Review: Danon disease: Review of natural history and recent advances.

Danon disease is a severe multisystem disorder clinically characterized by hypertrophic cardiomyopathy, skeletal myopathy and mental retardation in male patients, and by a milder phenotype (predominantly involving cardiac muscle) in female patients. The disease is inherited as an X-linked dominant trait. The primary deficiency of lysosome-associated membrane protein-2 (LAMP-2) causes disruption of autophagy, leading to an impaired fusion of lysosomes to autophagosomes and biogenesis of lysosomes. We surveyed over 500 Danon disease patients reported in the literature from the first description to the present, in order to summarize the clinical, pathological and molecular data and treatment perspectives. An early molecular diagnosis is of crucial importance for genetic counselling and for therapeutic interventions: in male patients, the prognosis is poor due to rapid progression towards heart failure, and only heart transplantation modifies the disease course.

Cerebral Mitochondrial Microangiopathy Leads to Leukoencephalopathy in Mitochondrial Neurogastrointestinal Encephalopathy.

BACKGROUND AND PURPOSE: Mitochondrial neurogastrointestinal encephalopathy is a rare disorder due to recessive mutations in the thymidine phosphorylase gene, encoding thymidine phosphorylase protein required for mitochondrial DNA replication. Clinical manifestations include gastrointestinal dysmotility and diffuse asymptomatic leukoencephalopathy. This study aimed to elucidate the mechanisms underlying brain leukoencephalopathy in patients with mitochondrial neurogastrointestinal encephalopathy by correlating multimodal neuroradiologic features to postmortem pathology. MATERIALS AND METHODS: Seven patients underwent brain MR imaging, including single-voxel proton MR spectroscopy and diffusion imaging. Absolute concentrations of metabolites calculated by acquiring unsuppressed water spectra at multiple TEs, along with diffusion metrics based on the tensor model, were compared with those of healthy controls using unpaired t tests in multiple white matters regions. Brain postmortem histologic, immunohistochemical, and molecular analyses were performed in 1 patient. RESULTS: All patients showed bilateral and nearly symmetric cerebral white matter hyperintensities on T2-weighted images, extending to the cerebellar white matter and brain stem in 4. White matter, N-acetylaspartate, creatine, and choline concentrations were significantly reduced compared with those in controls, with a prominent increase in the radial water diffusivity component. At postmortem examination, severe fibrosis of brain vessel smooth muscle was evident, along with mitochondrial DNA replication depletion in brain and vascular smooth-muscle and endothelial cells, without neuronal loss, myelin damage, or gliosis. Prominent periependymal cytochrome C oxidase deficiency was also observed. CONCLUSIONS: Vascular functional and histologic alterations account for leukoencephalopathy in mitochondrial neurogastrointestinal encephalopathy. Thymidine toxicity and mitochondrial DNA replication depletion may induce microangiopathy and blood-brain-barrier dysfunction, leading to increased water content in the white matter. Periependymal cytochrome C oxidase deficiency could explain prominent periventricular impairment.

Subpopulations of dermal skin fibroblasts secrete distinct extracellular matrix: implications for using skin substitutes in the clinic.

BACKGROUND: While several commercial dermoepidermal scaffolds can promote wound healing of the skin, the achievement of complete skin regeneration still represents a major challenge. OBJECTIVES: To perform biological characterization of self-assembled extracellular matrices (ECMs) from three different subpopulations of fibroblasts found in human skin: papillary fibroblasts (Pfi), reticular fibroblasts (Rfi) and dermal papilla fibroblasts (DPfi). METHODS: Fibroblast subpopulations were cultured with ascorbic acid to promote cell-assembled matrix production for 10 days. Subsequently, cells were removed and the remaining matrices characterized. Additionally, in another experiment, keratinocytes were seeded on the top of cell-depleted ECMs to generate epidermal-only skin constructs. RESULTS: We found that the ECM self-assembled by Pfi exhibited randomly oriented fibres associated with the highest interfibrillar space, reflecting ECM characteristics that are physiologically present within the papillary dermis. Mass spectrometry followed by validation with immunofluorescence analysis showed that thrombospondin 1 is preferentially expressed within the DPfi-derived matrix. Moreover, we observed that epidermal constructs grown on DPfi or Pfi matrices exhibited normal basement membrane formation, whereas Rfi matrices were unable to support membrane formation. CONCLUSIONS: We argue that inspiration can be taken from these different ECMs, to improve the design of therapeutic biomaterials in skin engineering applications.

