Annals of Diagnostic Paediatric Pathology

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1 Annals of Diagnostic Paediatric Pathology 2003, 7(4): Copyright by Polish Paediatric Pathology Society The neuronal ceroid lipofuscinoses Wies³awa Grajkowska 1, El bieta Czarnowska 1, Tomasz Kmieæ 2, Maciej Pronicki Annals of Diagnostic Paediatric Pathology 1 Department of Pathology 2 Department of Neurology and Epileptology The Children s Memorial Health Institute Warsaw, Poland Abstract The neuronal lipofuscinoses (NCLs) are described as a complex of inherited neurodegenerative disorders associated with intralysosomal accumulation of lipopigment in neuronal and extraneuronal cells. Morphologic pathology in NCL is marked by two processes: degeneration and loss of neuronal cells of central nervous system, resulting in atrophy of the cerebrum and cerebellum and widespread accumulation of autofluorescent lysosomal lipopigments of varying ultrastructure, the demonstration of which allows diagnostic recognition of NCL disease. Ultrastructural examinations of affected tissues may diclose 5 different patterns of lipopigments: usual lipofuscin, fingerprint deposits, granular profiles, curvilinear bodies, and microtubular aggregates, which may be found in conjunctiva, liver, lymphocytes, skin, full thickness rectal biopsies material. Deposits characteristic for NCL are ultrastructurally observed also in trophoblastic cells and amniotic fluid cells. Ultrastructural studies of biopsy tissues in order to identify pattern of pigment remains the gold standrad to identify NCL, together with clinical aspects and respective gene defects. Advances in molecular genetic techniques have allowed to identification of defective genes and their protein products in some NCL clinical forms. Key words: clinical characteristics lipopigments lysosomal disorders neurodegenerative diseases neuronal ceroid lipofuscinoses ultrastructural characteristics Introduction The childhood disorders, characterized by progressive myoclonic epilepsy, psychomotor deterioration with different motor disturbances, visual failure, familial occurrence and the intralysosomal accumulation of the autofluorescent lipopigment, lipofuscin (from Greek lipos - fat; from Latin fuscus - dusky) and ceroid (from Latin cera - wax) in neuronal and other cellular components of various tissues are described as the neuronal ceroid lipofuscinoses (NCLs). NCLs are a heterogenous group of inherited neurodegenerative diseases that display an autosomal recessive pattern of inheritance, however occasionally dominant forms also occur [1, 2]. Forms of the NCLs were first described in Norway by Stengel in 1826 and in 1903 by Batten, Spielmeyer and Vogt. The NCLs occur worldwide, but the gene frequency appears to be greater in the Northern European population. The disorders are relatively rare and an incidence is of 0,46 in West Germany and 0,36 in Italy per live births. The infantile NCL and late infantile NCL occur predominantly in Finland and form a part of the Finnish disease heritage [18]. Neurons in the central nervous system are the most severely affected, thus causing interrupted development of the brain and death [8, 16, 17]. NCL classification An important criterion for the classification of NCL disorders is the age of onset of the clinical symptoms and signs. In 1989 Dyken proposed a classification of NCL on the ground of the age, clinical symptoms, and ultrastructural aspects of the lipopigments, identifying 4 main and 6 minor types of NCL [5]. To these latter, 3 more minor types have been recently added (Table 1).The main childhood subtypes are in- Address for correspondence:

