Journal of Neuropathology and Experimental Neurology Vol. 62, No.

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Copyright q 2003 by the American Association of Neuropathologists January, 2003
pp. 1 13

The Neuronal Ceroid-Lipofuscinoses

MATTI HALTIA, MD, PHD

Abstract. The neuronal ceroid-lipofuscinoses (NCLs) collectively constitute the most common group of neurodegenerative
diseases in childhood and usually show an autosomal recessive mode of inheritance. Despite varying ages of onset and clinical
course characterized in most instances by progressive mental and motor deterioration, blindness, epileptic seizures, and
premature death, all forms of NCL show unifying histopathological features. There is accumulation of autofluorescent, periodic
acid-Schiff-, and Sudan black B-positive granules that are resistant to lipid solvents in the cytoplasm of most nerve cells and,
to a lesser degree, of many other cell types. The storage process is associated with progressive and selective neuronal loss
and gliosis with secondary white matter lesions. The ultrastructure of the storage deposits varies between different forms of
NCL and, along with the age of onset, has provided the basis for the traditional classification of NCLs. Recent molecular
genetic findings have established that defects in at least 7 different genes underlie the various forms of NCL. The purpose of
this paper is to provide an overview of the NCLs, review recent molecular genetic and biochemical findings, and discuss
their impact on our views on the classification and pathogenesis of these devastating brain disorders.

Key Words: Batten disease; Cell biology; Classification; Genetics; Neuronal ceroid-lipofuscinosis; Pathogenesis; Pathology.

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INTRODUCTION by intraneuronal accumulation of proteins (4, 5). Since
The neuronal ceroid-lipofuscinoses (NCLs) collective- 1995, molecular genetic and biochemical analyses have
ly constitute the most common type of inherited neuro- led to the identification of 7 different human or ovine
degenerative diseases in childhood (1). Their incidence NCL-associated genes. Discoveries of further NCL genes
in the US has been estimated at 1:12,500 (2) and they are to be expected.
usually show an autosomal recessive mode of inheritance. The purpose of the present paper is to provide an over-
The age of onset varies from infancy to late adult. Most view of this complex group of disorders, to review the
childhood forms are clinically characterized by progres- recent molecular genetic and biochemical findings, and
sive mental and motor deterioration, blindness, epileptic to discuss their impact on our views on the classification
seizures, and premature death, while the rare adult-onset and pathogenesis of NCLs.
forms are dominated by dementia. In addition to the hu- HISTORY AND CLASSIFICATION
man forms of NCL, a number of NCLs have also been
described in animals. Despite the varying ages of onset The clinical features characteristic of juvenile NCL
and clinical course, all forms of NCL share unifying were first reported by Stengel in 1826, based on his ob-
pathomorphological features. There is accumulation of servations of 4 Norwegian siblings with progressive
autofluorescent, periodic acid-Schiff (PAS)- and Sudan blindness, epilepsy, cognitive decline, and motor dys-
black B-positive granules that are resistant to lipid sol- function (6). Almost a century later, in 1903 and 1923,
vents in the cytoplasm of most nerve cells and, to a lesser Batten, Spielmeyer, and Vogt independently demonstrat-
degree, of many other cell types. The storage process is ed an intraneuronal storage process in juvenile patients
associated with progressive and selective neuronal loss with close clinical resemblance to those reported by Sten-
and gliosis with secondary white matter lesions. The ul- gel (3). Patients with a similar intraneuronal storage pro-
trastructure of the storage deposits varies between differ- cess but with late infantile onset were described by Ján-
ent forms of NCL and has provided the basis for the sky in 1908 and Bielschowsky in 1923, and patients with
traditional classification, along with the age of onset (3). adult onset by Kufs in 1925 (3). Because of histochemical
For most of the past century, NCLs had been con- resemblance of their storage material to ceroid or lipo-
ceived as lipidoses and grouped under the heading of the fuscin, Zeman and Dyken (1) coined the term neuronal
so-called amaurotic family idiocies (1, 3). However, stud- ceroid-lipofuscinosis in 1969 in order to separate the dis-
ies of an ovine form of NCL in the late 1980s led to the eases described by Batten, Spielmeyer, Vogt, Jánsky,
seminal discovery that NCLs are, in fact, characterized Bielschowsky, and Kufs from the gangliosidoses. An in-
fantile NCL was later described by Santavuori et al (7)
and Haltia et al (8, 9).
From the Departments of Pathology, University of Helsinki and Hel- A subclassification of the NCLs emerged that is based
sinki University Central Hospital, Helsinki, Finland. on the age of onset and the ultrastructure of the storage
Correspondence to: Matti Haltia, MD, PhD, Department of Patholo- material. Four main forms were recognized: infantile
gy, University of Helsinki, Haartmaninkatu 3, FIN-00290 Helsinki, Fin-
land. E-mail: matti.j.haltia@helsinki.fi
(INCL), late infantile (LINCL), juvenile (JNCL), and
This work was supported by the Academy of Finland, project number adult (ANCL) NCL. Recent molecular genetic and bio-
48173. chemical studies have revolutionized the classification.

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2 HALTIA

TABLE 1
The Human Neuronal Ceroid-Lipofuscinoses (NCL): Genetic Classification

Gene Clinical Ultrastructural Principle

Gene location Gene product phenotype phenotype stored protein

CLN1 1p32 Palmitoyl protein thioester- INCL (LINCL, JNCL, GROD SAP A & D
ase 1 ANCL)
CLN2 11p15 Tripeptidyl peptidase 1 classic LINCL CL SCMAS
CLN3 16p12 CLN3 protein (membrane JNCL FP (CL, RL) SCMAS
protein)
CLN4 ? ? ANCL FP, granular SCMAS
CLN5 13q22 CLN5 protein (membrane Finnish vLINCL RL, CL, FP SCMAS
protein)
CLN6 15q21–23 CLN6 protein vLINCL RL, CL, FP SCMAS
CLN7 8p23 CLN8 protein Turkish vLINCL RL, CL, FP SCMAS
CLN8 8p23 CLN8 protein (membrane Northern epilepsy CL-like, granular SCMAS
protein)
Abbreviations: INCL 5 infantile NCL; LINCL 5 late infantile NCL; vLINCL 5 variant late infantile NCL; JNCL 5 juvenile
NCL; ANCL 5 adult NCL; GROD 5 granular osmiophilic deposits; CL 5 curvilinear profiles; FP 5 fingerprint bodies; RL 5
rectilinear complex; SAP 5 saposins; SCMAS 5 subunit c of the mitochondrial ATP synthase.

