Electron Microscopy Of Skin Research Articles

MITO-FIND: A study in 390 patients to determine a diagnostic strategy for mitochondrial disease

Mitochondrial diseases, due to nuclear or mitochondrial genome mutations causing mitochondrial dysfunction, have a wide range of clinical features involving neurologic, muscular, cardiac, hepatic, visual, and auditory symptoms. Making a diagnosis of a mitochondrial disease is often challenging since there is no gold standard and traditional testing methods have required tissue biopsy which presents technical challenges and most patients prefer a non-invasive approach. Since a diagnosis invariably involves finding a disease-causing DNA variant, new approaches such as next generation sequencing (NGS) have the potential to make it easier to make a diagnosis. We evaluated the ability of our traditional diagnostic pathway (metabolite analysis, tissue neuropathology and respiratory chain enzyme activity) in 390 patients. The traditional diagnostic pathway provided a diagnosis of mitochondrial disease in 115 patients (29.50%). Analysis of mtDNA, tissue neuropathology, skin electron microscopy, respiratory chain enzyme analysis using inhibitor assays, blue native polyacrylamide gel electrophoresis were all statistically significant in distinguishing patients between a mitochondrial and non-mitochondrial diagnosis. From these 390 patients who underwent traditional analysis, we recruited 116 patients for the NGS part of the study (36 patients who had a mitochondrial diagnosis (MITO) and 80 patients who had no diagnosis (No-Dx)). In the group of 36 MITO patients, nuclear whole exome sequencing (nWES) provided a second diagnosis in 2 cases who already had a pathogenic variant in mtDNA, and a revised diagnosis (GLUL) in one case that had abnormal pathology but no pathogenic mtDNA variant. In the 80 NO-Dx patients, nWES found non-mitochondrial diagnosis in 26 patients and a mitochondrial diagnosis in 1 patient. A genetic diagnosis was obtained in 53/116 (45.70%) cases that were recruited for NGS, but not in 11/116 (9.48%) of cases with abnormal mitochondrial neuropathology. Our results show that a non-invasive, bigenomic sequencing (BGS) approach (using both a nWES and optimized mtDNA analysis to include large deletions) should be the first step in investigating for mitochondrial diseases. There may still be a role for tissue biopsy in unsolved cases or when the diagnosis is still not clear after NGS studies.

Systemic lupus erythematosus and antiphospholipid syndrome

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by the synthesis of antinuclear antibodies. Nucleosomes-the disc-like-structural unit of chromatin being composed of histones and DNA-are the primary antigenic structure that induces the formation of complement-activating antigen-antibody complexes in basement membranes. The autoreactivity in SLE has been elucidated in drug-induced SLE: Hypomethylation of DNA leads to overexpression of integrin CD 11a/CD 18, increased binding of T-cells to macrophages and B-cells, a higher rate of apoptosis of macrophages and elevated B-cell activity with consecutive production of autoantibodies. Disturbances of apoptosis (e.g. mutation of Fas gene) are relevant also in non drug-induced SLE. The morphologic diagnosis-by light microscopy, immunohistology and electron microscopy-of skin and renal biopsies can confirm the diagnosis and help in prognostic assessment as well as therapeutic decisions in SLE. The value of the morphologic diagnosis in SLE is enhanced by reporting semiquantitative scores of disease activity and chronicity. The antiphospholipid syndrome (APS) is characterized by thrombosis, thrombocytopenia, recurrent fetal loss and antiphospholipid antibodies. APS associated with SLE or other systemic autoimmune diseases has been termed secondary APS. Antibodies in APS are directed against phospholipids and phospholipid-protein complexes. One of these proteins is beta 2-glycoprotein 1. An important mechanism of the increased rate of thrombosis in APS is probably the inhibition of the protein C catalyzed inactivation of clotting factors V and VIII. Venous thrombosis with recurrent pulmonary emboli and arterial thrombi in brain, heart and kidney (thrombotic microangiopathy) can lead to divergent clinical manifestations in APS.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL)

