Neuronal ceroid lipofuscinosis: a clinicopathological study

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1 Seizure 2004; 13: doi: /s (03) Neuronal ceroid lipofuscinosis: a clinicopathological study SANJIB SINHA, P. SATISHCHANDRA, VANI SANTOSH,N.GAYATRI & S. K. SHANKAR Departments of Neurology and Neuropathology, National Institute of Mental Health & Neurosciences (NIMHANS), Bangalore, India Correspondence to: Dr P. Satishchandra, Professor and Head, Department of Neurology, National Institute of Mental Health & Neurosciences (NIMHANS), Bangalore, India. psatish@nimhans.kar.nic.in We report the clinical, electrophysiological, radiological and morphological features in a series of 12 patients of histopathologically confirmed cases (infantile, juvenile and adult onset) of neuronal ceroid lipofuscinosis (NCL) observed from 1979 to 1998 at National Institute of Mental Health & Neurosciences (NIMHANS), Bangalore (South India). The commonest type of NCL was juvenile (n = 8, 67%) while infantile and adult forms were two each (n = 2, 16.8%). The age at presentation ranged from 2 to 45 years (mean 12.6, 14.3 years; median 7 years; M:F ratio of 2:1). Four patients (33%) had positive family history and five patients had history of consanguineous parentage (41.6%). The commonest presenting symptoms were regression of milestones (83.3%) and/or seizures, myoclonus (83.8%) followed by involuntary choreiform movements (50%), visual loss (41.6%), ataxia (33.3%) and abnormal behaviour (16.6%). Neuro-ophthalmological abnormalities like optic atrophy (50%), macular degeneration (33.3%) and retinitis pigmentosa (8.3%) were seen in two thirds. Nerve conduction studies (n = 4) revealed abnormalities in two, suggestive of sensorimotor neuropathy. Scalp EEG (n = 9) showed slowing of background activity (BGA) of varying degrees with paroxysmal bursts of seizure discharges in majority. Cranial CT scan (n = 4) revealed varying degrees of diffuse atrophy. Diagnostic brain biopsy was carried out in 11 and brain was examined at autopsy in 1 case. Histological examination revealed characteristic PAS and Luxol Fast Blue (LFB) positive, autofluorescent (AF) intracellular ceroid material, both in neurons and astrocytes in the grey matter. Electron microscopy (n = 5) revealed curvilinear (n = 4), lamellar (n = 2) and electron dense (n = 2) inclusions in neurons, astrocytes and vascular endothelial cells. To conclude, this neurodegenerative disease had varied but characteristic clinical presentations and required histopathological confirmation of diagnosis BEA Trading Ltd. Published by Elsevier Ltd. All rights reserved. Key words: neuronal ceroid lipofuscinosis (NCL); infantile NCL; juvenile NCL; Kuf s variant. INTRODUCTION The neuronal ceroid lipofuscinosis (NCL) represents one of the common progressive neurodegenerative disorders during childhood in which the exact chemical nature of storage material and primary enzyme defect are still being defined 1, 3. The term NCL was coined by Zeman et al. to distinguish the familial cerebromacular degeneration clinically and pathologically distinctive from gangliosidoses 3. The disease group has an autosomal recessive pattern of inheritance and is commonly characterised by progressive myoclonic epilepsy with visual failure and accumulation of an autofluorescent lipopigment in the neurons and glial elements 1, 3 5. The recognised clinico-pathological forms of NCL are (1) infantile, (2) late infantile, (3) early juvenile, (4) juvenile, (5) adult, and (6) Finnish variant of late infantile form 5 7. The diagnosis is established by characteristic histopathological and ultrastructural features in brain and various other extracerebral tissues 1, 3, 8. Though it has characteristic clinico-pathological features, there are only two published case reports from India 14, 15. The aim of the present study is to review the presenting manifestations and clinical features and to review the histopathological characteristics in a series of neuronal ceroid lipofuscinosis cases seen at one center in South India /$ BEA Trading Ltd. Published by Elsevier Ltd. All rights reserved.

