Electron microscopic features of skin in neurometabolic disorders

Journal of the NeurologicalSciences, 112 (1992) 15- 29 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00

15

JNS 03842

Review article

Electron microscopic features of skin in neurometabolic disorders C. Ceuterick and J.-J. Martin Department of Neuropathology, Born-BungeFoundation, Universityof Antwerp, Antwerp, Belgium (Received 21 October, 1991) (Revised, received 6 April, i992) (Accepted 10 April, 1992)

Key words: Skin biopsy; Ultrastructure; Lysosomal diseases; Peroxisomal diseases; Diagnostic criteria; Neurometabolic disorders

Summary Skin biopsy may contribute to the clinical diagnosis of neurometabolic disorders. It is an easy and much less traumatic procedure than brain, rectal, peripheral nerve and skeletal muscle biopsies. The method is informative and not too time-consuming for an experienced examiner. Differential diagnosis is possible in most storage disorders since the ultrastructure of the storage is virtually typical in lysosomal and in nonlysosomal diseases. The storage has a particular distribution with characteristic ultrastructural patterns in the various cell types. Skin biopsy plays a major diagnostic role when clinical features are atypical for a storage disorder, to discover new phenotypic variants of known enzymatic deficiencies or when the biochemical defect has not yet been determined. It can be used as a screening procedure to orientate the investigations, to suggest specific biochemical assays on cultured fibroblasts or other tissues or body fluids. It can be applied to detect "presymptomatic" patients in affected families. Other disorders of the nervous system should be investigated in the future to ascertain whether skin biopsies could possibly be used for diagnostic purposes. Thorough knowledge of the morphological features of these disorders may also improve the understanding of their pathogenesis, shed some light on the underlying basic defects and control the results of therapy.

Introduction Major advances in the biochemical, electron microscopic and DNA features of neuro-metabolic disorders provide diagnostic tools for identifying neurometabolic disorders. For some of these genetic disorders, specific enzyme deficiencies are known and the morphological contribution of skin biopsy (Decloux and Friederici 1969; Carpenter et al. 1972, 1973, 1977; Kenyon et al. 1972; Bioulac et al. 1975; O'Brien et al. 1975; Dolman et al 1977; Kornfeld et al. 1977; Gebhart et al. 1978; Libert 1979; Ishii et al. 1981; Takebe et al 1981; Cable et al. 1982; Goebel et al. 1982; Renlund et al. 1983; Ikeda et al. 1986; Nag and Macleod 1986; Busard et al. 1987; Walter and Goebel 1988) has been established. "Reverse" genetics looking for the abnormal genes producing inherited disorders will clarify the underlying basic

Correspondence to: Dr. J.-J. Martin, Dept. of Neuropathology, Born-Bunge Foundation, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium.

defects. Although it is not our purpose to discuss the respective value of each method, it is obvious that they must be combined to allow a diagnosis in phenotypic variants of known enzymatic deficiencies and that morphology has still a role to play. Biochemical abnormalities have not yet been found in every metabolic disorder and until now only a few defective genes have been localized and identified (Rosenberg and Pettegrew 1991). To the best of our knowledge there are only a few papers (Martin and Ceuterick 1978; Arsenio-Nunes et al. 1981) and PhD theses by Libert (1979) and one of us (de Groote-Ceuterick 1988) which provide a review of the diagnostic applications of skin biopsies. Our present review was undertaken to summarize our 20year experience of skin biopsy in the diagnosis of neurometabolic disorders due to lysosomal defects, impairment of peroxisomal function(s) or to other unknown mechanisms. Additionally, we wanted to ascertain whether the electron microscopic (EM) of a skin biopsy remains useful. This retrospective study in conjunction with our earlier reports (Martin and Jacobs 1973a; Martin and Martin 1973b; Martin and de Groote 1974; Ceuterick

16 TABLE 1 NUMBER OF SKIN A N D / O R CONJUNCTIVAL BIOPSIES FROM PATIENTS WITH A NEUROMETABOLIC STORAGE DISORDER A: number of patients with skin and/or conjunctival biopsy(ies); B: number of patients with skin biopsy only; C: number of patients with skin and conjunctivai biopsies; D: number of patients with conjunctival biopsy only. In collaboration with Libert (Martin et al. 1981b), 108 additional conjunctival biopsies were used as comparison material. Since our last paper (Ceuterick and Martin 1984), the updated total number is 311. GAN = giant axonal neuropathy. Metabolic disorder Mucopolysaccharidoses Oligosaccharidoses Ceroido-lipofuscinoses Glycogenosis-type 2 Sphingolipidoses Peroxisomal diseases Infantile neuroaxonal dystrophy Various (Lafora disease (5), encephalopathy with astrocytic inclusions (1), cardiomyopathy (1), GAN (2), congenital hypomyelination neuropathy (1)) No definite diagnosis a Total

(A) No. of of patients

(B) Skin

(C) Skin and conjunctiva

(D) Conjunctiva

24 22 48 15 46 29 7 10

22 20 42 15 41 24 4 10

1 1 6 0 3 2 2 0

1 1 0 0 2 3 1 0

2

0

2

0

203

178

17

8

a No definite diagnosis: although clearcut morphological signs of storage have been found in the skin and conjunctival biopsies in 1 of the 2 patients, the disorder is not yet classified, pending the results of further biochemical and enzymatic studies.

et al. 1976; Martin et al. 1976a,b, 1977a,b, 1979a,b, 1980a,b,c, 1981a,b, 1984a,b; Martin and Ceuterick 1978; Dom et al. 1979; Ceuterick et al. 1980; Loonen et al. 1981 a,b; Libert et al. 1982; Vercruyssen et al. 1982; Ceuterick and Martin 1984, 1987; Cartigny et al. 1985; Lenders et al. 1986; de Groote-Ceuterick 1988; Martin and Ceuterick 1988), prompted us to outline specific morphological criteria and guidelines for EM diagnosis.