Juvenile dermatomyositis: A report of three cases.

Juvenile dermatomyositis (JDM), an autoimmune idiopathic myositis, is characterized by rash and proximal muscle weakness. Immunohistopathology typically shows perivascular inflammatory infiltrate with predominance of CD4+ T lymphocytes, perifascicular atrophy, and upregulation of major histocompatibility complex class I. JDM has been attributed to a humoral-driven muscle microangiopathy probably implicating the type I interferon pathway. Tubulo-reticular inclusions present in endothelial cell of muscle are biomarkers of interferon exposure, and so may be an indirect data of this myopathy especially in the absence of rash and inflammatory infiltrate. We report on three patients in which electron microscopy solves the differential diagnosis among infantile myositis showing peculiar inclusions.

Building blocks of the GIPU, Italian Group of Ultrastructural Pathology.

The Italian Group of Ultrastructural Pathology, GIPU, is a scientific organization committed to promote the art and science of Electron Microscopy (EM) in the pathology field in Italy, sharing its professional work with a public audience. The history of the GIPU goes back to 1990s when a founder group set up the Italian Group of Ultrastructural Diagnostic (GIDU) in Milan. The central focus of annual meetings was on EM, transmission and scanning one, about interesting cases in which it was instrumental in diagnosis. In the 1990s, ultrastructure was still the gold standard for cell/tissue morphology, biology, biochemistry, diagnostic pathology, and played an important role in tailored medicine. So, especially transmission EM, could play a critical role in the diagnosis of various diseases as in human as in animals. Best topics of the annual scientific meetings of the group were kidney, muscle, heart, and liver pathology, infertility, neuropathology, respiratory diseases, skin diseases, storage diseases, tumor pathology, infectious diseases, parasitology, veterinary pathology and more. Nowadays, EM is a method whose importance for diagnosis and pathology is well established: it is still essential in several pathologies, helpful in others, and welcome implemented in eclectic research pathology. Omission of EM likely makes the studies suboptimal and wasteful. So, from 2007 the name of the group has been changed to the Italian Group of Ultrastructural Pathology (GIPU) to favor broader applications of EM also to pathology research field. During last decades, GIDU/GIPU has interconnected with international (Society for Ultrastructural Pathology) and european (European Society of Pathology and Joint Meeting with the European Electron Microscopy Working Group) scientific society, according its statute. By 1991, GIPU has had 40 members: membership in this Group is still open and welcome to all pathologists, PhD, electron microscopy technologists, pathology trainees, and researchers interested in pathology and electron microscopy.

CD99 suppresses osteosarcoma cell migration through inhibition of ROCK2 activity.

CD99, a transmembrane protein encoded by MIC2 gene is involved in multiple cellular events including cell adhesion and migration, apoptosis, cell differentiation and regulation of protein trafficking either in physiological or pathological conditions. In osteosarcoma, CD99 is expressed at low levels and functions as a tumour suppressor. The full-length protein (CD99wt) and the short-form harbouring a deletion in the intracytoplasmic domain (CD99sh) have been associated with distinct functional outcomes with respect to tumour malignancy. In this study, we especially evaluated modulation of cell-cell contacts, reorganisation of the actin cytoskeleton and modulation of signalling pathways by comparing osteosarcoma cells characterised by different metastasis capabilities and CD99 expression, to identify molecular mechanisms responsible for metastasis. Our data indicate that forced expression of CD99wt induces recruitment of N-cadherin and ß-catenin to adherens junctions. In addition, transfection of CD99wt inhibits the expression of several molecules crucial to the remodelling of the actin cytoskeleton, such as ACTR2, ARPC1A, Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) as well as ezrin, an ezrin/radixin/moesin family member that has been clearly associated with tumour progression and metastatic spread in osteosarcoma. Functional studies point to ROCK2 as a crucial intracellular mediator regulating osteosarcoma migration. By maintaining c-Src in an inactive conformation, CD99wt inhibits ROCK2 signalling and this leads to ezrin decrease at cell membrane while N-cadherin and ß-catenin translocate to the plasma membrane and function as main molecular bridges for actin cytoskeleton. Taken together, we propose that the re-expression of CD99wt, which is generally present in osteoblasts but lost in osteosarcoma, through inhibition of c-Src and ROCK2 activity, manages to increase contact strength and reactivate stop-migration signals that counteract the otherwise dominant promigratory action of ezrin in osteosarcoma cells.

