Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix.

Deficiency of any component of the ER-resident collagen prolyl 3-hydroxylation complex causes recessive osteogenesis imperfecta (OI). The complex modifies the α1(I)Pro986 residue and contains cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and cyclophilin B (CyPB). Fibroblasts normally secrete about 10% of CRTAP. Most CRTAP mutations cause a null allele and lethal type VII OI. We identified a 7-year-old Egyptian boy with non-lethal type VII OI and investigated the effects of his null CRTAP mutation on collagen biochemistry, the prolyl 3-hydroxylation complex, and collagen in extracellular matrix. The proband is homozygous for an insertion/deletion in CRTAP (c.118_133del16insTACCC). His dermal fibroblasts synthesize fully overmodified type I collagen, and 3-hydroxylate only 5% of α1(I)Pro986. CRTAP transcripts are 10% of control. CRTAP protein is absent from proband cells, with residual P3H1 and normal CyPB levels. Dermal collagen fibril diameters are significantly increased. By immunofluorescence of long-term cultures, we identified a severe deficiency (10-15% of control) of collagen deposited in extracellular matrix, with disorganization of the minimal fibrillar network. Quantitative pulse-chase experiments corroborate deficiency of matrix deposition, rather than increased matrix turnover. We conclude that defects of extracellular matrix, as well as intracellular defects in collagen modification, contribute to the pathology of type VII OI.

The genetic features of 24 patients affected by familial and sporadic hemiplegic migraine.

Familial hemiplegic migraine (FHM) is the only migraine subtype for which a monogenic mode of inheritance, autosomal dominant has been clearly established. It is genetically heterogeneous and at least three different genes exist (CACNA1A, ATP1A2, and SCN1A), the so-called FHM1, FHM2, and FHM3 genes, respectively. Sporadic hemiplegic migraine (SHM) is a disorder, in which some patients may have their pathophysiology identical to FHM, but others, possibly the majority, may have different pathophysiology, probably related to the mechanisms of typical migraine with aura. In our study, we have screened the DNA of 24 patients affected by FHM and SHM. Only in three patients, 2 sporadic and 1 familial cases, we have described genetic mutations, all of them in the ATP1A2 gene. In our opinion, these results demonstrate a more frequent involvement of the ATP1A2 gene not only in the sporadic form, but probably also in the Italian FHM patients without permanent cerebellar signs. Moreover, the absence of CACNA1A, ATP1A2 and SCN1A mutations in the other 12 familial cases suggests the involvement of still unknown genes.

Amino acid changes in the amino terminus of the Na,K-adenosine triphosphatase alpha-2 subunit associated to familial and sporadic hemiplegic migraine.

Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura inherited with an autosomal dominant pattern. Here, we report the genetic analysis of four families and one sporadic case with hemiplegic migraine (HM) in whom we searched for mutations in the three genes associated with the disease CACNA1A, ATP1A2 and SCN1A. Two novel amino acid changes p.Arg65Trp and p.Tyr9Asn, in the Na,K-adenosine triphosphatase (ATPase) alpha-2 subunit encoded by the ATP1A2 gene, were found in one FHM family and in the sporadic case, respectively. These mutations are peculiar for their location in the extreme N-terminus, an uncommon mutation target in this protein. Low frequency of migraine attacks in all our mutant patients with low complexity of the associated aura symptoms in the sporadic case is also observed. Besides the two novel mutations, the data here reported confirm the involvement of ATP1A2 gene in the sporadic form of HM, while the negative results on the other families tested for all genes known in HM strengthen the hypothesis of the existence of at least another locus involved in FHM.

A CAV3 microdeletion differentially affects skeletal muscle and myocardium.