Material and methods

Standard electron microscopic techniques were used (de Groote-Ceuterick, 1988). The only special precaution was to preserve, during sectioning and fiat embedding of the skin specimens, such an orientation of the fragments that a full thickness section of epidermis, dermis and hypodermis could be examined. Cells representative of the different structures of the skin were examined each time. From 1973 until 1991, a total of 203 skin- a n d / o r conjunctival biopsies (Table 1) have been performed in 169 children and 34 adults (aged 21-60 years) with a neurometabolic storage disorder. A skin biopsy alone was taken in 178 patients. An additional conjunctival biopsy was performed in 17 patients; 8 patients underwent a conjunctival biopsy alone.

A thorough clinical evaluation and appropriate biochemical assays were realized in all cases. Skin biopsies were also studied in 37 parents and siblings presumed to be carriers or possible carriers. Skin- and conjunctival biopsies were obtained respectively from 393 and 80 "controls" (73% children, 27% adults) ranging from 10 days to 80 years of age. Controls were: (i) patients with non-progressive encephalopathies and a negative metabolic work-up; (ii) patients with other non-metabolic neurological diseases. Cultured fibroblasts obtained from skin biopsies of 15 patients with various disorders (oligosaccharidoses, ceroido-lipofuscinoses, glycogenosis-type 2, sphingolipidoses) and 25 controls were also examined with the electron microscope. Other tissues (mostly necropsy material) including central and peripheral nervous system, heart, skeletal muscle and visceral organs from patients were examined for comparison purposes.

Results

Normal morphologicalfeatures In our study of the normal skin in 393 controls, special attention was given to normal structures, artifacts and aspecific structures. Normal cellular or-

Fig. 1. Normal siractures. A: eccrine sweat duct wiih multivesicular dense bodies (--,). B: myelinated axon with an endomyelin Elholz granule (--*). Aspecific structures and specific storage. C: aspecific axonal structures: a presynaptic ending between smooth muscle cells shows a residual lamellar body (--~). D: specific storage: numerous membranous cytoplasmic bodies lying in a Schwann cell of unmyelinated axons in Tay-Sachs disease. Dystrophic axon (A) contains residual bodies. Scale: A, B, C -- 0.1 ~m; D -- 1/~m.

18 TABLE 2

ganelles may indeed be mistaken for abnormal inclusions: Birbeck granula in Langerhans cells, lipofuscin in various cells, dense core vesicles in Merkel's neuroreceptor cells, maturing melanin granules in melanoeytes and melanin in melanophages, multivesicular dense bodies (Fig. 1A) in eccrine sweat ducts, mast cell granules, rod-shaped bundles of Weibel-Palade's microtubules in endothelial cells and endomyelinic Elholz granules (Fig. 1B). Artifacts such as swelling of endoplasmic reticulum, Golgi apparatus or mitoehondria with disruption of the cristae may be confused with storage vacuoles. Sectioning artifacts may create pseudo-inclusions looking like inclusions found in eccrine sweat ducts in Lafora disease. The examination of skin biopsies from 393 "controis" demonstrated 6 main structural abnormalities: (1) Membrane-bound vacuoles were seen in eccrine sweat glands in 4% of our controls. Clear vacuoles were filled with a fibrillogranular material; heterogeneous vacuoles contained osmiophilic submembranous fingerprint profiles. (2) Axonal lesions consisting of electron-dense cristalloid tubular filaments, filamentous bodies looking like a corpus amylaceum or dense residual granular and/or lamellar bodies (Fig. 1C) were present in dermal nerve twigs in 3% of control specimens. (3) Electron-lucent clefts resembling rectilinear or curved spicular inclusions in Schwann cells of patients with adrenoleukomyeloneuropathy in less than 1% of our controls. (4) Curvilinear tubular deposits reminding of Farber disease were found in perivascular histiocytes of 7 controls, in the absence of any biochemical evidence of ceramidase deficiency. (5) Intramitochondrial cristalioids could be found in smooth muscle cells, in eccrine sweat ducts and in endothelial cells. (6) Dense spicular deposits occurred in mitochondria of eccrine sweat glands, smooth muscle cells, endothelial cells and fibroblasts in 3% of controls.