Gamma rays induce a p53-independent mitochondrial biogenesis that is counter-regulated by HIF1α.

Mitochondrial biogenesis is an orchestrated process that presides to the regulation of the organelles homeostasis within a cell. We show that γ-rays, at doses commonly used in the radiation therapy for cancer treatment, induce an increase in mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence, in the presence of a functional p53. Although the main effector of the response to γ-rays is the p53-p21 axis, we demonstrated that mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a murine double minute 2 (MDM2)-mediated hypoxia-inducible factor 1α (HIF1α) degradation, leading to the release of peroxisome-proliferator activated receptor gamma co-activator 1ß inhibition by HIF1α, thus promoting mitochondrial biogenesis. Mimicking hypoxia by HIF1α stabilization, in fact, blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally, we also show in vivo that post-radiotherapy mitochondrial DNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of cell senescence.

Myocardial adenomatoid tumor in eight cattle: evidence for mesothelial origin of bovine myocardial epithelial inclusions.

The adenomatoid tumor is an uncommon benign lesion, thus far described only in humans. Adenomatoid tumors typically arise in the genital tract, exceptionally in the heart, and usually represent an incidental finding. Microscopically, they are constituted by epithelioid cells that form tubular structures and anastomosing channels within a fibrous stroma. Mesothelial origin of these lesions is suggested by their immunohistochemical characteristics. In cattle, previously reported myocardial epithelial inclusions are morphologically similar in that the cells are immunoreactive for both cytokeratins and vimentin, and bear surface microvilli. Myocardial lesions found incidentally at slaughter in 8 cattle histologically resembled the so-called bovine myocardial epithelial inclusions and had morphologic and immunohistochemical features consistent with human adenomatoid tumor. All lesions were in the left ventricular myocardium, adjacent to the epicardium, and composed of epithelioid cells that formed cords and tubules, and were immunoreactive for pan-cytokeratins, cytokeratin 5/6, vimentin, calretinin, Wilms' tumor 1 suppressor gene, and CD30 antigen. By electron microscopy, numerous long slender microvilli were associated with desmosomes and tonofibrils. The immunohistochemical and ultrastructural features were considered consistent with mesothelial origin. These lesions, corresponding to the previously described myocardial epithelial inclusions in cattle, might be considered embryologic rests and could represent the bovine counterpart of the human adenomatoid tumor.

Early-onset dementia with prolonged occipital seizures: an atypical case of Kufs disease.

OBJECTIVE: Kufs disease is the adult-onset form of neuronal ceroid lipofuscinosis (NCL). Its two clinical phenotypes are type A (progressive myoclonus epilepsy with dementia) and type B (behavioral abnormalities and dementia, associated with pyramidal and extrapyramidal signs). METHODS: We describe the clinical evolution of an atypical case characterized by progressive dementia and focal occipital seizures. RESULTS: A healthy 37-year-old woman began showing memory deficits and behavioral disturbances (apathy, lack of inhibitions, untidiness). After 4 years, she developed rare clusters of tonic-clonic seizures, as well as focal seizures originating from the temporo-occipital regions, clinically associated with visual hallucinations, wandering, and agitation. When she was 44 years old, neuropsychological assessment revealed severe frontotemporal dementia. MRI showed cortical atrophy and, on T2-weighted images, hypointensity of the basal ganglia, and hyperintensity and reduction of the deep white matter. On the basis of these findings, a diagnosis of Kufs disease was hypothesized. A skin biopsy was negative, but electron microscopy examination of a right frontal lobe brain biopsy revealed the presence of typical storage material (fingerprint inclusions). The patient never developed myoclonus or extrapyramidal signs. DISCUSSION: Kufs disease is difficult to diagnose on account of its heterogeneous clinical pattern and pathologic features, and the lack of a specific genetic locus alteration. The neuropsychological pattern and MRI findings observed in patients with early-onset frontotemporal dementia and seizure disorder suggest that Kufs disease should be considered in their differential diagnosis. Extracerebral biopsy can be nondiagnostic, and when alternative diagnoses have been ruled out, cerebral biopsy should be considered.

Endoscopic appearance of GERD: putative role of cell proliferation.