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The human NCLs are now classified into 8 main genetic include brownish discoloration of the macula, retinal de-
forms (CLN1–8), based on the number of hitherto pre- generation with involution of retinal vessels, and optic
dicted gene loci (Table 1) (10, 11). This genetic classi- atrophy. By 3 yr of age the patients have lost their active
fication will be followed in the present article where the movements and visual contact with the environment and
different entities will be presented in their numerical or- they usually die at the age of 8 to 13 yr (7, 12). By
der. It is important to note that different mutations in a magnetic resonance imaging, the first brain abnormalities,
single gene may result in different phenotypes, including including hypointense thalami in T2-weighted images,
varying ages at onset. Common mutations that predom- may be detected at the age of 7 to 10 months, that is,
inate in a given form of NCL are usually associated with before the first clinical symptoms are observed. After 3
the classic clinical picture, while rare ‘‘private’’ muta- to 4 yr of age, magnetic resonance imaging shows ex-
tions may produce a deviant phenotype. treme cerebral and cerebellar atrophy with very high sig-
nal intensity in the white matter. The EEG becomes flat
CLN1 and the electroretinogram (ERG) extinguished by the age
Patients with mutations of the CLN1 gene occur world- of 3 yr (12).
wide. CLN1 mutations can give rise to 4 main phenotypes Clinical Features of Variant Forms: The variant forms
with varying ages of onset: infantile NCL (INCL, Haltia- with late infantile and juvenile onset clinically resemble
Santavuori disease, MIM 256730), and variant forms the classic forms of LINCL and JNCL (see CLN3, vide
with late infantile, juvenile, or adult onset. The classic infra), rather than INCL (11, 12). However, no vacuolat-
infantile form is by far the most common with close to ed lymphocytes are seen in the variant cases, in contrast
500 cases diagnosed worldwide. It is enriched in the to classic JNCL. The characteristic granular osmiophilic
Finnish population with an incidence of 1 in 20,000 and storage cytosomes occur in all clinical forms of CLN1
a carrier frequency of 1 in 70 (12). Patients with defects (see below).
of the CLN1 gene probably represent about 20% of all Neuropathological and Biochemical Features: INCL
NCL cases in the US, with only half presenting as infants was originally identified on the basis of frontal brain bi-
(11, 12). opsies carried out in order to establish the diagnosis in
Clinical Features of INCL: The affected children usu- clinically unsolved cases of rapidly progressive enceph-
ally seem to develop normally until approximately 1 yr alopathy (8). By the age of 1.5 yr, the cytoplasm of ce-
of age. However, the rate of head growth may begin to rebral cortical neurons shows moderate granular deposits
decrease by the age of 5 months. In addition to micro- that are PAS- (Fig. 1D) and Sudan black B-positive and
cephaly, muscular hypotonia and clumsiness of fine mo- autofluorescent in paraffin sections. There is progressive
tor control are further early signs. The development be- neuronal loss, infiltration of the cortex by macrophages
gins to slow during the second year of life. Hyperkinesias containing coarse PAS-positive and autofluorescent gran-
of the hands, myoclonic jerks, and seizures appear. There ules (Figs. 1E, 2A), and severe astrocytic hyperplasia and
is progressive loss of motor abilities, truncal ataxia, and hypertrophy (Fig. 1F). Even the astrocytes harbor small
visual failure. Ophthalmological findings after 2 yr of age granular storage deposits. By the age of 3 yr, almost all

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 THE NEURONAL CEROID-LIPOFUSCINOSES 3

cortical neurons are lost and the cortex is dominated by missense, splice-site mutations, and small deletions) are
numerous macrophages, often binucleate (8). infrequent (11, 12).
At autopsy, by about 10 yr of age, the strikingly atro-
phic brain (Fig. 1A) has a tough, rubbery consistency and CLN2
weighs approximately 250 to 350 g. Its cut surfaces are Mutations of the CLN2 gene are known to cause al-
grayish, without any clear demarcation between cortex most all cases of classic late infantile NCL (cLINCL,
and white matter. Both the cerebral (Fig. 1G) and cere- Jánsky-Bielschowsky disease, MIM 204500). cLINCL
bellar cortices (Fig. 1H) consist of a rim of hypertrophic probably has a worldwide distribution but seems to be
astrocytes and are depleted of neurons, with few char- more common in northern European populations than
acteristic exceptions: a few giant cells of Betz (Fig. 1G), elsewhere. An incidence figure of 0.46 per 100,000 live
and a very occasional hippocampal CA1 pyramidal cell births has been published from Germany (18), and the
or Purkinje cell may still be seen. The gliotic centrum prevalence in Sweden, Norway, and Finland has been
semiovale is almost devoid of axons and myelin sheaths, estimated at 0.6 to 0.7 per million inhabitants (19).
with the exception of a few myelinated axons derived Clinical Features: The onset of major symptoms is
from the precentral gyrus (Fig. 1J). The neurons of the usually heralded by seizures and occurs between 2 and 4
basal ganglia and upper brainstem are still partly pre- yr of life. The seizures may be partial, generalized tonic-
served but show characteristic intraneuronal storage. clonic, or secondarily generalized and are soon followed
There is active neuronophagy and macrophagocytosis in