2 6 Tabele 1 Clinical pattern of NCL features NCL syndrome Dynamics Age of onset Symptoms Major Batten-Spielmeyer-Vogt Chronic 4-10 years Bielschowsky Jansky Acute 2-4 years Kufs Subacute-chronic About age of 30 years Harberg Santavuori- Haltia (Finnish type) Acute 8-18 months Minor Visual, behavioral, dementia Seizures, psychomotor, visual Seizures or dementia, motor Psychomotor, visual, myoclonia Ultrastrucutral pattern Fingerprint, culvilinear Curvilinear, fingerprint Granular, fingerprint Granular Norman-Wood Static Congenital Seizures, vegetative Curvilinear Zeman-Dyken Acute Adult Seizures, visual, psychomotor Fingerprint, curvilinear Bielschowsky variant Subacute-acute Childhood Visual, psychomotor Fingerprint, curvilinear Edathodu Dyken Chronic Childhood Pervasiveness Fingerprint Infantile with autism Chronic Infantile Autism, hand movements Granular, fingerprint Juvenile with ataxia Chronic Juvenile Ataxia, spasticity Fingerprint Lake-Canavagh - Early juvenile Visual, psychomotor? Finnish variant - Late infantile Visual, psychomotor Curvilinear, finger Indian variant - Late infantile Visual, psychomotor? fantile (INCL, Harberg- Santavuori-Haltia disease, McKusick ), late infantile (LINCL, Jansky-Bielschowsky disease, early-onset Batten s disease, McKusick ), and juvenile (JNCL, late onset Batten s disease, Batten- Spielmeyer-Vogt disease, McKusick ). A fourth type, adult type, adult NCL (Kufs disease, McKusick ), is typically a disease of adult [18]. Genetics Recently a new genetic classification (CLN1-CLN8) is proposed (Table 2) [2, 8, 11]. In fact, 8 genes underlie the NCL syndromes, of which 5 have been isolated and mutations characterized: CLN1 (Infantile NCL, Haberg-Santavuori-Haltia disease), CLN2 (Late infantile NCL, Bielschowsky), CLN3 (JuvenileNCL, Batten dis.), CLN5 (Late infantile, Finnish variant dis.), and CLN8 (Protracted juvenile northern epilepsy/epmr) [1, 11, 14]. Genetic mutation and chromosome localization in adult NCL are not known [14]. Clinical features Clinical course of NCLs and symptoms vary with each person [17]. Following is an outline of the most typical symptology: visual impairment progressing to complete blindness, seizures, which may be frequent and difficult to control, decline in cognitive function, personality and behavioral changes, loss of communication skills, loss of motor skills and increasing spasticity, facial grimacing and abnormal body movements, a general, progressive deterioration to a vegetative state. Other symptoms what may develop include slowing of head growth with age, poor circulation in lower extremities, with legs and feet often cold and bluishred in color, decreased body fat and muscle mass, curvature of the spine, hyperventilation and/or breath-holding spells, teeth-grinding, and constipation. Infantile neuronal ceroidolipofuscinosis (INCL) INCL clinical signs are presented between 8 and 18 months of age. Affected children fail to thrive and have abnormally small heads. Also typical are short, sharp muscle contractions called myoclonic jerks. Initial signs of this disorder include delayed psychomotor development with progressive deterioration, other motor disorders, or seizures. The infantile form has the most rapid progression. Death occurs at between 3 and 10years [1]. Late infantile NCL (LINCL) The symptoms of classic LINCL appear between ages two and four years. All affected children present three stages of progressive disease with typical clinical symptoms. The major symptoms are seizures average at 3,2 yr, usually herald the onset of

3 7 further signs. Partial, generalized tonic-clonic or secondary generalized, absences may also occur. Myoclonus started at 3,7 yr, speech regression is observed at 4,8 yr, ataxia at 3,8 yr, visual failure increased at 4,2 yr, blindness is confirmed at 5,6 yr of age. Children generally become chair bound between 4and 6 years and death occurs in middle childhood [16]. Juvenile NCL (JNCL) Clinical signs of JNCL are presented between 4 and 10 years of age with progressive visual impairment caused by a characteristic retinitis pigmentosa. Most children become blind over 2-6 years. Dementia becomes evident in the second decade of life starting with compulsive speaking and characteristic stammer. Spastic tetraplegia leads to death in the late teens [16]. Adult NCL The disease starts mostly at age of 30 years, however later onset of clinical symptoms has been also described [4]. Clinical presentation includes a progressive myoclonal epilepsy with dementia, ataxia and late pyramidal and extrapyramidal features. The vision is normal. Some patients present photosensitivity. In literature are also reported cases of adult NCL type at paediaric age [17]. Pathogenesis In NCLs, a specific lysosomal defect can gradually lead to a general lysosomal dysfuntion, either by disruption of an essential metabolic route or by mechanic filling of the lysosomes due to accumulation of lipofuscin. Lysosomal