BACKGROUND: Erosive esophagitis is a frequent endoscopic feature in patients with gastro-oesophageal reflux disease. However, most of patients with heartburn/regurgitation have a non-erosive reflux disease. The reason for this heterogeneous impact of gastro-oesophageal reflux disease on oesophageal mucosa is unknown to date. AIM: To evaluate the cell proliferation status of oesophageal epithelium in both healthy normal subjects and patients with gastro-oesophageal reflux disease with or without erosions. MATERIALS AND METHODS: All the subjects underwent endoscopy and biopsies were taken at 5 cm from the squamo-columnar junction. Specimens were analysed both at histology and at transmission electron microscopy. Cell proliferation was evaluated by MIB1 immunostaining. Of the 85 subjects were studied, 10 were healthy controls with normal pH-testing and macroscopical, histological and ultrastructural patterns; 37 were patients with erosive esophagitis, and 38 patients with non-erosive reflux disease. RESULTS: At histology, of the 37 patients affected by erosive esophagitis, 30 had normal mucosa and 7 showed mild oesophagitis. One patient with non-erosive reflux disease showed signs of oesophagitis at histology. At TEM, all patients with gastro-oesophageal reflux disease had ultrastructural patterns of damage i.e. dilations of intercellular spaces (DIS), and all controls had a normal ultrastructural pattern. The mean (+/-SD) MIB1-LI values of normal subjects and non-erosive reflux disease and erosive oesophagitis patients were 62.2% (+/-9.1), 29.7% (+/-7.2) and 16.2% (+/-5.2), respectively; there were significant differences among the three groups (p<0.001). CONCLUSIONS: Oesophageal mucosa of patients with reflux symptoms presents a decrease in MIB1 immunostaining of 50% and 25% in non-erosive reflux disease and erosive esophagitis patients with respect to normal subjects.

Ultrastructural changes in dysferlinopathy support defective membrane repair mechanism.

BACKGROUND: The dysferlin gene has recently been shown to be involved in limb girdle muscular dystrophy type 2B and its allelic disease, Miyoshi myopathy, both of which are characterised by an active muscle degeneration and regeneration process. Dysferlin is known to play an essential role in skeletal muscle fibre repair, but the process underlying the pathogenetic mechanism of dysferlinopathy is not completely understood. AIMS: To define both specific alterations of muscle fibres and a possible sequential mechanism of myopathy development. METHODS: A histological, immunohistochemical, and ultrastructural analysis of 10 muscle biopsies from patients with molecularly diagnosed dysferlinopathy. RESULTS: An inflammatory response was seen in most of the muscle biopsies. The immunohistochemical pattern demonstrated active regeneration and inflammation. Non-necrotic fibres showed alterations at different submicroscopic levels, namely: the sarcolemma and basal lamina, subsarcolemmal region, and sarcoplasmic compartment. In the subsarcolemmal region there were prominent aggregations of small vesicles, probably derived from the Golgi apparatus, which consisted of empty, swollen cisternae. In the sarcolemma there were many gaps and microvilli-like projections, whereas the basal lamina was multilayered. CONCLUSIONS: The histopathological, immunohistochemical, and ultrastructural data show that dysferlinopathy is characterised by a very active inflammatory/degenerative process, possibly associated with an inefficient repair and regenerative system. The presence of many crowded vesicles just beneath the sarcolemma provides submicroscopical proof of a defective resealing mechanism, which fails to repair the sarcolemma.

A novel Notch3 gene mutation not involving a cysteine residue in an Italian family with CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary cerebrovascular disease leading to accumulating neurologic deficits and dementia. CADASIL has been linked to nucleotide substitutions and deletions in the Notch3 gene. All the mutations described until now lead to unpaired cysteine residue in the epidermal growth factor-like repeats. The authors report a family with CADASIL carrying a deletion in the Notch3 gene that did not involve a cysteine residue.

Amyloid deposition as a cause of atrial remodelling in persistent valvular atrial fibrillation.

