Mutations In CLN2 Research Articles

Pediatric onset neuronal ceroid lipofuscinoses: Unraveling clinical and genetic specifications.

To unravel the clinical and genetic specifications of Neuronal ceroid lipofuscinosis (NCL). This is a retrospective cross-sectional study conducted in the Department of Pediatric Neurology Children Hospital and University of Child Health Sciences, Lahore, Pakistan from March 2017 to March 2022. The primary outcome was to measure genotype-phenotype correlation by segregation of phenotypes according to genotype. The secondary outcomes included a correlation between genotype and distribution of age(s) of onset. One hundred fifty three patients clinically diagnosed with NCL underwent genetic testing and pathologic mutation was identified in 32.7% of patients. About 59.6% were male and 37.2% had an affected sibling. The median age was 5.46±1.95 years at the onset of the first symptom i.e., myoclonic seizures in 68%, and motor difficulty in 24%. Other features found were global developmental delay (56%), hypotonia (23%), visual impairment (80%), ataxia (22%), and disc pallor (56%). The most common type was CLN6 (Ceroid lipofuscinosis neuronal) (42%), CLN2 (16%) followed by CLN7 (12%). When 50 patients with recognized mutations were compared with 103 patients with no mutation, family history (p=0.049), early visual loss (p=0.016), hypotonia (p=0.001), white matter signals (p=0.026) and pan-atrophy(p=0.047) was statistically significant in the genetically confirmed NCL. Multiple pairwise comparisons indicated that the estimated age of onset for the CLN1 and CLN2 mutation group was significantly lower than other genotypes including CLN6 (p 0.012), CLN10 (p 0.007) and CLN12 (p 0.007). Following a detailed review of NCL symptomatology, a clinically-oriented approach should be used for a rapid diagnosis with confirmation by targeted molecular testing for future genetic counseling.

Enzyme replacement therapy attenuates disease progression in a canine model of late-infantile neuronal ceroid lipofuscinosis (CLN2 disease).

Using a canine model of classical late-infantile neuronal ceroid lipofuscinosis (CLN2 disease), a study was conducted to evaluate the potential pharmacological activity of recombinant human tripeptidyl peptidase-1 (rhTPP1) enzyme replacement therapy administered directly to the cerebrospinal fluid (CSF). CLN2 disease is a hereditary neurodegenerative disorder resulting from mutations in CLN2, which encodes the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Infants with mutations in both CLN2 alleles develop normally but in the late-infantile/early-childhood period undergo progressive neurological decline accompanied by pronounced brain atrophy. The disorder, a form of Batten disease, is uniformly fatal, with clinical signs starting between 2 and 4 years of age and death usually occurring by the early teenage years. Dachshunds homozygous for a null mutation in the canine ortholog of CLN2 (TPP1) exhibit a similar disorder that progresses to end stage at 10.5–11 months of age. Administration of rhTPP1 via infusion into the CSF every other week, starting at approximately 2.5 months of age, resulted in dose-dependent significant delays in disease progression, as measured by delayed onset of neurologic deficits, improved performance on a cognitive function test, reduced brain atrophy, and increased life span. Based on these findings, a clinical study evaluating the potential therapeutic value of rhTPP1 administration into the CSF of children with CLN2 disease has been initiated.

A Rare Case Report of Siblings with Infantile and Late Infantile Forms of Neuronal Ceroid Lipofuscinoses

Neuronal Ceroid Lipofuscinoses is a neurodegenerative disorder predominantly involving grey matter. The 4 classic forms are Infantile type, Late Infantile type, Juvenile type and Adult type. We present a case of 3 siblings of unrelated parents where the eldest had Infantile form of NCL, the second child had Late Infantile Form, and the third child is normal. Both children having different phenotypic presentations have curvilinear inclusion bodies on EM of axillary skin biopsy, low levels of tripeptidyl amino peptidase 1(TPP1) and normal levels of palmitoyl protein thioesterase 1(PPT1), a finding classically present in the Late Infantile variety involving CLN2 mutation. After extensive search we failed to find a case report describing a similar finding. DOI: http://dx.doi.org/10.3126/jnps.v33i3.7924 J. Nepal Paediatr. Soc. 2013;33(3):220-222