Description of electron microscopic features of neu"rometabolic disorders

Pathological data

Mucopolysaccharidoses (MPS)

General results Our findings in patients with progressive neurometabolic storage diseases are summarized in Table 2. Positive diagnostic information was obtained in 180 (89%) out of 203 patients from the presence of distinctive inclusions with characteristic ultrastructural features. Equivocal results which could possibly support the final diagnosis were obtained in 14 cases (7%). Nine biopsies (4%) were negative. One negative was from a patient with proven adrenomyeloneuropathy and could have been a false-negative since myelinated nerve twigs were not observed. From clinically healthy obligate carriers, only one biopsy out of 37 was positive. Lamellar inclusions were found in uncultured dermal fibroblasts from a father of 4 children with Niemann-Pick disease type C.

RESULTS OF SKIN A N D / O R CONJUNCTIVAL BIOPSIES FROM PATIENTS WITH A NEUROMETABOLIC STORAGE DISORDER Metabolic disorder Mucopolysaccharidoses Oligosaccharidoses Ceroido-lipofuscinoses Glycogenosis-type 2 Sphingolipidoses Peroxisomal diseases Infantile neuroaxonal dystrophy Various (Lafora disease, encephalopathy with astrocytic inclusions, cardiomyopathy, GAN, congenital hypomyelination neuropathy) No definite diagnosis a Total

No. of patients

Positive

Equivocal

Negative

24 22 48 15 46 29

24 21 46 15 36 23

0 1 2 0 7 3

0 0 0 0 3 3

7

7

0

0

10 2

7

1

2

1

0

1

203

180

14

9

a No definite diagnosis: although clearcut morphological signs of storage have been found in the skin and conjunctival biopsies in one of the two patients, the disorder is not yet classified, pending the results of further biochemical and enzymatic studies.

Cultured skin fibroblasts from 15 affected patients showed non-specific heterogeneous inclusions which strongly resembled the inclusions of cultured control fibroblasts. Other tissues obtained by biopsy or by autopsy of the patients confirmed the data gained from skin biopsies by revealing features identical to those found in the various cell types of the skin.

Electron microscopy was carried out in MPS I, MPS IH-IS, MPS II, MPS III and MPS IV. The findings consisted mainly in enlarged cells "gargoyle cells", with numerous vacuoles. In all our cases, clear membranebound vacuoles (0.2-4/~m) with a fibrillogranular (FG) material were numerous in epithelial cells, eccrine sweat glands (secretory tubule and ducts), fibroblasts, mast cells~ melanocytes and Schwann cells. The vacuoles were much less frequent in smooth muscle cells and in endothelial cells. Lamellated inclusions "zebra bodies" (0.5-1 /zm) with parallel arranged lamellae, were also found in Schwann cells. The vacuolation of the eccrine sweat glands which is also apparent upon semithin sections is a characteristic morphological feature in most types of MPS. The storage was similar and undistinguishable in all cell types but however more prominent in MPS I, MPS II and MPS III. We did not

Fig. 2. GMI-gangliosidosis (infantile form). A: heterogeneous inclusions in an eccrine sweat duct. B: electron-lucent vacuoles with fibrillogranutar material in a dark secretory cell of an eccrine sweat gland. C: dense circular lamellar profiles contained in a vacuole (* 1of an endothelial cell. D: granular inclusions sometimes intermixed with lamellar profiles in a Schwann cell of a myelinated axon. Scale: A, B, D = 1 pm; C = 0.1 Ccm.

20 find any changes in the severity of the storage as the disease progressed in one example of MPS II.

Oligosacchan'doses (OLS) This group of disorders is also characterized by the presence of clear vacuoles which may be seen by light microscopy (LM). Differential diagnosis is made possible by EM which demonstrates characteristic storage. Two sorts of membrane-bound inclusions were found in Schwann cells around unmyelinated axons in sialidosis type 2: clear vacuoles (0.3-0.5 /Lm) with a FG material and lamellar zebra-like bodies (0.3-1.4/~m). Heavily vacuolated fibroblasts showed membranebound vacuoles (0.5-2 ~m) with a FG material and a few lamellar profiles in I-Cell disease. Characteristic inclusions (0.3-0.8 /~m) with rectilinear, polygonal or ring-like lamellar profiles were demonstrated in endothelial cells. Less severely vacuolated fibroblasts were observed in pseudo-Hurler polydystrophy. Large amounts of clear vacuoles (0.2-2/~m) with a FG material were present in all cell types including basal epithelial cells, cells of Langerhans, eccrine sweat ducts, smooth muscle cells, fibroblasts, endothelial cells, Schwann cells and perineurial cells in the GoldbergWenger syndrome. Lamellar inclusions including zebra bodies and membranous cytoplasmic bodies with concentric lamellar arrangements were present in Schwann cells around unmyelinated axons and presynaptic endings. A few dystrophic axons were also observed as in many different OLS. All cell types including epithelial cells showed clear vacuoles (0.3-0.8 /~m) with FG material in infantile galactosialidosis. Aside from clear contents, some enlarged vacuoles (3-4 ~m) exhibited curved lamellar profiles in epithelial cells from o n e case. All cell types of eccrine sweat glands contained numerous clear vacuoles which could coalesce and enlarge (3 ~m). Zebra bodies and membranous cytoplasmic bodies (0.5-0.8/~m) occurred in the Schwann cells around unmyelinated axons. In GMl-gangliosidosis, a generalized storage of clear vacuoles (0.5-2/~m) was observed in epithelial cells, eccrine sweat glands, smooth muscle cells, endothelial cells, fibroblasts and Schwann cells. In addition to the clear vacuoles, more heterogeneous inclusions with lamellar and vesicular profiles were found in eccrine sweat ducts (Fig. 2A) while electron-lucent vacuoles with FG material were observed in the secretory cells (Fig. 2B). A careful