BACKGROUND: Caveolin-3 is the muscle-specific protein product of the caveolin gene family and an integral membrane component of caveolae. Mutations in the gene encoding caveolin-3 (CAV3) underlie four distinct disorders of skeletal muscle: the autosomal dominant form of limb-girdle muscular dystrophy type 1C (LGMD-1C), rippling muscle disease (RMD), sporadic and familial forms of hyperCKemia, and distal myopathy. OBJECTIVE: To characterize a multigenerational Italian family affected by an autosomal dominant myopathic disorder and to assess the expression of caveolin-3, dystrophin, dystrophin-associated glycoproteins, and neuronal nitric oxide synthase in the myocardium of an affected patient. METHODS: Clinical analysis involved 15 family members. Skeletal muscle expression of sarcolemmal proteins was evaluated by immunohistochemistry and western blot analysis in three affected individuals. Caveolar structures were analyzed through electron microscopy in muscle biopsies and in one heart biopsy. RESULTS: CAV3 genetic analysis showed a heterozygous 3-bp microdeletion (328-330del) in affected individuals, resulting in the loss of a phenylalanine (Phe97del) in the transmembrane domain. In the skeletal muscle, the mutation was associated with severe caveolin-3 deficiency and caveolar disorganization, whereas the expression of the other analyzed muscle proteins was unaltered. Remarkably, caveolin-3 was expressed in myocardium at a level corresponding to about 60% of that of control individuals and was correctly localized at the myocardial cell membranes, with preservation of cardiac myofiber caveolar structures. Clinical analysis revealed the concomitant presence in this family of the following phenotypes: RMD, LGMD, and hyperCKemia. CONCLUSIONS: Intrafamilial phenotypic heterogeneity is associated with caveolin-3 Phe97 microdeletion. The molecular network interacting with caveolin-3 in skeletal muscle and heart may differ.

Effect of enamel matrix derivative on human periodontal fibroblasts: proliferation, morphology and root surface colonization. An in vitro study.

BACKGROUND: Several clinical trials have shown the effectiveness of Emdogain(R) (EMD) in promoting tissue regeneration, even though the underlining biological mechanism is still poorly known. OBJECTIVES: The aim of the present study was to verify the effect of EMD on the proliferation of human periodontal ligament (PDL) fibroblasts and on their colonization and differentiation following contact with the root surface of extracted teeth in vitro. METHODS AND RESULTS: Fibroblasts from PDL were seeded on Petri dishes and cell growth was evaluated by cell counting in the presence and absence of EMD, after 1, 3 and 8 d of culture. A significant effect of EMD upon cellular proliferation at d 3 and 8 was detected. When PDL cells were grown for 12 d with EMD on etched human root surface, a change in cell morphology was observed. Scanning electron microscopy revealed that cells grown on root EMD-treated surface present a body with a flattened surface closely adherent to the substrate and an outer smooth surface rounded in shape. From the flattened surface some thin and elongated cellular processes connecting with the substrate were also observable. PDL cells grown on EMD-treated surface showed lack of alkaline phosphatase activity, as some authors noticed in cementoblasts in vitro. CONCLUSIONS: In conclusion, our data indicate that EMD enhances human PDL fibroblast proliferation. Furthermore, the cells in the presence of EMD show morphological changes that make them more similar to cementoblasts than to fibroblasts, suggesting a process of cellular differentiation that could play an important role in periodontal tissue repair.

Clinical, morphological and immunological evaluation of six patients with dysferlin deficiency.

Limb girdle muscular dystrophy (LGMD) type 2B and distal Miyoshi myopathy (MM) are caused by mutations in a recently discovered mammalian gene coding for a skeletal muscle protein called dysferlin. The protein is normally expressed at the skeletal muscle level and absent or reduced in affected patients. We selected a clinically heterogeneous population of Italian myopathic patients with clinical evidence of myopathy and/or hyperCKemia, EMG myopathic pattern, and no alterations of the dystrophin-sarcoglycan complex. Calpain, merosin, emerin and caveolin were also tested and found normal in all patients. Dysferlin immunohistochemical and Western blot analyses allowed us to identify six patients with dysferlin deficiency: one with distal myopathy, four with limb girdle myopathy and one with hyperCKemia. No apoptosis was found in any of the six muscle specimens, although expression of the pro-apoptotic Fas antigen was mildly increased in two cases. Inflammatory reactions were present in two of the six cases, but we found no evidence of immune-mediated processes.

Decreased EMG inhibition following electrical stimulation over muscle tendons in myopathies.

