The Journal of Dermatology Vol. 19: 892-896, 1992
WS VI-5
The Tuberous Sclerosis Complex, A Prototype of Hamartiosis and Hamartomatosis Manuel R. Gomez The tuberous sclerosis complex (TSC) is an autosomal dominant hamartiosis and hamartomatosis of one or more organs and presumably a focal disorder of cell migration, proliferation and differentiation after organogenesis begins. Its cause is unknown: no defect at molecular level has been found as yet in individuals with the TSC genotype. Morphological and behavioral cell changes have been described and will be dealt with but the true gene products are the hamartias and hamartomas recognized with the naked eye or with the aid of light microscopy in the patient's brain, retina, skin, kidney, heart, and other organs (1). Infrequently involved are the lungs, teeth, gums, rectum, pancreas, liver, spleen, lymph nodes, thyroid, thymus, adrenal glands, ovaries, testes, vagina, bones, synovia and very rarely the spinal cord (2-5). There are no reports of peripheral nerve or muscle involvement. The many possible combinations of organ involvement are the cause of a great variety of phenotypes. Expression of the TSC Genes The phenotypes are recognized by clinical, pathological, or imaging methods. The TSC lesions found in involved organs, frequently skin, brain, and retina are so characteristic that they constitute the mark of this disease. Other features by themselves may not be sufficient for diagnosis unless they are multiple. It should be noted that the hamartomas grow in different tissues at a certain age and not at any particular age. For instance cardiac rhabdomyomas are present at birth and tend to diminish in size and disappear in subsequent years while facial angiofibromas appear after 3 years of age. Their peak age of onset is around 10 years and rarely appear after 20 years. The SEGAs develop between 5 and 15 years and cease to grow after 20 years of age (see Fig. 1 also showing peak age of onset of RAML and LAMM). The skin is involved in about 90% of all patients, Professor of Pediatric Neurology, Mayo Clinic, Rochester, MN, U.S.A.
CAM
FAF SEGA
50 Fig. 1. Cartoon representing the age distribution of patients when different types of hamartomas appear for the first time in TSC. CRM=cardiac rhabdomyoma; FAF=faciai angiofibroma; SEGA= subependymal giant-cell astrocytoma; RAML= renal angiomyolipoma; LAMM=pulmonary Iymphangiomyomatosis. 10
20
30
40
but this is undoubtedly a biased figure because the skin is more accesible to direct inspection than any other affected organ. Several white spots on the skin (hypomelanotic macules) are often the first sign ofTSC. When this is the only finding in a carefully examined subject who has no direct relative with TSC, a definitive diagnosis of TSC should not be made. However, if the propositus has a direct relative with TSC, it is reasonable to make a provisional diagnosis of TSC. Young infants often present with seizures. Examination of the skin under natural or ultraviolet light (Wood's lamp) may disclose a number of white spots. Given this association, the diagnosis of TSC should only be presumptive unless a pathognomonic skin sign is present. Neuroimaging of the head with CT, MR!, or both may be necessary to establish the diagnosis ofTSC (and of cerebral involvement). Although skin hamartomas may not appear until the patient is a few years old or as late as in the second decade, the fibrous forehead plaques are often present at birth and their being pathognomonic gives their recognition much value. Technological advances have provided excellent imaging methods for the brain and other organs. The central nervous system (CNS) is the second most frequently affected tissue and may display 1) hamartias in the cerebral cortex and subjacent
Tuberous Sclerosis Complex Table I.