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leuco-encephalopathy (CADASIL) is a hereditary autosomal dominant non-atherosclerotic non-amyloid cerebral arteriopathy. The disease was identified in 1993. We are not aware of reports in the literature of its occurrence in South Africa, and we present the clinical and laboratory features of 5 patients with CADASIL. Patients with the characteristic radiological white matter disease and typical features (family history, ischaemic events, migraine or dementia) were evaluated for possible CADASIL by means of clinical examination, routine investigations for strokes, magnetic resonance imaging, skin biopsy electron microscopy, evoked potentials and electroencephalography. The clinical and laboratory features of our study largely correlate with reported studies. However, all of the skin biopsies were positive, and the onset of migraine in our patients was considerably earlier. A new finding, to our knowledge, was the normality of visual, somatosensory and auditory evoked potentials. Our study confirms the existence of CADASIL in South Africa, and also suggests that skin electron microscopy is useful, despite recent reports of its low sensitivity, and that evoked potentials in CADASIL are likely to be normal.

Mucolipidosis tipo IV en una paciente con ancestros mapuches. Caso clínico

We report a 7 year-old girl with mapuche ancestors, diagnosed as a cerebral palsy since infancy and on active rehabilitation. She acquired motor and cognitive skills at 3 years of age. At 5 years of age, a slow neurological deterioration started, associated to visual impairment. Optic atrophy was added to the typical neurological exam of ataxic cerebral palsy and the diagnosis was re-considered. Neuroimaging showed a slow and progressive atrophy of intracerebral structures and ultramicroscopy revealed intracytoplasmatic inclusions in conjunctiva and skin, compatible with mucolipidoses type IV (ML-IV). ML-IV must be included in the differential diagnosis of cerebral palsy associated with loss of acquired skills and progressive visual impairment. Electron microscopy of skin or conjunctiva is a useful diagnostic test. Suspicion of ML-IV must not be restricted to Ashkenazi Jewish population.

Juvenile neuronal ceroid lipofuscinosis.

A case of juvenile neuronal ceroid lipofuscinosis (JNCL) diagnosed on the basis of clinical features, electrophysiologic studies and skin electron microscopy is reported. JNCL was suspected on the basis of characteristic symptoms including progressive loss of vision, seizures, mental retardation and motor disabilities. Diagnosis was confirmed by neurophysiological and biopsy studies. The disease is caused by 23 different mutations in a gene recently isolated on chromosome 16 p11.2-12.1. Although universally fatal, characterisation of mutations can help in prenatal diagnosis in future pregnancies.

Systemic lupus erythematosus and antiphospholipid syndrome

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by the synthesis of antinuclear antibodies. Nucleosomes- the disc-like structural units of chromatin, composed of histones and DNA-are the primary antigenic structure that induces the formation of complement-activating antigen-antibody complexes in basement membranes. The autoreactivity in SLE has been elucidated in drug-induced SLE: hypomethylation of DNA leads to overexpression of integrin CD11a/ CD18, increased binding of T-cells to macrophages and B-cells, a higher rate of apoptosis of macrophages, and elevated B-cell activity with consecutive production of autoantibodies. Disturbances of apoptosis (e.g. mutation of Fas gene) are relevant also in non-drug-induced SLE. The morphologic diagnosis-by light microscopy, immunohistology and electron microscopy-of skin and renal biopsies can confirm the diagnosis and help in prognostic assessment as well as therapeutic decisions in SLE. The value of the morphologic diagnosis in SLE is enhanced by reporting semiquantitative scores of disease activity and chronicity. The antiphospholipid syndrome (APS) is characterized by thrombosis, thrombocytopenia, recurrent fetal loss and antiphospholipid antibodies. APS associated with SLE or other systemic autoimmune diseases has been termed secondary APS. Antibodies in APS are directed against phospholipids and phospholipid-protein complexes. One of these proteins is beta 2-glycoprotein 1. An important mechanism of the increased rate of thrombosis in APS is probably the inhibition of the protein C-catalyzed inactivation of clotting factors V and VIII. Venous thrombosis with recurrent pulmonary emboli and arterial thrombi in brain, heart and kidney (thrombotic microangiopathy) can lead to divergent clinical manifestations in APS.

Generalized acquired cutis laxa associated with coeliac disease: evidence of immunoglobulin A deposits on the dermal elastic fibres

Acquired cutis laxa (ACL) is an uncommon elastolytic disorder of unknown aetiology. In rare instances, ACL has been reported in association with autoimmune diseases and dermal deposit of immunoglobulins, suggesting that destruction of elastic tissue may be immunologically mediated. We report a 35-year-old man with generalized acquired cutis laxa (GACL) associated with a persistent papular erythematous eruption that histopathologically showed some resemblance to dermatitis herpetiformis. A marked reduction and degeneration of dermal elastic fibres was noted in biopsies from loose-hanging skin. Direct immunofluorescence from non-inflammatory loose skin revealed granular immunoglobulin A (IgA) deposits at the basement membrane zone and fibrillar IgA deposits in the dermal papillae. IgA deposits were also observed on the elastic fibres of the reticular dermis. Electron microscopy of skin from the submammary fold revealed fragmented elastic fibres, partial absence of peripheral microfibrils and abundant neutrophils, some of which were degranulated and adjacent to elastic fibres. Immunoelectron microscopy of an erythematous papule revealed IgA deposits around dermal elastic fibres. Antigliadin, antireticulin and antiendomysium antibodies were present. Jejunal biopsies showed a gluten-sensitive enteropathy. A possible IgA-mediated immune mechanism for the development of GACL in our patient is suggested.

Ultrastructural Findings in Epidermolysis Bullosa

Electron microscopy of skin provides diagnostic criteria for distinguishing the simplex, junctional, and dystrophic forms of inherited epidermolysis bullosa (EB). The plane of cleavage in blister formation indicates the localization of structural weakness within the epidermis and basement membrane zone, and, together with ultrastructural changes in affected skin, these are clues to the underlying genetic bases for these disorders. Skin biopsy specimens from individuals with EB were evaluated by electron microscopy to identify structural changes and determine the subtype of EB. Discrete, circumscribed clumps of keratin filaments present in the basal keratinocytes are pathognomonic for EB simplex Dowling-Meara. These and other observations of keratin filament disruption have led to the identification of mutations in keratin genes in Dowling-Meara and Koebner forms of EB simplex. Changes in the density and structure of anchoring fibrils and the relative amount of type VII collagen detected by immunostaining of the dermoepidermal junction in dystrophic EB have led to sequencing of mutations in the type VII collagen gene. Although mutations in junctional EB have not been reported, findings of structural alterations in hemidesmosomes and immunohistochemical studies of kalinin (BM600 and epiligrin), and in junctional EB with pyloric atresia alterations in the integrin alpha 6 beta 4, indicate molecules involved in basal keratinocyte adhesion to the basement membrane that are candidate genes for junctional EB. Electron microscopy of skin when correlated with mutations in EB will help us understand the significance of these structural molecules in normal skin and the pathogenesis of EB.

Juvenile dystonic lipidosis (variant of Niemann-Pick disease type C)

In two siblings affected with dementia, epilepsy and vertical supranuclear ophthalmoplegia, foam cells and sea-blue histiocytes were found in the bone marrow. Electron microscopy of skin and neuromuscular biopsies gave presumptive evidence in favour of a storage disorder. Postmortem examination of both cases revealed an intraneuronal polymorphous lysosomal storage in the central nervous system (in the cortex and in many nuclei e.g. the substantia nigra and the reticular formation of the brain stem). In the visceral organs with the spleen most severely affected, the inclusions had a different ultrastructure, being composed of tightly apposed leaflets. The biochemical study revealed accumulation of sphingomyelin and other lipids in liver and spleen, with normal sphingomyelinase activities, which is consistent with the diagnosis of Niemann-Pick disease type C. In the brain, the most striking abnormalities involved the glycolipids. Sphingomyelinase activities were unchanged in cultivated skin fibroblasts. These data compared with those of reported cases, allowed the following conclusions to be made: (1) although the combination of clinical features appears to be unique, none of them, when considered separately, is pathognomonic for juvenile dystonic lipidosis; (2) diagnosis during life can be suggested by careful examination of nerve bundles and fibroblasts with the electron microscope, although the method of choice appears to be the study of bone marrow; but final assessment of the diagnosis, in the absence of demonstrable enzymic deficiency, requires in most cases a study of the lipid profile in a liver biopsy (or better, spleen tissue whenever available); (3) the intralysosomal storage is different, both morphologically and biochemically, in the central nervous system and in the spleen; (4) juvenile dystonic lipidosis represents a juvenile variant of Niemann-Pick disease type C, pending the discovery of the primary defect responsible for this disorder.