examination of the storage in the endothelial cells revealed more characteristic inclusions (0.5-1/~m) with small circular lamellar profiles which could also be densified (Fig. 2C). Granular inclusions intermixed with lamellar profiles (Fig. 2D) were seen in the Schwann cells of myelinated axons. The Schwann cells surrounding unmyelinated axons were filled with a variety of pleomorphic bodies which mainly consisted of parallel and concentrically arranged lamellae. Nearly all cell types including basal epithelial cells and eccrine sweat glands contained clear vacuoles (diameter 0.5-1 ~m) with a FG material in the infantile form of Salla disease. Clear vacuoles with FG material, membranous cytoplasmic bodies and polymorphous inclusions (0.32.5 /Lm) were found in all cell types of the eccrine sweat glands in the Berman syndrome (ML IV). The heterogeneous inclusions showed whorled lamellar profiles, dense paracrystalline-like aggregates and electron-lucent zones. Smaller membranous cytoplasmic bodies were also present in epithelial cells, smooth muscle cells, fibroblasts, endothelial cells and Schwann cells. Inclusions (0.5-1/~m) with clear and dark fibrillogranular material were both found in nearly all cell types in fucosidosis. Eccrine sweat glands in which no secretory granules were found and endothelial cells were heavily vacuolated. A narrowing of the lumen could be observed. Heterogeneous lamellar inclusions with concentric or parallel lamellar structures were observed in Schwann cells around unmyelinated axons. A few fibrillogranular inclusions were also demonstrated. Unfortunately, no myelinated axons were found. In mucosulfatidosis, clear vacuoles (0.2-1 ~m) with FG material occurred in nearly all cell types and tuffstone bodies in Schwann cells. Inclusions (0.5-2 ~m) with curved tubular profiles mainly in dermal histiocytes, lameilar inclusions (zebra bodies) (0.3-1.5 ~m) in eccrine sweat glands and needle-shaped inclusions in Schwann cells were observed in Farber disease. Small lamellar inclusions were occasionally encountered in epithelial cells and eccrine sweat glands.

Ceroido-lipofuscinoses (CLF) Granular osmiophilic deposits (GROD) were seen in the different structures of the skin in the infantile form (Hagberg-Santavuori). Inclusions (0.3-1 ~m) were numerous in epithelial cells, eccrine sweat glands,

Fig. 3. Ceroid-lipofuscinosis, juvenile type (Spielmeyer-Vogt-Sj6gren). A: specific inclusions (--,) showing characteristic curvilinear profiles, rectilinear profiles, fingerprints and lipid droplets within an eccrine sweat gland. B: clear vacuoles with a few lameUar profiles in a smooth muscle cell by the same patient. Glycogenosis-type 2. C: fibroblast with numerous membrane-bound /3 glycogen particles in the infantile for~a. D: less numerous membrane-bound /3 glycogen particles (--,) in a fibroblast of an adult patient with the muscular form. Scale: A = 0.1 /tm; B, C, D -- 1/Lm.

4

~w'~

22 smooth muscle cells, vessel walls, fibroblasts, melanocytes and Schwann cells. The membrane-bound inclusions had an electron-dense aspect with a granular matrix and electron-dense aggregates. Higher magnifications of the aggregates showed densely packed short lamellar profiles. GROD showed rectilinear, polygonal, or ring-like short lamellar profiles in the endothelial cells. Inclusions (0.5-1 ~m) with curvilinear profiles were easily found in all cell types in the late infantile form (Jansky-Bielschowsky): epithelial cells, eccrine sweat glands, smooth muscle cells, vessels, fibroblasts, Schwann cells and neuroreceptor cells (Merkel type). The clear cells of the eccrine sweat glands contained clusters of numerous curvilinear bodies. Occasional inclusions were seen in sebaceous glands and in some axons. Less abundant and more pleomorphic inclusions (0.3-1 /zm) with curvilinear, rectilinear and fingerprint profiles (Fig. 3A) sometimes associated with lipid droplets were observed in the juvenile form (Spielmeyer-Vogt-Sj6gren). They were most easily found in eccrine sweat glands, vessels and histiocytes. Their occurrence in epithelial cells, smooth muscle cells, melanocytes and Schwann cells appeared to be more inconsistent. Apart from these characteristic inclusions, occasional vacuolar inclusions (Fig. 3B) in which the lamellar profiles were masked and lamellar bodies (zebra-like and membranous-cytoplasmic-like bodies) could be encountered mostly in Schwann cells and perivascular cells. Vacuoles with granulofilamentar material, a few lamellar profiles, fingerprint profiles and lipid droplets were found in the eccrine sweat glands of two affected brothers with Kufs disease. Lipopigmentary granules with less well-defined lamellar profiles were found in fibroblasts and smooth muscle cells. The ultrastructural findings were also useful when dealing with much less typical forms of CLF: (1) e.g. pleomorphic inclusions (0.5-1.5/zm) with curvilinear, rectilinear and fingerprint profiles were easily found in nearly all cell types including neuroreceptor cells (Pacini) in an example of the intermediate form of CLF (Lake-Cavanagh), (2) generalized granular osmiophilic deposits were found in all cell types in the skin of a 10-year-old patient with a juvenile cerebromacular degeneration.

