Biomedical subjects

L Morandi

Publications and source records attributed to L Morandi.

At least 37 records · Page 2Linked to original sources

PubMed2001

Mild muscular dystrophy due to a nonsense mutation in the LAMA2 gene resulting in exon skipping.

Nonsense mutations outside the splicing consensus sequence have been reported to cause skipping of the nonsense-containing exon in several human diseases. We describe, for the first time, nonsense-mediated exon skipping in the laminin alpha2 (LAMA2) gene. Two siblings from a consanguineous family had altered expression of the laminin alpha2 chain and moderate clinical manifestations. In both we identified the new nonsense mutation Arg744Stop, which we expected to result in a totally non-functional polypeptide. However, analysis of the transcript revealed skipping of exon 15, containing the mutation, even though the consensus sequences for splicing at both ends of the exon and the beginning of intron 15 were unaltered. Exon skipping restored the open reading frame of the mutant transcript and resulted in a truncated protein. In cases where the genetic findings do not elucidate the phenotype, mRNA analysis is necessary to clarify the primary effect of mutations. Our findings also point to the necessity of immunochemical screening for expression of laminin alpha2 chain in atypical dystrophic adults as well as children.

Adult↗

PubMed2001

[Demonstration of TT virus in liver tissue fixed in formalin and embedded in paraffin].

INTRODUCTION: TT virus has been recently isolated in Japan in patients with acute and chronic non-A/non-G hepatitis. Its possible etiopathogenetic role in causing hepatitis has been initially taken in consideration. On the contrary, more recent studies deny the importance of TT virus in causing liver damage. Most of the studies are based on serological data or on viral detection from frozen liver tissue. AIM OF THE STUDY: In the present paper we describe a method to detect viral genome from formalin-fixed and paraffin-embedded liver tissue. MATERIALS AND METHODS: Twelve needle biopsies from liver were studied. Six cases were selected on the basis of serological negativity for HBV and HCV markers. Five cases of HCV-related chronic hepatitis and one HCV- and HIV-positive intravenous drug abuser were also included. All patients underwent liver biopsy, performed with a 14-G needle. Liver specimens were formalin-fixed and paraffin-embedded as routine. From each block, sections were cut and stained for histopathologic examination. Additional 5 microns sections were employed to extract DNA for nested PCR. RESULTS: In 2 of 12 cases studied, TT virus genome was found. In both cases the presence of viral DNA was confirmed by sequencing. Both patients were male. The first patient was a 39-year-old HIV- and HCV-positive intravenous drug abuser. The second patient was a 60-year-old heavy alcohol drinker. In both cases the presence of TT virus apparently did not affect the histological picture. CONCLUSION: It is possible to detect TT virus genome from formalin-fixed and paraffin-embedded tissue. This method offers the possibility to perform retrospective studies.

Adult↗

PubMed2000

Different mutations in the LMNA gene cause autosomal dominant and autosomal recessive Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EMD) is a condition characterized by the clinical triad of early-onset contractures, progressive weakness in humeroperoneal muscles, and cardiomyopathy with conduction block. The disease was described for the first time as an X-linked muscular dystrophy, but autosomal dominant and autosomal recessive forms were reported. The genes for X-linked EMD and autosomal dominant EMD (AD-EMD) were identified. We report here that heterozygote mutations in LMNA, the gene for AD-EMD, may cause diverse phenotypes ranging from typical EMD to no phenotypic effect. Our results show that LMNA mutations are also responsible for the recessive form of the disease. Our results give further support to the notion that different genetic forms of EMD have a common pathophysiological background. The distribution of the mutations in AD-EMD patients (in the tail and in the 2A rod domain) suggests that unique interactions between lamin A/C and other nuclear components exist that have an important role in cardiac and skeletal muscle function.

Adolescent↗

PubMed2000

Congenital muscular dystrophy associated with calf hypertrophy, microcephaly and severe mental retardation in three Italian families: evidence for a novel CMD syndrome.