proteins are known to have a tendency to compensate a defective enzyme with an increased activity of the others, as the example of NCL diseases shows. Six forms of NCL (CLN2, CLN3, CLN4, CLN5, CLN6, CLN8) accumulate subunit c of mitochondrial ATP synthase as a component of the storage lipopigment [13, 15, 18]. CLN2 is associated with a deficiency of lysosomal pepstain-insensitive peptidase (PPP1). CLN3 is believed to be associated with mutations of a lysosomal membrane protein of unkown function [15]. Three lysosomal enzymes are responsible for three early-onset forms of NCLs: PPT1 in infantile NCL (INCL, CLIN1), tripeptidyl peptidase 1 (TPP1) also called pepstatininsensitive protease in late-infantile NCL (LINCL, CLN2), and cathepsin D in congenital ovine NCL [15,18]. The mutations of catalytic site of PPT1, substrate binding, or folding result in inactive enzyme and lead to severe clinical phenotypes. TTP1 is an exopeptidase that cleaves N-terminal tripeptides from peptides or proteins with a preference for the N-terminal GlyProX sequence. Cathepsin D is a well-known aspartyl protease. Activity of PPT1 and TPP1 can be measured in white blood cells. JNCL is caused by the deficiency of transmembrane protein of lysosomal membrane, what is of unknown function and with no diagnostic assay [3, 18]. Ultrastructural features NCL is generally included with the lysosomal storage disorders because the lipopigment inclusions are bound by the lysosomal membrane [13]. With electron microscopy it is possible to distinguish 5 different type of osmiophilic lipopigments: usual lipofuscin, fingerprint deposits, curvilinear bodies, granular profiles (GRODs) and microtubular aggregates [2, 3]. The abnormal deposits result from a shortage of enzymes normally responsible for the breakdown of lipopigments [16]. The GRODs are deposits composed of conglomerates of spherical globules of nm in diameter, which can be thigtly or loosely bound [12]. Occasionally single or paired membranous structures within the matrix are observed (Fig. 1). Ultrastructural analysis of blood lymphocytes can be often false negative [2]. The curvilinear bodies are enclosed by single membrane, however in some areas membrane may not be clearly seen (Fig. 2). They do not contain granular component. The fingerprint bodies may be found within membrane bound vacuoles or non-vacuolar lysosomes (Fig. 3) and occasionally mixed with curvilinear components (Fig. 4) [17]. In the past years there was thought that each ultrastructural pattern was the counterpart of a specific clinical type of NCLs. In fact, there seemed to be a prodominance of fingerprint bodies in chronic juvenile forms, the curvilinear bodies occurred more frequently in the late infantile forms and GRODs in infantile cases. In adult NCL type a granular component is frequently seen, however both fingerprint and curvilinear profiles also observed [2, 3]. The accumulation of deposits is not restricted to nervous system but is systemic, and may be observed also in peripherial blood lymphocytes, epithelial cells, fibroblasts, endothelium, myocytes, Schwann cells, eccrine sweat gland epithelial cells, and trophoblastic cells. The most explored for diagnosis of NCL were conjunctiva, rectum and liver biopsy material [7]. Ultrastructural examination of a pellet of lymphocytes is sufficient to identity the deposits, however there must be examined not less than 100 cells. It must be pointed that ultrastructural analysis of blood lymphocytes can be often false negative. Ultrastructurally characteristic deposits in lymphocytes include GRODs in NCL1, curvinlinear bodies in CLN2, fingerprtints in CLN3 and single inclusions with a condensed fingerprint pattern in CLN6 [2, 3]. In conjunctiva deposits are typically seen in fibroblasts, endothelial cells and nerves. It is reliable tissue for diagnosis of all type of NCL. In rectal samples, the neurons of submucosal plexuses storage GRODs in CLN1, curvilinear bodies in CLN2, and fingerprint inclusions in CLN3, CLN5 and CLN6 [17]. The diagnostic of skin biopsy material is reliable if it is taken enough deep to include sweat glands. The secretory eccrine epithelial cells are the most important for diagnosis exhibiting NCL deposits, which are not founded in the sebaceous gland and apocrine glands.