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by ataxia, myoclonus, and developmental regression,
these regions. In contrast, the spinal neurons are well pre- with patients becoming unable to walk or sit unsupported
served despite marked intraneuronal storage (Fig. 1I), as and losing speech. A gradual decline of vision leads to
are the neurons of the spinal and autonomic ganglia (9). blindness by 5 or 6 yr. The patients usually die in middle
In the retina, the visual, bipolar, and ganglion cells are childhood (11, 20). Findings at brain imaging are rela-
completely lost (Fig. 1K) with optic atrophy and gliosis tively nonspecific, but the neurophysiological findings are
(13). Small storage granules can be seen even in many characteristic. The EEG shows an occipital photosensitive
extraneural cell types, including epithelial cells such as response using flash rates at 1 to 2 Hz. The ERG is di-
eccrine sweat glands or thyroid follicles, testes, skeletal, minished or extinguished and the visual evoked potentials
cardiac and smooth muscle cells, endothelial cells, and (VEPs) grossly enhanced, as are the somatosensory
macrophages in lymphatic tissues (Fig. 1L). Such periph- evoked potentials (SEPs) (11, 20).
eral deposits are usually minor and not associated with Neuropathological and Biochemical Features: At au-
tissue destruction (9). topsy, the brain, particularly the cerebellum, is severely
The intraneuronal and peripheral cytoplasmic deposits atrophic with brain weights of the order of 500 to 700 g
show a characteristic electron microscopic appearance. (20, 21). On cut surfaces the cortex is thin and the ven-
They consist of membrane-bound conglomerates of tricular system enlarged. By light microscopy there is se-
roundish electron-dense globules (Fig. 3A) with a finely vere, partially laminar, loss of cerebral cortical neurons,
granular internal ultrastructure (8, 9), called granular os- with activation of the micro- and macroglia. Even the
miophilic deposits (GROD) (14). Enzyme histochemistry cerebellar Purkinje cells and granule cells are markedly
reveals acid phosphatase activity (8). The deposits show reduced in number. The cytoplasm of the remaining neu-
immunoreactivity for sphingolipid activator proteins A ronal perikarya in the cerebral cortex and subcortical
and D (Fig. 2B). Amino terminal sequence analysis and structures is slightly to moderately distended with storage
Western blotting of purified storage granules has identi- granules. So-called meganeurites (i.e. spindle-shaped ac-
fied sphingolipid activator proteins A and D as the major cumulations of storage granules in the proximal axonal
storage proteins (15). segments of cortical neurons) are a characteristic feature,
Molecular Genetic Findings: The CLN1 gene on chro- particularly in lamina III. So-called myoclonus bodies, or
mosome 1p32 (16) was identified by a positional candi- spheroids, may occur in the neurons of the basal ganglia
date gene approach. It encodes palmitoyl protein thioes- and dentate nucleus (21). The neuronal loss is accom-
terase 1 (PPT1) (17). To date, almost 40 different PPT1 panied by a secondary loss of axons and myelin in the
mutations are known. Almost all Finnish INCL patients white matter. There is retinal atrophy, which is more se-
are homozygous for the same missense mutation at vere in the peripheral than central part and begins at the
Arg122Trp. By far the most common PPT1 mutation in photoreceptor layer (20, 21).
the US population resulting in infantile onset is a pre- The storage granules are autofluorescent and stain with
mature stop codon at arginine 151. A common missense Luxol fast blue, PAS, and Sudan black B in paraffin sec-
mutation, Thr75Pro, accounts for most of the variant ju- tions. The granules are strongly immunoreactive for sub-
venile cases with GROD in the US and for the cluster of unit c of the mitochondrial ATP synthase (SCMAS) (22)
such cases in Scotland. Other mutations (i.e. nonsense, but also for SAP-A and SAP-D (23). Minor deposits of

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Fig. 1. A–C: Generalized brain atrophy is a striking feature in all childhood forms of neuronal ceroid-lipofuscinosis (scale in
millimeters). It is extreme in the infantile form CLN1 (brain weight 290 g) (A), severe in the Finnish variant late infantile form CLN5
(brain weight 650 g) (B), and less pronounced in the juvenile form CLN3 (brain weight 960 g) (C). D–L: Characteristic aspects of
the pathology of the infantile form of CLN1, the most severe of the established forms of neuronal ceroid-lipofuscinosis. D: Periodic
acid-Schiff (PAS)-positive storage material in the cytoplasm of cortical neurons of a 1.5-yr-old child. Biopsy, PAS stain, 3400. E:
Total neuronal loss and infiltration of the cortex by numerous macrophages harboring PAS-positive granules in a 3-yr-old patient. Some

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Fig. 2. A: The storage material in all forms of neuronal ceroid-lipofuscinosis shows autofluorescence in ultraviolet light, as
seen here in cortical macrophages of a 3-yr-old patient with the infantile form CLN1. Biopsy, unstained section viewed in
ultraviolet light, 3300. B: In the infantile form of neuronal ceroid-lipofuscinosis, saposins A and D constitute the main protein
component of the storage material. Biopsy section of a 1.5-yr-old patient. Immunostain for saposin D, 3150. C: The autoflu-
orescent eccentric intraneuronal storage material, as seen here in the Finnish variant late infantile neuronal ceroid-lipofuscinosis
CLN5, forms moon-like semicircles. Autopsy, unstained section viewed in ultraviolet light, 3300. D: A section of the previous
specimen shows immunoreactivity for subunit c of the mitochondrial ATP synthase, 3300. E: In several forms of neuronal ceroid-
lipofuscinosis, particularly in the upper part of the cortical lamina III, the neurons may show accumulations of the storage material
in the proximal axon (axonal spindles or meganeurites). Immunostain for subunit c of the mitochondrial ATP synthase in a case
of Finnish variant late infantile neuronal ceroid-lipofuscinosis, 3300. F–H: In all forms of the neuronal ceroid-lipofuscinoses,
the abnormal storage granules in the cytoplasm of neurons stain positively with the Luxol fast blue (F), PAS (G), and Sudan
black B (H) methods, as seen here in a patient with Northern epilepsy CLN8, 31,000.