Molecular epidemiology of childhood neuronal ceroid-lipofuscinosis in Italy

BackgroundTo review the descriptive epidemiological data on neuronal ceroid lipofuscinoses (NCLs) in Italy, identify the spectrum of mutations in the causative genes, and analyze possible genotype-phenotype relations.MethodsA cohort of NCL patients was recruited through CLNet, a nationwide network of child neurology units. Diagnosis was based on clinical and pathological criteria following ultrastructural investigation of peripheral tissues. Molecular confirmation was obtained during the diagnostic procedure or, when possible, retrospectively.ResultsOne hundred eighty-three NCL patients from 156 families were recruited between 1966 and 2010; 124 of these patients (from 88 families) were tested for known NCL genes, with 9.7% of the patients in this sample having not a genetic diagnosis. Late infantile onset NCL (LINCL) accounted for 75.8% of molecularly confirmed cases, the most frequent form being secondary to mutations in CLN2 (23.5%). Juvenile onset NCL patients accounted for 17.7% of this cohort, a smaller proportion than found in other European countries. Gene mutations predicted severe protein alterations in 65.5% of the CLN2 and 78.6% of the CLN7 cases. An incidence rate of 0.98/100,000 live births was found in 69 NCL patients born between 1992 and 2004, predicting 5 new cases a year. Prevalence was 1.2/1,000,000.ConclusionsDescriptive epidemiology data indicate a lower incidence of NCLs in Italy as compared to other European countries. A relatively high number of private mutations affecting all NCL genes might explain the genetic heterogeneity. Specific gene mutations were associated with severe clinical courses in selected NCL forms only.

Late Infantile Neuronal Ceroid Lipofuscinosis and Dopamine Deficiency

Neuronal ceroid lipofuscinosis is a severe neurodegenerative lysosomal storage disorder. Gamma-aminobutyric acid and glutamate deficiency have been reported with CLN1, CLN3, and CLN6. Isolated biopterin/neopterin without dopamine deficiency has been reported in 1 patient with a CLN2 mutation. This report describes a patient with a CLN2 mutation with symptomatic biopterin and dopamine deficiency. A 4-year-old boy presented with intractable epilepsy and developmental regression starting 1 year previously. His exam showed retinopathy, scanning speech, dysmetria, and ataxic fenestrating gait with stooped posture. Electroencephalogram showed generalized spikes with occipital spikes on slow photic stimulation. Brain magnetic resonance images 1 year apart showed significant diffuse atrophy. CLN2 gene sequencing showed pathogenic compound heterozygous mutations. Cerebrospinal fluid neurotransmitters showed low homovanillic acid and tetrahydrobiopterin. Levodopa-carbidopa resulted in dramatic improvement of gait. Dopamine/biopterin deficiency is a possible secondary manifestation of CLN2 mutations. Levodopa and dopamine agonists might be useful in treating these secondary abnormalities and improving quality of life in these patients.

Late Infantile Neuronal Ceroid Lipofuscinosis: A New Mutation in Arabs

The neuronal ceroid lipofuscinoses are a group of dominant neurodegenerative, progressive, and fatal disorders characterized clinically by myoclonic epilepsy, in variable association with dementia, ataxia, and visual loss. Neuronal ceroid lipofuscinoses were classified into several phenotypes according to their age of onset: infantile, late infantile, juvenile, and adult. A specific phenotype was named "northern epilepsy," and its onset of signs occurs between ages 5-10 years. Deficiencies in the lysosomal activity of two specific enzymes were found in several types of neuronal ceroid lipofuscinosis: palmitoyl-protein thioesterase 1, encoded by the CLN1 gene, and tripeptidyl-peptidase 1, encoded by the CLN2 gene. Several mutations in CLN2 were described previously. We describe a novel mutation in two siblings of Israeli-Arab origin, with a clinical picture compatible with late infantile neuronal ceroid lipofuscinosis. Both siblings were found to be homozygous for a deletion of a C nucleotide at position 775 in exon 7 of the CLN2 gene. These findings have implications for the worldwide epidemiology of neuronal ceroid lipofuscinosis.