OBJECTIVE: To assess the central EMG inhibitory action of tendon afferent input in muscle diseases. METHODS: The EMG inhibition elicited by electrical stimulation over muscle tendons was tested in 13 healthy voluntary subjects and 8 patients who had a primary muscle disease with a mild force deficit. Electrical stimuli were delivered to the tendon of the extensor carpi radialis muscle at the wrist during tonic voluntary isometric contraction at 50% of the maximum EMG level. The EMG signal was recorded by surface electrodes over the extensor carpi radialis muscle. RESULTS: The prestimulus background EMG level was reduced in 7 out 8 of the patients. Both groups had the same phases of EMG modulation following tendon stimulation (TE1, TI1, TE2) and their latency and amplitude did not differ significantly. Conversely, the area of TI1 was significantly larger (i.e. the inhibition decreased) in patients ([mean+/-SD] absolute area: controls=4.1+/-1.6 mVms, patients=6.9+/-2.9 mVms, P<0.05). CONCLUSIONS: In muscle dysfunction there are serial 'upstream' changes of central inhibitory systems, probably to maximize the residual muscle power of the affected muscle.

Volatile components of cigarette smoke: effect of acrolein and acetaldehyde on human gingival fibroblasts in vitro.

BACKGROUND: Tobacco and some of its volatile and non-volatile components have been found to affect many types of cells including gingival fibroblasts. Since normal gingival fibroblast functioning is fundamental to the maintenance of the periodontal connective tissue, as well as to wound healing, we examined the effect of acrolein and acetaldehyde, volatile components of cigarette smoke, on proliferation, attachment, and ultrastructure of human gingival fibroblasts (HGFs) in culture. METHODS: Human gingival fibroblast (HGF) strains derived from healthy individuals with non-inflamed gingiva were used in this study. The cells were incubated in the presence of different concentrations of acrolein and acetaldehyde. Cell attachment and proliferation were evaluated after incubation for 3 hours and 5 days, respectively. In addition, the cells were examined with a transmission electron microscope in order to evaluate their morphology. RESULTS: The results show that acrolein and acetaldehyde produced dose-dependent inhibition of HGF attachment and proliferation. The cytotoxic effect was, however, reversible when both substances were removed, after 3 days, from the medium. The main ultrastructural finding for the HGF cytoplasm was the presence of vacuoles and lysosomal structures that became prominent with increasing concentration of acrolein and acetaldehyde. CONCLUSIONS: Our experimental data suggest that acrolein and acetaldehyde, volatile components of tobacco smoke, are detrimental to HGF survival and consequently to the oral connective tissue. According to our morpho-functional evidence, these findings corroborate clinical and epidemiological investigations demonstrating smoke as a risk factor in the development of periodontal disease.

Beta-1,3-galactosyltransferase and alpha-1,2-fucosyltransferase involved in the biosynthesis of type-1-chain carbohydrate antigens in human colon adenocarcinoma cell lines.

We studied the beta-1,3-galactosyltransferase (GalT) and alpha-1,2-fucosyltansferase (FT) involved in the biosynthesis of type-1-chain carbohydrate antigens in human colon adenocarcinoma cell lines. We detected a GalT activity able to use GlcNAc as acceptor and found that lacto-N-biose I (Galbeta1-3GlcNAc) is the only reaction product. Such beta1,3GalT is kinetically similar to a pig trachea enzyme involved in mucin synthesis. The specific activity is high in cells that react strongly with anti-Lewis a and anti-Lewis b antibodies, and undetectable in a cell line that lacks antibody reaction. Reverse-transcriptase-mediated PCR analysis followed by DNA sequencing indicated that secretor-type alpha1,2FT is expressed in the cells, while the H type alpha1,2FT is not. The apparent Km values for donor and acceptor substrates determined for alpha1,2FT are similar to those of secretor-type alpha1,2FT and the specific activity measured correlates with Lewis b antigen expression on the cell surface. Moreover, some of the cell lines express Lewis y and H type 2 antigens, indicating that secretor type alpha1,2FT is responsible for their synthesis. Results suggest that biosynthesis of type-1-chain tumor-associated antigens in human colon carcinoma cells is operated by secretor-type alpha1,2FT, as reported in normal mucosa, and that beta1,3GalT activity may play a relevant role in its control.

