120 onset (late infantile) and having speciWc neurological symptoms and electrophysiological Wndings [24]. There are only three reports referring to variants with protracted courses [11, 19, 25]. This is only the fourth report of atypi- cal CLN2 having one novel mutation in the CLN2/TPP1 gene and diVering clinically from previously published cases with respect to the absence of epilepsy. It indicates that the phenotypic spectrum of this type of NCL may be unexpectedly broad. It is also the Wrst neuropathology report showing diVerent sensitivities of various neuronal systems to TPP1 deWciency. Clinical history A boy with an unremarkable family history (mother healthy, father treated for diabetes, one healthy sister) was born in 1960. The Wrst problem reported by his parents was at the age of 3 years when the child experienced diYculty in trying to run. According to the parents, the child was quite clumsy and awkward. Nevertheless, at this age, he was able to feed himself using cutlery. He spoke in sen- tences, but with a clear lisp; speech therapy started at age 4. The boy’s intellect was above average. During kindergarten he exhibited behavioral problems and had diYculty in com- municating with other children. He detested noise (vacuum cleaners, washing machines) and was afraid of them. He was unable to learn to ride a bicycle during his preschool years. An investigation for regression of motor functions and mental diYculties was undertaken by neurologists and psychologists during the Wrst class of primary school; a slight cerebral palsy with normal intellect was diagnosed. The child’s ability to draw corresponded to the age of 3 years. At the age of 7 years, following a measles vaccina- tion, his condition worsened. He was sent to hospital at the age of 8 years for thorough investigations. Routine bio- chemical tests of the blood, urine, and cerebrospinal Xuid revealed nothing abnormal. The EEG revealed diVuse abnormalities with a parieto-occipital maximum. Episodic high-voltage, slow waves were induced by hyperventila- tion. A neurologist found paleocerebellar, neocerebellar, and extrapyramidal syndromes and dementia. Vision was 7/ 18, eye grounds were normal and skull X-rays were normal. Mental deterioration, speech, and motor diYculties pro- gressed gradually and over time, and eventually total blind- ness and spasticity developed. Therapy was limited to Chlorpromazine (2 £ 10 mg) and to vitamin B12. After several years, he became wheelchair bound and shortly thereafter, he became bedridden. The patient never experi- enced myoclonic or any other types of convulsions. He died at the age of 15 years (1975) in extreme cachexia from aspiration bronchopneumonia while residing in a social care institution. 123 Acta Neuropathol (2008) 116:119–124 The autopsy revealed pronounced cerebral and cerebellar atrophy (850 g). The substantia nigra was unpigmented. Materials and methods Samples of central nervous system and visceral organs (heart, kidney, liver, spleen, and lungs) were Wxed in 10% formalin and processed for histology and after osmiWcation for electron microscopy. For histology, the sections were stained using a battery of techniques used for analysis of tissues in neuronal ceroid lipofuscinoses [4, 7, 8]. In our review of the archived tissue samples, cathepsin D was detected with rabbit polyclonal antibodies (DAKO, Copen- hagen, Denmark; dilution 1:2,000 or 4,000). Subunit c of mitochondrial ATP synthase (SCMAS) and tripeptidylpepti- dase 1 (TPP1) enzyme protein were also detected using anti- bodies kindly supplied by Dr. Ezaki (Juntedo University, Tokyo, Japan). After dewaxing, the sections were incubated with the primary antibodies (diluted 1:100–200) overnight in the refrigerator, washed and exposed to the appropriate Envision™+kit (DAKO, Copenhagen, Denmark). DNA extraction from the archived paraYn blocks of pro- band’s spleen, brain and heart was unsuccessful. Total geno- mic DNA from proband’s parents was isolated from Ficoll separated lymphocytes using the QIAamp Blood Mini Kit. Total RNA was isolated from the maternal blood according to the method described [2] with Trizol lysis (Invitrogen). cDNA was prepared by reverse transcription using Super- Script II Reverse Transcriptase (Invitrogen) and oligo dT18 primer (Invitrogen). The entire gene-coding region (13 exons) as well as 5 and 3 untranslated regions and exon/ intron boundaries of the CLN 2 gene were examined by direct sequencing of PCR and RT/PCR products. Primers for PCR were designed according to GenBank reference sequence (NC_000011.8; NM_000391.2). PCR and RT/PCR products were prepared using thermostable DNA polymerase PPP Master Mix (Top Bio) and puriWed on 1% agarose gel by Wizard SV Gel and PCR clean-up System (Promega). PCR products were used as a template for cycle sequencing on capillary sequencer (ABI 3100-Avant Genetic Analyzer). TPP1 activity was measured in leukocytes Xuorimetri- cally using the synthetic substrate Ala-Ala-Phe-7-amido-4- methylcoumarin (Sigma A3401) accordingly [28]. Results Storage process Regardless of cell types, storage was characterized by accumulation of autoXuorescent granules, and for the most part, was maximally expressed in central neurons which