←
of the macrophages are binucleated. Biopsy, PAS stain, 3400. F: The cortex of a 10-yr-old patient consists of a dense network of
hypertrophic astrocytes. Autopsy, Cajal stain, 3300. G: The precentral cortex of a 10-yr-old patient shows a few ballooned giant cells
of Betz (arrows) while all other neurons have disappeared. The white matter (WM) in the lower left corner does not essentially differ
from the cortex. Autopsy, PAS stain, 350. H: The cerebellar cortex shows complete loss of both granular and Purkinje cells replaced
by a rim of hypertrophic Bergmann astrocytes and occasional macrophages. Autopsy, PAS stain, 350. I: The spinal anterior horn
neurons are preserved but show eccentric cytoplasmic accumulation of Luxol fast blue-positive material. Autopsy, Luxol fast blue-
cresyl violet stain, 3200. J: White matter of the precentral gyrus showing a few preserved isolated myelinated axons, apparently
derived from the remaining Betz cells. Autopsy, Luxol fast blue-cresyl violet stain, 350. K: The neuroretina (between arrows) has
been completely destroyed and replaced by gliotic scar tissue and occasional macrophages. Autopsy, PAS stain, 3100. L: Spleen tissue
showing a group of large macrophages harboring PAS-positive storage material. Autopsy, PAS stain, 3200.

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6 HALTIA

Fig. 3. The ultrastructural appearances of the abnormal intraneuronal deposits vary between different forms of the neuronal
ceroid-lipofuscinoses. Four basic types can be delineated: (A) granular osmiophilic deposits are characteristic of the infantile and
other forms of CLN1, 310,000; (B) curvilinear profiles are typical of classic late infantile neuronal ceroid-lipofuscinosis CLN2,
320,000; (C) fingerprint bodies are the predominant type of intraneuronal inclusions in the juvenile form CLN3, 330,000; and
(D) many inclusions in the variant forms of late infantile neuronal ceroid-lipofuscinosis correspond to the rectilinear complex,
315,000.

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similar granules also occur in astrocytes and in many oth- cognitive functions usually starts by 8 or 9 yr of age.
er cell types throughout the body. The storage bodies Speech becomes dysarthric usually after the age of 15 yr.
have a fairly uniform ultrastructure in all cells involved. Seizures appear in most patients between 7 and 18 yr of
They are bound by a unit membrane and consist of ac- age and are predominantly of the generalized tonic-clonic
cumulations of so-called curvilinear profiles (Fig. 3B), or the complex partial type. Many patients show signs of
i.e. uniformly curved short thin lamellar stacks of alter- parkinsonism by their mid-teens (11, 31). The patients
nating dark and light lines. Dimensions of the lines range usually die in the third or fourth decade but patients with
from 1.9 to 2.4 nm (14). Granular or fingerprint com- a more protracted course have been described (32). The
ponents do not occur. SCMAS has been shown to be the EEG shows nonspecific abnormalities. ERG shows se-
major protein component of purified storage cytosomes vere changes, and a reduced b-wave may be seen even
(24). at the earliest stage. VEPs are markedly reduced or abol-
Molecular Genetics: CLN2 was mapped to chromo- ished and SEPs often enhanced. After the age of 12 yr,
some 11p15 by homozygosity mapping in consanguine- neuroimaging usually shows progressive brain atrophy,
ous families (25), but the gene product and the gene were mainly affecting the cerebral hemispheres. Vacuolated
identified using a biochemical approach (26). The CLN2 lymphocytes can be regularly demonstrated on peripheral
gene encodes a ubiquitously expressed lysosomal prote- blood films, a unique finding in NCL (11, 31).
ase with tripeptidyl-peptidase 1 activity (TPP1) (26–28). Neuropathological Features of JNCL: At autopsy, the
brain shows moderate generalized atrophy (Fig. 1C) with
Over 40 different mutations of the TPP1 gene have been
brain weight of approximately 800 to 1,000 g. On cut
reported. Two mutations, IVS5-1G-.C affecting splicing
surfaces, the cortical ribbon is slightly reduced in thick-
and the nonsense mutation R208X, are particularly com-
ness and may have a slightly brownish hue. Nigral pig-
mon (29, 30).
mentation is reduced. The white matter has a relatively
CLN3 normal appearance but the ventricular system is slightly
to moderately dilated. By histological examination there
Juvenile NCL (JNCL, Batten-Spielmeyer-Vogt disease, is variable neuronal depletion that may not be very ob-
MIM 304200) is the most common form of NCL world- vious in routinely stained sections. By special techniques
wide and is usually caused by defects in the CLN3 gene. selective loss of neurons has been found in the cerebral
The incidence of JNCL varies in different countries, with cortical layers II and V, as well as in the corpus striatum
the highest figures (up to 7 per 100,000 live births) hav- and amygdala. In the cerebellar cortex there is severe loss
ing been reported from Scandinavia (19). of the granule cells while the Purkinje cells may be better
Clinical Features of JNCL: The first symptom is onset preserved. The neuronal loss is associated with reactive
of progressive visual failure between 4 to 7 yr of age, astrocytic proliferation and hypertrophy and microglial
leading to blindness within 2 to 10 yr. Funduscopy shows activation. The remaining nerve cell perikarya are slight-
macular and retinal degeneration, optic atrophy, and pig- ly to moderately distended by intracytoplasmic accumu-
ment accumulation in the peripheral retina. Slowly pro- lation of strongly autofluorescent granular storage mate-
gressive deterioration of short-term memory and other rial. The entire neuroretina is usually largely destroyed

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 THE NEURONAL CEROID-LIPOFUSCINOSES 7

and replaced by scar tissue, composed of proliferated with dementia, ataxia, and late pyramidal and extrapy-
Mueller cells and other astrocytes. There is migration of ramidal symptoms. With the progression of the disease
cells of the pigment epithelium through the subretinal the seizures may become intractable. Phenotype B ini-
space into the retina. Granular storage material also oc- tially shows behavioral problems, including depression
curs in peripheral neurons and in many extraneural cell and progressive dementia associated with motor problems
types, including the epithelial cells of the sweat glands, such as dysarthria, ataxia, and extrapyramidal symptoms.
the endothelial cells, as well as smooth, skeletal, and car- In both phenotypes, vision is normal without evidence of
diac muscle cells (31). pigmentary retinal degeneration. Some patients may dis-
The intraneuronal storage granules have a pale yellow play features of both main phenotypes. The course of the
brown color in unstained sections and stain with Luxol disease is slowly progressive and leads to death after an
fast blue, PAS, and Sudan black B. They show strong average duration of 12.5 yr. Neuroimaging usually dem-
acid phosphatase activity and are immunoreactive for onstrates cortical brain atrophy (35, 36).
SCMAS and also for SAP A and SAP D (24). By elec- Neuropathological Features: There is mild to moderate
tron microscopy, they consist of membrane-bound, elec- cerebral atrophy with frontoparietal accentuation (37). At
tron-dense fingerprint bodies (Fig. 3C) composed of light microscopic examination of paraffin sections, cere-
paired parallel dark lines of 7.6- to 9.6-nm width and the bral cortical neurons show intracytoplasmic accumulation
central lucent line ranging between 1 and 3 nm (14). The of autofluorescent granules stained with the Luxol fast
paired parallel dark lines are separated from each other blue, PAS, and Sudan black B methods. In addition to