AIM: The spectrum of histological alterations, namely atrial amyloidosis, in the right and left atria of patients with chronic persistent atrial fibrillation (AF) and rheumatic heart disease is not completely known. METHODS AND RESULTS: One hundred and twenty-eight atrial appendages (66 left and 62 right), obtained from 72 patients with rheumatic valve disease and chronic AF undergoing cardiac surgery for valve replacement or repair and AF treatment were histologically evaluated for the presence of amyloid deposits. One hundred and four specimens of left and right auricles from 52 patients in sinus rhythm with severe chronic heart failure undergoing heart transplant were also analyzed (controls). Amyloid was found in 33 (46%) valvular patients with chronic persistent AF and in 6 (12%) controls. Amyloid was related to the presence and duration of AF, was more frequently found in left atrial samples and was independent of age. On stepwise logistic regression analysis, AF duration and female gender were independently related to amyloid deposition. CONCLUSIONS: Patients with long-standing AF and rheumatic heart disease have a very high prevalence of atrial amyloidosis. Amyloid deposition is more frequent in left than in right atrial appendage and correlates with AF duration and female gender. Amyloid deposition could constitute an additional histological feature in the structural remodeling of atria during long-standing AF, at least in rheumatic valve disease. Persistence of AF might play a pivotal role in promoting amyloid deposition.

Cell proliferation and ultrastructural changes of the duodenal mucosa of patients affected by familial adenomatous polyposis.

Patients affected by familial adenomatous polyposis (FAP) are at risk of developing duodenal neoplasia. Our objective was to detect early abnormalities of the epithelial cell proliferation and ultrastructure of apparently normal duodenal mucosa of FAP patients. Biopsy specimens were taken from the duodenal mucosa. Cell proliferation was studied by immunohistochemistry with proliferating cell nuclear antigen (PCNA), and ultrastructure, by transmission electron microscopy. We found that the PCNA labeling index for duodenal mucosa of patients with FAP was higher in comparison to the case of hospital controls without cancer risk (P = 0.019). Moreover, ultrastructural changes related to an impairment of cell adhesion function were found in all biopsies of FAP patients but not in the duodenal mucosa of the controls. We conclude that alterations of cell proliferation kinetics and epithelial adherens junction structures were phenotypic characteristics of histologically normal duodenal mucosa of FAP patients. These abnormalities may be considered as intermediate biomarkers of neoplasia and potential surrogate endpoints in chemoprevention studies.

The problematic issue of Kufs disease diagnosis as performed on rectal biopsies: a case report.

Kufs disease, the late-onset form of a group of neurodegenerative disorders, known as the neuronal ceroid-lipofuscinoses, is characterized by intraneuronal/extraneuronal accumulation of proteins that are visible as fingerprint inclusions and granular osmiophilic deposits (GRODs) at the ultrastructural level. A problematic case of Kufs disease in a 53-year-old female affected by progressive gait disturbances, myoclonus, epilepsy, and profound dementia is presented. Laboratory, biochemical, and molecular genetic tests were unremarkable. A magnetic resonance imaging of the brain revealed a moderate atrophy over both hemispheres with no white matter changes. Ultrastructural examination of rectal mucosa biopsies showed fingerprint inclusions in perivascular smooth muscle cells. Pathological examination of autoptic tissues showed numerous intraneuronal PAS-positive, diastase-resistant inclusions corresponding to GRODs at the ultrastructural examination, but no fingerprint inclusions. Cerebellum, skeletal, and cardiac muscles, skin, and liver were unaffected. The present case illustrates the diagnostic difficulties encountered while examining Kufs disease pathological samples. Main problematic issues include (1) specificity and diagnostic value of fingerprint inclusions when found exclusively in perivascular smooth muscle cells; (2) safe distinction of GRODs from lipofuscin inclusions in cerebral tissue; and (3) reliability in using extraneural tissues and, in particular, rectal mucosa biopsies for diagnostic purposes.

An Italian case of CADASIL with mutation CGC-TCG in codon 1006, exon 19 Notch3 gene.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is commonly overlooked or misdiagnosed owing to its recent identification. It is characterized clinically by recurrent cerebral infarcts, usually appearing between the ages of 30 and 50 years, subcortical dementia, and pseudobulbar palsy. It begins with migraine with aura in approximately one-third of patients. The pathological hallmark of angiopathy is the presence of characteristic granular osmiophilic material (GOM) within the basal lamina of smooth muscle cells. The defective gene in CADASIL is Notch3, which encodes a large transmembrane receptor, and 70% of missense mutations are in exons 3 and 4. Each gene defect leads to either a gain or loss of a cysteine residue in the extracellular N-terminal domain of the molecule. We report the case of a 53-year-old woman admitted to the hospital for transient ischemic attack and stroke-like episodes recurrent since age 43 years. The patient had pseudobulbar palsy, pyramidal signs, and cognitive impairment but not frank dementia. Cerebral MRI showed periventricular diffuse and confluent ischemic lesions. Ultrastructural study revealed an abnormal deposition of granular osmiophilic material (GOM) within the basal lamina in skin capillaries. Direct sequence analysis of the Notch3 gene was performed. Since no mutation was detected in exons 3 and 4, the remaining exons were sequenced and a missense mutation, CGC-TGC in codon 1006 of exon 19 was found. The mutation led to a gain of a cysteine residue. This is the first missense mutation in codon 1006 of exon 19 of the Notch3 gene to be described in Italy and the second reported in the literature.