Glycogenosis type 2 EM revealed membrane-bound vacuoles filled with /3 glycogen particles, which are characteristic for the disorder. The inclusions were numerous and widespread in the infantile form (Fig. 3C). Glycogen-filled vacuoles were observed in basal epithelial cells, in the myo-epithelial cells, clear and dark cells of eccrine sweat glands, fibroblasts, vessel walls and Schwann cells. Clusters of inclusions were prominent in smooth muscle cells. In addition to the typical deposits, varying features could however be seen. Some glycogen de-

posits were packed with finely amorphous granular material. "Empty" vacuoles with a reduced number of glycogen particles were also observed. Glycogen-filled vacuoles were demonstrated in lesser amounts in fibroblasts, smooth muscle cells and Schwann cells in the muscular form by adult patients (Fig. 3D).

Sphingolipidoses (SPH) GM2-gangliosidoses. Numerous pleomorphic membranous cytoplasmic bodies (MCB) (0.2-0.5/zm) were demonstrated almost exclusively in the Schwann cells (Fig. 1D) of unmyelinated axons in Tay-Sachs disease (GM2 gangliosidosis type I). Some inclusions showed finely granular material. Fingerprint patterns, submembranous lamellar aggregates as well as membranous concentric- and zebra-like bodies were also seen. Several MCB have also been seen in a few endothelial cells, fibroblasts, dermal presynaptic endings and in Schwann cells of myelinated axons, Dystrophic axons were present and contained nonspecific electron-dense granular and lamellar bodies with numerous mitochondria. Qualitatively similar but widespread lamellar inclusions (0.2-1/~m) were encountered in the infantile form of Sandhoff disease (GM2 gangliosidosis type If). Granular bodies, zebra bodies, MCB and fingerprints were found in eccrine sweat ducts, smooth muscle cells, fibroblasts, vessel walls and Schwann cells of myelinated and unmyelinated axons. The latter were usually severely affected. Dystrophic axons were also present. In the adult form of Sandhoff disease, small MCB-like bodies (0.2-0.4 /~m) limited to some Schwann cells or axons and dystrophic axons were observed. Fabry disease. Ultrastructurally, three sorts of inclusions (0.4-1.5 /zm) were recognized in fibroblasts, perineurial cells, vessel walls and in eccrine sweat glands. Membranous cytoplasmic bodies with concentric lamellae, zebra bodies with parallel lamellae which had sometimes a washed-out aspect and pleomorphic inclusions with sinuous, parallel or concentric lamellar structures, prismatic figures, fingerprints and granular material were demonstrated. Most inclusions were membrane-bound. Some very large inclusions (2.5 x 5 /~m) were found in the endothelial cells and smooth cells of vessel walls. Narrowing of the capillaries could be observed but no obstruction was seen despite the extensive storage. The involvement of the perineurial cells was obvious while Schwann cells remained normal. Metachromatic leukodystrophy. Examination of cutaneous nerve bundles in the infantile form, juvenile form and adult form revealed three main types of membrane-bound inclusions: tuffstone bodies (0.3-1 /~m), lamellar zebra-like bodies (0.3-1.8 ~m) and prismatic inclusions (0.8-2/~m). Tuffstone bodies, which were most abundant and

23 lamellar inclusions were frequently found in the Schwann ceils. Tuffstone bodies were found chiefly in Schwann cells of myelinated axons in adult patients while they were more frequent in Schwann cells of

unmyelinated axons in our younger patients. Lamellar inclusions were less numerous. They were common in Schwann cells of unmyelinated axons. However, they seemed more prominent in Schwann cells of myeli-

Fig. 4. A: cardiomyopathy with accumulation of intermediate filaments in skeletal muscles. Large number of tubulo-filamentar structures in a Schwann cell of a myelinated axon. Inset: higher magnification. B: giant axonal neuropathy. Accumulation of intermediate filaments (*) in a fibroblast. Scale: A, B = l~m; inset in A = 0.1/~m.

24 nated axons in the adult form. Prismatic inclusions were much less frequently observed and were mainly found in endoneurial fibroblasts but for rare exceptions. These subtle differences were not helpful in differentiating the clinical subtypes. Krabbe disease. Straight or curved needle-like inclusions were demonstrated in Schwann cells mostly around myelinated axons and in endoneurial fibroblasts. The inclusions were free in the cytoplasm or were enclosed in vacuoles in which they were sometimes negatively outlined. The deposits were found near normal myelinated axons or were associated with myelin debris. No dermal globoid cells were ever observed. Niemann-Pick disease. In type A, numerous lamellar inclusions (0.7-1.5/~m) with parallel or concentric lameUae were found in fibroblasts, vessel walls and nerve bundles (Schwann cells of myelinated and unmyelinated axons, endoneurial fibroblasts). Some inclusions were demonstrated in eccrine sweat ducts and in some axons. Dystrophic axons were seen. In type C, osmiophilic pleomorphic lamellar inclusions (0.3-2 /zm) were mainly found in perivascular histiocytes. A careful examination demonstrated however, a few inclusions in epithelial cells, eccrine sweat glands, endothelial cells, Schwann cells and melanocytes. One or more concentric lamellar arrangements, electron-lucent area, dense lamellar figures and annular profiles were associated in a granular matrix. Distended dystrophic axons were present. We found a few mixed granular and concentrically arranged lamellar bodies (1 /zm) in fibroblasts and pericytes and a few intra-axonai polymorphous cytoplasmic bodies in unmyelinated fibers in juvenile dystonic lipidosis.