The clinical and genetic epidemiology of neuronal ceroid lipofuscinosis in Newfoundland

The neuronal ceroid lipofuscinoses (NCLs) are the commonest neurodegenerative disorders of children. The aims of this study were to determine the incidence of NCL in Newfoundland, identify the causative genes, and analyze the relationship between phenotype and genotype. Patients with NCL diagnosed between 1960 and 2005 were ascertained through the provincial genetics and pediatric neurology clinics. Fifty-two patients from 34 families were identified. DNA was obtained from 28/34 (82%) families; 18 families had mutations in the CLN2 gene, comprising five different mutations of which two were novel. One family had a CLN3 mutation, another had a novel mutation in CLN5, and five families shared the same mutation in CLN6. One family was misdiagnosed, and in two, molecular testing was inconclusive. Disease from CLN2 mutations had an earlier presentation (p = 0.003) and seizure onset (p < 0.001) compared with CLN6 mutation. There was a slower clinical course for those with CLN5 mutation compared with CLN2 mutation. NCL in Newfoundland has a high incidence, 1 in 7353 live births, and shows extensive genetic heterogeneity. The incidence of late infantile NCL, 9.0 per 100,000 (or 1 in 11,161) live births, is the highest reported in the world.

Intraventricular Enzyme Replacement Improves Disease Phenotypes in a Mouse Model of Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL) is an autosomal recessive neurodegenerative disease caused by mutations in CLN2, which encodes the lysosomal protease tripeptidyl peptidase 1 (TPP1). LINCL is characterized clinically by progressive motor and cognitive decline, and premature death. Enzyme-replacement therapy (ERT) is currently available for lysosomal storage diseases affecting peripheral tissues, but has not been used in patients with central nervous system (CNS) involvement. Enzyme delivery through the cerebrospinal fluid is a potential alternative route to the CNS, but has not been studied for LINCL. In this study, we identified relevant neuropathological and behavioral hallmarks of disease in a mouse model of LINCL and correlated those findings with tissues from LINCL patients. Subsequently, we tested if intraventricular delivery of TPP1 to the LINCL mouse was efficacious. We found that infusion of recombinant human TPP1 through an intraventricular cannula led to enzyme distribution in several regions of the brain of treated mice. In vitro activity assays confirm increased TPP1 activity throughout the rostral-caudal extent of the brain. Importantly, treated mice showed attenuated neuropathology, and decreased resting tremor relative to vehicle-treated mice. This data demonstrates that intraventricular enzyme delivery to the CNS is feasible and may be of therapeutic value.

Timing of Therapeutic Intervention Determines Functional and Survival Outcomes in a Mouse Model of Late Infantile Batten Disease

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a monogenic disorder caused by the loss of tripeptidyl peptidase 1 (TPP1) activity as a result of mutations in CLN2. Absence of TPP1 results in lysosomal storage with an accompanying axonal degeneration throughout the central nervous system (CNS), which leads to progressive neurodegeneration and early death. In this study, we compared the efficacies of pre- and post-symptomatic injections of recombinant adeno-associated virus (AAV) for treating the cellular and functional abnormalities of CLN2 mutant mice. Intracranial injection of AAV1-hCLN2 resulted in widespread human TPP1 (hTPP1) activity in the brain that was 10-100-fold above wild-type levels. Injections before disease onset prevented storage and spared neurons from axonal degeneration, reflected by the preservation of motor function. Furthermore, the majority of CLN2 mutant mice treated pre-symptomatically lived for at least 330 days, compared with a median survival of 151 days in untreated CLN2 mutant controls. In contrast, although injection after disease onset ameliorated lysosomal storage, there was evidence of axonal degeneration, motor function showed limited recovery, and the animals had a median lifespan of 216 days. These data illustrate the importance of early intervention for enhanced therapeutic benefit, which may provide guidance in designing novel treatment strategies for cLINCL patients.

Neurological deterioration in late infantile neuronal ceroid lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL) is associated with progressive degeneration of the brain and retina starting in early childhood. Thirty-two individual neurologic, ophthalmologic, and CNS imaging (MRI and MRS) assessments of 18 children with LINCL were analyzed. Disease severity was followed by two rating scales, one previously established but modified to solely assess the brain and exclude the retinal disease (modified Hamburg LINCL scale), and a newly developed scale, with expanded evaluation of the CNS impairment (Weill Cornell LINCL scale). For the 18 children, the Weill Cornell scale yielded a closer correlation with both age and time since initial clinical manifestation of the disease than did the modified Hamburg scale. There were no significant differences as a function of age or time since initial manifestation of the disease in the rating scales among the most frequent CLN2 mutations (G3556C, 56% of all alleles or C3670T, 22% of all alleles). Measurements of cortical MRS N-acetyl-aspartate content, MRI ventricular, gray matter and white matter volume, and cortical apparent diffusion coefficient correlated to a variable degree with the age of the children and the time since initial clinical manifestation of the disease. All imaging measurements correlated better with the Weill Cornell CNS scale compared to the modified Hamburg LINCL scale. The data suggest that the Weill Cornell late infantile neuronal ceroid lipofuscinosis (LINCL) scale, together with several of the MRI measurements, may be useful in the assessment of severity and progression of LINCL and for the evaluation of novel therapeutic strategies.