We describe four Italian patients (aged 3, 4, 12, and 13 years ) affected by a novel autosomal form of recessive congenital muscular dystrophy. These patients were from three non-consanguineous families and presented an almost identical phenotype. This was characterized by hypotonia at birth, joint contractures associated with severe psychomotor retardation, absent speech, inability to walk and almost no interest in their surroundings. In addition, all patients had a striking enlargement of the calf and quadriceps muscles. Ophthalmologic examination revealed no structural ocular abnormalities in any of the children; one patient had severe myopia. In all cases a magnetic resonance imaging of the brain showed an abnormal posterior cranial fossa with enlargement of the cisterna magna and variable hypoplasia of the vermis of the cerebellum. Abnormality of the white matter was also present in all patients, in the form of patchy signal most evident in the periventricular areas. Serum CK was grossly elevated in all. The muscle biopsy from all cases showed dystrophic changes compatible with congenital muscular dystrophy. Immunofluorescence studies showed mild to moderate partial deficiency of laminin alpha 2 chain. Linkage analysis in the only informative family excluded the known loci for congenital muscular dystrophy, including laminin alpha 2 chain on chromosome 6q2, the Fukuyama congenital muscular dystrophy locus on 9q3 and the muscle-eye-brain disease on chromosome 1p3. We propose that this represent a novel severe variant of congenital muscular dystrophy, with associated central nervous system involvement.

Adolescent↗

PubMed2000

Unusual expression of emerin in a patient with X-linked Emery-Dreifuss muscular dystrophy.

We report on a patient with the typical clinical findings of Emery-Dreifuss muscular dystrophy due to a mutation in the emerin gene that should have produced a higher molecular weight protein. Immunohistochemical analysis showed emerin localized only in the cytoplasm of muscle fibres and lymphoblastoid cells. The emerin molecule contained the nucleoplasmic domain and the transmembrane domain responsible for nuclear membrane targeting, so its incorrect localization and lack of function could be due to abnormal folding resulting in rapid degradation or inability to bind other nuclear proteins.

Adolescent↗

PubMed2000

Disruption of heart sarcoglycan complex and severe cardiomyopathy caused by beta sarcoglycan mutations.

Two young males with limb-girdle muscular dystrophy (LGMD) resulting from sarcoglycan deficiency died at 27 (patient 1) and 18 years (patient 2) of severe cardiomyopathy. Genetic analysis showed that they were compound heterozygotes for mutations in the beta sarcoglycan gene. One of these mutations, an 8 bp duplication in exon 3, was common to both patients. The second mutation in patient 2 was a 4 bp deletion at the splice donor site of intron 2, not reported previously. Patient 2 had more severe heart and skeletal muscle defects with faster deterioration; no sarcoglycans were detected in his skeletal muscle. The second mutation in patient 1, inferred because the unaffected father carries the 8 bp duplication, was not found. In patient 1, both heart and skeletal muscle were analysed and showed reduction of all sarcoglycans in both tissues and incorrect localisation of alpha and gamma sarcoglycans in heart. Therefore mutations in one sarcoglycan gene can disrupt the entire sarcoglycan complex in both skeletal and cardiac muscle. Differing expression patterns of sarcoglycan components in heart and skeletal muscle could be the result of alternatively spliced transcripts in these tissues. By sequencing an alternative transcript, highly expressed in the heart and skeletal muscle of patient 1, we found an 87 bp cryptic exon not previously reported. Although cardiomyopathy can result from mutations in alpha and gamma sarcoglycans, we show for the first time that the condition can also be caused by mutations in the beta sarcoglycan gene. This report therefore expands the phenotype of sarcoglycanopathies and suggests that cardiac function in LGMD patients with defective sarcoglycan expression should be monitored.

Adolescent↗

PubMed2000

Partial laminin alpha2 chain deficiency in a patient with myopathy resembling inclusion body myositis.