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by a lighter intervening layer of varying thickness. While the neuronal perikarya, storage is also frequently seen in
fingerprint bodies are found in pure form in gastrointes- the proximal axonal segments (axon spindles). The gran-
tinal intraneuronal storage bodies, they may be admixed ules are immunoreactive for SCMAS and also for SAP
with curvilinear or rectilinear profiles even within the A and SAP D. The ultrastructural pattern is variable. The
same storage cytosome in endothelial and smooth muscle abnormal deposits frequently show a granular component
cells in rectal and skin biopsies and in the epithelial cells either exclusively or in combination with fingerprint and
of the sweat glands of JNCL patients. The characteristic curvilinear profiles (35–37).
electron-dense storage inclusions may also be found The deeper cortical layers IIIc, V, and VI usually show
within membrane-bound vacuoles in blood lymphocytes the most pronounced enlargement of the neuronal peri-
and in the epithelial cells of eccrine sweat glands (31). karya, whereas axonal spindles are most conspicuous in
Biochemical analysis of purified JNCL storage granules layers IIIa and IIIb. Neuronal loss is variable with the
has shown that SCMAS is the main storage protein (24). stellate cells of layers II and III being severely affected.
Molecular Genetics: The CLN3 gene was initially Even the cerebellar Purkinje cells may be considerably
linked to the haptoglobin locus on the long arm of chro- reduced in number. Storage is also observed in the neu-
mosome 16 (33), and collaboration of 5 research groups rons of the basal ganglia, thalamus, reticular formation
finally led to the isolation of the gene on 16p12 by po- of the brainstem, and spinal anterior horn neurons. Al-
sitional cloning (34). To date, over 30 CLN3 mutations though retinal architecture is usually preserved at the
have been reported, the most common being a 1.02-kb light microscopic level, ultrastructural studies in some
deletion leading to skipping of exons 7 and 8 and early cases have revealed deposits of abnormal storage material
truncation of the CLN3 protein (11, 31). in the ganglion cells. Biopsy and autopsy studies of other
extracerebral tissues have given variable results without
CLN4 a consistent diagnostic pattern (35–37).
The CLN4 locus corresponds to the hypothetic gene Molecular Genetic and Biochemical Findings: To date,
implicated in adult onset NCL (ANCL, Kufs/Parry dis- no ANCL gene has been cloned or even mapped. Eleva-
ease, MIM 204300/162350). However, ANCL seems to tion in the levels of 2 mannose 6-phosphate glycoproteins
be genetically heterogeneous (35), and although autoso- in an individual with ANCL may indicate perturbation of
mal recessive inheritance is usually observed, several lysosomal function (38).
families with autosomal dominant inheritance have been
reported. ANCL is a very rare condition, and the diag- CLN5
nosis should be accepted only after ultrastructural studies Mutations in the CLN5 gene result in the so-called
of affected tissues and careful exclusion of other alter- Finnish variant LINCL (MIM 256731) (39), which is
natives. clinically and neuropathologically distinct from classic
Clinical Features: The clinical onset of the disease oc- LINCL. This variant LINCL has so far been almost ex-
curs, on the average, at 30 yr of age, with a range of 11 clusively found in Finland.
to 50 yr (35). The clinical phenotype is heterogeneous Clinical Features: The Finnish vLINCL has its clinical
and 2 main forms can be delineated. Phenotype A is char- onset between the ages of 4.5 and 6 yr, with slight motor
acterized by progressive myoclonus epilepsy associated clumsiness and muscular hypotonia followed by impaired

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8 HALTIA

concentration, learning problems, and mental retardation. CLN6

Visuomotor problems may be an early sign. Epileptic sei-
Defects in the CLN6 gene result in a variant form of
zures are usually generalized and become manifest by the
LINCL, also called early juvenile NCL (Lake-Cavanagh)
age of 7 to 8 yr, followed by ataxia and myoclonia be-
(MIM 601780) (45). Most patients with this disease are
tween 7 and 10 yr of life. Athetosis may occur somewhat
from Southern Europe, particularly Portugal, or of Indian
later. At an early stage, ophthalmological investigation or Pakistani extraction, including Romany people in the
reveals macular dystrophy and progressive optic atrophy Czech Republic (46).
is found after the age of 7 to 9 yr with functional blind- Clinical Features: The clinical features closely resem-
ness. The patients usually lose their walking ability by ble those of classic LINCL. However, approximately one
the age of approximately 10 yr and survive until 14 to third of the patients have a slightly later onset and a
32 yr of age. Characteristic neurophysiological findings somewhat more protracted course, with seizures, ataxia,
are observed by the age of 7 to 10 yr and include pos- and myoclonus as the leading symptoms. Even the neu-
terior spikes to low-frequency photic stimulation in the rophysiological characteristics resemble those found in
EEG, giant VEP and SEP, and abolished ERG. Neuro- classic LINCL. The EEG is abnormal, usually with a pos-
imaging shows early cerebellar involvement with severe itive response to slow rate photic stimulation at early
cerebral and cerebellar atrophy later. Vacuolated lympho- stages. The ERG becomes extinguished early on and a
cytes are not found (39, 40). giant VEP may be seen before it becomes diminished or