Skin findings leading to a definitive diagnosis
893
Table 2. Pathologic/imaging features leading to a definitive diagnosis
One of the following: Facial angiofibromas Fibrous forehead plaque Ungual fibroma Shagreen patch (needs histopathologic verification)
white matter (cortical tubers), 2) radial foci of' heterotopic undifferentiated cells associated with hypomyelinated white matter, and 3) hamartomas located either in the subependymal region (subependymal nodules), protruding into the ventricles, or embedded in the basal ganglia or cerebral white matter. The hamartomas are subependymal giantcell astrocytomas (SEGA) and may grow from the subependymal nodules invading the ventricles and by obstructing the foramina of Monro cause intracranial hypertension. These tumors may also grow within the white matter from radial heterotopic cell foci. Individuals with or without neurologic symptoms may display imaging signs of TSC in head CT or MR. Relatives of patients and other persons at risk of having TSC should have neuroimaging studies. A . negative head CT does not exclude the need for and MR and vice versa. On rare occasions, individuals carrying the TSC gene have neither cutaneous nor neuroimagting signs: a renal CT or ultrasound of the adult at risk may reveal multiple angiomyolipomas. In infants and children an echocardiogram may reveal rhabdomyomas. Renal imaging in infants may disclose renal cysts which, associated to other signs, may confirm the diagnosis. Table 1 is a list of the skin lesions, and on Table 2 the neuropathologic or neuroimaging features for definitive diagnosis are listed. On Table 3 the visceral hamartomas (angiomyolipomas and rhabdomyomas) and the hamartias (renal cysts) are listed for a definitive diagnosis of TSC to be made by clinical or pathologic examination, or by imaging technology. Detailed descriptions of these lesions have been published elsewhere (1, 2). The following clinical features commonly found in TSC patients or their relatives are non-pathognomonic; if a multiplicity one of them is the only feature in the propositus, the clinician should suspect the diagnosis of TSC: hypomelanotic skin macules, generalized seizures in infants, and particularly infantile spasms, complex partial seizures at any age, dental enamel pits, recurrent spon-
Cortical tuber* Subependymal glial nod/SEGA* Retinal hamartoma' A single lesion is sufficient if pathologically verified; it must be multiple if found by imaging* or by ophthalmoscopyf
taneous pneumothorax, chylothorax, recurrent hematuria with or without lumbar pain, renal failure, calcified subependymal nodules evidenced by CT scan, multiple subcortical areas of hypomyelination evidenced by MRI, multiple subcortical areas of decreased attenuation evidenced by CT scan, multiple hyperechoic areas evidenced by renal ultrasound, cardiac tumors in fetus or newborn recognized by echocardiography, or having a direct relative with TSC. The presence of two of the aforementioned features in an individual warrants only a provisional diagnosis. The diagnosis ofTSC is unquestionable in any individual who has one or more of the pathognomonic signs listed in Tables 1-3. Gene Expression at CeDular Level in the Skin The stroma of facial angiofibromas, ungual fibromas, and shagreen patches contains polygonal cells with pointed prolongations reminiscent of a starfish. Ishibashi et al. found these cells to be larger than ordinary fibroblasts and to have characteristics halfway between fibroblasts and histioeytes (6). Nickel and Reed observed them intermingled with multinucleated giant cells in facial angiofibromas and suggested they might be glial cells (7). Ishibashi et at found they stain for neuron-specific enolase and, more strikingly, react with monoclonal antibody to glial fibrillary acidic protein (GFAP), thus expressing neural rather than fibroblastic features. Table 3. Visceral lesions leading to a definitive diagnosis One of the following: Mwtipleangiomyolipomas (AML) Multiple cardiac rhabdomyomas or Two of the following: SingleAML Multiple renal cysts Renal hamartoblastoma Lymphangioleiomyomatosis
894
Gomez
When cultured in vitro, these cells have a distinct pleomorphism, and some have long cytoplasmic processes like glial cells (8). Several investigators have observed abnormal division of these cells in culture (9) but we do not know whether this is an aberration of the cells or an in vitro artefact (see below). Davidson et al. (10) also found neuronal-markers in the unusual neuron-like cells obtained from angiofibromas and a renal angiomyolipoma and called them N-cells.Johnson et al. (11) reporting on the N-cells emphasized their similarity with cells found in cortical tubers and in SEGAs. Examination with the electron microscope displayed mixed features of astrocytes and neurons with predominance of one or the other. Johnson et al. then proposed that N-cells may result from dedifferentiation of a normal occurring cell in the skin? perhaps a fibroblast or a melanocyte that regresses to a more primordial state, or from failure of certain dermis cells to achieve terminal differentiation. Onodera et al. (12) observed these cells stain with antibody against Tubulin, Actin, Vimentin, Fibronectin, and GFAP and in addition express a unique 55kd protein in their cytoskeleton (13). Since the neuronlike cells may express neuronal and occasionally oligodendrogial markers, their failure to differentiatevmust have occurred before migration to the periphery. Chromosomal Abnormalities in Cultured Cells Scappaticci et al. found that in cell cultures derived from facial angiofibromas there are variable but consistent karyotypic abnormalities: premature centromere dysjunction of all or part of the chromosomes, increased incidence of breaks, dicentric chromosomes, micronucleii and polyploid metaphases. Chromosome 3 was more frequently involved than the other chromosomes in these aggregations. The authors concluded that "karyotypic variation can be considered a cellular phenotypic characteristic of TS in fibroblasts cultured from the skin lesions" (14). Dietrich et al. found increased frequencies of unstable chromosomal anomalies in lymphocytes and in fibroblasts from unaffected skin of the patients with TSC. There were partial trisomes of the long arms of chromosomes 1, 3, 7, 10, and 15. These authors also confirmed the premature centromere dysjunction affecting all or only part of chromosomes of cultured fibroblasts derived from angiofibromas (15). Ishibashi et al. (16) had observed cell division of cultured nonepithelial cells from facial angio-
fibroma and noticed chromosomal aggregations during metaphase. They concluded there is disturbance of the centromere-microtubules-centriole system, and particularly the function of microtubules differs from that of ordinary fibroblasts of healthy subjects. Unequal quantitites of DNA found in the nuclei of the abnormal cells was attributed to the disturbance during cell division causing daughter cells to contain unequal quantities of DNA. Further, during mitosis some chromosomes do not line up, others do it at a site far from the equator or lag in the process of division, suggesting that something prevents an orderly arrangement of chromosomes at the equator during metaphase (16). The question remains whether or not some or all the observations in cultures are merely artefacts. Gene Expression at Cellular Level in the Central Nervous System The cortical tubers lack the normal lamination of the cerebral cortex and contain misaligned, bizarre stellate neurons intermingled with giant cells sometimes polygonal in shape, often multinucleated and having prominent nucleoli and pale acidophilic cytoplasm. Some contain nuclear inclusions. These cells are believed to be undifferentiated bipotential cells precursors of both glia and neurons. Giant cells are also found in the cerebral white matter of TSC patients.where they form heterotopic nests or rows radially oriented as if for a distance they followed the radial glial guides of neuronal migration but failed to reach the cortical plate. Giant cells are also present in subependymal nodules, SEGAs and in retinal astrocytic hamartomas. The fine structure of the atypical cells in the cortical tubers has been studied in cerebral biopsy with the Golgi-Cox method and with electron microscopy (EM). With the Golgi-Cox (17) method, the giant cells are not evident but in the subpial region of the tuber there are clumps of glial cells. Elsewhere in cortex predominate a cell type not usually seen in normal cortex: small, bearing beaded dendrites, sparse in spines, and multipolar or stellate in shape. These cells are believed to be neurons of a primitive type, similar to those found in the normal caudate and the geniculate nuclei. In the depth of small tubers, cells may be arranged in layers including radially oriented pyramidal cells forming a rudimentary cortex as if it had begun but failed to competely develop. The cerebral cortex contiguous to a tuber displays no cytoarchitectonic alteration but the dendritic arborizations are less extensive than those of normal cortex suggesting
895
Tuberous Sclerosis Complex that the tuber interferes with the full development of its neighboring cortex (17) or that the extracellular matrix here is less deficient in a protein necessary for migration and differentiation than more complete cortical tubers. Speculations on the Pathogenesis of TSC The hamartias ofTSC or cortical tubers constitute a multifocal disturbance of cytoarchitectonic organization where large and bizarre neurons or stellate neurons lack laminar and columnar arrangement The focal distribution of the tubers is paralleled by scattering of subependymal hamartomas over the ependymal surface and both with rostral preponderance. Cutaneous, renal, cardiac and retinal hamartomas are also isolated or focal, a fact characteristic of TSC and other hamartomatoses. We do not know if the abnormal undifferentiated large cells in the brain resulted from an uneven mitotic division just as the N-cells grown in culture from facial angiofibroma stroma but it is possible that during cerebral embryogenesis a disturbance of the centromere-microtubule-centriole system prevents proper chromosomal arrangement during mitosis, resulting in abnormally large cells that are unable to migrate and differentiate normally. Perhaps a defective contractile protein that forms microtubules in neuron-like cells plays a role in the disturbed cytogenesis, post-mitotic differentiation and neuronal migration prior to organogenesis. If so, we could call the normal protein missing in TSC T-sclerin, a constituent of