2 236 S. Sinha et al. MATERIALS AND METHODS The case records of 12 patients of neuronal ceroid lipofuscinosis (NCL) confirmed pathologically obtained from the medical records were reviewed to evaluate the clinical, electrophysiological and pathological characteristics. The family history and the clinical manifestations of the disease were carefully noted. CSF results were available in 10 patients. Scalp EEG (n = 9), nerve conduction studies (n = 4), cranial CT scan (n = 4) findings were noted. Histopathological study of brain Eleven patients underwent diagnostic right frontal brain biopsy after obtaining the informed consent. In addition partial autopsy confined to the brain was performed in one patient who succumbed to the illness. One half of the tissue was fixed in 10% of buffered neutral formalin for light microscopy (LM) and the other half in 3% gluteraldehyde for electron microscopic (EM) studies. Paraffin embedded sections from each case were routinely stained with Haematoxylin and Eosin (H & E), Luxol Fast Blue (LFB) and Periodic Acid Schiff (PAS) to identify the stored lipofuscin pigments. The unstained sections were examined under fluorescent microscopy by epi-illumination (HBO 50, DMRB, Leica) to record autofluorescent (AF) nature of the intracellular adventitious material. For EM study, the grey and white matter from the biopsied tissue, (fixed earlier) were processed separately. The ultra-thin sections were stained with uramyl acetate/lead citrate and viewed under JEOL 100 CX at 60 kv 16. RESULTS The age of the patients at the time of presentation ranged from 2 to 45 years (mean 12.6 ± 14.3 years, median 7 years). There were eight male and four fe- male patients. The mean age of onset of symptoms was 10.8 ± 14.5 years (median 4.75 years). The duration of illness ranged from 1 to 60 months (21.8 ± 18.3 months). Five patients (33%) had positive family history with autosomal recessive pattern of Mendelian inheritance. Consanguineous parentage was noted in five (41.6%) and two among them had positive family history. Based on the age of onset of illness, three clinical forms could be categorised infantile NCL = 2 cases (mean age 1.25±0.4 years), juvenile NCL = 8 cases (mean age 5.6 ± 9 years) and adult NCL = 2 cases (mean age 41.5±2.1 years). The presenting clinical features in infantile NCL group were regressed milestones, chorea, seizures and myoclonus, while juvenile NCL patients had regression of acquired milestones (100%) followed by chorea and choreoathetosis (3/8), seizure and myoclonus (3/8) and ataxia (3/8). The two cases with adult variant of NCL presented with abnormal behaviour and extra pyramidal features, one of them in addition had pyramidal signs, ataxia and dementia (Table 1). Only one case of infantile NCL had primary optic atrophy. The patients in juvenile group had ophthalmic abnormalities in the form of primary optic atrophy (6/8), macular degeneration (3/8) and retinitis pigmentosa (1/8). The adult forms did not have any ophthalmic abnormality. Cranial CT scan (n = 4) revealed severe diffuse atrophy in three patients and mild to moderate diffuse atrophy in one. CSF examinations carried out in 10 patients revealed mean cell count of 2 mm 3 ; the CSF protein was raised in one case of juvenile NCL (220 mg%) and in one adult NCL case (114 mg%). Scalp EEG was abnormal in majority of the patients (8/9). The background activity (BGA) revealed paucity of alpha waves (n = 8) with varying degree of diffuse slowing. Paroxysmal generalised bursts of spike and wave (n = 4), polyspikes and wave (n = 3) and or or bursts of slow waves (n = 2) were observed. Nerve conduction studies carried out in four were abnormal in two (50%). One had decreased compound muscle action potential (CMAP) when recorded over right Table 1: Clinical manifestations in NCL. Features INCL (n = 2) JNCL (n = 8) ANCL (n = 2) Total (n = 12) (%) Regressed milestones (91.6) Seizures (83.3) Myoclonus (41.6) GTCS (33.3) Partial seizures 1 1 (8.3) Involuntary movements (75) Visual abnormalities (66.6) Pyramidal signs (58.3) Ataxia (33.3) Dementia (16.6) Abnormal behaviour 2 2 (16.6)