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Neuropathological and Biochemical Features: There is absent. The findings at neuroimaging are nonspecific.
severe generalized cerebral and extreme cerebellar atro- Vacuolated lymphocytes are not found (45–47).
phy at autopsy (Fig. 1B), with total brain weight of 450 Neuropathological Features: There is severe general-
to 650 g (41). In an early cortical biopsy the general ized brain atrophy, with the brain weights at autopsy
cytoarchitecture is well preserved, but all neurons show ranging between 600 and 900 g. vLINCL is histologically
moderate amounts of intracytoplasmic autofluorescent characterized by ubiquitous intraneuronal storage of au-
storage granules (Fig. 2C) positively stained with the tofluorescent granules, which stain positively with Luxol
Luxol fast blue, PAS, and Sudan black B methods. The fast blue, PAS, and Sudan black methods and are im-
granules are strongly immunoreactive for SCMAS (Fig. munoreactive for SCMAS. There is cortical neuronal
2D) and also for SAP A and D (41). In older autopsy loss, most marked in the occipital lobe and in layer V.
cases, the most intense storage is seen in the deeper cor- The granular layer of the cerebellum is depleted while
tical layers while the superficial part of layer III harbors part of the Purkinje cells may persist. As in classic
numerous axonal spindles (Fig. 2E). There is progressive LINCL, there are small storage deposits in many extra-
neuronal loss, partly in a laminar pattern and particularly neural tissues. The ultrastructure of the storage cytosomes
involving layers III and V. The cerebellar Purkinje and in cerebral neurons corresponds to a mixture of rectilinear
granular cells are almost completely destroyed while complex and fingerprint patterns while intestinal neurons
most subcortical structures show moderate to pronounced only show pure fingerprint profiles. The abnormal cyto-
storage but relatively modest neuronal loss. There is se- somes in eccrine sweat gland epithelium, smooth muscle,
vere cortical astrocytosis and moderate to severe second- and endothelial cells may contain both curvilinear, fin-
ary loss of myelin in the white matter (41). The ultra- gerprint, and rectilinear complex profiles (45–47).
structure of the cerebral intraneuronal storage bodies Molecular Genetic Findings: The CLN6 gene on chro-
(Fig. 3D) corresponds largely to the rectilinear complex mosome 15q21–23 was recently identified by positional
(14). However, autonomic ganglion cells of the gut wall cloning (48, 49). It encodes a novel protein with several
have shown cytosomes with pure fingerprint patterns. predicted transmembrane domains. To date, 7 mutations
Storage granules are also found in many extraneural tis- have been reported, with E72X nonsense mutation in
sues (e.g. skin and rectal mucosa) where their ultrastruc- exon 3 occurring in several Costa Rican families.
ture corresponds to classic curvilinear profiles or to the
rectilinear complex (39, 40). SCMAS was found to be CLN7 and CLN8
the major protein species in isolated storage cytosomes A defect of the CLN8 gene underlies Northern epilepsy
(41). (NE), also known as progressive epilepsy with mental
Molecular Genetic Findings: The CLN5 gene is located retardation (50, 51). It is the most protracted form of
on chromosome 13q22 (42) and was identified by posi- human NCL (52, 53), to date only described in Finland.
tional cloning (43). To date, 4 mutations of the CLN5 However, a number of patients with the so-called Turkish
gene have been identified (43, 44). The Finnish major variant LINCL (54), originally thought to represent a dis-
mutation, a 2-bp deletion in exon 4, has been identified tinct genetic locus designated CLN7, were recently linked
in over 90% of Finnish disease chromosomes. to CLN8 (55).

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 THE NEURONAL CEROID-LIPOFUSCINOSES 9
TABLE 2
Spontaneous and Genetically Engineered Animal Models for Human Neuronal Ceroid-Lipofuscinoses (NCL)

Model Animal Animal disease/modification

Congenital NCL? Sheep (Swedish Landrace) Congenital ovine NCL with mutation
of the cathepsin D gene (60, 61)
Mouse Cathepsin D knockout (70, 71)
CLN1/INCL Mouse PPT1 knockout (66)
CLN3/JNCL Mouse CLN3 knockout (67–69)
CLN6/vLINCL Sheep (New Hampshire) Ovine NCL (63)
Mouse nclf mouse (62)
CLN8/Northern epilepsy Mouse mnd mouse (57, 64, 65)

Clinical Features of NE: The clinical onset of Northern observable neuronal loss, most evident in the hippocam-
epilepsy occurs by the age of 5 to 10 yr with generalized pal CA 2 sector where it is coupled with some astrocytic
tonic-clonic seizures. Some patients may also show com- and microglial reaction. Limited electron microscopic
plex partial seizures during the first few years. The fre- studies of the storage material have shown membrane-

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quency of the seizures increases towards puberty but de- bound, electron-dense cytosomes with structures resem-
creases spontaneously during adulthood. Mental bling curvilinear profiles and occasional foci with a gran-
retardation becomes manifest 2 to 5 yr after the onset of ular ultrastructure. Western blotting and N-terminal
epilepsy and slowly progresses. In young adulthood, the sequence analysis of purified storage granules indicates
patients show progressive slowness and clumsiness in that SCMAS is the major storage protein (52, 53).
fine motor tasks, and balance problems become obvious Turkish Variant LINCL: Only very limited clinical and
after the age of 30. Some patients have shown diminished morphological data are available on the Turkish variant
visual acuity without any observed ocular abnormality. LINCL patients with onset of symptoms in the late in-
At brain imaging, cortical atrophy has been seen in all fantile age range. Seizures and poor motility are promi-
patients over 40 yr of age but is rare before the age of nent early features while visual impairment may begin
30 yr. EEG consistently shows slowing of the background simultaneously or later. The subsequent course is char-
activity. Rhythmic delta activity is abundant while spe- acterized by motor and cognitive deterioration. Electron
cific sleep patterns can be missing. Epileptiform activity microscopic studies have shown rectilinear inclusions in
is frequently observed, but its amount is scant. No con- a skeletal muscle biopsy, predominant curvilinear bodies
sistent VEP abnormalities have been reported and no vac- with or without rectilinear profiles, and no fingerprint in-
uolated lymphocytes have been seen (50–52). clusions in rectal or skin biopsies (54).
Neuropathological and Biochemical Features of NE: Molecular Genetic Findings: The CLN8 gene is located
No specific macroscopic changes have been observed. on chromosome 8p23 (56) and encodes a novel 286 ami-
The most striking histopathological abnormality is the no acid transmembrane protein (57). All Finnish patients
presence of granular cytoplasmic storage material partic- share a missense mutation in codon 24 (R24G) (57).
ularly within nerve cells but also, to a lesser extent, in
OTHER FORMS OF HUMAN NCL AND
many other cell types throughout the body. The storage
ANIMAL MODELS
granules are autofluorescent and Luxol fast blue-, PAS-,
and Sudan black B-positive in paraffin sections (Fig. 2F– In addition to CLN1–8 there are reports on atypical
H) and immunoreactive for SCMAS. The amount of stor- cases of NCL. These include 3 patients with juvenile on-
age varies greatly between different neuronal popula- set and late visual impairment whose findings did not fit
tions, resulting in a strikingly distinct distribution pattern. into any of the established categories of NCL. These pa-
The most prominent storage occurs in the hippocampal tients were suggested to represent CLN9. Two reports
CA 2–4 sectors, while the CA1 sector and fascia dentata describe a congenital form of human NCL, however
are almost spared. In the isocortex, the deep part of lam- without molecular genetic documentation (58, 59).
ina III shows the most pronounced perikaryal ballooning
of the large pyramidal cells while conspicuous axonal Spontaneous Animal Models (Table 2)
spindles are frequent in the upper parts of the same lam- A congenital ovine NCL in Swedish Landrace lambs
ina. The cerebellar granule cells and Purkinje cells are (60) was recently shown to be caused by a mutation of
relatively well preserved. The basal ganglia, thalamus, the cathepsin D gene (61). The immunohistochemical and
brainstem, and spinal cord show mostly slight to mod- ultrastructural features of the affected lambs closely re-
erate intraneuronal storage. There is no or only slight semble those found in 1 human case of congenital NCL

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10 HALTIA

where cathepsin D deficiency has not been excluded. Two TPP1 (26), are soluble lysosomal enzymes, as is cathep-
animal models are available for CLN6: the nclf mouse sin D, an aspartyl proteinase (61). In contrast, the pre-
(48, 49, 62) and ovine NCL in South Hampshire sheep dicted amino acid sequences of the products of the CLN3
(63). The so-called motor neuron degeneration (mnd) and CLN5 genes suggest that they are integral transmem-
mouse is syntenic to CLN8 (57, 64). While the mnd mice brane proteins, reported to have a lysosomal location in
show characteristic morphological features of NCL (65), non-neuronal cells (73). However, mitochondrial (74),
their clinical manifestations are distinct from Northern Golgi compartment (75), and nuclear and cytoplasmic
epilepsy. The mice develop early spastic paresis and (76) localizations have also been suggested for the CLN3
blindness but no epilepsy. A variety of further sponta- protein. The CLN8 gene also encodes a novel transmem-
neous animal forms of NCL have been described, how- brane protein (57). The CLN8 protein contains an ER
ever without molecular genetic characterization. retrieval signal and has been found both in the ER and
the ER-Golgi intermediate compartment in non-neuronal
Genetically Engineered Models of NCL (Table 2)
cells (77).
Gupta et al (66) recently reported a knockout mouse PPT1 removes fatty acid groups from several S-acyl-
model of INCL created by targeted disruptions of the ated proteins such as oncogene H-ras (78). The function
PPT1 gene. The mice were viable and fertile but devel- of TPP1, a serine protease, is to remove N-terminal tri-
oped myoclonic jerking, epileptic seizures, spasticity, and peptides from substrates with free amino termini (79). In
progressive motor abnormalities leading to death by 10 vitro, this enzyme has been reported to participate in the

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months of age. There was prominent accumulation of au- lysosomal degradation of SCMAS (80). However, the in
tofluorescent storage material, neuronal loss, and apopto- vivo substrates of PPT1 and TPP1 are unknown. CLN3
sis in the brains of these mice, providing the first animal as well as Btn 1, the yeast homolog of CLN3, may have
model for CLN1. Also CLN3 knockout mouse models a role in the regulation of vacuolar pH or vesicular traf-
have been published (67–69). Despite characteristic in- ficking (81–84). The exact function of the human CLN3
traneuronal storage these mice show no obvious clinical still remains elusive, and no data are yet available on the
phenotype. Cathepsin D-deficient mice (70) show typical functions of the CLN5, CLN6, and CLN8 proteins. It is
pathomorphological features of NCL and a severe clinical of interest in this context to note that altered lysosomal
phenotype, including early seizures (71). Efforts to create
pH has been reported even in the fibroblasts of NCL pa-
nematode models of NCL are in progress.
tients (85).
PATHOGENETIC CONSIDERATIONS The localization and functions of the NCL-associated
proteins in neurons are not necessarily the same as in
The concept of NCL is based on a relatively uniform
non-neuronal cells. In fact, there is growing evidence that
morphological phenotype, characterized by the accumu-
at least PPT1 and CLN3 are involved in synaptic func-
lation in neurons and, to a much lesser extent, in many
tion. In mouse and rat brain, PPT1 expression is under
other cells of intracytoplasmic autofluorescent deposits
developmental control and closely follows the temporal
with typical cytochemical properties and ultrastructural
and spatial pattern of synaptogenesis (86, 87). In the
patterns. Despite ubiquitous storage, only the neurons of
mouse brain, PPT1 is enriched in the synaptosome and
the CNS are selectively destroyed. The storage material
is largely proteinaceous and, depending on the identity synaptic vesicle fractions (88) and colocalizes with pre-
of its main protein component, the NCL can be divided synaptic synaptophysin and the synaptic vesicle marker
into 2 broad categories, those storing SCMAS and those SV2 in transfected murine cortical neurons (89). Like-
storing SAP (Table 1). High concentrations of SAP ex- wise, CLN3 is mainly found in the synaptosomes of pre-
clusively associate with GRODs, suggesting that the ul- synaptic nerve terminals of mouse primary neurons (90).
trastructure is determined by the stored protein. Immu- Only limited information is available on the effects of
noreactivity of the intraneuronal deposits for amyloid the pathogenic mutations in the various forms of NCL.
beta has also been reported (72). However, the mecha- CLN1 is best studied in this respect and may serve as an
nisms of accumulation of these highly hydrophobic pro- example. The PPT1 enzyme has been crystallized (91)
teins and their relation, if any, to clinical symptomatology and its known 3-dimensional structure allows the estab-
and neuronal death remain unsolved. lishment of phenotype-genotype correlations. Mutations
The morphological uniformity of the NCL contrasts associated with the severe infantile phenotype are located
with their newly discovered genetic heterogeneity. More near the active site (Ser 155) and profoundly disturb the
than 115 mutations in at least 7 different genes (CLN1– structure and function of the enzyme. In contrast, muta-
3, CLN5–6, CLN8 and cathepsin D) underlie different tions causing late-onset forms of CLN1 are remote from
forms of NCL in man and animals. The products of most the catalytic triad, leading only to limited changes in pro-
of these genes are ubiquitously expressed and not neuron- tein structure (91). The most common mutations causing
specific. The CLN1 and CLN2 proteins, PPT1 (17) and the severe infantile phenotype, a premature stop-codon at

