of left-right body asymmetry. Ependymal cells lining the brain ventricles also carry motile cilia, but their biological function has remained obscure. Here, we show that ependymal cilia generate a laminar flow of cerebrospinal fluid through the cerebral aqueduct, which we term as 'ependymal flow'. The axonemal dynein heavy chain gene Mdnah5 is specifically expressed in ependymal cells, and is essential for ultrastructural and functional integrity of ependymal cilia. In Mdnah5-mutant mice, lack of ependymal flow causes closure of the aqueduct and subsequent formation of triventricular hydrocephalus during early postnatal brain development. The higher incidence of aqueduct stenosis and hydrocephalus formation in patients with ciliary defects proves the relevance of this novel mechanism in humans.

Time-dependent and tissue-specific accumulation of mtDNA and respiratory chain defects in chronic doxorubicin cardiomyopathy.

BACKGROUND: Doxorubicin causes a chronic cardiomyopathy of unknown pathogenesis. We investigated whether acquired defects in mitochondrial DNA (mtDNA) and interconnected respiratory chain dysfunction may represent a molecular mechanism for its late onset. METHODS AND RESULTS: Rats were treated weekly with intravenous doxorubicin (1 mg/kg) for 7 weeks, starting at 11 weeks of age (group B). Controls received saline. Group C received doxorubicin identically to group B, but the course was started at 41 weeks of age. All rats were killed at week 48. Doxorubicin was also injected once, either 6 days (group D) or 2 hours (group E) before euthanasia. Heart and skeletal muscle were examined. Only group B rats developed a significant clinical, macroscopic, histological, and ultrastructural cardiomyopathy. Group B hearts had the lowest cytochrome c oxidase (COX) activity (24% of controls; P=0.003), the highest citrate synthase activity (135% of controls; P=0.005), and the highest production of superoxide. In group B, the respiratory subunit COXI, which is encoded by mtDNA, was reduced (P<0.001), as was mtDNA (49% of controls, P<0.001). Group C hearts differed from group B in their lower cardiomyopathy score (P=0.006), higher COX activity (P=0.02), and higher mtDNA content (P=0.04). Group B and to a lesser extent group C hearts contained deleted mtDNA. There was no detectable mitochondrial toxicity in group D and E hearts or in skeletal muscle. CONCLUSIONS: In doxorubicin cardiomyopathy, mtDNA alterations, superoxide, and respiratory chain dysfunction accumulate long-term in the absence of the drug and are associated with a late onset.

Mutation of the SBF2 gene, encoding a novel member of the myotubularin family, in Charcot-Marie-Tooth neuropathy type 4B2/11p15.

Autosomal recessive hereditary motor and sensory neuropathy or Charcot-Marie-Tooth disease (CMT) is a severe childhood-onset neuromuscular disorder. Autosomal recessive CMT is genetically heterogeneous with one locus mapped to chromosome 11p15 (CMT4B2). The histopathological hallmarks of CMT4B2 are focal outfoldings of myelin in nerve biopsies. Homozygosity mapping, in a Turkish inbred family with four children affected by CMT characterized by focally folded myelin, provided linkage to the CMT4B2 locus. We identified a large, novel gene, named SET binding factor 2 (SBF2), that lies within this interval and is expressed in various tissues, including spinal cord and peripheral nerve. SBF2 is a member of the pseudo-phosphatase branch of myotubularins and was an obvious candidate for CMT4B2 by virtue of its striking homology to myotubularin-related protein 2 (MTMR2), causing another form of autosomal recessive CMT with outfoldings of the myelin sheaths. Molecular study of the SBF2 gene in the CMT4B family demonstrated the presence of a homozygous inframe deletion of SBF2 exons 11 and 12 in all four affected individuals. On the protein level, this mutation is predicted to disrupt an N-terminal domain that is conserved in SBF2 and its orthologues across species. Myotubularin-related proteins have been suggested to work in phosphoinositide-mediated signalling events that may also convey control of myelination. Localization of SBF2 within the candidate interval, cosegregation with the disease, expression in the peripheral nervous system, and resemblance of the histopathological phenotype to that related to mutations in its paralogue MTMR2 indicate that this gene is the CMT4B2 gene.

Evidence of nucleoside analogue reverse transcriptase inhibitor--associated genetic and structural defects of mitochondria in adipose tissue of HIV-infected patients.

To investigate if possible mitochondrial injury can be found in adipose tissue of nucleoside analogue reverse transcriptase inhibitor (NRTI)-treated patients, subcutaneous fat was taken from the buttocks of 24 HIV-positive patients and 8 HIV-negative controls. The content of mitochondrial DNA (mtDNA) was quantified using a Southern blot technique. Fat biopsies were examined by electron microscopy and screened by restriction fragment length polymorphism analysis for the presence of the nt 8344 and 3243 mtDNA point mutations. Age, sex, and body mass index did not differ between the HIV-negative controls, the HIV-positive patients currently treated with NRTIs (NRTI group, n = 19), and the HIV-positive patients without NRTIs (no-NRTI group, n = 5). The mean mtDNA content was 44% lower in the NRTI group compared with the no-NRTI group ( p =.01) but did not differ between the control group and the no-NRTI group. When the HIV-infected patients were stratified to a group with clinical signs of lipoatrophy at the biopsy site (LA group, n = 11) and a group without lipoatrophy (no-LA group, n = 13), the mean mtDNA content in the LA group was 39% lower than that in the no-LA group ( p =.02). No point mutations or deletions were observed. The adipocytes of patients with lipoatrophy contained multiple small lipid vacuoles, and the mitochondria harbored inclusions reminiscent of mtDNA cytopathies. mtDNA depletion and ultrastructural abnormalities of adipocytes suggest a link between mitochondrial damage, the use of NRTIs, and lipoatrophy in HIV-infected patients.