4 8 Fig. 1 Granular bodies deposits (GRODs) in the cytoplasm of fibroblast from conjunctiva. Mag. x Fig. 2 Curvilinear bodies in the cytoplasm of fibroblast from conjunctiva. Mag. x Fig. 3 Fingerprint like inclusions in the cytoplasm of hepatocytes. Mag. x Fig. 4 Mixed inclusions in the cytoplasm of endothelial cell. Mag. x In skeletal muscle biopsy material are found GRODs of CLN1 and heavy condensed curvilinear bodies of CLN2. Deposits of fingerprints are not presented and due to that reason the muscle biopsy is not recommended for NCL diagnosis. Prenatal diagnosis of NCLs is possible in amniotic fluid cells (fetal lymphocytes and fetal skin at later gestational age) or chorionic villus samples by a combination of ultrastructural, biochemical and/or molecular analysis [6, 10]. Under electron microscopy were found deposits characteristic for CLN1, CLN3 [3]. cal studies of the brain, including EEG, ERG, and visual evoked response, neuroradiological studies including MRI and CT of the head, prenatal diagnosis in amniotic fluid cultures or chorionic villus samples by a combination of biochemical and/ or molecular analysis[6, 7, 14, 15]. Although considerable progress in understanding and diagnosis of NCL has been achieved recently, the metabolic basis of the NCL disorders has remained unexplained. Still must be clarify why lipopigments accumulate. An understanding of the initial biochemical defect is essential for diagnostic purposes an possibly specific therapeutic strategies [9, 13]. Presently, there is not known treatment or cure of NCLs. The seizures can be reduced or controlled with anticonvulsant drugs. Physical and occupational therapy may help patients retain function as long as possible. Some reports have described a slowing of the disease progression in children with JNCL who treated with vitamins C and E and with diets low in vitamin A. Summary At present the diagnosis of NCL is a complicated process requiring a complete clinical evaluation, laboratory testing, and periodic reevaluations. These may include: ultrastructural studies of blood or skin, or conjunctival biopsy, biochemical studies of blood and fibroblast cultures for enzyme studies (PPT1, TPP1), molecular genetic studies of blood or fibroblast cultures for identification of the gene mutation, electrophysiologi-

5 9 Tabele 2 Genetic classifications and molecular features of NCL Clinical forms Genetic type Chromosome location Gene product Infantile NCL (Harberg-Santavuori- Haltia) CLN1 1p32 Lysosomal palmitoyl protein thioesterase Late infantile NCL (Bielschowsky) CLN2 11p15 Lysosomal pepstain-insensitive peptidase Juvenile NCL (Batten) CLN3 16p12 Lysosomal transmembrance CLN3 protein Adult NCL (Kufs) CLN4 Not know Not know Late infantile (Finnish variant) CLN5 13q31-32 Lysosomal transmembrance CLN5 protein Late infantile (Indian variant) CLN6 15q21-23 Not know Late infantile (Turkish variant) CLN7 Not know Not know Northern epilepsy/epmr CLN8 8p23 Membrane protein References 1. Bennet MJ, Hofmann SL (1999) The neuronal lipofuscinoses (Batten disease): a new class of lysosomal storage diseases. J Inher Metab Dis 22: Boldrini R, Biselli R, Santorelli F, Bosman C (2001) Neuronal Ceroid Lipofuscinosis: an ultrastructural, genetic, and clinical study report. Ultrast Pathol 25: Carlen B, Englund E (2001) Diagnostic value of electron microscopy in a case of juvenile neuronal ceroid lipofuscinosis. Ultrastr Pathol 25: Constantinidis J, et al (1992) The adult and latent adult form of neuronal ceroidlipofuscinosis. Acta Neuropathol (Berl) 83: Dyken P (1989) The neuronal ceroid lipofuscinoses J Child Neurol 4: Goebel HH (1994) Prenatal ultrastructural diagnosis in the neuronal ceropidlipofuscinoses. Pathol Res Pract 190: Goebel HH (2000) Morphological aspects of the neuronal ceroid lipofuscinoses. Neurol Sci 21: Goebel HH, Schochet SS, Jaynes M, Bruck W, Kohlsschutter A, Hentati F (1999) Progress in neuropatholgy of the neuronal ceroid lipofuscinoses. Mol Genet Metab 66: Goebel HH, Sharp JD(1998) The neuronal lipofuscinoses: recent advances. Brain Pathol 8: Goebel HH, Vesa J, Reitter B, Goecke T, Schneider-Ratzke B, Merz E (1995) Prenatal diagnosis of infantile neuronal lipofuscinosis: a combined electron microscopic and molecular genetic approch. Brain Dev 17: Gradiner R (2000) The molecular genetic basis of the neuronal ceroid lipofuscinoses Neurol Sci 21: Hofman IL, Taschner PEM (1995) Late onset juvenile neuronal ceroid lipofuscinosis with granular osmiophilic deposists (GROD). Am J Med Genet 57: Jolly RD, Brown S Das AM, Walkley SU(2002) Mitochondrial dysfunction in the neuronal ceroid-lipofuscinoses (Batten disease) Neurochem Int 40: Rapola J, Jarvela I, Peltonen L (1991) The neuronal ceroid lipofuscinoses: unfolding the genetic defect. Pediatric Pathol 11: Suopanki J, Partanen S, Ezaki J, Baumann M, Kominami E, Tyynela J (2000) Developmental changes in the expression of neuronal ceroid lipofuscinoses-linked proteins. Molec Genet Metab 71: Surtees R (2002) Understanding neurodegenerative disorders. Curr Paediatr 12: Wisniewski KE, et al (1992) Variability in the clinical and pathological findings in the neuronal ceroid lipofuscinosis; review of data and observations. A. J Med Genet 42: Zhong N (2000) Neuronal ceroid lipofuscinoses and possible pathogenic mechanism. Mol Genet Metab 71: