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Handbook of Clinical Neurology, Vol.

87 (3rd series)
Malformations of the Nervous System
H. B. Sarnat, P. Curatolo, Editors
# 2008 Elsevier B. V. All rights reserved

Hamartomatous disorders of cellular lineage


Chapter 9

Tuberous sclerosis
PAOLO CURATOLO* AND ROBERTA BOMBARDIERI

Pediatric Neurology Unit, Tor Vergata University of Rome, Rome, Italy

9.1. Overview number of scleroses’. Von Recklinghausen’s brief


report contains the first description of the two patho-
9.1.1. Definition
logical lesions most often observed in newborns with
TSC: cardiac rhabdomyomas and cortical tubers.
Tuberous sclerosis complex (TSC) is a genetically
Bourneville (1880) gave the first detailed report of its
determined, variably expressed, multisystem disorder
neurological symptoms and gross cerebral pathology.
that may affect any human organ with well circum-
On postmortem examination, Bourneville found that
scribed, benign, noninvasive lesions. The skin, brain,
many cerebral convolutions had hard, raised, whitish
retina, heart, kidney and lung are the organs most often
areas of greater density than the surrounding cortex.
involved. This devastating disorder, resulting from
He concluded that the seizures had a focal origin and
mutations in one of two genes (TSC1 and TSC2), affects
progressed to generalized attacks, attributing their par-
as many as 1:5800 live births (Curatolo, 2003). The
tial onset to the conspicuous sclerotic lesions in the
importance of CNS involvement in TSC is emphasized
ascending frontal and parietal convolutions of the left
by the fact that the condition has retained its name for
cerebral hemisphere (Fig. 9.1). One year later Bourne-
over a century. ‘Tuberous sclerosis of the cerebral con-
ville and Brissaud (1881) made a new clinicopathologi-
volutions’ was the term used by Bourneville (1880) to
cal observation, emphasizing the association between
describe the unique and distinctive cerebral pathology
the cerebral lesions and the kidney tumors.
he found in a patient with seizures and mental subnor-
In 1901 Pellizzi reported a precise microscopic
mality. The term ‘tuberous sclerosis complex’, pro-
description of the cortical tubers, finely illustrated with
posed in 1942 by Moolten, persists as a designation for
ink drawings of atypical neurons, subcortical areas of
all forms and variants of the disease. The majority of
hypomyelination and subependymal nodules. (Fig. 9.2).
patients identified as having the disorder experience
He also reported the frequent association of the cerebral,
symptoms referable to the CNS. Even in subjects with-
renal and cardiac lesions with the ‘cutaneous adenomas’
out neurological symptoms, CNS lesions are present in
in TSC patients.
all brains studied. CNS abnormalities therefore remain
In 1908 Vogt described the association between the
the hallmark of TSC and its most common and clinically
cerebral sclerosis of the circumvolutions and the facial
serious manifestations.
adenoma sebaceum. Through Vogt’s work, the syndrome
consisting of seizures, mental retardation and ‘adenoma
9.1.2. Historical background sebaceum’ was established as the classic clinical triad
of features for the diagnosis of TSC. The dominant inher-
Friedrich Daniel von Recklinghausen (1862) presented itance of TSC and its high mutation rate were first
to the Obstetrical Society of Berlin the pathological described by Gunther and Penrose (1935). Lagos and
findings in a newborn who had ‘died after taking a Gomez (1967) documented a family in which five gen-
few breaths’. The heart had several tumors protruding erations were affected by TSC, reporting that all the
on the cardiac surface and the brain contained a ‘great mentally retarded patients had previously had seizures.

*Correspondence to: Dr Paolo Curatolo, Paediatric Neurosciences, Tor Vergata University of Rome, Via Montpellier 1,
1–00133, Rome, Italy. curatolo@uniroma2.it.
130 P. CURATOLO AND R. BOMBARDIERI
9.2. Neuropathology
9.2.1. Brain abnormalities

A number of brain lesions can be associated with TSC,


and more than 95% of patients demonstrate at least
one of the abnormalities listed in Table 9.1 (DiMario,
2004). The most commonly identified are cortical
tubers, subependymal nodules and subependymal giant
cell astrocytomas.
Cortical tubers are variable in size, occur most often at
the gray–white matter junction and are usually multiple.
Tubers are typically observed in the cerebrum but can
be seen in the cerebellum as well. Less frequently,
Fig. 9.1. Cerebral tuberous sclerosis showing sclerotic, hyper- tubers can degenerate into cystic lesions associated with
trophic circumvolutions. (From Bourneville and Brissaud, 1881.) subcortical component adjacent to the cortical lesion
(Griffiths and Martland, 1997). Tubers themselves can
be limited to the cortex or the subcortical white matter
and occasionally can have a more ‘wedge-shaped’
appearance and a focal area of calcification. The overly-
ing cortex of a tuber can also be dysplastic and sugges-
tive of pachygyria or polymicrogyria.
On gross pathological examination, tubers are firm,
well circumscribed nodules that span flattened gyri
and sulci (Fig. 9.3). On microscopic examination, the
normal hexalaminar structure of neocortex is lost with-
in the tuber and the gray–white junction is blurred
(Fig. 9.4). Interestingly, the cytoarchitecture of cere-
bral cortex surrounding tubers is typically normal, sug-
gesting that tubers result from a developmental defect
affecting a restricted population of neuronal precursor
cells during corticogenesis. The most prominent
abnormal cell types in tubers are large dysplastic neu-
rons and giant cells as well as bizarrely shaped astro-
Fig. 9.2. Ink drawings of atypical neurons and cortical cytes (Fig. 9.5). Dysplastic neurons exhibit disrupted
cytoarchitectural disorganization. (From Pelizzi, 1901.) radial orientation in cortex and abnormal dendritic
arborization. Heterotopic neurons scattered in the deep
white matter are a frequent finding as well.
In 1979 Gomez observed in a large series that only
Tubers are identified as low-signal lesions on mag-
29% of all patients presented all three of Vogt’s triad
netic resonance imaging (MRI) T1-weighted sequences
characteristics. In 6% of the patients none of the clas-
and as high-signal lesions on T2-weighted and
sic features was diagnosed. These data determined the
weakening of the classic triad of Vogt and gave rise to
the revision of the diagnostic criteria that followed.
Table 9.1
Until the late 1980s, very little real progress was
made toward uncovering the molecular basis of tuber- Brain lesions in TSC (modified by DiMario, 2004)
ous sclerosis. The first report of genetic linkage analysis
Cortical/subcortical tubers
identifying a probable TSC gene on chromosome 9q34
Subependymal nodules
appeared in 1987. This gene was given the designation Subependymal giant cell astrocytomas
TSC1 (van Slegtenhorst et al., 1997). Subsequent studies White matter (30%): linear migration lines, ‘cystlike’ lesions
indicated that not all TSC families demonstrated linkage Corpus callosum agenesis/dysplasia
to the 9q34 region. Later, chromosome 16p13 was iden- Transmantle cortical dysplasia
tified as the site of a second TSC locus denoted TSC2. Association with hemimegalencephaly,
(Kandt et al., 1992, European Chromosome 16 Tuber- schizencephaly, intracranial arterial aneurysms (60%)
ous Sclerosis Consortium, 1993).
TUBEROUS SCLEROSIS 131
in the myelinated brain. Instead the tuber may appear
relatively hypointense compared to the adjacent unmy-
elinated white matter on T2-weighted sequences (Baron
and Barkovich, 1999). Tubers may be detected by MRI
in the neonatal period and have been identified in fetal
life as early as 26 weeks of gestation (Fig. 9.7).
Subependymal nodules (SENs) are hamartomatous
lesions that are typically located along the walls of
the lateral ventricles (Fig. 9.8); they have never been
observed in the third ventricle. SENs, found in the vast
majority of patients, are often multiple and generally
small (1 cm or less). Although SENs can occur any-
where along the ventricular surface, they have a partic-
Fig. 9.3. View of a cortical tuber. ular predilection for the region around the foramen of
Monro. The biological behavior of SENs in this region
is to enlarge (Inoe et al. 1998).
SENs must develop during fetal life and grow in
proportion to the remainder of the brain. Calcification
can be present in infancy or evolve more gradually
over childhood into puberty. Growth tends to stop after
the first decade (Hosoya et al., 1999). SENs are pre-
dominantly composed of dysplastic astrocytes and
mixed-lineage astrocytic or neuronal cell components.
MRI of SENs shows these lesions to have a signal iso-
intense to white matter on T1-weighted sequences and
iso- or hypointense on T2-weighted sequences. Large
calcifications are seen with T2-weighted MRI as a signal
void, whereas smaller SENs are more readily imaged
Fig. 9.4. Cerebral cortex showing disorganization of the nor- with computed tomography (CT), especially if calcified.
mal cortical pattern. Abnormal neurons have lost their nor- Some SENs enhance following intravenous gadolinium
mal orientation. administration. Any SENs located near the foramen of
Monro that enhance must be carefully evaluated by
serial imaging studies. SENs are largely asymptomatic
and seem to be unrelated to neurological symptoms
(Fig. 9.9).
Subependymal giant cell astrocytomas (SEGAs)
enlarge over a long period of time and are typically
identifiable by the end of the first decade of life, but
may be detected in infancy (Tien et al., 1990). MRI of
a EGA often shows an heterogeneous mass with lower
signal than normal white matter on T1 and higher than
the adjacent white and gray matter on T2. Hypointense
areas within these tumors may be secondary to calcifica-
tions, tumor vessels or intratumoral hemorrhage. All
SEGAs enhance following gadolinium administration
Fig. 9.5. Cortical tubers showing architectural disarray with
(Nabbout et al., 1999).
large, abnormal neurons. Many of the cellular constituents of features of SENs
and SEGAs, such as giant cells, are similar to those in
tubers sharing a similar profile of protein expression
fluid-attenuated inversion recovery (FLAIR) sequences (Jozwiak et al., 2006). However, the cellular packing
(Fig. 9.6). In very young patients with immature white density in SENs/SEGAs is greater than in tubers and
matter, the MRI appearance of tubers varies. In early these lesions have the cytopathological appearance
infancy, many tubers are not detectable. When detected of a tumor. For example, in SEGAs, multinucleated
the tubers do not show the characteristic findings noted cells may be seen with rare mitotic figures and cellular
132 P. CURATOLO AND R. BOMBARDIERI

Fig. 9.6. Subcortical tubers (arrows) shown on (A) FLAIR and (B) T2-weighted images. Small tubers are much more evident
on FLAIR images.

cells and are indicative of a disorder of migration asso-


ciated with abnormal cell differentiation. Wedge-
shaped lesions are triangular in configuration and have
their apex near the ventricle and their base at the cortex.
This pattern is usually evident in the T2-weighted imaging.
The site, shape and histopathological findings of white
matter lesions confirm that TSC is a disorder of both his-
togenesis and cell migration. In particular, the different
location between the lateral ventricles and the cerebral
cortex could be the result of different timing of neuronal
and glial migration arrest (Houser et al., 1991). Linear
migration streaks can be observed in up to 30% of
patients when examined very carefully on T2-weighted
MRI sequences.
Fig. 9.7. Axial fetal MRI at 27 weeks of gestation showing Further evidence of brain dysplasia can be observed
multiple SENs and tubers. on neuroimaging, including partial agenesis of the cor-
pus callosum, cortical dysplasia with focal gyriform
abnormalities and heterotopias, transmantle cortical
pleomorphism. In addition to giant cells, a heteroge- dysplasia and hemimegalencephaly. Additional, though
neous array of astrocytic, polygonal, epithelioid and rarer, CNS manifestations within the infratentorial com-
spindle cell populations may also be seen. Vascular partment include cerebellar folia atrophy, cerebellar
endothelial proliferation and necrosis may be observed nodular white matter calcifications and tuber-like
(Scheithauer and Reagan, 1999). Other brain abnormal- hamartomas in the cerebellum.
ities in patients with TSC are also detectable on neuro- Therefore, TSC should be considered not merely as
imaging (Bozzao et al., 2003). These include a number collections of isolated CNS abnormalities but more
of white-matter linear lesions, referred to as migration cohesively as a neuronal migration disorder. The essen-
lines. White matter lesions appears as radial bands, tial features of a neuronal migration disorder include
wedge-shaped and nodular foci mostly affecting the 1) abnormal number, size, and thickness of cortical gyri;
supratentorial white matter. Radial white matter lesions 2) heterotopic neuronal aggregates; and 3) variable
run from the ventricles through the cerebral mantle to degrees of cortical cytoarchitectural disorganization
the normal cortex or to the cortical tuber (Fig. 9.10). and aberrant columnar and laminar arrangement.
From an histological point of view these anomalies are The fundamental pathogenesis of TSC must incor-
almost identical to those observed in the cortical tubers. porate a logical explanation for the lesions detectable
Radial bands are composed of clusters of heterotopic both outside and within the central nervous system.
TUBEROUS SCLEROSIS 133

Fig. 9.8. Cortical tubers and subependymal nodules (SENs). Tubers are more evident on FLAIR images (B) than on T2-
weighted (A) and T1-weighted (C) images. SENs are shown as hypointense nodules in the ependymal layer on T2-weighted
sequences (A), because of calcification, and protrude inside the ventricle on T1-weighted images (C). Calcification is shown
by CT both in the tubers (arrows) and in the SENs in the same patient (D).

The brain lesions seen in TSC are, histologically, tissue and neuronal migration. In TSC two populations of
hamartomas. These are focal ‘tumor-like’ malforma- neuroepithelial cells are generated by the germinal
tions composed of abnormal proportions of cellular ele- matrix. The first consists of normal neuroblasts that
ments normally present in the brain. These elements form normal neurons and astroglia, which migrate to
have an abnormal cellular morphology, are in abnormal the cortical plate to form histologically normal cere-
quantities and locations, and are in a disorganized bral cortex. The second is an abnormal cell population
cytoarchitectural arrangement (dysplasia). Tubers, sub- that forms primitive cells, which often fail to show
ependymal nodules and subependymal giant cell astro- clear neuronal and glial differentiation. Some of these
cytomas represent focal tissue dysplasia. Transmantle cells, named ‘neuroastrocytes’, remain in the germinal
cortical dysplasia, hemimegalencephaly and cerebellar matrix zone where they form subependymal nodules
hemisphere megalencephaly represent more regional and giant cell tumors. Some neuroastrocytes show par-
tissue dysplasia. Linear migration streaks represent tial migration, forming heterotopias in the subcortical
trails of abnormal cell migration (DiMario, 2004). white matter. More differentiated cells migrate to the
cortical plate, where they form aggregates of dysplas-
9.2.2. Molecular pathogenesis tic cortex, the cortical tubers (Fig. 9.11).
Two tumor-suppressor genes have been associated
Present evidence suggests that the CNS lesions of TSC with the disorder. TSC1 , located on chromosome
are due to a developmental disorder of neurogenesis 9q34, and TSC2, on chromosome 16p13.3, respectively
134 P. CURATOLO AND R. BOMBARDIERI

Fig. 9.9. Progressive growth of an enhancing lesion at the foramen of Monro in a TSC patient. (A) Coronal T1-weighted image
with gadolinium shows a small enhancing lesion (arrow). This has grown markedly at the 3-year follow-up (B).

Fig. 9.10. Axial MRI demonstrating white matter bands in both hemispheres.

encode the proteins tuberin and hamartin. At the moment, Major advances in the understanding of the functions
about 500 different mutations in the two TSC genes are of tuberin and hamartin have shown that in normal
known. The mechanism of how mutations in these two cells the tuberin–hamartin complex acts as an inhibitor
separate genes lead to tuber formation is not clear. It is of the kinase mTOR, an important element in a signal-
likely that hamartin and tuberin participate in the same ing pathway involved in the control of cell growth and
pathways of cellular growth control and share common proliferation, controlled by the protein-kinase Akt.
biochemical properties that are important in early devel- When stimulated by growth factors or other agents,
opment and maturation of the brain. The proteins appear the activation of the oncoprotein Akt leads to phos-
to colocalize at the cellular level and recent evidence sug- phorylation of TSC2 and the consequent inactivation
gests a direct binding between tuberin and hamartin. of the inhibitory activity of the tuberin–hamartin com-
These two proteins form a cytosolic complex interacting plex. In cells lacking both TSC1 and TSC2, mTOR/S6
at the N-terminal ends. This interaction is abolished in kinase activity is increased severalfold and is no longer
some TSC-associated mutations (Nellist et al., 1999). dependent upon signaling, which normally regulates its
TUBEROUS SCLEROSIS 135

Fig. 9.11. Inactivation of the tuberin–hamartin complex (THC) alters cellular differentiation in the central nervous system.
Neurons and astrocytes arise from neuroglial precursors during development. Central nervous system lesions associated with
TSC contain giant cells that express a variety of cellular markers, including those characteristic of immature nervous system
cells. This suggests that disruption of the THC by tuberin or hamartin loss interferes with the normal maturation of precursor
cells and impairs their ability to differentiate into more developmentally mature neurons or astrocytes. (Modified from
Scheidenhelm and Gutmann, 2004.)

activity, including through the PI3 kinase pathway. This predominant cellular phenotype of the subependymal
fundamental biochemical defect in cells lacking TSC1 nodules in the Eker rat model of TSC is astrocytic
or TSC2 seems to contribute to the growth of TSC (Yeung et al., 1997). Immunohistochemical and molec-
hamartoma (Kwiatkowski et al., 2004) (Fig. 9.12). ular analyses have identified neuronal mRNAs and pro-
Giant cells are the hallmark histological cell type teins such as neuron-specific enolase, tubulin,
within tubers, SENs, and SEGAs that are unique microtubule associated proteins (MAPs) and intermedi-
to TSC. They are large (80–150 mm in diameter), poly- ate filaments in giant cells within tubers (Hirose et al.,
gonal or ovoid eosinophilic cells that extend short, 1995; Crino et al., 1996; Lopes et al., 1996). In addition,
thickened processes of unclear identity (i.e. axons or a small proportion of giant cells in SEGAs express met-
dendrites). Giant cells are distributed from the pial sur- enkephalin, b-endorphin, serotonin and neuropeptide Y,
face to the subcortical white matter without clear radial suggesting a neural lineage (Hirose et al., 1995). CD44,
or laminar orientation, and may appear in clusters. They a cell adhesion molecule, mediates cell-cell and cell-
do not exhibit preference for superficial or deep parts of matrix interactions is expressed near giant cells in both
cortex. tubers and SEGAs (Arai et al., 2000). CD44 is expressed
The cell lineage and phenotype of giant cells has in astrocytic processes, predominantly in white matter
been a matter of some debate since giant cells have been and subpial regions, suggesting its involvement in the
shown to express both glial and astrocytic markers. maintenance of a stable central nervous system
A fundamental issue regarding the pathogenesis of cytoarchitecture. Tuber sections probed with antibodies
CNS lesions in TSC is which cell precursors, i.e. glial recognizing the neural marker NeuN, a DNA binding
or neuronal, give rise to giant cells in tubers, SENs and protein present in mature neurons, exhibit a hetero-
SEGAs during early brain development. Early evidence geneous staining pattern (Crino, 2003). Some of the
argued independently for neuronal or glial lineage of giant cells and most of the dysplastic neurons are labeled
giant cells based on identification of neuronal or glial but a small proportion are unlabeled, suggesting either a
structural features using electron microscopy (Tromb- mixed neuroglial phenotype or that these cell types
ley and Mirra, 1981; Yamanouchi et al., 1997). For are phenotypically immature. Dysmorphic neurons
example, the identification of rough endoplasmic retic- express a variety of neuronal markers, e.g. cytoskeletal
ulum, intermediate filaments extending into processes elements, neurotransmitter receptor subunits, which
of ambiguous morphology, prominent paranuclear suggest they are indeed of neuronal phenotype. These
Golgi zones and dense-core granules (suggestive of neural markers have been identified within a subpopula-
secretory vesicles) in giant cells suggested neuronal tion of giant cells in SEGAs and SENs, suggesting that
features. In contrast, a subpopulation of giant cells in giant cells in tubers are more closely akin to neurons
SENs and SEGAs is immunoreactive for GFAP, vimen- whereas those in SENs/SEGAs may reflect a more mixed
tin or S-100, which suggests a glial phenotype. The population. Synaptophysin immunoreactivity has been
136 P. CURATOLO AND R. BOMBARDIERI
gene mutations in developing cortex occur at least as
early as mid- to late corticogenesis, since nascent cor-
tical tubers have been identified in embryonic brains
as early as 20 weeks gestation (Park et al., 1997). Sim-
ilarly, SENs may be formed during embryogenesis or,
like SEGAs, may appear in childhood or adulthood.
The link between altered function of tuberin and
hamartin and the genesis of tubers, SENs and SEGAs,
however, remains to be defined. Tubers probably
derive from early progenitor cells in the ventricular
zone while SENs and SEGAs may derive from neuroe-
pithelial cell precursors in the subventricular zone or
ventricular zone. Current data suggest that these pro-
teins probably regulate cellular proliferation and
potentially cellular movement, and thus the morpho-
logical appearance of tubers and SEGAs suggests
alterations in these cellular domains. For example,
antisense inhibition of tuberin expression in cultured
cells fosters cell proliferation (Soucek et al., 1997)
and hamartin probably contributes to the formation of
focal adhesions necessary for cellular migration (Lamb
et al., 2000). Further understanding of the downstream
cellular events resulting from TSC1 or TSC2 mutations
may highlight specific cellular pathways or select cell
types that account for the histological manifestations
Fig. 9.12. Functions of the tuberin–hamartin complex of these lesions. It remains to be shown that the same
(THC). The THC negatively regulates Rheb, a small guano- processes are altered in tubers, SENs and SEGAs.
sine triphosphatase that activates the mammalian target in There have been several theories proposed to
the rapamycin (mTOR)/S6 kinase (S6K)/factor 4E binding explain how tubers, SENs and SEGAs may form dur-
protein (4E-BP) 1 signaling cascade, which is important for ing cortical development. For example, it is believed
the regulation of cell size and proliferation. In addition, the that all neuroepithelial progenitor cells carry a single
THC may regulate expression of the cyclin D kinase inhibi- allelic TSC gene mutation. In view of the focal nature
tor p27kip1, which is involved in both cell growth and size of these lesions, one hypothesis is that a ‘second-hit’
control. To relieve the THC-mediated inhibition of cell size
mutation in the existing normal TSC allele occurs
and proliferation, phosphatidylinositol-3 kinase (PI3K) and
within a single neuroepithelial cell in the ventricular
Akt are activated following stimulation of the insulin-like
growth factor (IGF) receptor. Akt phosphorylates tuberin, zone or subventricular zone as a result of inherent sus-
inactivating the complex and resulting in increased cell size ceptibility or random mutation. As a consequence of
and proliferation. (Modified from Scheidenhelm and the two mutations within this precursor cell, there is
Gutmann, 2004.) loss of hamartin or tuberin function and the ensuing
downstream events may alter one of several steps
involved in cellular proliferation or differentiation.
reported along the cell membrane of giant cells (Lippa This model suggests that all the cells within a tuber,
et al., 1993) and single-cell mRNA analysis from micro- SEN or SEGA derive from the initial cell sustaining
dissected giant cell has shown that these cells express a ‘second-hit’ TSC mutation.
NMDA, GluR and GABAA receptor subunit mRNAs, A problem with this hypothesis is that while loss of
suggesting the possibility of synaptic connectivity heterozygosity may account for SEN and SEGA for-
between giant cells and surrounding neurons. mation, allelic loss occurs rarely, if ever, in tubers
(Henske et al., 1996; Niida et al., 2001). In fact, it is
9.2.3. Developmental pathogenesis difficult to account for the overt cellular heterogeneity
observed in tubers if all cells are derived from a com-
Tubers probably result from the effects of TSC1 or mon precursor cell. One possibility is that the precur-
TSC2 mutations on one of several steps during cortical sor cell sustaining the ‘second hit’ can yield progeny
development, including cell proliferation, differentia- of disparate cell phenotypes via intrinsic programs or
tion and migration. The cellular effects of the TSC stochastic events. Similarly, loss of heterozygosity at
TUBEROUS SCLEROSIS 137
either TSC1 or TSC2 loci was not identified in glio- plastic neurons, may be merely ‘innocent bystanders’
neuronal malformations with cellular morphology that whose functional attributes, such as locating the appro-
was similar to tubers (Norman et al., 1997). The fail- priate laminar destination, have been altered by the
ure to detect loss of heterozygosity in tubers suggests presence of aberrant giant cells. In this view, the giant
either an experimental artifact that disrupts the allelic cells would be the ‘two-hit’ cells that migrate albeit
loss or that tubers form by a distinct mechanism from abnormally into the cortical plate and disrupt migra-
other lesions in TSC. tion of adjacent ‘single-hit’ neurons. The cortical dys-
Variable mutational events occurring in disparate plasia observed in tubers would reflect a secondary or
precursor cell populations or at different developmen- bystander effect in which ‘normal’ (single mutation)
tal timepoints could result in cells populating tubers neurons fail to migrate appropriately because of the
or SEGAs with remarkably distinct phenotypes. Thus, disruption of migratory pathways by giant cells. Con-
cellular heterogeneity in tubers and SEGAs may also versely, dysplastic neurons may reflect the ‘two-hit’
reflect TSC mutations occurring at distinct time- cells that cannot migrate correctly and that induce
points. If a mutation were to occur in an early progen- cytomegaly in giant cells through the effects of a
itor cell, then the progeny of that cell might be more released growth factor. Indeed, the mutational status
diverse than if such a mutation were to occur in a more of other cells within tubers or SEGAs, i.e. endothelial
mature precursor cell, where the progeny would be a cells or macrophages, remains unknown. These con-
more restricted phenotype. Recent studies using retro- siderations are vital towards understanding the patho-
viral tagging strategies have demonstrated that clonally genesis of tubers and SEGAs they warrant a clear
derived cells may give rise to quite diverse cell popu- view of how tuberin or hamartin dysfunction results
lations, including pyramidal and multipolar cells and in abnormal cytoarchitecture and potentially epilepto-
even astrocytes (Reid et al., 1995). An alternative pos- genesis. In contrast, in the latter scenario, the effects
sible explanation for the observed heterogeneity of of altered tuberin or hamartin function would be evi-
protein expression and cell morphology of dysplastic dent in both neurons and giant cells and thus TSC1
neurons and giant cells may be that in some cases a and TSC2 may serve as epilepsy susceptibility genes.
second-hit mutation occurs in a cell destined to
become a pyramidal cell while in others it occurs with- 9.3. Molecular genetics
in a cell destined to become an astrocyte. These dis-
parities may explain why giant cells have been Tuberous sclerosis results from mutations in TSC1, the
reported to express a variety of often conflicting neural gene on chromosome 9q34, and TSC2, the gene on
and glial protein lineage markers. In contrast, only chromosome 16p13 (European Chromosome 16 Tuber-
specific precursor cells may be capable of becoming ous Sclerosis Consortium, 1993; van Slegtenhorst et al.,
a giant cell or dysplastic neuron and hence a require- 1997). Frequent loss of heterozygosity for alleles in
ment might be that second-hit mutations occur in spe- 16p13.3 and rare loss in 9q34 has been found in hamar-
cific precursor cells. Thus, the phenotypic pathological tomas from TSC patients, indicating that a second
variability reported in tubers and SEGAs may suggest somatic mutation may be required to produce the TSC
that, in some patients, giant cells in tubers are largely phenotype at the cellular level (Green et al., 1994a,
derived from an astrocytic precursor cell whereas, in 1994b). These findings are consistent with TSC1 and
other patients, tubers are populated by giant cells that TSC2 acting as growth suppressor genes (Carbonara
derive from neuronal precursors. In fact, these differ- et al., 1994).
ences might be observed within the same patient, so The TSC2 gene maps to a gene-rich region of
that a proportion of tubers may be neuronal and a por- 16p13.3, approximately 2.25 Mb from the telomere
tion astrocytic. These possibilities have important the- and immediately adjacent to the PKD1 gene. The
oretical functional implications: for example, tubers 5.5 kb transcript spans an estimated 43 kb of genomic
derived from neural precursors might be more epilep- sequence and comprises 42 known exons, of which 41
togenic than those derived from astrocytes, which are coding, and encodes an 1807 amino-acid protein,
would explain why in some TSC patients only one of called tuberin, with little similarity to other known
several tubers is epileptogenic (Crino, 2004). genes. 163 amino acids near the COOH terminus are
An important additional issue is whether all the cel- homologous to the catalytic domain of a guanosine tri-
lular constituents of tubers or SEGAs directly result phosphatase (GTPase) activating protein, GAP3 (rap1-
from TSC gene mutations. According to Crino GAP). GAPs are regulators of the GTP binding and
(2003), all cells within these lesions may necessarily hydrolysing activity of the Ras superfamily of pro-
reflect downstream effects of TSC gene mutations, or teins, which help to regulate cell growth, proliferation
alternatively some cell types, e.g. astrocytes or dys- and differentiation.
138 P. CURATOLO AND R. BOMBARDIERI
The TSC1 gene consists of 23 exons, of which the in Table 9.3. Most of the findings traditionally regarded
last 21 contain coding sequence. The TSC1 protein, as among the most specific for TSC become apparent
which is called hamartin, consists of 1164 amino acids only in late childhood, limiting the usefulness for early
with a calculated mass of 130 kDa. The protein is gen- diagnosis in infants.
erally hydrophilic and has a single potential transmem-
brane domain at amino acids 127–144 as well as a 9.4.1. Neurological manifestations
probable 266-aminoacid coiled-coil region beginning
at position 730 (van Slegtenhorst et al., 1997; Vinters 9.4.1.1. Epilepsy
et al., 1998). The characterization of the TSC1 and Seizures are the most common neurological symptom
TSC2 genes is summarized in Table 9.2. of TSC, occurring in about 90% of patients (Curatolo
The mutations observed in TSC1 consist of small et al., 2002). Epilepsy in TSC often begins during
deletions, small insertions and point mutations. the first year of life and, in most cases, in the very first
The majority of mutations are likely to inactivate pro- months. The high incidence of epileptic spasms
tein function and these findings support the hypothesis and hypsarrhythmia has long been emphasized but it
that TSC1 functions as a tumor suppressor gene. is now clear that epileptic spasms in infants with
At the moment, about 500 different mutations in both TSC are clinically and electroencephalographically
TSC genes are known. There is an equal distribution of different from the classic epileptic spasms and hypsar-
mutations between TSC1 and TSC2 among familial rhythmia of West syndrome (Curatolo et al., 2001a).
cases, while among sporadic cases TSC2 mutations are In the same child focal seizures may precede, coexist
much more frequent than TSC1 mutations. The wide with or evolve into epileptic spasms. Subtle focal
range of tissues in which TSC associated hamartomas seizures, such as unilateral tonic or clonic phenomena
develop implies a fundamental role for both TSC genes mainly localized in the face or limbs, and other
in regulating cell proliferation and differentiation. seizures with subtle lateralizing features, such as tonic
eye deviation, head turning and unilateral grimacing,
9.4. Clinical features can occur but may be missed by the parents until the
third or fourth month of life. The EEG at onset usually
Clinical features of TSC are most commonly observed shows focal or multifocal spike discharges and irregu-
in the brain, skin, kidneys, heart, eyes and lungs. The lar focal slow activity. Video-EEG monitoring and
physical findings can vary greatly, since TSC can polygraphic recordings of the epileptic spasms have
affect different organ systems in different ways at dif- shown that each spasm consists of a combination of
ferent times in the individual’s life. The frequency of both focal and bilateral manifestations. Although the
clinical signs in different age groups is summarized pathophysiological mechanisms responsible for the