Clinical variability in Becker muscular dystrophy. Genetic, biochemical and immunohistochemical correlates.

We have investigated 59 Becker muscular dystrophy patients, representing 56 independent mutations, to test the hypothesis of predictability of muscle dystrophin expression and clinical phenotype based on location of dystrophin gene mutations. Partial intragenic deletions and duplications account for 82% of the independent mutations, of which 76.7% were deletions and 5.3% duplications. Mutations in which boundaries could be defined, were of in-frame type (35 out of 37, 94.6%, with two exceptions. Eighty-two percent of mutations were located at the distal part of the rod domain (exons 45-60), 9% at domain I (promoter through exon 9) and 9% at proximal and central parts of domain II. Domain I deleted patients tended to have a worse clinical phenotype, with earlier presentation, faster progression rate and lower dystrophin expression, while distal rod domain deleted patients showed a more classic Becker muscular dystrophy phenotype. Between these two groups, only the differences in the immunohistochemical patterns of dystrophin expression and disease progression rate were statistically significant. Partial clinical and biochemical heterogeneity was observed in the distal domain II patient group, due to the presence of few patients covering the extremities of clinical severity. Two asymptomatic patients had deletions located in the central (exons 41-44) and distal parts (exons 50-53) of the rod domain. Severe myalgia and cramps were often reported as early onset symptoms (18 out of 59): no correlation was found between this symptomatology and the location of the mutation. Relative levels of muscle dystrophin correlated with immunohistochemical patterns of subsarcolemma staining. Dystrophin levels (as estimated by 30 kDa antibody immuno-reactivity) correlated with age of reaching a moderate degree of muscle involvement as well as with delay in reaching that stage, a parameter of disease progression rate. Our data confirm that different Becker muscular dystrophy gene in-frame mutations have different effects on dystrophin expression and clinical severity, indicating several functional roles of the dystrophin domains.

Clinical and biochemical evidence of skeletal muscle involvement in galactose-1-phosphate uridyl transferase deficiency.

An 8-year-old boy with galactose-1-phosphate uridyl transferase (GALT) deficiency presented with hypotonia, muscle hypotrophy, hepatomegaly, bilateral cataract and mild mental retardation. Two brothers showed a GALT activity consistent with a homozygotic condition and both parents were found to be heterozygotes for this defect. Histological and ultrastructural examination of muscle biopsy specimens showed several necrotic fibres. GALT activity was undetectable in skeletal muscle and muscle tissue cultures; myotubes converted galactose to CO2 at a lower rate than controls. Galactose-1-phosphate was increased in the patient's red cells and muscle tissue. GALT deficiency, not previously described in muscle, may be of pathogenic relevance in determining the myopathic features present in GALT deficiency syndrome.

Lack of anionic phospholipid calcium binding sites in Duchenne muscular dystrophy.

We studied membrane ultrastructural localization of anionic phospholipids (AP) and sialic acid (SA) calcium binding sites in muscle biopsies from Duchenne muscular dystrophy (DMD) and 3 Becker's muscular dystrophy (BMD) patients using polymyxin B (PXB) and limulus polyphemus (LP) as cytochemical markers. We found that AP calcium binding sites are lacking at muscle cell surface in all DMD muscle tissues, in both intact and degenerating muscle fibers. In BMD, AP have an unusual distribution along plasma membrane. Sialic acid calcium binding sites have the same localization along plasma membrane and basal lamina in DMD, BMD, and control muscles. The absence or alterations of structures involved in calcium binding in DMD and BMD may alter membrane calcium permeability, leading to abnormal Ca2+ influx into cells causing muscle necrosis.

Congenital myopathy with type 2A muscle fiber uniformity and smallness.

We describe a 12-year-old girl with congenital myopathy. ATPase histochemical reactions and immunocytochemical analysis of muscle fiber-type composition with monoclonal antibodies against slow, fast (2A and 2B) and fetal myosin demonstrate that this congenital disease is characterized by type 2A muscle fiber uniformity and smallness. This is an unusual feature for a congenital myopathy in which the fiber type predominance, when present, is confined to type I.

Dystrophin deficiency in a case of congenital myopathy.