3 NCL 237 Fig. 1: Microphotograph showing granules within the neurons and astrocytes exhibiting autofluorescence; this feature is diagnostic (AF 480). extensor digitorum brevis (EDB) and decreased sensory nerve action potential (SNAP) when recorded over right sural nerve while the second patient had increased distal latency of 9.0 millliseconds (ankle to EDB). On histopathological examination, the brunt of the disease was noted in the grey matter with relatively unaffected white matter. There was evidence of variable degree of focal neuronal loss in the superficial layers, while in the deeper layer neuronal swelling was noted. In the zones of neuronal depletion and swelling, mild to moderate reactive astrocytosis with prominent processes was noted. Most of the neurons and reactive astrocytes have intracytophasic LFB positive, autofluorescent ceroid lipofuscin material displacing the Nissl s substance (Fig. 1). Similar material was also noted in the neuropil as free granules, infiltrating granules and occasionally in endothelial cells. Ultrastructural studies carried out in five cases revealed characteristic inclusions within the neuronal cytoplasm. Such inclusions were also noted in astrocytes and endothelial cells. They were curvilinear inclusion in four, lamellar inclusions in two and electron dense inclusions in another two cases (Fig. 2). Four patients were followed up regularly from 3 months to 4 years (18.7 ± 19.9 months). Patients with seizure and myoclonus were treated with antiepileptic drugs. One adult onset NCL patient who had a protracted course died in the hospital after 3 months of illness. One of the juvenile NCL patient had a follow up of 4 years during which period his jerks were not controlled with anticonvulsants and developed progressive loss of acquired milestones and optic atrophy. DISCUSSION NCL was more frequently seen among males (M:F 2:1). The mean duration of illness prior to presentation was 22 months. Similar data had been reported in literature 3 5, 7. Three clinical forms of NCL could be identified in the present study i.e. infantile NCL (n = 2), juvenile NCL (n = 8) and adult NCL (n = 2). Most of the literature revealed juvenile NCL to be the commonest 3. However, Sjogren (1931) and Raynes (1962) had reported equal occurrence of juvenile NCL and late infantile NCL 3. The adult variety of NCL remarks to be a rare entity in most series as in the present one 3, 4, 7. To the best of our knowledge, this is the first series of NCL from India. In the present series neuro-ophthalmic abnormalities were seen in two thirds of cases with primary optic atrophy as the commonest abnormality 4, 7 followed by macular degeneration (n = 4) and retinitis

4 238 S. Sinha et al. Fig. 2: Electron micrograph showing characteristic curvilinear inclusions (marked as C ) within the neuronal cytoplasm ( ). pigmentosa (n = 1). None of the adult NCL patients had any ophthalmological findings. Zeman et al. had found pigmentary changes in 25, macular degeneration in 12 and optic atrophy in 5 patients giving clue to the diagnosis when associated with regression of milestones and myoclonus 3. Both adult NCL patients in this study had manifested in early part of the fifth decade. One of these adult NCL had been reported earlier 17. The oldest published case of NCL in literature was at the age of 63 years 18. The two Kuf s adult variant cases in our series could be categorised into type B with predominant behavioural changes, dementia, ataxia and rigidity as described in , 19. Since long-term follow up is not available in our series, it is difficult to comment on the natural history. Generally the infantile and late infantile NCL have a rapid course and child usually dies within 5 10 and 12 years, respectively. In juvenile NCL death occurs by years, while the adult group has a variable course 3 5. The response to anticonvulsants has been unsatisfactory 3, 4, 7. The juvenile NCL may have circulating vacuolated lymphocytes in 10 50% of smears 3, 4, 7. The peripheral blood film did not reveal any vacuolated lymphocytes in this series. The significance of raised CSF protein noted in two of our cases remains unexplained and has not been reported in the literature. MRI of brain in these patients reveals diffuse cerebral and cerebellar atrophy and it tends to increase with disease progression 21. MRI could not be performed in this study. The cerebral atrophy noted in the cranial CT scan had been observed to worsen as the disease progresses on serial neuroimaging studies Nerve conduction studies are usually within normal limits though subclinical peripheral neuropathy had been reported 2, 3. Nerve conduction performed in our patient showed features of neuropathy in two 4, 7. Photic response in EEG is characteristically reported with low frequency stimulation in NCL 3, 21, 25. However, this was not noticed in any of our cases in this series as in any other form of progressive myoclonic epilepsy reported from India 8. The cornerstone in the diagnosis of NCL is histopathological examination of the brain tissues with demonstration of autofluorescence exhibited by the lipopigments 3, 5. This was evident in all of our cases. Apart from brain, extra cerebral tissues may also show inclusions, for example, lymphocytes, skin, rectal mucosa, peripheral nerves 1, 5, The sensitivity of finding inclusions in these extracerebral tissues, which is easily accessible, is variable. We did not examine these peripheral tissues in our study and hence cannot be commented. Electron microscopy in our

5 NCL 239 series revealed that the inclusions were in neuronal cytoplasm (100%) and endothelial cell (80%). The commonest type of inclusion material was curvilinear inclusion seen mainly in juvenile NCL patients. Recently, based on the occurrence of morphological forms of lymphocytic inclusions in electro microscopy (EM), NCL had been further categorised as (1) infantile NCL granular bodies/grods, (2) late infantile NCL curvilinear bodies, (3) juvenile NCL finger print bodies, and (4) adult onset NCL with varied forms and combination of inclusions 1, 5. However, in the present study characteristic fingerprint or GRODs inclusions were not seen and instead curvilinear inclusions were seen in JNCL cases. A combination of fingerprint and curvilinear inclusions in JNCL cases had been documented in other studies 29. Overlapping of morphological features of inclusions, formation of inclusions of similar morphologies in different stages of disease can lead to difficulties in diagnosis by ultrastructural studies 30. Recently, the genotypes and the chromosomal locus had been determined in the various subtypes of NCL and in some of them the gene products have also been identified 9 11 (Table 2). Attempts are made to understand the pathogenesis by finding the primary abnormality and assist in the prenatal and heterozygote detection for counselling 1, 12, 13. Genetic analysis could not be done in the present series, as facilities for this are not available in India. However, in future if genetic analysis is done the genetic classification can become the method of choice along with the possible gene therapy. To conclude, a clinical diagnosis of NCL should be considered in all children with characteristic clinical features of progressive myoclonic epilepsy and visual failure and could be confirmed by histopathological examination. Less invasive extracerebral tissue biopsies of skin, rectal mucosa, peripheral nerves could be helpful and its role needs to be further evaluated in larger number of cases. Electron microscopic stud- Table 2: Genetic classification of NCL a. Clinical types Genetic types Chromosomal loci Gene product INCL CLN1 Ip32 Lysosomal palmitoyl Protein thioesterase LINCL CLN2 11p15 Lysosomal pepstatin Insensitive peptidase LINCL CLN5 13q31-32 Not known JNCL CLN3 16p12 Transmembranous (?) ANCL CLN4 Not known Not known EJNCL CLN6 15q21-23 Not known INCL = infantile NCL, LINCL = late infantile NCL, JNCL = juvenile NCL, ANCL = adult NCL, EJNCL = early juvenile NCL. a Adapted from Goebel and Sharp 1. ies are definitely complementary to further classify NCL according to the characteristic forms of inclusions. However, the main focus should be on the genetic studies of the NCL, which is also useful in prenatal diagnosis, genetic counselling and possible gene therapy in near future. REFERENCES 1. Goebel, H. H. and Sharp, J. D. The neuronal ceroid lipofuscinoses recent advances. Brain Pathology 1998; 8: Gutteridge, J. M. C., Westermarck, T. and Santavuori, P. Iron and oxygen radicals in tissue damage implication for the NCL. Acta Neurologica Scandinavica 1983; 6: Zeman, W., Donanne, S., Dyken, P. and Green, J. The NCL (Batten-Vogt syndrome). In: Handbook of Neurology, Vol. 10 (Eds P. J. Vinken and G. W. Bruyn). Amsterdam, North Holland Publishers, 1970: pp Swick, H. M. 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