It is becoming evident that clinical phenotypes associated with partial laminin alpha2 chain deficiency are variable. We recently observed a 29-year-old man with leukoencephalopathy and vacuolar myopathy resembling inclusion body myositis. Laminin alpha2 immunohistochemical analysis showed reduction of the protein on muscle fiber surfaces. Molecular analysis revealed two novel compound heterozygous mutations in the LAMA2 gene. This is the first report linking a mutation in the LaMA2 gene with leukoencephalopathy and inclusion body-like myositis.

Adult↗

PubMed1999

Clinical correlations in 16 patients with total or partial laminin alpha2 deficiency characterized using antibodies against 2 fragments of the protein.

BACKGROUND: Many patients with classic congenital muscular dystrophy have been found to have partial or total deficiency of the alpha2 chain of laminin 2 (merosin). This deficiency has mostly been studied using only 1 antibody against a fragment of the protein. OBJECTIVES: To characterize the expression of laminin alpha2 in the skeletal muscle of patients with laminin alpha2 deficiency using antibodies against 2 different portions of the protein and to correlate the immunochemical findings with clinical phenotype. METHODS: We studied 4 patients with total lack of laminin alpha2 and 12 with partial laminin alpha2 deficiency with immunohistochemical techniques and Western blot analysis. We used antibodies recognizing an 80-kd fragment toward the C-terminus and a 300-kd fragment toward the amino-terminal. Patient characteristics examined were functional compromise, magnetic resonance imaging or computed tomography of the brain, electromyography, evoked potentials, and creatine kinase levels. RESULTS: In 4 patients, immunohistochemical analysis revealed no reactivity to either antibody; in 2 patients, the 300-kd fragment alone was partially expressed; in 2 patients, the 80-kd fragment alone was partially expressed; and in 8 patients, both fragments were partially expressed. Immunoblot analysis revealed bands of reduced intensity and normal molecular weight generally corresponding to the immunohistochemical findings. Absence of both fragments or of one with reduction of the other always produced a severe clinical phenotype, while a milder clinical phenotype was observed when both fragments were partially expressed. CONCLUSIONS: Extent of laminin alpha2 deficiency in most cases correlates with clinical phenotype but not with peripheral and central white matter abnormalities. Skin biopsy specimens may reveal laminin alpha2 deficiency in patients who have normal laminin alpha2 levels in muscle biopsy specimens.

Adolescent↗

PubMed1999

Development of muscle pathology in canine X-linked muscular dystrophy. I. Delayed postnatal maturation of affected and normal muscle as revealed by myosin isoform analysis and utrophin expression.

Canine X-linked muscular dystrophy (CXMD) is genetically homologous to Duchenne muscular dystrophy and shares the severe myopathy and lethal clinical development of the human disease. We used immunohistochemistry to characterize the time course of postnatal expression of adult fast, adult slow and developmental myosin in the muscle of CXMD dogs, carriers and healthy controls. We also characterized the expression of utrophin and dystrophin. This detailed immunolocalization study confirmed that postnatal muscle maturation is delayed in normal dogs compared to other animals and humans, and is only achieved at around 60 days. In CXMD dogs major derangement of myosin expression became evident from about 15 days; there was a selective loss of fibers expressing fast myosin and persistence of developmental fibers compared to controls. In carriers, the proportion of dystrophin-deficient fibers, which mainly expressed fast myosin, decreased with age. In controls and carriers utrophin was absent from muscle fiber surfaces in 2-day-old animals but present between 15 and 30 days, to mostly disappear by 60 days. In dystrophic animals, sarcolemmal expression of utrophin was more marked and persistent. That immature neonatal muscle from control dogs normally contains sarcolemmal utrophin may have implications for the success of utrophin up-regulation therapy to correct the dystrophic phenotype. The data of this study provide important baseline information for further studies on the development and progression of pathological changes in the muscle of CXMD dogs.

Aging↗

PubMed1999

Transforming growth factor-beta1 and fibrosis in congenital muscular dystrophies.

We evaluated transforming growth factor-beta1 (TGF-beta1) expression in the muscle of four laminin alpha2-negative, four laminin alpha2-positive and seven partial laminin alpha2-deficient congenital muscular dystrophy (CMD) patients, and compared it to Duchenne muscular dystrophy (DMD) patients and controls. TGF-beta1 mRNA levels in skeletal muscle from laminin alpha2-negative and laminin alpha2-positive CMD patients were significantly greater than in controls (P < 0.05 and P < 0.005, respectively), while in partial laminin alpha2-deficient muscular dystrophy patients the amount was not significantly higher than in controls (P > 0.1). The TGF-beta1 values were lower than those found in DMD, although the extent of fibrosis was greater in CMD than in DMD and controls. Our findings suggest that TGF-beta1 is involved in CMD muscle fibrosis, but differently from what we observed in DMD muscles as it seems not to be the major player in connective tissue proliferation.

Adult↗

PubMed1999

Detection of Helicobacter pylori cagA gene by polymerase chain reaction in faecal samples.

OBJECTIVE: The polymerase chain reaction (PCR) has been extensively and successfully used to detect Helicobacter pylori in gastric juice and gastric biopsies. In contrast, the results obtained using faeces as biological samples for PCR are rather conflicting. This may be due to the presence of faecal inhibitory compounds (polysaccharides) which can inhibit the amplification reaction. The aim of this study was to characterize the H. pylori genotype in faecal samples by using specific primers for the cagA gene. To overcome the problem of contamination by polysaccharides, we used a filter-based extraction technique already applied in a previous study. METHODS: Antral and body biopsies were obtained from 30 symptomatic patients undergoing upper endoscopy. PCR was used to detect the presence of H. pylori organisms in faecal samples by using primers selected for the urease gene A. In addition, H. pylori organisms were characterized both in faecal samples and paraffin-embedded biopsies by PCR with specific primers for the cagA gene. RESULTS: All patients showed a positive CLO test (rapid urease test) and evidence of H. pylori by Warthin-Starry stain. PCR detected the urease A gene in the faecal samples of all patients. The cagA gene was detected in the faecal and biopsy samples of 18 subjects (60%). Duodenal ulcer and/or antral erosions were observed in 15 of the 18 cagA-positive patients (83.3%) and in five of the 12 cagA-negative patients (41.7%). Endoscopic features of normal mucosa or gastritis were observed in three cagA-positive patients (16.7%) and in seven cagA-negative patients (56.3%). cagA-positive status was found to be significantly related to the endoscopic features of duodenal ulceration and/or antral erosions. CONCLUSIONS: Our findings prove that faeces are suitable samples for the detection of cagA status. Moreover, they confirm the existence of a significant relationship between cagA-positive status and duodenal ulcer and/or antral erosions.

Adult↗

PubMed1998

Congenital muscular dystrophy with merosin deficiency: MRI findings in five patients.

We present the MRI findings in five patients with congenital muscular dystrophy (CMD) and merosin (laminin alpha2) deficiency, which was total in one and partial in four. In one patient with partial merosin deficiency, MRI was normal. The other four patients had supratentorial white matter abnormalities. In three, T2-weighted images revealed subcortical, deep lobar and periventricular high signal in white matter, while in the other there were only small peritrigonal areas of increased signal. On T1-weighted images, there was slightly low signal. Cortical abnormalities were absent. None of these changes were accompanied by symptoms or signs of central nervous system involvement. White matter abnormalities in a patient with CMD should prompt investigation of merosin.

Adult↗

PubMed1998

A new non-radioactive method for the screening and prenatal diagnosis of myotonic dystrophy patients.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease with an estimated incidence of 1 in 8000 and is the most common form of muscular dystrophy affecting adults. An unstable, untranslated part of the myotonic dystrophy protein kinase gene on the long arm of chromosome 19, composed of CTG repeats, is a genetic marker for DM. We have developed a fast non-radioactive polymerase chain reaction (PCR) procedure to detect the (CTG)n repeat expansion in DM patients and their relatives. Genomic DNA extracted from peripheral blood lymphocytes was amplified by PCR using specific primers to flank the region containing the triplets. To improve the amplification of this CG-rich region, either 10% glycerol or rTth DNA polymerase XL (extra long) was added to the reaction mixture, allowing amplification of huge expansions otherwise not polymerized by PCR. The PCR products were Southern blotted and the expansion revealed using a fluorescein-labelled (CTG)10 probe. We compared our results with those obtained in 24 patients and relatives using genomic digestion followed by radioactive Southern blot; in all cases the results overlapped. The same technique was used for prenatal diagnosis in pregnant DM mothers. We conclude that this new method is reliable for the genetic testing of DM patients.

Adult↗

PubMed1997

X-linked Emery-Dreifuss muscular dystrophy can be diagnosed from skin biopsy or blood sample.

We have raised an anti-emerin polyclonal antibody against a fusion protein encompassing most of the hydrophilic portion of emerin. Using this antibody, we have analyzed emerin expression in Emery-Dreifuss muscular dystrophy (EDMD) patients and controls, by immunocytochemistry, in skeletal muscle and skin, and by immunoblot, in peripheral blood mononuclear cells and lymphoblasts. Emerin was localized on the surfaces of nuclei in control skeletal muscle and skin but was absent or reduced in patient skeletal muscle, was absent from the skin of patients, and was expressed only in a few nuclei in a patient's mother. Immunoblot of peripheral blood cells from EDMD patients showed absence of the emerin band, altered-size emerin, or a protein of normal molecular mass but slightly reduced quantity. The diagnosis of X-linked EDMD is normally confirmed by genetic analysis of the STA gene coding for emerin. We propose immunocytochemical evaluation of emerin expression in skin biopsies as a sensitive and more convenient tool for diagnosing X-linked EDMD and, in particular, for distinguishing it from the autosomal dominant form. This technique may be applied to suspected EDMD patients, especially sporadic cases or those with incomplete clinical phenotype, and also suspected carriers. Immunoblot of peripheral blood cells is also useful, but it may not unequivocally identify carriers and some patients.

Adolescent↗

PubMed1997

Concomitant deficiency of beta- and gamma-sarcoglycans in 20 alpha-sarcoglycan (adhalin)-deficient patients: immunohistochemical analysis and clinical aspects.

We have investigated the expression, using immunohistochemistry, of beta- and gamma-sarcoglycans in the muscles of 20 patients in whom previous screening had revealed a deficiency of alpha-sarcoglycan. alpha-, beta- and gamma-sarcoglycans were absent in 7 patients and variably reduced in 8 patients, in 2 of whom beta-sarcoglycan was more reduced than the alpha- and gamma-proteins. In 5 other patients with variably reduced alpha- and beta-sarcoglycans, gamma-sarcoglycan was completely absent. In all patients the distribution of hyposthenia at disease onset was similar, and predominantly involved pelvic girdle muscles; however, the age at onset and rate of disease progression were highly variable. In severely compromised patients, the onset of disease was before 10 years of age and gamma-sarcoglycan or all three sarcoglycans were absent from muscles. Immunohistochemical analysis of sarcoglycans should be part of routine screening for muscle dystrophies to identify patients with sarcoglycanopathy. Gene analysis is necessary to identify the primary defect; however, sarcoglycan immunohistochemistry may be useful for indicating which gene to investigate. Further biochemical characterization of the interactions between these proteins is required to fully elucidate their roles in causing severe, moderate or mild muscular dystrophy.

Adolescent↗

PubMed1996

Developmental expression of dystrophin, dystrophin-associated glycoproteins and other membrane cytoskeletal proteins in human skeletal and heart muscle.

Dystrophin, utrophin and the dystrophin-associated glycoproteins, beta-dystroglycan and adhalin, were analyzed, together with the membrane cytoskeletal proteins beta-spectrin, vinculin and talin, and adult and fetal myosin heavy chains, in 25 normal human fetuses from 8 to 24 weeks of gestation. Dystrophin was present in heart and skeletal muscle from 8 weeks although in the latter was mainly in the cytoplasm at this stage. Utrophin expression increased until around gestational weeks 19/21, but by 24 weeks immunostaining and immunoblot band intensities had reduced. Beta-dystroglycan was scarce in skeletal muscle at 8 weeks, increased with maturation and was more abundant in heart of the same age. Adhalin appeared later than beta-dystroglycan on skeletal muscle fiber surfaces, positivity became more intense as the fibers matured. In heart adhalin was detectable only in groups of cells at 12-16 weeks. From 8 weeks all fetal myotubes expressed beta-spectrin on their surfaces, while vinculin and talin positivity was mainly at the periphery of the fascicles, increasing with age. Adult slow myosin was seen in most myotubes at 10 weeks. Secondary myotubes then formed which increasingly expressed adult fast myosin, while still retaining fetal myosin. By 24 weeks most fibers expressing adult slow myosin had lost fetal myosin and were more mature in the expression of most membrane proteins. Muscle membrane organization during human fetal development is a complex process and takes place earlier in heart than skeletal muscle.

Blotting, Western↗

PubMed1996

Clinical heterogeneity of adhalin deficiency.

We report adhalin deficiency in 8 patients with clinically diagnosed muscular dystrophy, dystrophic histopathological features, high plasma creatine kinase levels, normal expression of dystrophin, and marked variability of symptoms. Although the distribution of hyposthenia was similar in all 8 patients and predominantly involved muscles in the pelvic girdle, age at onset and rate of disease progression were highly variable: In 2 patients onset, at ages 24 and 25, was later than has been previously observed. We found no apparent relation between disease severity and the quantity of adhalin expressed. Two kinds of myopathy with adhalin deficiency have been reported: one caused by a mutation in the adhalin gene on chromosome 17 (primary adhalinopathy) and the other linked to chromosome 13. The product of the gene on chromosome 13 is probably associated with adhalin and its deficiency results in secondary adhalinopathy. The severity of clinical phenotypes in these adhalinopathies seems to relate more to the kind and site of the mutations than to the residual amount of the protein. We also detected a variable reduction in the laminin beta 1 subunit by immunohistochemistry in most patients, confirming that this is commonly associated with adhalin deficiency.

Adolescent↗

PubMed1996

Dystrophin-associated protein abnormalities in dystrophin-deficient muscle fibers from symptomatic and asymptomatic Duchenne/Becker muscular dystrophy carriers.

The absence of dystrophin in muscle fibers is associated with a major reduction in dystrophin-associated proteins (DAPs) and disruption of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. We investigated the expression of the DAPs beta-dystroglycan, alpha-sarcoglycan, gamma-sarcoglycan and syntrophin as well as utrophin in the muscles of 13 Duchenne muscular dystrophy (DMD) carriers (with variable percentages of dystrophin-deficient fibers and with a range of clinical symptoms), 2 Becker muscular dystrophy (BMD) carriers (expressing a highly truncated protein in some fibers), 2 girls with a DMD-like phenotype, and 11 BMD carriers with almost normal dystrophin expression (reduced or patchy distribution in a few fibers only and rare dystrophin-deficient fibers). DAPs were highly reduced in all fibers lacking dystrophin in the DMD carriers, but were almost normal in the dystrophin-deficient fibers of the 2 BMD carriers with highly truncated dystrophin. In the 11 BMD carriers with nearly normal dystrophin, the few fibers with reduced or patchy dystrophin immunostaining also showed reduced DAP expression in correlation with dystrophin expression. Immunoblot for beta-dystroglycan and alpha-sarcoglycan confirmed the immunohistochemical findings. Utrophin expression was slightly increased in a proportion of fibers in the DMD and BMD carriers with dystrophin mosaicism. We found no correlation between utrophin expression and DAP expression. We conclude that absence or reduction of dystrophin in muscle fibers of DMD and BMD carriers causes a reduction of DAPs in the same fibers, as observed in DMD and BMD patients, while utrophin does not seem to play a role in DAP expression in adult muscle.

Adolescent↗