We studied a 5-year-old boy who had the "floppy infant syndrome" and a dystrophic pattern on muscle biopsy. According to the clinical presentation and the histopathological findings the diagnosis of congenital muscular dystrophy with associated intellectual retardation was made. Immunohistochemical and immunoblot studies using anti-dystrophin antibodies showed complete absence of the protein in the patient's muscle. DNA analysis using cDNA probes showed a deletion at the 5' end of the dystrophin gene. Our observations on this patient suggest a new phenotypical variant of Duchenne muscular dystrophy.

Congenital myopathy associated with abnormal accumulation of desmin and dystrophin.

We studied a 5-yr-old boy clinically presenting congenital myopathy. Muscle biopsy showed sarcoplasmic accumulation of desmin filaments leading to diagnosis of desmin storage myopathy. An immunohistochemical study of other cytoskeletal proteins (actin, alpha-actinin, vimentin and dystrophin) was performed. Desmin positive areas reacted strongly with anti-mid-rod and C-terminus dystrophin antibodies. Probed with the same antibodies by Western blot, desmin and dystrophin showed normal molecular size but densitometric analysis demonstrated a parallel increase of both proteins. Our results indicate that intrasarcoplasmic desmin storage is associated with an abnormal accumulation of dystrophin. Since no other cytoskeletal proteins are accumulated this finding seems to be specific and suggests a possible structural and functional association between these two proteins in striated muscle.

Desmin and vimentin as markers of regeneration in muscle diseases.

Localization and distribution of desmin and vimentin have been studied in different neuromuscular disorders using monoclonal antibodies. We have demonstrated that vimentin, although virtually absent in normal human muscle fibers, is expressed in regenerating fibers in different neuromuscular disorders. Moreover, these fibers showed a strong positivity with desmin antibodies. In normal muscle fibers desmin is only localized at Z-line level. These results suggest that desmin and vimentin may be over-expressed during muscle regeneration processes, probably because of their importance in the structural organization of the sarcomere.

Muscle mitochondrial DNA deletion and 31P-NMR spectroscopy alterations in a migraine patient.

A 40-year-old female suffering from recurrent migrainous strokes is reported. She did not show any muscle weakness or wasting. Ragged red and cytochrome c oxidase negative fibers were present in the muscle biopsy. Muscle mitochondrial DNA analysis showed a 5 kb deletion, without a point mutation at nucleotide pair 3243 in the mitochondrial tRNALeu(UUR) gene. Phosphorus nuclear magnetic resonance spectroscopy of brain and gastrocnemius muscle showed a defective energy metabolism in both organs. An increased inorganic phosphate to phosphocreatine ratio due to a decreased phosphocreatine content was found in the occipital lobes, while an abnormal work-energy cost transfer function and a low rate of phosphocreatine post-exercise recovery were found in the muscle.

Appearance and localization of dystrophin in normal human fetal muscle.

We studied the localization of dystrophin in normal human fetal muscle by immunohistochemistry. Our results show the appearance of dystrophin at week 11 and a progressive organization of the protein along membrane in the following weeks of gestation. At week 22 almost all fibers show a clear membrane immunostaining. Concomitant analysis of muscle fiber-type composition reveals no correlation between progressive appearance of dystrophin and muscle fiber-type differentiation. Our findings suggest that synthesis and localization of dystrophin in developing human skeletal muscle is time-related and probably independent of neuronal influences.

Ubidecarenone in the treatment of mitochondrial myopathies: a multi-center double-blind trial.

Forty-four patients with mitochondrial myopathies were treated with Ubidecarenone (CoQ10) for 6 months in an open multi-center trial. No side effects of the drug were observed. Sixteen patients showing at least 25% decrease of post-exercise lactate levels were selected as responders. Responsiveness was apparently not related to CoQ10 level in serum and platelets or to the presence or absence of mtDNA deletions. The responders were treated for a further 3 months with CoQ10 or placebo in the second blind part of the trial; no significant differences were observed between the 2 groups. It is not clear why CoQ10 had therapeutic effects in some patients and not in others with the same clinical presentation and biochemical defect, and we failed to identify candidate responders before treatment. At the dose of CoQ10 used in this study (2 mg/kg/day) the therapy requires a long administration time before a response is seen.