the radial glial guides for neuronal migration. Alternatively the T-sclerin could be part of the membrane receptors of migrating neurons. It has been established that cells destined to form the neocortex migrate from the germinal matrix zone to the cortex in sequence, the first to migrate forming the deep layer of the cortex and the last ones to migrate reaching the more superficial layers. They are guided in their journey by bipolar radial glial cells with processes extending to the ventricular zone and to the external limiting membrane of the hemisphere. The motility of primordial cells could be affected either by glial guide constituents to which the neuron might become firmly or persistently attached or by abnormal cell membrane receptors in the migrating neuron (18). Certainly, the interior "milieu" must play an important role in the growth of TSC hamartomas. Specifically, during the gestational period there is rapid growth of the cardiac rhabdomyomas that ceases after birth when the tumors become smaller
in size to the extent that many disappear. Between 3 and 8 years of age, facial angiofibromas appear in about half of the patients with TSC (see Fig. 1) but as a rule only for the next 10 years may continue to grow. SECAs may grow from subependymal nodules and become tumors but only in about 6 percent of patients between 5 and 20 years of age will obstruct the CSF circulation (19). Renal angiomyolipomas (RAML) and pulmonary lymphangiomyomatosis (LAMM) usually appear predominantly in female patients past their third and fourth decades of life respectively. It is as if a hormonal timetable determines when these different hamartomas are turned on or off. To explain the multifocality of the hamartias and hamartomas is not easy butJohnson et al. (11) have proposed the two-hit hypothesis as follows: the first hit is a mutational change. affecting a molecule for cell migration either inherited or the result of a new mutation which occurs before segregation of germline from somatic cells. This mutation causes an abnormal migration of precursor cells. A second hit occurs in embryonic development to some cells during division as a random mutation and this causes loss of a suppressor gene leading to the development of hamartomas at random in focal parts of one or more organs. References 1) Gomez MR: Tuberous Sclerosis, 2nd Ed, Raven Press, New York, NY, 1988. 2) johnson WG, Gomez MR (ed): Tuberous Sclerosis and Allied Disorders, Ann NYAcad Sci, 615: 1-148,1991. 3) Devroede G, Lemieux B, Masse S, Lamarche j, Herman PS: Colonic hamartomas in tuberous sclerosis, Gastroenterology, 94: 182-188,1988. 4) Drut R: Multivisceral dysplastic lesions in a patient with tuberous sclerosis and Langerhans cell histiocytosis, Pediatr Pathol, 10: 633-639, 1990. 5) Koprowski C, Rorke LB: Spinal cord lesions in tuberous sclerosis, PediatrPathol, 1: 475-480, 1983. 6) Ishibashi Y, Matsukawa A, Yu H-S, et al: An ultrastructural observation of the facial lesion in Pringle's disease, Nishinikonj Dermatol, 43: 1029-1043, 1981. 7) Nickel WR.Reed WB: Tuberous sclerosis, ArchDermatol, 85:209..226, 1962. 8) Ishibalilli Y, Watanabe R, Nogita T, Takahashi T, Onodera K, Kimura G: Abnormal gene expressions ofstroma cells inpatients with tuberous sclerosis, in joltn$oJlW(;, Gomez MR (ed): Tuberous Sclerosis and 1JIsorders, Ann NYAcad Sci, 615: 228-242, 1991. 9) IshibashiY, Inoue Y,Takehara K, Fume M, Kukita A: Disturbed-mitotic processes of stroma cells in patients with tuberous sclerosis,jpnj Dermatol, 93: 1045-1058, 1983.' (in Japanese)
Allied
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10) Davidson MM, Yoshidome H, Stenroos E, Johnson WB: Neuron-like cells in culture of tuberous sclerosis tissue, in Johnson WG, Gomez MR (ed): Tuberous Sclerosis and Allied Disorders, Ann NY Acad Sci, 615: 196-210, 1991. 11) Johnson WG, Yoshidome H, Stenroos ES, Davidson MM: Origin of the neuron-like cells in tuberous sclerosis tissue, in johnson WG, Gomez MR (ed): Tuberous Sclerosis and Allied Disorders, Ann NY Acad Sci, 615: 211-219, 1991. 12) Onodera K, Takahashi T, Ito S, Kimura G, Watanabe R, Ishibashi Y: Molecular biology of cultured cells from a patient with tuberous sclerosis, in johnson WG, Gomez MR (ed): Tuberous Sclerosis and Allied Disorders, Ann NYAcad Sci, 615: 372-374, 1991. 13) Umeda T, Niijima T, Tashiro T, Komiya Y, Onodera K, Ishibashi Y: 55KD protein unique to cultured cells from angiofibroma of tuberous sclerosis, Cell Mol Biology, 33: 483-493, 1987.
14) Scappaticci S, Cerimele D, Tondi M, Vivarelli R, Fois A, Fraccaro M: Chromosome abnormalities in tuberous sclerosis, Hum Genet, 79: 151-156, 1988. 15) Dietrich CU, Krone W, Hochsattel R: Cytogenetic studies in tuberous sclerosis, Cancer Genet Cytogenet, 45: 161-177, 1990. 16) Ishibashi Y,Inoue Y,Takehara K, Fume M, Kukita A: Disturbed mitotic process of stroma cells in a patient with tuberous sclerosis, ] Dermatol (Tokyo), 11: 236-252,1984. 17) Huttenlocher PR, Wollmann RL: Cellular neuropathology of tuberous sclerosis, in johnson WB, Gomez MR (ed): Tuberous Sclerosis and AlliedDisorders, Ann NYAcad Sci, 33: 140-145,1991. 18) Caviness VS,Takahashi T: Cerebral lesions of tuberous sclerosis in relation to normal histogenesis, in johnson WB, Gomez MR (ed): Tuberous Sclerosis and AlliedDIsorders, Ann NYAcad Sci, 33: 157-195, 1991.