Dilated intercellular spaces as a marker of oesophageal damage: comparative results in gastro-oesophageal reflux disease with or without bile reflux.

BACKGROUND: The dilation of oesophageal intercellular spaces, clearly apparent in transmission electron microscopy images, is a marker of cellular damage induced by acid. AIM: To analyse the presence of dilated intercellular spaces and to quantify the scores in controls and in patients with gastro-oesophageal reflux disease or duodenal gastro-oesophageal reflux accompanied by erosive or non-erosive reflux disease. METHODS: Thirty-eight symptomatic patients with gastro-oesophageal reflux disease or duodenal gastro-oesophageal reflux and 12 asymptomatic controls, classified on the basis of pH-metry and bilimetry, underwent endoscopy. Six tissue biopsies were taken from the normal mucosa for light microscopy and transmission electron microscopy evaluation. Dilated intercellular spaces were measured on photomicrographs of the specimens (at least 100 transects were measured for each patient). RESULTS: Twenty-two patients with gastro-oesophageal reflux disease had normal macroscopic mucosa but, at histology, five patients with erosive gastro-oesophageal reflux disease had mild oesophagitis and one had moderate oesophagitis. Seven patients with duodenal gastro-oesophageal reflux had normal mucosa, whilst three with erosive duodenal gastro-oesophageal reflux had mild oesophagitis at histology. At transmission electron microscopy, all controls had dilated intercellular spaces of less than 1.69 microm. Each symptomatic patient had a mean dilated intercellular space value and a mean value of the maximum dilated intercellular space at least three or more times greater than that in controls (P < 0.001). No statistical differences were observed between erosive and non-erosive oesophagitis. CONCLUSIONS: The dilated intercellular space is an extremely sensitive marker of damage in gastro-oesophageal reflux disease, duodenal gastro-oesophageal reflux and non-erosive reflux disease, and serves as the most appropriate marker of damage evaluation in non-erosive reflux disease reported to date. A mean dilated intercellular space of 0.74 micro m provides a cut-off score for damage. No quantitative or qualitative differences in dilated intercellular space scores were found between pure and mixed acid reflux.

Morphological changes in late onset acid Maltase deficient patients with splicing gene mutation.

Clinical and morphological features have been studied in 11 late onset Acid Maltase Deficient (AMD) patients. All patients have been diagnosed on biochemical evidence of acid maltase deficiency on muscle biopsy. Molecular studies showed a heterozygous mutation (IVS1-13 T > G transversion resulting in aberrant splicing) in the GSDII gene, which is the most common mutation in late onset AMD patients. Morphological features in muscle biopsy showed a vacuolar myopathy and Golgi apparatus proliferation within fibres. The peripheral areas of autophagic vacuoles were positive for caveolin-3 and dystrophin, documenting an extensive membrane turnover. The ultrastructural study of muscle biopsy showed randomly distributed or isolated vesicles sometimes derived from the Golgi apparatus. In subsarcolemmal region, lipofucsin bodies and abnormal mitochondria with crystalline inclusions were observed. Primary and secondary lysosomes were typically filled with glycogen. These data suggest a predominant role of Golgi in vesicle proliferation and extensive intra-fibral membrane remodelling. The pathological changes observed are selective for muscle fibres (mostly in type 1) and for muscle groups (mainly proximal). An attractive hypothesis for the variability of clinical phenotype in adult and infantile onset AMD patients is that in the former, an aberrant transcript of smaller size may have originated from alternative splicing (exon 2 skipping). A residual enzyme activity is detectable in muscle, but the intracellular processing of the enzyme precursor from Golgi to the mature form in lysosomes might be blocked.