Table 9.2
Characterization of the TSC1 and TSC2 genes

TSC1 TSC2

Localization 9q34 16p13.3


Structure 23 exons – 8.6 kb transcript alternate 41 exons – 5.5 kb transcript exons 25, 26 and 31
splicing in the 5 UTR alternately spliced
Mutations Small truncating mutations Large deletions/rearrangementsSmall
truncating mutationsMissense mutations
Occurrence 10–15% of sporadic cases 70% of sporadic cases
Phenotype ?less mental impairment ?more likely to be mentally retarded
Contiguous gene deletion syndrome with PKD1
Loss of heterozygosity Rare Frequent
in hamartomas
Product Hamartin Tuberin
Function(s) ?Regulates cell adhesion through ?GTPase activating proteinRole in the cell cycle
interaction with ezrin and rho
Regulator/modulator of tuberin activity
Subcellular localization Cytoplasmic, ?cortical Cytoplasmic, ?Golgi-associated
Animal models Knockout mice under development Eker rat, knockout mice, Drosophila (gigas)
TUBEROUS SCLEROSIS 139
Table 9.3
Frequency of clinical signs in children with definite tuberous sclerosis in different age groups

Less than 2 years 2–5 years 5–9 years 9–14 years

Criterion % Criterion % Criterion % Criterion %

Hypomelanotic macules 89 Hypomelanotic macules 95 Hypomelanotic macules 97 Hypomelanotic macules 97


Cardiac rhabdomyoma 83 Epilepsy 91 Epilepsy 94 Epilepsy 96
Epilepsy, usually IS 83 Facial angiofibromas 63 Facial angiofibromas 73 Facial angiofibromas 77
Renal AMLs 16 Renal AMLs 41 Renal AMLs 63 Renal AMLs 64
Facial angiofibromas 10 Cardiac rhabdomyoma 21 Shagreen patch 34 Shagreen patch 47
Retinal hamartoma 8 Shagreen patch 20 Cardiac rhabdomyoma 21 Cardiac rhabdomyoma 41
Retinal hamartoma 17 Retinal hamartoma 20 Liver AMLs 23
Forehead plaque 9 Forehead plaque 16 Forehead plaque 18
Liver AMLs 11 Retinal hamartoma 17
Periungual fibromas 11

AML, angiomyolipoma.
Modified from Jozwiak et al., 2000.

coexistence of spasms and focal motor seizures are and epilepsy referred to tuberous sclerosis clinics
still uncertain, epileptic spasms associated with TSC who achieve prolonged seizure remission is small.
may show focal features at onset, followed by a rapid Usually, seizure remission is associated with mild
secondary generalization. The age at seizure onset neurological deficits, and sustained remission is more
and the age when epileptiform activity becomes appar- likely to be associated with normal intelligence, a
ent on the EEG is largely dependent on the location of greater likelihood of having a normal finding on elec-
the cortical tubers detected by MRI and may coincide troencephalogram at the time of discontinuation, and
with functional maturation of the cortex, with an earlier fewer cortical and subcortical tubers on neuroimaging
expression for temporo-occipital regions than for frontal (Sparagana et al., 2003). Unfavorable prognostic
ones (Curatolo et al., 1991). factors include onset before 1 year of age, presence
A number of young children with TSC, who present of multiple seizure types (spasms and focal motor or
with focal seizures or epileptic spasms at onset, later complex partial seizure, drop attacks and atypical
develop intractable seizures with multifocal EEG absences), multifocal discharges in the awake state
abnormalities associated with bilateral and more syn- that tend to bilateralize in sleep (Fig. 9.11) and/or
chronous slow spike-wave complexes and an electrocli- secondary bilateral synchrony and occurrence of new
nical pattern that resembles Lennox–Gastaut syndrome. electroencephalographic (EEG) foci during evolution
In patients with TSC, the differential diagnosis between (Curatolo and Verdecchia, 2004).
Lennox–Gastaut syndrome and localization related In TSC, seizures have a focal or multifocal origin
symptomatic epilepsy originating in the frontal lobe with a topographic correspondence between EEG foci
may be extremely difficult and only in few cases long- and MRI high-signal lesions, demonstrating the prepon-
term video-EEG monitoring can reveal subtle electro- derant role of cortical tubers as epileptogenic foci.
clinical manifestations suggestive of a focal seizure Immunohistochemical and molecular analysis have
onset. In these patients, high time-resolution topographic indicated that the neuronal populations within cortical
EEG analysis and dipole localization methods may tubers might have intrinsic epileptogenicity and actively
detect secondary bilateral synchrony, often originating participate in the generation of partial seizures, through
in frontal regions and corresponding to prominent corti- the release of neurotransmitters or neuromodulators into
cal tubers detected by MRI in the mesial surface of the the adjacent brain tissue (Wolf et al., 1995). Giant cells
frontal or the anterior temporal lobes (Seri et al., 1998) in tubers express neurotransmitter-producing enzymes
(Fig. 9.13). and neurotransmitter receptors, such as NMDA receptor
The natural history of epilepsy in patients with subunit 1 and GABAA receptor subunits (White et al.,
TSC from infancy into childhood tends to be one of 2001). It has recently been suggested that changes in
increasing seizure frequency and severity, with poor the properties of GABAA receptors, possibly related to
response to antiepileptic drug treatment (Curatolo plastic changes in subunit combinations, may result in
et al., 2005). The proportion of children with TSC an altered regulation of inhibitory function. On the basis
140 P. CURATOLO AND R. BOMBARDIERI

Fig. 9.13. MRI demonstrates multiple cortical tubers in both hemispheres and subependymal nodules in a 7-year-old girl with
intractable seizures and severe learning disability. Interictal EEG showing apparently generalized spike and wave discharges.
A lateralized onset from the right frontal tuber was detected by EEG mapping.

of these findings it is possible to hypothesize that epilep- TSC have normal intelligence, while the other half
togenesis in TSC may result from ‘aberrant plasticity’, have mental disabilities ranging from mild learning
that may involve a subunit switch of GABAA receptors disabilities to severe mental retardation (Prather and
and changes in a regulatory system. Concas et al. de Vries, 2004). It seems that the majority of indivi-
(1999) reported that the changes in the plasticity of duals with TSC either fall into a severely disabled
GABAA receptors are related to the physiological group or have normal intelligence. This ‘bimodal
changes in plasma and brain concentration of neuroster- distribution’ is important, because the behavioral and
oids, which may act as endogenous modulators of psychiatric problems associated with ‘able’ and the
GABAA receptors. TSC patients with epilepsy may ‘severely disabled’ group are different. The likelihood
present a dysregulation of enzymes deputed to the syn- of learning disability developing appears to be asso-
thesis of these neuroactive steroids, possibly resulting ciated with the nature of the genetic mutation, the
in a decreased GABAergic transmission. These modifi- extent of brain abnormality and the age of onset and
cations could be related to a genetically determined type of seizure disorder (Goodman et al., 1997; Jones
mechanism that regulates 3b-tetrahydroprogesterone et al., 1997; Dabora et al., 2001; Joinson et al.,
synthesis or metabolism, and/or to a functional regula- 2003). However, the mechanisms involved are likely
tion linked to the expression of receptors to which these to be different according to the nature of the cognitive
neuroactive steroids bind (Di Michele et al., 2003). impairment, with perhaps structural and functional
The mechanism involved in drug resistance in TSC disruptions to the development of different neural
is unknown. Recently, Lazarowski et al. (2004) investi- networks resulting in distinct, partly overlapping
gated the expression of the proteins MDR-1 (multidrug cognitive impairments (de Vries et al., 2005).
resistance gene) and MRP-1 (multidrug resistance-asso- Individuals with normal overall intellectual abilities
ciated protein-1) in epileptogenic cortical tubers of three are at increased risk of specific cognitive deficits
pediatric patients with TSC and refractory epilepsy (Harrison et al., 1999; de Vries et al., 2001). These
obtained after surgical resection. In these specimens, include attention deficits (e.g. difficulties sustaining
MDR-1 and MRP-1 proteins were strongly immunore- attention, switching of tasks), executive control
active in abnormal balloon cells, dysplastic neurons, problems (e.g. planning, sequencing), language
astrocytes, microglial cells and blood–brain barrier (e.g. expressive or receptive language delay), learning
vessels. A more extensive MDR-1 immunoreactivity and memory problems, and visual-spatial difficulties.
was also observed, suggesting that refractory epilepsy Children with TSC who have such neuropsychological
phenotype in TSC may be associated with the expres- deficits often present with specific scholastic diffi-
sion of both multi-drug-resistance MDR-1 and MRP-1 culties in reading, writing, spelling and arithmetic.
transporters in epileptogenic cortical tubers. Because of these difficulties, a number of children
with TSC are often diagnosed with dyspraxia or
9.4.1.2. Cognitive and behavioral disorders dyslexia (Bolton, 2003).
Tuberous sclerosis is associated with a wide range of A very significant proportion of children and adults
cognitive, behavioral and psychiatric manifestations with TSC will present with a range of behavioral and
(Curatolo et al., 1991). About half of individuals with psychiatric difficulties. In children with severe global
TUBEROUS SCLEROSIS 141
disabilities (or mental retardation), infantile autism 2003). The TSC2 gene product tuberin is highly
and other autism spectrum disorders are seen in more expressed in brain regions involved in the behavioral
than 50% of cases (Curatolo and Verdecchia, 2004). phenotypes of the autistic disorder. The genetic dissec-
Despite the great deal of progress in the last few tion of the short arm of chromosome 16 in autism
years, the neural basis of autism in TSC is still largely could help to localize more precisely a susceptibility
unknown and represents a major challenge for child gene and clarify its position with respect to the TSC2
neurologists (Wiznitzer, 2004). The epidemiological locus. Several factors support role for the dopamine
studies of autism, despite differing tuberous sclerosis b-hydroxylase (DBH) gene, which has been mapped
populations and varying diagnostic criteria, all arrive to 9q34 in the etiology of autism. Some families with
at a surprisingly high incidence of autism, between autistic children have a low level of serum dopamine
17% and 61%. The incidence of autism may be signif- DBH, which catalyzes the conversion of dopamine to
icantly higher than the rates of cardiac and renal norepinephrine (Robinson et al., 2001). The DBH gene
abnormalities, for which screening is routinely con- is closely linked to one of the loci for TSC.
ducted in this population. It is to be hoped that early Attention deficit hyperactivity disorder (ADHD)
diagnosis of autism will allow for earlier treatment and related behaviors (such as impulsivity, overactivity
and the potential for better outcome. From the current and attention problems in daily life) are also seen in
evidence, no one factor (learning disability, tuber about half of children with TSC. ADHD-related beha-
localization, occurrence of infantile spasms, focal viors are more likely to be seen in those with severe
EEG abnormalities or seizure) can be causally linked disabilities but are highly over-represented in those
with the abnormal behavior. Rather, it may be that with normal ability as well.
all the etiological categories detailed above may be Self-injurious behaviors, aggressive outbursts, diffi-
causal for autism in TSC. Some patients may exhibit cult temper tantrums and chronic sleep problems are
autistic behavior secondary to specifically located cor- often seen in children with TSC with severe disabilities.
tical tubers, others may be affected by specific epilep- In older children, adolescents and adults, anxiety
tic phenomena, with others perhaps harboring a and mood-related disorders become increasingly prev-
deletion in an adjacent but otherwise independent gene alent. Anxiety and mood symptoms are seen in indivi-
that is critical in the genesis of autism. The better iden- duals with and without global cognitive difficulties
tification of patients, especially the identification of and can be very debilitating to higher-functioning
less severely affected patients, presumably with a lower young people and adults.
cortical lesion burden, combined with improved imag- Sleep disorders, such as night waking, prolonged
ing with MRI or functional imaging techniques, may sleep latency and seizure-related sleep problems, are
aid in studies of the localization of behavior in the considered one of the most common behavioral mani-
developing brain. Similarly, the improved treatments festations in children with TSC. Prolonged sleep laten-
for epilepsy in TSC patients, including the remarkable cy and frequent awakenings due to epileptic seizures
response of infantile spasms to vigabatrin, as well as need to be differentiated using polysomnography
the continuing success of epilepsy surgery, may allow (Bruni et al., 1995).
for the elimination or reduction of epilepsy as a vari-
able in attempts to distinguish between the various 9.4.2. Non neurologic manifestations
possible etiologies of autism in TSC. Improved seizure
control may allow for a more independent analysis 9.4.2.1. Cutaneous manifestations
of the effects of lesion localization, learning disabil- The most well-known cutaneous manifestations of
ity or genotype on behavior. Finally, as the underly- TSC are facial angiofibromas, which don’t tend to
ing genetic mutations in more and more patients appear until late childhood or early adolescence. They
with tuberous sclerosis are characterized, genotype/ start out as flat, reddish macular lesions that, at an ear-
phenotype correlations with autistic behavior can ly stage, may seem to be freckles. As time passes,
be made. angiofibromas become increasingly erythematous and
Autism may also reflect a direct effect of the abnor- nodular and sometimes present with a friable surface
mal genetic program. The terminal 2 megabases of the that may bleed easily. Facial angiofibromas are usually
short arm of human 16 contain the TSC2 locus and noted first in childhood and progress during puberty
there is linkage evidence for bipolar affective disorder and adolescence. Other skin lesions consist of hypo-
and epilepsy, as well as for autism, in this same area melanotic macules (i.e. ash leaf), ungula or gingival
(Daniels et al., 2001). fibromas, and thickened, firm areas of subcutaneous
A locus of susceptibility to autism has been identi- tissue often on the lower back or on the buttocks or
fied in the 16p13 chromosomal region (Lucarelli et al., torso (shagreen patch) or forehead and face (fibrous
142 P. CURATOLO AND R. BOMBARDIERI
plaques). The hypomelanotic macules tend to be round and often result in hemorrhage, particularly if promi-
or oval in shape and range from a few millimeters to as nent abnormal vasculature is present. AMLs with
much as 5 cm in length. Occasionally they have an fewer dysplastic vessels may have a smaller risk of
irregular, reticulated appearance, as if white confetti catastrophic hemorrhage but can present problems
paper had been strewn over the skin (confetti lesions). from their sheer size. If there are too many AMLs to
Whenever the scalp is involved, an area of poliosis can count, they are not surgically respectable, which can
result. Hypomelanotic macules may be present at birth lead to hypertension and renal failure if too much
or not show up until later in life. Their location and normal kidney tissue is destroyed between the large
number varies extensively from person to person. AMLs. Renal ultrasounds are performed to assess
Once the criterion of ‘three or more hypomelanotic changes in the size of AMLs or cysts, in the hope that
macules’ has been met, the number or size is not sig- this will allow intervention prior to development of
nificant. A widespread technique for enhancing their renal failure. Small renal cysts and AMLs may
visualization involves an examination of the skin not grow significantly until after puberty, but exces-
under ultraviolet light using a Wood’s lamp. Fibromas sive growth has been sometimes observed in young
of the skin arise in multiple locations. When present in children.
the lumbar region, they are called a ‘shagreen patch’. Renal cysts are found in 20% of males and 9% of
These are connective tissue hamartomas consisting of females with TSC. They are rarely if ever symptomatic.
various amounts of vascular structures, fat, collagen, Simple renal cysts often occur with AMLs and this
elastic tissue, smooth muscle and skin. Seldom found combination should suggest the diagnosis of TSC.
in infants, they become more common after the first Sometimes multiple renal cysts can be confused with
decade of life and persist throughout adult life. Fibro- true PKD. PKD occurs in 2–3% of persons with TSC
mas can also occur in the periungual regions, gingivae and usually presents early in life with hypertension,
or potentially anywhere in cutaneous or mucosal hematuria or renal failure. This occurs as the result
tissues. The underlying tissue may be hypertrophic or of a genetic abnormality (usually a single large
hamartomatous. deletion) affecting both the TSC2 gene and the PKD1
gene immediately adjacent to it. Renal cell carcinoma
9.4.2.2. Renal manifestations seems to occur more frequently in individuals with
Renal manifestations of TSC are the third most com- TSC than in the general population, and at an earlier
age.
mon clinical feature. Four types of lesion can occur:
angiomyolipomas (AMLs), isolated renal cyst(s), auto-
somal dominant polycystic kidney disease (PKD) and 9.4.2.3. Cardiac manifestations
renal cell carcinoma. AMLs are observed in as many Cardiac involvement is typically maximal at birth or
as 80% of individuals with TSC, usually detected after early in life and it may be the presenting sign of
the third year of age. AMLs tend to grow very slowly TSC, particularly in early infancy. Some 50–60% of
over several years. Growth spurts can be observed in individuals with TSC show evidence of cardiac
preadolescent boys and postmenarche girls. AMLs involvement, usually in the form of rhabdomyomas.
consist of abnormal blood vessels, smooth muscle cells On the other hand, anywhere from 50–85% of infants
and fat, with each present in varying degrees. Indivi- with isolated cardiac rhabdomyomas have been
duals with TSC may have either multiple small AMLs observed to later show definite evidence of TSC.
studding the surface of the kidney, multiple small Rhabdomyomas are benign tumors that may be focal
AMLs throughout the kidney or one or more larger or diffuse and infiltrating in character. They produce
lesions. These larger lesions are more apt to be symp- symptoms predominantly through outflow tract
tomatic, particularly when greater than 4–6 cm in their obstruction or by interfering with valvular function.
largest diameter. They frequently produce nonspecific Additionally, they can disrupt electrical conductivity
complaints such as flank pain. Of greater concern and cause arrhythmias. Rhabdomyomas develop dur-
are potentially life-threatening retroperitoneal hemor- ing intrauterine life (usually between weeks 22 and
rhages from rupture of dysplastic, aneurysmal blood 26 of gestation). The lesions usually undergo sponta-
vessels that feed the AMLs. These hemorrhages also neous regression in the first few years of life, although
can destroy adjacent normal renal parenchyma or pro- residual areas of histologically abnormal myocardium
duce abdominal distention and obstruction due to mass may persist. These lesions can involve the cardiac
effects. Some studies indicate that as many as 75% of conducting system and thus may predispose an affected
AMLs will increase in size over time. Very large individual to ventricular pre-excitation or other
AMLs (> 6–8 cm in diameter) are likely to progress arrhythmias both in infancy and later in life.
TUBEROUS SCLEROSIS 143
9.4.2.4. Ophthalmic manifestations either TSC1 or TSC2, which produce the characteristic
At least 50% of patients have ocular abnormalities; smooth muscle hypertrophy and destruction of normal
some studies have reported the prevalence as high as lung.
80%. These lesions are again characterized as hamarto-
mas, specifically retinal astrocytomas, which typically 9.4.3. Genotype/phenotype correlations
become calcified over time. Initially appearing as
rounded, nodular or lobulated areas on funduscopic Although TSC2 mutations are about five times more
examination, they become whitish in color as they calci- common than TSC1 mutations in sporadic TSC
fy. They tend to be indolent and rarely produce symp- patients, the two genes appear to account for an equal
toms or require intervention. On the rare occasion that proportion of mutations in large TSC families suitable
visual acuity is affected, a retinal hamartoma may be for linkage analysis (Povey et al., 1994; Jones et al.,
found impinging upon and compromising the retinal 1997). Smaller TSC families with only two or three
fovea and/or optic nerve. Hypopigmented areas of the affected individuals are intermediate in that TSC1
retina, iris and even eyelashes have also been reported mutation frequency is 15–50% and TSC2 accounts
(Jozwiak, 2003). for the majority of the remainder (Niida et al., 1999;
van Slegtenhorst et al., 1999; Dabora et al., 2001).
These observations suggest that, on average, patients
9.4.2.5. Pulmonary manifestations with TSC1 mutations may be less severely affected
Three forms of symptomatic pulmonary involvement than those with TSC2 mutations, so that each new spo-
in TSC have been described: multifocal microno- radic TSC1 patient has a better chance of founding a
dular pneumocytic hyperplasia, pulmonary cysts and family than a new TSC2 patient.
lymphangioleiomyomatosis (LAM) (Franz, 2004). Jones et al. (1997, 1999) assessed the frequency of
Involvement occurs mainly in adult women, common- intellectual disability in sporadic cases as a reliable
ly aged 30 or older. It was long thought to be distinctly and important aspect of disease severity. Unlike many
uncommon, affecting 1% or less of women with other components of the TSC phenotype, which show
TSC. However, recent prospective and retrospective age-dependent penetrance, intellectual disability is
studies have found cystic pulmonary abnormalities in almost invariably present from early childhood if it
as many as 40% of women with TSC, although most develops at all, and rarely escapes detection. Moderate
of those women remain asymptomatic. About 60% of to severe intellectual disability also clearly limits
women with sporadic LAM also have renal AMLs reproductive capacity. Intellectual disability was sig-
but do not have other characteristics of TSC. Smooth nificantly more frequent among sporadic cases with
muscle cells undergo abnormal proliferation with TSC2 than TSC1 mutations (Jones et al., 1999) How-
secondary compromise of bronchioles, venules and ever, other series have not replicated this finding
lymphatic structures. Slowly, normal pulmonary (Kwiatkowska et al., 1998; Young et al., 1998; Niida
elasticity is lost, with the resultant decrease in vital et al., 1999; van Slegtenhorst et al., 1999).
capacity and increase in residual volume. Pulmonary Sporadic patients with TSC1 mutations have, on
hypertension and worsening hypoxia and hypercapnia average, a milder phenotype than patients with TSC2
eventually supervene. When LAM is suspected clini- mutations. They have a lower seizure frequency, moder-
cally, high-resolution CT of the chest is the most ate to severe mental retardation, fewer subependymal
sensitive diagnostic modality. Because of the over- nodules and cortical tubers, less severe kidney involve-
whelming predominance of LAM in women, it is ments, no retinal hamartomas and less severe facial
possible that estrogen accelerates the progression of angiofibroma (Dabora et al., 2001). Moreover, TSC2
the condition. LAM may be progressive in a small mutations are associated with a significantly earlier epi-
percentage of individuals with TSC, particularly women. lepsy presentation than TSC1 mutations, which results
Recent studies have shown that around 40% of women in frequent epileptic spasms (Jozwiak et al., 2001).
with TSC have subtle signs of LAM; a baseline CT of In this patient set, those patients without an identified
the lungs should be carried out. In cases where LAM mutation had clinical features that were on average sig-
is progressive, a lung transplant may be necessary. nificantly milder than TSC2 patients and generally sim-
Interestingly, LAM has occasionally recurred in trans- ilar to those of TSC1 mutations, apart from kidney
planted lungs. Karbowniczek et al. (2004) demon- disease, which was similar to the TSC2 patient set
strated the migration of cells from renal AMLs to the (Sancak et al., 2005). These observations suggest that
lungs of women with LAM regardless of whether they a significant proportion of these patients have mosai-
had TSC, and the cells almost certainly cause the dis- cism for a TSC2 mutation, causing a generally milder
order. These cells frequently present abnormalities of phenotype. Some of these patients might possibly have
144 P. CURATOLO AND R. BOMBARDIERI
mutations in a third TSC gene, accounting for a small and typical hypomelanotic macules can be early recog-
proportion of all cases. nized by most clinicians who are knowledgeable about
TSC. Generally, a dermatological examination might
9.5. Diagnostic criteria be important when the skin lesions are atypical or when
the diagnosis of TSC is uncertain.
At the time of initial diagnosis diagnostic studies are A scrupulous age-appropriate screening for behav-
commonly performed, either to confirm the presence ioral, cognitive and neurodevelopmental dysfunction
of TSC or to evaluate presenting symptoms such as should also be performed at the time of diagnosis.
seizures or cardiac dysfunction. In addition, it is often Unfortunately, children with apparently normal initial
useful to establish a baseline assessment in areas that testing and developmental milestones can still suffer
could develop complications later. Unless specific milder deficits that interfere with their learning. It is
areas of concern are identified by these initial studies, advisable that each child is reassessed around the time
later tests are aimed towards areas with a high risk of school begins, even if no abnormalities were detected
dysfunction and towards lesions that might be possibly by the previous screenings. Children with abnormal
treated (Roach and Sparagana, 2004). behavior or cognitive function should be periodically
Moreover, at the time of initial presentation, the retested, and re-evaluation is also appropriate when
majority of the patients with TSC undergo MRI to there is a significant change in behavioral or cognitive
search for additional evidence of TSC already sus- function. Newly diagnosed adolescents or young adults
pected on the basis of other signs (Roach et al., with a well established pattern of completely normal
1998). The number, size and perhaps the location of social and cognitive function as determined by educa-
the dysplastic cortical lesions detected by MRI tend tional achievement sometimes do not require formal
to correlate with the severity of the clinical neurologi- testing.
cal dysfunction (Curatolo et al., 1991; Shepherd et al., Gene characterization could ultimately identify
1995; Goodman et al., 1997). EEG is useful when the patients at a greater or lesser risk of particular complica-
initial presentation includes epileptic seizures. Never- tions. Moreover, further diagnostic studies could be per-
theless, children who never manifested seizures and formed selectively on individuals at greatest risk for
are not suspected of having epileptic seizures generally certain specific complications, thereby decreasing the
do not need to undergo baseline EEG. number of useless investigations. It is possible that, in
Usually, adolescents and adults have a greater the near future, gene typing at the time of initial diagnosis
chance of developing symptomatic renal angiomyoli- will reveal its usefulness once molecular testing becomes
pomas than children, but it sometimes happens even readily available and there are sufficient data to deter-
in childhood (Jozwiak et al., 1998). For each patient mine which clinical phenotypes correlate with which.
renal ultrasonography is carried out at the time of diag- The diagnostic criteria for TSC are summarized in
nosis. Furthermore, cardiac arrhythmias occasionally Table 9.4.
occur even in patients with TSC who do not have a
demonstrable cardiac rhabdomyoma. An arrhythmia 9.5.1. Evaluation of family members
can be present at birth or develop later. Wolff–Parkin-
son–White syndrome appears to be the most commonly Since a number of affected individuals show only sub-
noted arrhythmia in patients with TSC. Therefore, a tle clinical features of TSC, diagnosis of family mem-
baseline study must be performed at the time of diag- bers may be difficult. Such a problem is crucial for
nosis. Echocardiography may reveal one or more accurate genetic counseling in this autosomal domi-
cardiac rhabdomyomas in more than half of the youn- nant disorder. The majority of affected individuals
ger individuals with TSC (Gibbs, 1985; Jozwiak et al., who are subject to a thorough physical examination,
1994). However, these cardiac tumors are more innoc- including a skin examination with ultraviolet light
uous than they might seem, as they tend to involute and a retinal examination through dilated pupils, have
over the years, often disappearing completely by adult- at least subtle physical findings of TSC. Furthermore,
hood. Moreover, the most rapid reduction in lesion a completely normal physical examination and exten-
size occurs during the first 3 years of life, a period sive radiographic testing, including high definition
after which rhabdomyomas tend to change less MRI, still cannot definitely exclude TSC, since there
dramatically (DiMario et al., 1996). is always a chance of germline mosaicism.
Each patient affected by TSC should have an accu- Occasionally, MRI may be helpful, in parents with
rate ophthalmic examination at the time of diagnosis. few physical findings and other normal diagnostic
Children usually do not suffer from facial angiofibro- studies. The usefulness of MRI often lies in its ability
mas or ungual fibromas at the time of initial diagnosis, to help confirm a diagnosis that is already suspected.
TUBEROUS SCLEROSIS 145
Table 9.4 Testing recommendations at the time of diagnosis
and recommended frequency of follow-up investiga-
Revised clinical diagnostic criteria for tuberous sclerosis
tion are summarized in Table 9.5.
complex
Confirmatory testing by DNA analysis for TSC is
Major features Minor features helpful in individuals who fail to meet the criteria for
definite TSC and to improve genetic counseling. Pre-
Facial angiofibromas or Multiple, randomly natal genetic testing for TSC is also possible when
forehead plaque distributed pits in dental there is a defined TSC mutation in a specific family.
enamel Preimplantation genetic diagnosis is a method of deter-
Nontraumatic ungueal or Hamartomatous rectal
mining the genetic characteristics of an embryo creat-
periungual fibroma polyps
ed by in vitro fertilization. Although preimplantation
Hypomelanotic macules Bone cysts
(three or more) genetic diagnosis is feasible for TSC, its use is not
Shagreen patch (connective Cerebral white matter radial widespread.
tissue nevus) migration lines Several issues limit the usefulness of confirmatory
Multiple retinal nodular Gingival fibromas testing for TSC, beginning with the fact that most
hamartomas patients develop the disorder via a spontaneous muta-
Cortical tuber Nonrenal hamartoma tion. And, although there are few false-positive tests,
Subependymal nodule Retinal achromic patch as much as 15–20% of the time the test fails to demon-
Subependymal giant cell ‘Confetti’ skin lesions strate a disease-causing mutation. Nevertheless, confir-
astrocytoma matory testing in an individual who already fulfills the
Cardiac rhabdomyoma, Multiple renal cysts diagnostic criteria for defining disease can help to
single or multiple
identify a mutation that can then be sought in other
Lymphangiomyomatosis and
or renal angiomyolipoma family members or for subsequent prenatal diagnosis.
Somatic and germline mosaicism also complicate
Modified from Roach et al., 1998. confirmatory testing for TSC (Au et al., 2004). A con-
servative recurrence risk for seemingly unaffected

No systematic studies have addressed the advantage


of renal ultrasonography in identifying minimally Table 9.5
affected individuals with TSC. Nonetheless, ultrasonog- Testing recommendations
raphy clearly demonstrates renal angiomyolipomas and
is broadly available and certainly cheaper than other Assessment Initial testing Repeat testing
studies. This is why renal ultrasonography is recom-
mended where diagnostic studies are carried out on fam- Neurodevelopmental At diagnosis As clinically
ily members who might potentially be affected. testing and at indicated
school
Molecular diagnosis will be used increasingly to
entry
differentiate TSC patients from clinically normal fam- Ophthalmic At diagnosis As clinically
ily members. Molecular testing for TSC can identify examination indicated
certain individuals with TSC who do not satisfy the Electroencephalography At diagnosis As clinically
clinical diagnostic criteria (Roach et al., 1998). Occa- indicated
sionally, couples have more than one child with TSC, Electrocardiography At diagnosis As clinically
in spite of the fact that neither patient has either physi- indicated
cal or radiological evidence of the disease. In these Echocardiography If cardiac If cardiac
families, TSC probably results from germline mosai- symptoms dysfunction
cism. Unfortunately, neither routine diagnostic studies occur occurs
nor DNA-based testing is likely to detect germline Renal ultrasonography At diagnosis Every 1–3 years
Chest CT At adulthood If pulmonary
mosaicism in these individuals, because the parent
(women dysfunction
who carries the mutation will not have a detectable only) occurs
mutation in DNA extracted from leukocytes (Rose Cranial MRI At diagnosis Children/
et al., 1999; Yates et al., 1997). Therefore, genetic adolescents:
counseling for families with one affected child should every 1–3
include a small (1–2%) possibility of recurrence, even years
in parents who have no evidence of TSC after a thor-
ough diagnostic evaluation (Roach et al., 1998). Modified from Roach et al., 1999.
146 P. CURATOLO AND R. BOMBARDIERI
couples with a single affected child, even when they When a patient is diagnosed with TSC in infancy or
have no demonstrable TSC mutation, is 2% (Rose early childhood, testing should be repeated around
et al., 1999). the time the child enters school. Older children,
instead, should be reassessed periodically in response
9.6. Clinical management to educational or behavioral concerns.

Long-term surveillance testing should be directed 9.6.2. Kidney


toward lesions that are frequent, lesions that can be
treated if early identified, and lesions that carry a sig- By the age of 10 years, almost 75% of children with
nificant risk of dysfunction or death. A surveillance TSC have sonographic evidence of one or more renal
protocol based on the natural history of TSC provides angiomyolipomas (Ewalt et al., 1998; Weiner et al.,
some practical basis for driving follow-up tests. Any 1998). During the first decade of life, the number
effort should be made to minimize costly testing of and size of the renal angiomyolipomas tend to increase
asymptomatic patients and to maximize the likelihood but large renal angiomyolipomas are more likely to
of early identification of a treatable lesion. The guide- cause symptoms than smaller lesions (Steiner et al.,
lines that follow are designed for long-term clinical 1993). It is therefore wise to monitor patients with
management of an asymptomatic patient whose diag- large tumors more closely. Renal ultrasonography
nosis is perfectly assured (Roach et al., 1998). should be generally undertaken every 1–3 years. How
frequently patients are tested depends mostly on the
9.6.1. Central nervous system degree of concern for them and on the results of previ-
ous examinations. Regardless of age, patients who
Subependymal giant cell astrocytomas are locally have large renal lesions or lesions that seem to have
invasive and can cause hydrocephalus because of their grown substantially should have more frequent fol-
typical occurrence in the anterior lateral ventricle. low-up examinations. MRI might be necessary for
Enlarging giant cell tumors can be removed through these patients to define the extent of the kidney disease
early identification, before symptoms develop and with greater precision.
before they become locally invasive. Children should
undergo periodic cranial imaging with either CT or 9.6.3. Heart
MRI scans every 1–3 years, depending on the level
of clinical suspicion in each child. In general, there is Although about two-thirds of infants with TSC have
a greater likelihood for children to develop subependy- echocardiographic evidence of one or more cardiac
mal giant cell astrocytomas than for adults. Usually the rhabdomyomas, these tumors tend to regress over time
need for EEG is dictated by the clinical features and and can disappear altogether by adulthood (Alkalay
response of seizures to antiepileptic drugs. As a rule, et al., 1987; Smythe et al., 1990; Webb et al., 1993).
EEG is not required for adults with TSC who do not The majority of the patients with TSC who have a car-
have epileptic seizures. However, since seizures are diac rhabdomyoma remain asymptomatic (Jozwiak
not regularly clinically obvious, EEG should always et al., 1994). Continuous cardiac evaluations are not
be considered in the evaluation of a patient with an required and are even unnecessary for most asymp-
unexplained decline of cognitive or behavioral func- tomatic TSC patients. However some patients may
tion in whom epileptic seizures are suspected. During occasionally develop arrhythmias during adolescence
early infancy, the seizure pattern can change rapidly or adulthood. A cardiologist should follow all those
and it may sometimes be necessary to repeat the stud- patients who have new symptoms that might indicate
ies at frequent intervals. cardiac dysfunction and those with previous symp-
A number of neurodevelopmental and behavioral toms, benefiting from periodic studies to evaluate
dysfunction patterns occur as a result of TSC. In con- heart function.
trast to mental retardation, which is a classic and com-
mon feature of TSC, learning disabilities, autism, 9.6.4. Lungs
attentional deficits and other difficulties are probably
under-recognized (Hunt and Dennis, 1987; Curatolo Pulmonary disease (lymphangiomyomatosis) due to
et al., 1991). The children who suffer such problems TSC is uncommon. In the rare cases in which it occurs,
can benefit from early recognition, specific education it is suffered almost exclusively by women (Smolarek
and treatment plans. Formal cognitive testing is not et al., 1998). The average age of onset is 32–34 years.
necessary for adolescents and adults with well estab- The best method to use to investigate the pulmonary
lished patterns of normal social and cognitive function. abnormalities of TSC is the chest CT scan. The panel
TUBEROUS SCLEROSIS 147
recommended no routine testing in either asymptomatic et al., 2001; Curatolo et al., 2006). The major advan-
children or adolescents. Women should undergo chest tages of vigabatrin are the ability to rapidly escalate
CT scans at least once on reaching adulthood. If pulmo- the dosage at the initiation of treatment, rapid efficacy,
nary symptoms develop, this test should be repeated. suitability for outpatient treatment and particularly
good tolerability with generally only minor adverse
9.6.5. Retina effects, with the exception of visual field loss. Vigaba-
trin is a novel drug that works by preventing the break-
Around 75% of patients with TSC have retinal lesions down of the neurotransmitter GABA.
(Kiribuchi et al., 1986). In a severely impaired child, The safety of vigabatrin has caused concern since a
ophthalmic examinations are sometimes difficult to specific visual field loss has been documented in trea-
perform without sedation, and are unlikely to identify ted adults and some children (Harding et al., 2002).
impending visual loss from a treatable lesion. Repeated Visual field loss is usually asymptomatic and can be
ophthalmological evaluations are usually unnecessary, detected only by perimetric visual field studies. In very
unless there is some specific reason for concern. young children it is difficult if not impossible to detect
the visual field loss. Until a clear answer about the
9.6.6. Skin occurrence of this adverse effect in children has been
establish through randomized studies, VGB may still
Facial angiofibromas are benign skin tumors that be considered first-line therapy in epileptic spasms.
can have major consequences for some patients. Laser We recommend a 10-day course of VGB as initial
therapy is one way in which skin tumor growth can therapy Adrenocorticotropic hormone (ACTH) should
be limited, although the treatments often need to be be the first drug of choice if VGB is not available
repeated periodically as the lesions tend to regrow (Curatolo et al., 2001b). When VGB or ACTH fails
gradually after the treatment is over. Ungual fibromas to control spasms, then topiramate can be prescribed.
sometimes cause severe problems, which can be effec- Recently, some authors reported that the use of levetir-
tively treated. acetam as adjunctive antiepileptic therapy in patients
with TSC can reduce seizure frequency (Collins
9.7. Treatment et al., 2006).
Alternative treatments to antiepileptic drugs should
The pathological manifestations of TSC can cause var- be considered in patients with TSC whose seizures
iable symptoms based on the size and location of the cannot be effectively controlled. Current nonpharma-
hamartomas. Therefore, a variety of management con- cological treatments included vagus nerve stimulation,
siderations are necessary. Table 9.6 describes the vari- ketogenic diet and resective epilepsy surgery (Thiele,
ous treatment considerations for each feature. 2004). Epilepsy surgery has a very important role in
The goals of treatment for individuals with TSC are the management of children when an epileptogenic
the same as for all individuals with a multisystem tuber or epileptogenic zone is identified (Romanelli
chronic condition: providing the best possible quality et al., 2004). Converging information between video-
of life with the fewest complications from the underly- EEG monitoring and extracranial localization with
ing disease process, fewest adverse treatment effects MRI fusioning can improve our ability to select candi-
and fewest medications. Appropriate and effective dates who could benefit from surgical treatment. There
therapy is not only aggressive but also relies upon is little doubt that the treatment of TSC will benefit
recognition of the natural history of the various lesions from present and future research on the molecular
of TSC. mechanisms involved in the pathogenesis of this
Unfortunately, no one medical treatment usually devastating disease. Knowledge regarding the function
results in satisfactory relief for all or even most indivi- of the tuberin–hamartin complex has led to therapeutic
duals with TSC. A combination of medical treatment intervention trials (Lee et al., 2005).
modalities is then frequently required. Rapamycin, a commercially available immunosup-
The choice of specific antiepileptic drugs for treat- pressant, inhibits the ability of mTOR to phosphorylate
ing seizures in individuals with TSC is based on the downstream substrates, such as the S6Ks and 4EBPs.
seizure type(s), epilepsy syndrome(s), other involved Rapamycin is generally a well tolerated medication.
organ system, age of the individual, and antiepileptic Common, typically self-limiting side effects include
drug side effect profiles and formulations available. aphthous ulcers, acneiform rash, diarrhea and arthral-
Treatment of children with TSC and infantile gias, as well as potentially dramatic elevation of serum
spasms with vigabatrin is very effective (Chiron cholesterol and lipoproteins. Oral rapamycin therapy
et al., 1991, 1997; Jambaque et al., 2000; Elterman can induce regression of astrocytomas associated with
148 P. CURATOLO AND R. BOMBARDIERI
Table 9.6
Recommendation for treatment

Pathological manifestations Recommendations for management

Dermatological Facial angiofibromas Repeated laser treatment, dermal abrasion and surgical removal
Neurological Ungular fibromas/shagreen patch Surgical or laser removal
Cortical tubers Anticonvulsants, resective epilepsy surgery
Subependymal giant cell Surgical removal with or without ventriculoperitoneal shunt
astrocytomas
Neuropsychological Learning disabilities, affective Early childhood programs and ongoing evaluations and
and cognitive disorders, autism spectrum treatment
disorders
Pulmonary Lymphangioleiomyomatosis Pulmonary decorticationHormone therapy
(medroxyprogesterone acetate, surgical estrogen ablation,
tamoxifen)Lung transplantation
Renal Angiomyolipomas Renal cell Aggressive approach to AMLs > 3.5 cm with either arterial
carcinoma Polycystic kidney embolization or surgical resection, exploration with renal
disease conserving surgery, treatment of hypertension with
medication, renal transplant
Cardiac Rhabdomyoma Antiarrhythmic agents and diureticsSurgical intervention

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