WS VI-5
The Tuberous Sclerosis Complex, A Prototype of Hamartiosis and Hamartomatosis Manuel R. Gomez The tuberous sclerosis complex (TSC) is an autosomal dominant hamartiosis and hamartomatosis of one or more organs and presumably a focal disorder of cell migration, proliferation and differentiation after organogenesis begins. Its cause is unknown: no defect at molecular level has been found as yet in individuals with the TSC genotype. Morphological and behavioral cell changes have been described and will be dealt with but the true gene products are the hamartias and hamartomas recognized with the naked eye or with the aid of light microscopy in the patient's brain, retina, skin, kidney, heart, and other organs (1). Infrequently involved are the lungs, teeth, gums, rectum, pancreas, liver, spleen, lymph nodes, thyroid, thymus, adrenal glands, ovaries, testes, vagina, bones, synovia and very rarely the spinal cord (2-5). There are no reports of peripheral nerve or muscle involvement. The many possible combinations of organ involvement are the cause of a great variety of phenotypes. Expression of the TSC Genes The phenotypes are recognized by clinical, pathological, or imaging methods. The TSC lesions found in involved organs, frequently skin, brain, and retina are so characteristic that they constitute the mark of this disease. Other features by themselves may not be sufficient for diagnosis unless they are multiple. It should be noted that the hamartomas grow in different tissues at a certain age and not at any particular age. For instance cardiac rhabdomyomas are present at birth and tend to diminish in size and disappear in subsequent years while facial angiofibromas appear after 3 years of age. Their peak age of onset is around 10 years and rarely appear after 20 years. The SEGAs develop between 5 and 15 years and cease to grow after 20 years of age (see Fig. 1 also showing peak age of onset of RAML and LAMM). The skin is involved in about 90% of all patients, Professor of Pediatric Neurology, Mayo Clinic, Rochester, MN, U.S.A.
CAM
FAF SEGA
50 Fig. 1. Cartoon representing the age distribution of patients when different types of hamartomas appear for the first time in TSC. CRM=cardiac rhabdomyoma; FAF=faciai angiofibroma; SEGA= subependymal giant-cell astrocytoma; RAML= renal angiomyolipoma; LAMM=pulmonary Iymphangiomyomatosis. 10
20
30
40
but this is undoubtedly a biased figure because the skin is more accesible to direct inspection than any other affected organ. Several white spots on the skin (hypomelanotic macules) are often the first sign ofTSC. When this is the only finding in a carefully examined subject who has no direct relative with TSC, a definitive diagnosis of TSC should not be made. However, if the propositus has a direct relative with TSC, it is reasonable to make a provisional diagnosis of TSC. Young infants often present with seizures. Examination of the skin under natural or ultraviolet light (Wood's lamp) may disclose a number of white spots. Given this association, the diagnosis of TSC should only be presumptive unless a pathognomonic skin sign is present. Neuroimaging of the head with CT, MR!, or both may be necessary to establish the diagnosis ofTSC (and of cerebral involvement). Although skin hamartomas may not appear until the patient is a few years old or as late as in the second decade, the fibrous forehead plaques are often present at birth and their being pathognomonic gives their recognition much value. Technological advances have provided excellent imaging methods for the brain and other organs. The central nervous system (CNS) is the second most frequently affected tissue and may display 1) hamartias in the cerebral cortex and subjacent
Tuberous Sclerosis Complex Table I.
Skin findings leading to a definitive diagnosis
893
Table 2. Pathologic/imaging features leading to a definitive diagnosis
One of the following: Facial angiofibromas Fibrous forehead plaque Ungual fibroma Shagreen patch (needs histopathologic verification)
white matter (cortical tubers), 2) radial foci of' heterotopic undifferentiated cells associated with hypomyelinated white matter, and 3) hamartomas located either in the subependymal region (subependymal nodules), protruding into the ventricles, or embedded in the basal ganglia or cerebral white matter. The hamartomas are subependymal giantcell astrocytomas (SEGA) and may grow from the subependymal nodules invading the ventricles and by obstructing the foramina of Monro cause intracranial hypertension. These tumors may also grow within the white matter from radial heterotopic cell foci. Individuals with or without neurologic symptoms may display imaging signs of TSC in head CT or MR. Relatives of patients and other persons at risk of having TSC should have neuroimaging studies. A . negative head CT does not exclude the need for and MR and vice versa. On rare occasions, individuals carrying the TSC gene have neither cutaneous nor neuroimagting signs: a renal CT or ultrasound of the adult at risk may reveal multiple angiomyolipomas. In infants and children an echocardiogram may reveal rhabdomyomas. Renal imaging in infants may disclose renal cysts which, associated to other signs, may confirm the diagnosis. Table 1 is a list of the skin lesions, and on Table 2 the neuropathologic or neuroimaging features for definitive diagnosis are listed. On Table 3 the visceral hamartomas (angiomyolipomas and rhabdomyomas) and the hamartias (renal cysts) are listed for a definitive diagnosis of TSC to be made by clinical or pathologic examination, or by imaging technology. Detailed descriptions of these lesions have been published elsewhere (1, 2). The following clinical features commonly found in TSC patients or their relatives are non-pathognomonic; if a multiplicity one of them is the only feature in the propositus, the clinician should suspect the diagnosis of TSC: hypomelanotic skin macules, generalized seizures in infants, and particularly infantile spasms, complex partial seizures at any age, dental enamel pits, recurrent spon-
Cortical tuber* Subependymal glial nod/SEGA* Retinal hamartoma' A single lesion is sufficient if pathologically verified; it must be multiple if found by imaging* or by ophthalmoscopyf
taneous pneumothorax, chylothorax, recurrent hematuria with or without lumbar pain, renal failure, calcified subependymal nodules evidenced by CT scan, multiple subcortical areas of hypomyelination evidenced by MRI, multiple subcortical areas of decreased attenuation evidenced by CT scan, multiple hyperechoic areas evidenced by renal ultrasound, cardiac tumors in fetus or newborn recognized by echocardiography, or having a direct relative with TSC. The presence of two of the aforementioned features in an individual warrants only a provisional diagnosis. The diagnosis ofTSC is unquestionable in any individual who has one or more of the pathognomonic signs listed in Tables 1-3. Gene Expression at CeDular Level in the Skin The stroma of facial angiofibromas, ungual fibromas, and shagreen patches contains polygonal cells with pointed prolongations reminiscent of a starfish. Ishibashi et al. found these cells to be larger than ordinary fibroblasts and to have characteristics halfway between fibroblasts and histioeytes (6). Nickel and Reed observed them intermingled with multinucleated giant cells in facial angiofibromas and suggested they might be glial cells (7). Ishibashi et at found they stain for neuron-specific enolase and, more strikingly, react with monoclonal antibody to glial fibrillary acidic protein (GFAP), thus expressing neural rather than fibroblastic features. Table 3. Visceral lesions leading to a definitive diagnosis One of the following: Mwtipleangiomyolipomas (AML) Multiple cardiac rhabdomyomas or Two of the following: SingleAML Multiple renal cysts Renal hamartoblastoma Lymphangioleiomyomatosis
894
Gomez
When cultured in vitro, these cells have a distinct pleomorphism, and some have long cytoplasmic processes like glial cells (8). Several investigators have observed abnormal division of these cells in culture (9) but we do not know whether this is an aberration of the cells or an in vitro artefact (see below). Davidson et al. (10) also found neuronal-markers in the unusual neuron-like cells obtained from angiofibromas and a renal angiomyolipoma and called them N-cells.Johnson et al. (11) reporting on the N-cells emphasized their similarity with cells found in cortical tubers and in SEGAs. Examination with the electron microscope displayed mixed features of astrocytes and neurons with predominance of one or the other. Johnson et al. then proposed that N-cells may result from dedifferentiation of a normal occurring cell in the skin? perhaps a fibroblast or a melanocyte that regresses to a more primordial state, or from failure of certain dermis cells to achieve terminal differentiation. Onodera et al. (12) observed these cells stain with antibody against Tubulin, Actin, Vimentin, Fibronectin, and GFAP and in addition express a unique 55kd protein in their cytoskeleton (13). Since the neuronlike cells may express neuronal and occasionally oligodendrogial markers, their failure to differentiatevmust have occurred before migration to the periphery. Chromosomal Abnormalities in Cultured Cells Scappaticci et al. found that in cell cultures derived from facial angiofibromas there are variable but consistent karyotypic abnormalities: premature centromere dysjunction of all or part of the chromosomes, increased incidence of breaks, dicentric chromosomes, micronucleii and polyploid metaphases. Chromosome 3 was more frequently involved than the other chromosomes in these aggregations. The authors concluded that "karyotypic variation can be considered a cellular phenotypic characteristic of TS in fibroblasts cultured from the skin lesions" (14). Dietrich et al. found increased frequencies of unstable chromosomal anomalies in lymphocytes and in fibroblasts from unaffected skin of the patients with TSC. There were partial trisomes of the long arms of chromosomes 1, 3, 7, 10, and 15. These authors also confirmed the premature centromere dysjunction affecting all or only part of chromosomes of cultured fibroblasts derived from angiofibromas (15). Ishibashi et al. (16) had observed cell division of cultured nonepithelial cells from facial angio-
fibroma and noticed chromosomal aggregations during metaphase. They concluded there is disturbance of the centromere-microtubules-centriole system, and particularly the function of microtubules differs from that of ordinary fibroblasts of healthy subjects. Unequal quantitites of DNA found in the nuclei of the abnormal cells was attributed to the disturbance during cell division causing daughter cells to contain unequal quantities of DNA. Further, during mitosis some chromosomes do not line up, others do it at a site far from the equator or lag in the process of division, suggesting that something prevents an orderly arrangement of chromosomes at the equator during metaphase (16). The question remains whether or not some or all the observations in cultures are merely artefacts. Gene Expression at Cellular Level in the Central Nervous System The cortical tubers lack the normal lamination of the cerebral cortex and contain misaligned, bizarre stellate neurons intermingled with giant cells sometimes polygonal in shape, often multinucleated and having prominent nucleoli and pale acidophilic cytoplasm. Some contain nuclear inclusions. These cells are believed to be undifferentiated bipotential cells precursors of both glia and neurons. Giant cells are also found in the cerebral white matter of TSC patients.where they form heterotopic nests or rows radially oriented as if for a distance they followed the radial glial guides of neuronal migration but failed to reach the cortical plate. Giant cells are also present in subependymal nodules, SEGAs and in retinal astrocytic hamartomas. The fine structure of the atypical cells in the cortical tubers has been studied in cerebral biopsy with the Golgi-Cox method and with electron microscopy (EM). With the Golgi-Cox (17) method, the giant cells are not evident but in the subpial region of the tuber there are clumps of glial cells. Elsewhere in cortex predominate a cell type not usually seen in normal cortex: small, bearing beaded dendrites, sparse in spines, and multipolar or stellate in shape. These cells are believed to be neurons of a primitive type, similar to those found in the normal caudate and the geniculate nuclei. In the depth of small tubers, cells may be arranged in layers including radially oriented pyramidal cells forming a rudimentary cortex as if it had begun but failed to competely develop. The cerebral cortex contiguous to a tuber displays no cytoarchitectonic alteration but the dendritic arborizations are less extensive than those of normal cortex suggesting
895
Tuberous Sclerosis Complex that the tuber interferes with the full development of its neighboring cortex (17) or that the extracellular matrix here is less deficient in a protein necessary for migration and differentiation than more complete cortical tubers. Speculations on the Pathogenesis of TSC The hamartias ofTSC or cortical tubers constitute a multifocal disturbance of cytoarchitectonic organization where large and bizarre neurons or stellate neurons lack laminar and columnar arrangement The focal distribution of the tubers is paralleled by scattering of subependymal hamartomas over the ependymal surface and both with rostral preponderance. Cutaneous, renal, cardiac and retinal hamartomas are also isolated or focal, a fact characteristic of TSC and other hamartomatoses. We do not know if the abnormal undifferentiated large cells in the brain resulted from an uneven mitotic division just as the N-cells grown in culture from facial angiofibroma stroma but it is possible that during cerebral embryogenesis a disturbance of the centromere-microtubule-centriole system prevents proper chromosomal arrangement during mitosis, resulting in abnormally large cells that are unable to migrate and differentiate normally. Perhaps a defective contractile protein that forms microtubules in neuron-like cells plays a role in the disturbed cytogenesis, post-mitotic differentiation and neuronal migration prior to organogenesis. If so, we could call the normal protein missing in TSC T-sclerin, a constituent of the radial glial guides for neuronal migration. Alternatively the T-sclerin could be part of the membrane receptors of migrating neurons. It has been established that cells destined to form the neocortex migrate from the germinal matrix zone to the cortex in sequence, the first to migrate forming the deep layer of the cortex and the last ones to migrate reaching the more superficial layers. They are guided in their journey by bipolar radial glial cells with processes extending to the ventricular zone and to the external limiting membrane of the hemisphere. The motility of primordial cells could be affected either by glial guide constituents to which the neuron might become firmly or persistently attached or by abnormal cell membrane receptors in the migrating neuron (18). Certainly, the interior "milieu" must play an important role in the growth of TSC hamartomas. Specifically, during the gestational period there is rapid growth of the cardiac rhabdomyomas that ceases after birth when the tumors become smaller
in size to the extent that many disappear. Between 3 and 8 years of age, facial angiofibromas appear in about half of the patients with TSC (see Fig. 1) but as a rule only for the next 10 years may continue to grow. SECAs may grow from subependymal nodules and become tumors but only in about 6 percent of patients between 5 and 20 years of age will obstruct the CSF circulation (19). Renal angiomyolipomas (RAML) and pulmonary lymphangiomyomatosis (LAMM) usually appear predominantly in female patients past their third and fourth decades of life respectively. It is as if a hormonal timetable determines when these different hamartomas are turned on or off. To explain the multifocality of the hamartias and hamartomas is not easy butJohnson et al. (11) have proposed the two-hit hypothesis as follows: the first hit is a mutational change. affecting a molecule for cell migration either inherited or the result of a new mutation which occurs before segregation of germline from somatic cells. This mutation causes an abnormal migration of precursor cells. A second hit occurs in embryonic development to some cells during division as a random mutation and this causes loss of a suppressor gene leading to the development of hamartomas at random in focal parts of one or more organs. References 1) Gomez MR: Tuberous Sclerosis, 2nd Ed, Raven Press, New York, NY, 1988. 2) johnson WG, Gomez MR (ed): Tuberous Sclerosis and Allied Disorders, Ann NYAcad Sci, 615: 1-148,1991. 3) Devroede G, Lemieux B, Masse S, Lamarche j, Herman PS: Colonic hamartomas in tuberous sclerosis, Gastroenterology, 94: 182-188,1988. 4) Drut R: Multivisceral dysplastic lesions in a patient with tuberous sclerosis and Langerhans cell histiocytosis, Pediatr Pathol, 10: 633-639, 1990. 5) Koprowski C, Rorke LB: Spinal cord lesions in tuberous sclerosis, PediatrPathol, 1: 475-480, 1983. 6) Ishibashi Y, Matsukawa A, Yu H-S, et al: An ultrastructural observation of the facial lesion in Pringle's disease, Nishinikonj Dermatol, 43: 1029-1043, 1981. 7) Nickel WR.Reed WB: Tuberous sclerosis, ArchDermatol, 85:209..226, 1962. 8) Ishibalilli Y, Watanabe R, Nogita T, Takahashi T, Onodera K, Kimura G: Abnormal gene expressions ofstroma cells inpatients with tuberous sclerosis, in joltn$oJlW(;, Gomez MR (ed): Tuberous Sclerosis and 1JIsorders, Ann NYAcad Sci, 615: 228-242, 1991. 9) IshibashiY, Inoue Y,Takehara K, Fume M, Kukita A: Disturbed-mitotic processes of stroma cells in patients with tuberous sclerosis,jpnj Dermatol, 93: 1045-1058, 1983.' (in Japanese)
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10) Davidson MM, Yoshidome H, Stenroos E, Johnson WB: Neuron-like cells in culture of tuberous sclerosis tissue, in Johnson WG, Gomez MR (ed): Tuberous Sclerosis and Allied Disorders, Ann NY Acad Sci, 615: 196-210, 1991. 11) Johnson WG, Yoshidome H, Stenroos ES, Davidson MM: Origin of the neuron-like cells in tuberous sclerosis tissue, in johnson WG, Gomez MR (ed): Tuberous Sclerosis and Allied Disorders, Ann NY Acad Sci, 615: 211-219, 1991. 12) Onodera K, Takahashi T, Ito S, Kimura G, Watanabe R, Ishibashi Y: Molecular biology of cultured cells from a patient with tuberous sclerosis, in johnson WG, Gomez MR (ed): Tuberous Sclerosis and Allied Disorders, Ann NYAcad Sci, 615: 372-374, 1991. 13) Umeda T, Niijima T, Tashiro T, Komiya Y, Onodera K, Ishibashi Y: 55KD protein unique to cultured cells from angiofibroma of tuberous sclerosis, Cell Mol Biology, 33: 483-493, 1987.
14) Scappaticci S, Cerimele D, Tondi M, Vivarelli R, Fois A, Fraccaro M: Chromosome abnormalities in tuberous sclerosis, Hum Genet, 79: 151-156, 1988. 15) Dietrich CU, Krone W, Hochsattel R: Cytogenetic studies in tuberous sclerosis, Cancer Genet Cytogenet, 45: 161-177, 1990. 16) Ishibashi Y,Inoue Y,Takehara K, Fume M, Kukita A: Disturbed mitotic process of stroma cells in a patient with tuberous sclerosis, ] Dermatol (Tokyo), 11: 236-252,1984. 17) Huttenlocher PR, Wollmann RL: Cellular neuropathology of tuberous sclerosis, in johnson WB, Gomez MR (ed): Tuberous Sclerosis and AlliedDisorders, Ann NYAcad Sci, 33: 140-145,1991. 18) Caviness VS,Takahashi T: Cerebral lesions of tuberous sclerosis in relation to normal histogenesis, in johnson WB, Gomez MR (ed): Tuberous Sclerosis and AlliedDIsorders, Ann NYAcad Sci, 33: 157-195, 1991.