Homogeneous PCR nucleobase quenching assays to detect four mutations that cause neuronal ceroid lipofuscinosis: T75P and R151X in CLN1, and IVS5-1G > C and R208X in CLN2

The neuronal ceroid lipofuscinoses (NCLs) are a family of autosomal recessive lysosomal storage diseases characterized by progressive epilepsy, dementia and visual loss. The juvenile form of the disease (onset age 4–8 years with visual loss) is usually caused by mutations in the CLN3 gene, but some cases have been shown to be due to specific mutations in the CLN1 or CLN2 genes, which are usually associated with NCL with onset in infancy or late infancy, respectively. The CLN1 mutations T75P and R151X, and the CLN2 mutations R208X and IVS5-1G > C, are found in many NCL patients with a juvenile presentation that is not due to CLN3 mutation. We have developed and validated a set of assays for these mutations using PCR followed by differential melting of a fluorescently labeled oligo probe, on a Roche LightCycler platform. The nucleobase quenching phenomenon was used to detect probe hybridization. The tests were validated using alternate assays: PCR followed by allele specific restriction enzyme digestion for the CLN1 mutations, and PCR followed by sequencing for the CLN2 mutations. The homogeneous PCR method gave 100% concordance of results with the alternate methods. This new assay, combined with a test for the common 1 kbp deletion in the CLN3 gene, provides a set of DNA-based assays suitable for detection of the most common mutations causing NCL with onset in the juvenile age range.

A frame shift mutation in canine TPP1 (the ortholog of human CLN2) in a juvenile Dachshund with neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage diseases characterized by progressive neuropathy and the accumulation of autofluorescent cytoplasmic granules. Clinical signs of a new canine NCL began in a 9-month-old male Dachshund with vomiting, mental dullness, and loss of previously learned commands and rapidly progressed to include disorientation, ataxia, visual deficits, generalized myoclonic seizures, and death at 12 months of age. Neurons throughout the CNS contained autofluorescent storage granules that stained with periodic acid-Schiff and Luxol fast blue stains. Electron microscopy revealed that the storage granule contents consisted of curvilinear-appearing material characteristic of human late infantile NCL caused by CLN2 mutations. Nucleotide sequence analysis of canine TPP1, the ortholog of human CLN2, revealed a single nucleotide deletion in exon 4 which predicted a frame shift with a premature stop codon. Brain tissue from the affected dog lacked detectable activity of the tripeptidyl-peptidase enzyme encoded by TPP1, whereas the specific activities of 15 other lysosomal enzymes were higher than those in the brains of three control dogs. The affected Dachshund was homozygous for the mutant c.325delC allele, his sire and dam were heterozygotes, and 181 unrelated dogs, including 77 Dachshunds, were all homozygous for the wild-type allele. A DNA assay that detects the mutant allele will help Dachshund breeders avoid producing affected puppies in future generations. Furthermore, this Dachshund NCL may prove to be a useful model for studying the pathogenesis of neurodegeneration in human late infantile NCL and for evaluating novel therapeutic interventions for this disease.

Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1' and P2' residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.