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 THE NEURONAL CEROID-LIPOFUSCINOSES 11

arginine 151 and the missense mutation Arg122Trp, re- 6. Stengel C. Beretning om et maerkeligt Sygdomstilfaelde hos fire
sult in a severely truncated or unstable protein that is Soedskende i Naerheden af Roeraas. Eyr et medicinsk Tidskrift
1826;1:347–52
degraded in the ER. In contrast, the missense mutation 7. Santavuori P, Haltia M, Rapola J, Raitta C. Infantile type of so-
Thr75Pro leads to a milder juvenile phenotype (12). called neuronal ceroid-lipofuscinosis. 1. A clinical study of 15 pa-
While all these mutations lead to deficient activity of the tients. J Neurol Sci 1973;18:257–67
PPT1 enzyme, they may also have an effect on its intra- 8. Haltia M, Rapola J, Santavuori P, Keränen A. Infantile type of so-
cellular trafficking, and this effect may differ between called neuronal ceroid-lipofuscinosis. 2. Morphological and bio-
chemical studies. J Neurol Sci 1973;18:269–85
neurons and non-neuronal cells. In non-neural cell lines,
9. Haltia M, Rapola J, Santavuori P. Infantile type of so-called neu-
mutant PPT1 polypeptides were retained in the ER while ronal ceroid-lipofuscinosis. Histological and electron microscopic
the wild-type protein was localized to the lysosomes (92). studies. Acta Neuropathol 1973;26:157–70
However, in primary neurons both wild type and mutant 10. Goebel HH, Mole SE, Lake BD. The neuronal ceroid lipofuscinoses
PPT1 polypeptides causing mild phenotypes were trans- (Batten disease). Amsterdam: IOS Press, 1999
11. Wisniewski KE, Kida E, Golabek AA, Kaczmarski W, Connell F,
ported along the neurites and colocalized with synaptic
Zhong N. Neuronal ceroid lipofuscinoses: Classification and diag-
markers. Only mutant PPT1 polypeptides causing severe nosis. In: Wisniewski KE, Zhong N, eds. Batten disease: Diagnosis,
phenotypes were retained in the ER (92). treatment and research. San Diego: Academic Press, 2001:1–34
The deranged metabolic pathways leading to neuronal 12. Santavuori P, Gottlob I, Haltia M, et al. CLN1. Infantile and other
degeneration and death in NCL remain to be elucidated. types of NCL with GROD. In: Goebel HH, Mole SE, Lake BD,
eds. The neuronal ceroid lipofuscinoses (Batten disease). Amster-

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Both apoptotic (93, 94) and excitotoxic mechanisms have
dam: IOS Press, 1999:16–36
been proposed (95). It seems possible that the products 13. Tarkkanen A, Haltia M, Merenmies L. Ocular pathology in infantile
of all NCL genes have either direct or indirect interac- type of neuronal ceroid-lipofuscinosis. J Pediatr Ophthalmol 1977;
tions, participating in the same trafficking pathway of vi- 14:235–41
tal importance for neurons (96). The recent advances in 14. Elleder M, Lake BD, Goebel HH, Rapola J, Haltia M, Carpenter
S. Definitions of the ultrastructural patterns found in NCL. In: Goe-
the field of molecular genetics presented above have ex-
bel HH, Mole SE, Lake BD, eds. The neuronal ceroid lipofusci-
posed the striking genetic heterogeneity of NCL and form noses (Batten disease). Amsterdam: IOS Press, 1999:5–15
a solid basis for further studies. Identifying the molecular 15. Tyynelä J, Palmer DN, Baumann M, Haltia M. Storage of saposins
pathogenesis of these devastating neuronal disorders will A and D in infantile neuronal ceroid-lipofuscinosis. FEBS Lett
likely deepen our understanding of the basic mechanism 1993;330:8–12
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ceroid lipofuscinosis (CLN1) maps to the short arm of chromosome
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ACKNOWLEDGMENTS protein thioesterase gene causing infantile neuronal ceroid-lipofus-
The author would like to thank Docents Pirkko Santavuori, MD, PhD, cinosis. Nature 1995;376:584–87
Jaana Tyynelä, PhD, Riitta Herva, MD, PhD, and Anders Paetau, MD, 18. Claussen M, Heim P, Knispel J, Goebel HH, Kohlschuetter A. In-
PhD for close collaboration in NCL research over many years, and cidence of neuronal ceroid-lipofuscinoses in West Germany: Vari-
Professor Anna-Elina Lehesjoki, MD, PhD, and Docent Jaana Tyynelä, ation of a method for studying autosomal recessive disorders. Am
PhD, for their constructive comments on this manuscript. Finally, the J Med Genet 1992:42:536–38
author would like to acknowledge the work of many investigators of 19. Uvebrant P, Hagberg B. Neuronal ceroid-lipofuscinoses in Scandi-
the NCL whose work could not be cited due to space limitations. navia. Epidemiology and clinical pictures. Neuropediatrics 1997;
28:6–8
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