Adrenoleukomyeloneuropathy

phous material. The myelin sheaths of these axons were often thin and irregular. Myelin debris were not seen. Less affected myelinated axons contained enlarged mitochondria, few lamellar structures and focal small accumulations of interconnected vesicles and tubules. Vesiculo-tubular profiles were ~ever observed in the Schwann cells. Unmyelinated axons often contained compact intra-axonal aggregates of vesiculo-tubular profiles which were more loosely packed or connected in myelinated axons. All other skin appendages were normal.

Encephalopathy with astrocytic lamellar inclusions (Towfighi et al. 1975) The main feature was the presence of membranebound spicular clefts and elongated straight or slightly curved lamellar profiles within a few fibroblasts and some Schwann cells of unmyelinated axons.

Lafora disease Numerous filamentous and finely granular deposits (1.5-5/~m) were observed in eccrine sweat ducts. They stained positively with periodic acid thiocarbohydrazide silver proteinate (Thi6ry 1967) and were not membrane-bound. One such deposit was usually found close to the nucleus of basal or peripheral cells. The other skin structures including nerve twigs were intact. In one patient with a few Lafora bodies in eccrine sweat ducts from an upper skin leg biopsy, we had expected more significant results in the sweat ducts from an

axilla skin biopsy. However despite serial sections and careful search, no Lafora bodies could be demonstrated at axillar level.

Cardiomyopathy with accumulation of non-desmin intermediate filaments in skeletal muscle (Stoeckel et al. 1981)

Spicular, rectilinear or slightly curved electron-lucent clefts were demonstrated in Schwann cells surrounding normal myelinated axons in adrenoleukodystrophy. The deposits were not membrane-bound but lined by free glycogen particles, osmiophilic lamellar structures and mitochondria. Prominent lesions occurred in clusters. Serial sections were sometimes necessary. Unmyelinated axons were normal. Similar but less numerous lesions were found in myelinated nerve twigs in adrenomyeloneuropathy.

Accumulation of 10-nm thick filaments was demonstrated in Schwann cells (Fig. 4A) of myelinated and unmyelinated axons, of terminal perivascular axons and of presynaptic endings between smooth muscle cells. The deposits (0.5-4/zm) were not membrane-bound. The filaments resembled tubules which were packed in parallel bundles and showed a twist-like aspect in longitu0inal section. Circular profiles with a hollow structure were observed in transverse section. Intraaxonal alterations were not found.

Infantile neuro-axonal dystrophy

Giant axonal neuropathy (GAN) Markedly increased intermediate filaments within

Vesiculo-tubular profiles split by lamellae and clear clefts were demonstrated in myelinated- and unmyelinated axons. Dermal presynaptic endings between smooth muscle cells, terminal axons around sweat glands and perivascular terminal endings showed similar lesions. More severely affected myelinated axons contained large networks of vesiculo-tubular profiles, numerous mitochondria, glycogen granules and amor-

fibroblasts (Fig. 4B), endothelial cells and Schwann cells were found in the skin of one patient in which the results of sural nerve biopsy confirmed GAN. In contrast, no dermal giant axons were seen. In another case in which a peripheral nerve biopsy could not be realized, similar results in the skin were highly suggestive of GAb/.

25

Congenital hypomyelination neuropathy A tentative diagnosis of congenital hypomyelination neuropathy was made in a 3-week-old patient. As compared with control cases, no myelin, very thin myelin sheaths or incomplete compaction of myelin lamellae were found in a skin biopsy. These findings were confirmed two weeks later by sural nerve biopsy.

Discussion

Specific results Significant morphological abnormalities in uncultured skin reside essentially in the characteristic combination of membrane-bound inclusions or deposits in the different sorts of cells. Clear vacuoles with a fibrillogranular (FG) material and zebra-like bodies in Schwann cells are characteristic for mucopolysaccharidoses (MPS) and oligosaccharidoses (OLS). A differential diagnosis between MPS and OLS can be made on the basis of a careful examination of the inclusions. For example, characteristic intravacuolar small ring-like lamellar profiles are present in endothelial cells from patients with I-Cell disease or inclusions with clear and dark FG material are found in fucosidosis. Many sorts of inclusions with specific patterns are virtually typical for the different sorts of ceroido-lipofuscinoses (CLF), sphingolipidoses (SPH), glycogenosis type 2 and ALMN. Granular osmiophilic deposits are observed in the infantile CLF, curvilinear bodies in the late infantile CLF, inclusions with curvilinear, rectilinear and fingerprints profiles in the juvenile CLF and membranebound vacuoles with a few osmiophilic profiles and fingerprint profiles in eccrine sweat glands in the adult CLF. Intermediate forms such as Lake-Cavanagh disease or juvenile cerebromacular degeneration (Carpenter et al. 1973) have special features. Some factors such as the orientation of the inclusions in relation to the plane of sectioning or the solubility of the storage material during fixation and embedding may affect its configuration. In the SPH and especially in Niemann-Pick disease the different lamellar arrangements which characterize the storage depend upon such factors. Finally skin lesions can be characteristic or highly suggestive for infantile neuroaxonal dystrophy (INAD), Lafora disease, giant axonal neuropathy (GAN) and congenital hypomyelination neuropathy. Our results show that electron microscopy of skin biopsy is a useful diagnostic tool in many neurometabolic disorders. Various points deserve comment. (1) Three main requirements are necessary to avoid false-positive diagnoses in neurometabolic disorders:

(i) an adequate technical processing of the tissue for electron microscopy; (ii) a thorough knowledge of normal cellular organelles (Zelickson 1967) and (iii) a critical evaluation of the many aspecific structures. The latter point has generally not received enough attention and is especially important since such inclusions can mislead the unexperienced observer. For example, electron-lucent vacuoles in eccrine sweat glands can mimic the ones of MPS or the ones with heterogeneous contents those of Kufs disease. Axonal lesions such as groups of tubular filaments or corpus amylaceum-like bodies must be distinguished from the membranovesicular tubular profiles, lamellar structures and clear clefts found in infantile neuro-axonal dystrophy. Dense residual granular a n d / o r lamellar bodies must be distinguished from the membranous cytoplasmic bodies of Tay-Sachs disease. Dilatation of smooth endoplasmic reticulum or of Golgi apparatus forming clear clefts in Schwann cells must be differentiated from the electron-lucent rectilinear or curved spicular profiles of ALMN. Spicular inclusions in ALMN are associated with osmiophilic lamellar structures, free glycogen granules and mitochondria and this is rather characteristic. Curvilinear deposits in dermal histiocytes may look like the inclusions found in Farber disease. The storage is however widespread and present in most dermal cells in the latter condition. Intramitochondrial cristalloids have been reported in controls and in mitochondrial myopathies. In the latter, the mitochondria are abnormally numerous or large with distorted and concentric cristae. Dense mitochondrial spicular deposits have been sometimes considered as staining artifacts. However, Munoz et al. (1987), reporting similar mitochondriai deposits in a rectal biopsy of 2 children with sporadic spinocerebellar degeneration, identified them as calcium hydroxyapatite crystals. (2) General characteristics of the process of storage can be useful for the interpretation of the skin biopsies. (a) The frequency and the number of inclusions vary according to age. Numerous inclusions are easily found in most cells of young patients while their presence in older patients is more difficult to detect because they are less numerous or limited to a few cell types. Therefore the absence of inclusions in older patients does not by any means rule out a diagnosis of storage disorder, since sampling artifacts may lead to false negative results. In glycogenosis type 2 for example, abundant membrane-bound glycogen granules are present in all cell types in the infantile Pompe disease while few inclusions are present in fibroblasts or endothelial cells in adult patients. (b) Inclusions are not necessarily similar in one cell type, in all cell types of the skin or in all tissues. Examples are the electron-lucent vacuoles in nearly all cell types including eccrine sweat glands and the lamel-

26 lar inclusions in Schwann cells in the skin of patients with MPS or the small ring-like lamellar profiles in the endothelial cells from patients with 1-Cell disease while clear vacuoles are present in the other cell types. Comparative studies of dermal nerve twigs and mesenchymal cells with neurons and mesenchymal cells of central nervous (CNS) tissue may also show quantitative and qualitative similarities or differences (Ceuterick and Martin 1984, Martin and Ceuterick 1988). Among many examples, the vacuoles with submembranous osmiophilic material present in eccrine sweat glands of adult CLF contrast with the massive intraneuronal storage made of dense ct~rvilinear and rectilinear lamellar profiles. In Krabbe disease, spicular inclusions are found in skin and CNS but globoid cells are limited to the CNS. (3) Skin biopsies are useful in the preliminary identification of a new disorder or in the preliminary stages of the diagnostic work-up, mainly when dealing with phenotypical variants of known metabolic diseases. In personal examples of Berman syndrome (mucolipidosis IV), typical storage was found in the skin before the biochemical diagnosis had been made (Goebel et al. 1982). In the infantile form of SaUa disease, morphological signs of storage were demonstrated before the disease had been formally identified by biochemists. It is also known that among gangliosidoses, chronic GM2 gangliosidosis may simulate Friedreich's ataxia (Willner et al. 1981), making therefore a skin biopsy very useful to detect a typical but unexpected storage in a heredo-ataxia (Oonk et al. 1979). (4) Skin biopsies are extremely useful to make a diagnosis when the metabolic error is not yet known or not yet fully identified such as in the ceroido-lipofuscinoses. Skin biopsies in which typical storage is easily found in eccrine sweat glands as in many other dermal cells, are an easy way to diagnose CLF. Inclusions specific for each type such as curvilinear bodies in Jansk3'-Bielschowsky disease are easily found without any difficulty in all cell types. Skin biopsy remains a method of choice even if the new developments concerning the accumulation of subunit c of mitochondrial ATP synthase in the inclusion bodies open the way for new promising etiopathogenic developments (Hall et al. 1991). (5) Skin biopsies can also be used for diagnostic purposes in other conditions than the lysosomal diseases. Such conditions are at the present time: (1) infantile neuroaxonal dystrophy (Martin et al. 1979b; Ceuterick and Martin 1990); (2) Lafora disease (Busard et al. 1987); (3) cardiomyopathies with accumulation of nondesmin intermediate filaments in skeletal muscle; (4) giant axonal neuropathy; and (5) congenital hypomyeli-