Intracranial Delivery of CLN2 Reduces Brain Pathology in a Mouse Model of Classical Late Infantile Neuronal Ceroid Lipofuscinosis

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a lysosomal storage disorder caused by mutations in CLN2, which encodes lysosomal tripeptidyl peptidase I (TPP1). Lack of TPP1 results in accumulation of autofluorescent storage material and curvilinear bodies in cells throughout the CNS, leading to progressive neurodegeneration and death typically in childhood. In this study, we injected adeno-associated virus (AAV) vectors containing the human CLN2 cDNA into the brains of CLN2(-/-) mice to determine therapeutic efficacy. AAV2CUhCLN2 or AAV5CUhCLN2 were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age, and mice were then killed at 13 weeks after injection. Mice treated with AAV2CUhCLN2 and AAV5CUhCLN2 contained TPP1 activity at each injection tract that was equivalent to 0.5- and 2-fold that of CLN2(+/+) control mice, respectively. Lysosome-associated membrane protein 1 immunostaining and confocal microscopy showed intracellular targeting of TPP1 to the lysosomal compartment. Compared with control animals, there was a marked reduction of autofluorescent storage in the AAV2CUhCLN2 and AAV5CUhCLN2 injected brain regions, as well as adjacent regions, including the striatum and hippocampus. Analysis by electron microscopy confirmed a significant decrease in pathological curvilinear bodies in cells. This study demonstrates that AAV-mediated TPP1 enzyme replacement corrects the hallmark cellular pathologies of cLINCL in the mouse model and raises the possibility of using AAV gene therapy to treat cLINCL patients.

427. AAV2- and AAV5-Mediated CNS Delivery of Human CLN2 Reduces Lysosomal Storage in a Mouse Model of Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a neurometabolic disorder caused by mutations in CLN2, which encodes lysosomal tripeptidyl peptidase (TPP-1). Lack of TPP-1 results in intracellular accumulation of autofluorescent storage material within the CNS, neurodegeneration and death typically in childhood. In this study, we injected clinical-grade AAV vectors containing the human CLN2 cDNA into the LINCL mouse brain to determine therapeutic efficacy. A total of 3.6 |[times]| 109 genome copies of AAV2CUhCLN2 (n=5) or AAV5CUhCLN2 (n=6) were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age. At 13 weeks post-injection (19 weeks of age), mice were sacrificed and brains hemispheres were separated from each other. The left hemisphere was analyzed for TPP-1 enzyme activity, and the right hemisphere was processed for immunohistochemistry and detection of autoflourescent storage material. Brains treated with AAV2CUhCLN2 contained TPP-1 activity at each of three injection tracts equivalent to that of levels in the brains of heterozygote which are phenotypically normal. In contrast, AAV5CUhCLN2-injected brains had approximately two fold wild-type levels of enzyme activity within these tracts. Immunohistochemistry and confocal microscopy of the right hemispheres showed the presence of TPP-1 protein in the lysosomes and neuronal processes of cells at the injection sites. Compared to control animals (AAV2CUNull, sham, uninjected), there was a marked reduction of autofluorescent storage material with AAV2CUCLN2 and AAV5CUCLN2 in the injected regions which include motor cortex, thalamus, cerebellum, as well as other regions such as the striatum and the hippocampus. There was no statistical difference in the amount of autofluorescent reduction mediated by the two serotype vectors despite the higher levels of enzyme activity with AAV5CUCLN2. Behavioral testing on the rotorod showed improvement of cerebellar motor function at 8 and 10 weeks post-injection with both viral vectors. This study demonstrates that the LINCL brain is responsive to TPP-1 replacement with different AAV serotype vectors, and that heterozygote levels of enzyme activity is sufficient to reverse lysosomal pathology and partially restore behavioral function.

Functional consequence and clinical importance of distinct mutations in CLN2, the gene associated with classical late infantile ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCL) are lysosomal diseases characterized by progressive neurodegeneration resulting in loss of vision, seizures, mental regression, behavioral changes, movement disorders and premature death. One of the most frequent of these diseases is classical late infantile NCL caused by mutations in the CLN2 gene and is related to defects of lysosomal tripeptidyl peptidase I (TPP-I).

Mutations in classical late infantile neuronal ceroid lipofuscinosis disrupt transport of tripeptidyl-peptidase I to lysosomes.

Classical late infantile neuronal ceroid lipofuscinosis is an autosomal recessive disease caused by mutations in the CLN2 gene resulting in functional defects of the gene product tripeptidyl-peptidase I. This disease is associated with a progressive neurodegenerative course beginning at the age of two years with developmental stagnation, finally leading to a complete loss of motor function, vision and speech by the age of 10 years. We analyzed the functional consequences of the mutations R127Q, R208X, N286S, I287N, T353P and Q422H, which were previously identified in patients with late infantile ceroid lipofuscinosis, with regard to enzymatic activity, stability, post-translational processing and intracellular localization of tripeptidyl-peptidase I. We could not detect any translational product for the mutant R208X. We found that four missense mutations, N286S, I287N, T353P and Q422H, which are located in conserved protein regions of tripeptidyl-peptidase I, decreased the enzymatic activity dramatically, blocked processing to mature size peptidase and led to protein retention in the endoplasmatic reticulum and rapid degradation in non-lysosomal compartments. We conclude that these amino-acid substitutions induce major misfolding of the precursor peptidase and hence prevent post-translational processing and lysosomal targeting of tripeptidyl-peptidase I. In contrast, the amino-acid substitution R127Q within a non-conserved protein region did not significantly affect enzymatic activity, stability, processing and lysosomal targeting of tripetidyl-peptidase I. Thus, our functional analyses of CLN2 mutations reveal novel insight into the molecular defect underlying dysfunction of tripeptidyl-peptidase I.

Late infantile neuronal ceroid lipofuscinosis: quantitative description of the clinical course in patients with CLN2 mutations.

We examined 26 individuals with clinical and electron microscopic signs of late infantile neuronal ceroid lipofuscinosis (LINCL). In 22 cases, we found both pathogenic alleles. Sixteen patients exclusively carried either one or a combination of the two common mutations R208X and IVS5-1G > C. In the remaining cases, four missense mutations could be detected, of which R127Q, N286S, and T353P represent novel, previously not described alleles. A clinical performance score was developed by rating motor, visual, and verbal functions and the incidence of cerebral seizures in 3-month intervals during the course of the disease. A Total Disability Score was derived by summing up the single scores for motor, visual, and verbal functions. The 16 individuals with the two common mutations were grouped together (referred to as standard patients), and the 5th, 50th, and 95th centiles were calculated and graphically depicted over time. The scores for motor function and language ability dropped earliest and progressed very similarly in the standard patients. The performance curves of two children with the N286S mutation slightly diverged from the 95th centile. However, the performance curves of one patient with atypical LINCL carrying the R127Q mutation fell far beyond the 95th centile. The presented performance rating clearly and quantitatively delineates the disease course of the LINCL patients and hence offers a useful tool for clinical evaluation of future therapeutic interventions. In addition, the described performance score system can be applied to other types of neuronal ceroid lipofuscinoses and could be adapted to various other neurodegenerative diseases of childhood.

Heterogeneity of late-infantile neuronal ceroid lipofuscinosis

Late-infantile neuronal ceroid lipofuscinosis (LINCL), an autosomal recessively inherited lysosomal storage disorder characterized by autofluorescent inclusions and rapid progression of neurodegeneration, is due to CLN2 gene mutations. However, CLN2 mutation analysis has failed to identify some clinically diagnosed "late-infantile" NCL cases. This study was conducted to further characterize genetic heterogeneity in families affected by LINCL. DNA mutations in the CLN1, CLN2, and CLN3 genes that underlie INCL (infantile NCL), LINCL, and JNCL (juvenile NCL), respectively, were studied with molecular analyses. A total of 252 families affected by childhood NCL were studied. Of 109 families clinically diagnosed as having LINCL, 3 were determined to have either INCL or JNCL by identification of mutation(s) in CLN1 or CLN3. Six families diagnosed initially as having JNCL were found to have LINCL based on the finding of mutations in the CLN2 gene. In addition, several novel mutations were identified. Clinical and genetic heterogeneity of LINCL was demonstrated in nine LINCL families studied.

A novel nonsense mutation (Q509X) in three Italian late-infantile neuronal ceroid-lipofuscinosis children.

We identified a novel nonsense CLN2 mutation (Q509X) in three Italian children with classical late-infantile neuronal ceroid lipofuscinosis (LINCL) from two unrelated families. The mutation introduced a premature stop codon in exon 12 of the CLN2 gene, resulting in a protein lacking the last 54 residues. Haplotype analysis suggested independent origin of the mutation in our families. The protein truncation test was employed to verify the functional consequences of the novel Q509X mutation. In Patient 1, the mutant alleles were transcribed but translated in a shorted peptide suggesting that the Q509X mutation is likely to interfere with CLN2p function. While expanding the list of genetic variants in LINCL, our findings represent the first molecular characterization of LINCL patients in South Europe.