Diagnostic role of skin or conjunctival biopsies in neurological disorders: An update

Updated figures from our reports on electron microscopy of skin or conjunctival biopsies include 256 patients, mostly suffering from lysosomal diseases. Significant morphological data supportive of the diagnosis and additional to enzyme assay (when and if an assay is available for the disorder) were discovered in 95% of the cases. Equivocal or negative data amounted to 5%. The present paper deals with some metabolic disorders which had not been fully dealt with in our previous publications and with an extension of the indications of skin biopsies: adult form and atypical variants of ceroid-lipofuscinoses, galactosialidosis, mucolipidosis IV, infantile neuroaxonal dystrophy, Lafora's disease, cardiomyopathy with generalized accumulation of intermediate filaments and congenital hypomyelination neuropathy. A comparison between biopsy and autopsy material in storage diseases shows that the storage of inclusions does not remain limited to one cell type or to one tissue even if no clinical signs are detectable. This ubiquitous character of the storage can be used for diagnostic purposes. On the other hand, the membrane-bound inclusions are not necessarily similar in all cell types and the search for characteristic features can be difficult in adult patients. Finally it is evident that skin biopsies can be used in other conditions than lysosomal disorders. The applicability of this procedure to other diseases needs further exploratory work.

Electron Microscopy of Skin and Peripheral Blood Lymphocytes in Infantile (Santavuori) Neuronal Ceroid Lipofuscinosis

Skin punch biopsies and peripheral blood lymphocyte preparations from two siblings with early infantile (Santavuori) neuronal ceroid lipofuscinosis have been examined by electron microscopy. In both cases characteristic osmiophilic inclusion bodies were found in various cells in the skin and in lymphocytes. In one case, lymphocyte inclusions were detected before the onset of any symptoms as a result of a family study. This indicates the possibility of screening lymphocytes of siblings of affected patients as a result of a family study. This indicates the possibility of screening lymphocytes of siblings of affected patients with a view to early detection of the disorder.

Trends in Electron Microscopy of Skin

Some trends in electron microscopy of skin have emerged and should be pursued in the future. The fine structure and some basic cellular reaction patterns of epidermal cells are discussed to illustrate the interplay of morphologic, cytochemical, and tracer studies. Intracytoplasmic membranes and secretory granules, lysosomes and endocytic mechanisms, cytomembranes and cell surface specialization are discussed to show how these can be used to arrive at a more meaningful interpretation of structure. Despite all advances, however, a great deal more needs to be done before the details of skin structure are completely elucidated.

Ultrastructural features of mast cells in systemic mastocytosis

A 3-year-old boy with systemic mastocytosis has been observed since the age of 4 months when he was first diagnosed as suffering from urticaria pigmentosa. Involvement of skin, liver, spleen and bones was observed. The electron microscopy of skin and liver revealed varied alterations in the morphology of mast cells. The most important findings were irregularly-shaped cells and unusual long and interdigitated cytoplasmic villi, with consequent aggregation of mast cells which was more prominent in the dermis. Proliferation and accumulation of mitochondria in one part of the cell and deeply indented nuclei were frequent. The problem, whether the morphological changes encountered--especially the complex interdigitation of villi--should be interpreted as a sign of expected neoplastic development, is discussed.

Electron Microscopy of Skin and Mucous Membrane

Electron Microscopy of Skin and Mucous Membrane