nation neuropathy (Guzetta et al. 1982). Skin biopsies may be used as the first choice morphologic procedure in most of these conditions. (6) Finally, skin biopsies may be used to evaluate other affected family members in a presymptomatic or symptomatic stage of the disorder. Biopsies are less frequently realized to detect clinically healthy obligate carriers of a neurometabolic storage disorder. It is difficult to determine whether this is really useful. Our limited series of patients (37) tends to suggest that it is not a very rewarding procedure. We have found only one positive example in the skin of the clinically healthy father of 4 affected children with Niemann-Pick disease type C (Ceuterick et al. 1986).

Limitations of a skin biopsy The procedure is innocuous and there are no formal contra-indications. The size of the biopsy (2-3 mm), its superficial character, the use of steristrips, make that the only relative contra-indication would be to biopsy someone with a coagulation diathesis. The morphological investigations must be performed in a rigourous way with respect to procedure and examination requirements (Sipe and O'Brien 1979; de GrooteCeuterick 1988). It is evident that positive diagnostic information is favourably influenced by a careful selection based on clinical data and appropriate paraclinical investigations. As stated by Arsenio-Nunes et al. (1981), morphological diagnostic information may be expected only from progressive encephalopathies. When the storage is striking and highly suggestive, it is of course much easier to support the diagnosis than when less obvious alterations are present. Equivocal results are mostly due to inadequate sampling or processing of the material. Personal experience plays a major role in the interpretation of results and helps to avoid false positive results. Negative or false-negative results are unavoidable and do not rule out a storage disorder. This is especially so in some peroxisomal diseases (Wanders et al. 1988). In most of these conditions, it is yet preferable to visualize the peroxisomes and their enzymatic equipment by immunocytochemistry in a liver biopsy. Negative results have also been observed in skin biopsies of Gaucher disease or in a special encephalopathy associated with astrocytic lamellar inclusions (Towfighi et al. 1975; Martin et al. 1977b).

Skin or conjunctival biopsy, which choice? In our experience, skin and conjunctival biopsies offer comparative results in most neurometabolic disorders. However, more material and more structures such as sweat glands, neuroreceptor cells and smooth muscle cells are available from a skin biopsy and this may reduce false-negative information. Examination of

27 sweat glands is an important diagnostic tool in ceroidlipofuscinosis and in Lafora disease (Busard et al. 1987). If specific features have been missed, one can repeat the biopsy since it is not an invasive procedure. Finally, skin offers also the advantage to cultivate fibroblasts for further biochemical and enzymatic investigations. Future prospects

Skin biopsy keeps its value as a highly diagnostic tool in metabolic disorders in which the biochemical defect has not yet been determined or when dealing with phenotypic variants or with aberrant clinical presentations. In cases offering considerable diagnostic difficulties, simple biopsy procedures using skin may be required as a screening test. In patients with spinocerebellar degeneration (Oonk et al. 1975; Willner et al. 1981) or presenting with progressive myoclonus epilepsy (Roger et al. 1990), an underlying metabolic defect may be documented by a skin biopsy. Ruling out a storage disorder in such cases by showing normal or aspecific skin structures is also an important diagnostic step. At-risk members of the family may also be screened. Skin biopsies may be used prospectively to assess their potential diagnostic value in other neurological diseases affecting (i) the CNS as in Alexander disease, Hallervorden-Spatz disease, juvenile neuroaxonal dystrophy, Leigh syndrome, Menkes disease, PelizaeusMerzbacher disease, Rett syndrome (Nag and MacLeod 1986) and sudanophilic leukodystrophies; (ii) the peripheral nervous system in various hereditary sensory and motor neuropathies and (iii) in encephalomyopathies with mitochondrial abnormalities. Skin biopsies may also be used, in some storage disorders, to control the value of specific therapies (Navarro et al. 1991). In conclusion, skin biopsy nowadays still represents a useful addition to enzyme assay and to DNA technology in the field of metabolic diseases. Acknowledgements We are very grateful to all physicians who referred the patients and to the laboratories performing the biochemical assays (Dr. R.J.A. Wanders, Amsterdam, Dr. P. Willems, Antwerp, and Prof. Dr. A. van Elsen, Antwerp). We also thank Prof. Dr. B.D. Lake, Prof. Dr. H.H. Goebel and Dr. C. Catsman who submitted patients with diagnostic problems. The excellent technical assistance provided by Mrs L. Dewit (electron microscopy) and Mrs. I. Bats (photography) is also gratefully acknowledged. This work was supported by the "Fonds voor Geneeskundig Wetenschappelijk Onderzoek" (grants 9.0011.89 and 3.0020.90).

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