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Background: Neuronal Ceroid Lipofuscinosis (NCL) disorders, recognized as the primary cause of childhood dementia globally, constitute a spectrum of genetic abnormalities. CLN8, a subtype within NCL, is characterized by cognitive decline, motor impairment, and visual deterioration. This study focuses on an atypical case with congenital onset and a remarkably slow disease progression. Methods: Whole-genome sequencing at 30× coverage was employed as part of a national genomics program to investigate the genetic underpinnings of rare diseases. This genomic approach aimed to challenge established classifications (vLINCL and EPMR) and explore the presence of a continuous phenotypic spectrum associated with CLN8. Results: The whole-genome sequencing revealed two novel likely pathogenic mutations in the CLN8 gene on chromosome 8p23.3. These mutations were not previously associated with CLN8-related NCL. Contrary to established classifications (vLINCL and EPMR), our findings suggest a continuous phenotypic spectrum associated with CLN8. Pathological subcellular markers further validated the genomic insights. Discussion: The identification of two previously undescribed likely pathogenic CLN8 gene mutations challenges traditional classifications and highlights a more nuanced phenotypic spectrum associated with CLN8. Our findings underscore the significance of genetic modifiers and interactions with unrelated genes in shaping variable phenotypic outcomes. The inclusion of pathological subcellular markers further strengthens the validity of our genomic insights. This research enhances our understanding of CLN8 disorders, emphasizing the need for comprehensive genomic analyses to elucidate the complexity of phenotypic presentations and guide tailored therapeutic strategies. The identification of new likely pathogenic mutations underscores the dynamic nature of CLN8-related NCL and the importance of individualized approaches to patient management.

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BACKGROUND: The fat deposition has a crucial role in animal meat flavor, and fat deposition-related traits are vital for breeding in the commercial duck industry. Avian fat-related traits are typical complex phenotypes, which need a large amount of data to analyze the genetic loci. RESULTS: In this study, we performed a new phenotypic analysis of fat traits and genotyped whole-genome variations for 1,246 ducks, and combed with previous GWAS data to reach 1,880 ducks for following analysis. The carcass composition traits, subcutaneous fat weight (SFW), subcutaneous fat percentage (SFP), abdominal fat weight (AFW), abdominal fat percentage (AFP) and the body weight of day 42 (BW42) for each duck were collected. We identified a set of new loci that affect the traits related to fat deposition in avian. Among these loci, ceroid-lipofuscinosis, neuronal 8 (CLN8) is a novel candidate gene controlling fat deposition. We investigated its novel function and regulation in avian adipogenesis. Five significant SNPs (the most significant SNP, P-value = 21.37E-12) and a single haplotype were detected in the upstream of CLN8 for subcutaneous fat percentage. Subsequently, luciferase assay demonstrated that 5 linked SNPs in the upstream of the CLN8 gene significantly decreased the transcriptional activity of CLN8. Further, ATAC-seq analysis showed that transcription factor binding sites were identified in a region close to the haplotype. A set of luciferase reporter gene vectors that contained different deletion fragments of the CLN8 promoter were constructed, and the core promoter area of CLN8 was finally identified in the -1,884/-1,207 bp region of the 5' flanking sequences, which contains adipogenesis-related transcription factors binding sites. Moreover, the over-expression of CLN8 can remarkably facilitate adipocyte differentiation in ICPs. Consistent with these, the global transcriptome profiling and functional analysis of the over-expressed CLN8 in the cell line further revealed that the lipid biosynthetic process during the adipogenesis was significantly enriched. CONCLUSIONS: Our results demonstrated that CLN8 is a positive regulator of avian adipocyte differentiation. These findings identify a novel function of CLN8 in adipocyte differentiation, which provides important clues for the further study of the mechanism of avian fat deposition.

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BACKGROUND: CLN8-Batten disease (CLN8 disease) is a rare neurodegenerative disorder characterized phenotypically by progressive deterioration of motor and cognitive abilities, visual symptoms, epileptic seizures, and premature death. Mutations in CLN8 results in characteristic Batten disease symptoms and brain-wide pathology including accumulation of lysosomal storage material, gliosis, and neurodegeneration. Recent investigations of other subforms of Batten disease (CLN1, CLN3, CLN6) have emphasized the influence of biological sex on disease and treatment outcomes; however, little is known about sex differences in the CLN8 subtype. To determine the impact of sex on CLN8 disease burden and progression, we utilized a Cln8mnd mouse model to measure the impact and progression of histopathological and behavioral outcomes between sexes. RESULTS: Several notable sex differences were observed in the presentation of brain pathology, including Cln8mnd female mice consistently presenting with greater GFAP+ astrocytosis and CD68+ microgliosis in the somatosensory cortex, ventral posteromedial/ventral posterolateral nuclei of the thalamus, striatum, and hippocampus when compared to Cln8mnd male mice. Furthermore, sex differences in motor-behavioral assessments revealed Cln8mnd female mice experience poorer motor performance and earlier death than their male counterparts. Cln8mnd mice treated with an AAV9-mediated gene therapy were also examined to assess sex differences on therapeutics outcomes, which revealed no appreciable differences between the sexes when responding to the therapy. CONCLUSIONS: Taken together, our results provide further evidence of biologic sex as a modifier of Batten disease progression and outcome, thus warranting consideration when conducting investigations and monitoring therapeutic impact.

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The CLN8 disease type refers to one of the neuronal ceroid lipofuscinoses (NCLs) which are the most common group of neurodegenerative diseases in childhood. The clinical phenotypes of this disease are progressive neurological deterioration that could lead to seizures, dementia, ataxia, visual failure, and various forms of abnormal movement. In the current study, we describe two patients who presented with atypical phenotypic manifestation and protracted clinical course of CLN8 carrying a novel compound heterozygous variant at the CLN8 gene. Our patients developed a mild phenotype of CLN8 disease: as they presented mild epilepsy, cognitive decline, mild learning disability, attention-deficit/hyperactivity disorder (ADHD), they developed a markedly protracted course of motor decline. Bioinformatic analyses of the compound heterozygous CLN8 gene variants were carried out. Most of the variants seem likely to act by compromising the structural integrity of regions within the protein. This in turn is expected to reduce the overall stability of the protein and render the protein less active to various degrees. The cases in our study confirmed and expanded the effect of compound heterozygous variants in CLN8 disease.

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CLN8 is a ubiquitously expressed membrane-spanning protein that localizes primarily in the ER, with partial localization in the ER-Golgi intermediate compartment. Mutations in CLN8 cause late-infantile neuronal ceroid lipofuscinosis (LINCL). We describe a female pediatric patient with LINCL. She exhibited a typical phenotype associated with LINCL, except she did not present spontaneous myoclonus, her symptoms occurrence was slower and developed focal sensory visual seizures. In addition, whole-exome sequencing identified a novel homozygous variant in CLN8, c.531G>T, resulting in p.Trp177Cys. Ultrastructural examination featured abundant lipofuscin deposits within mucosal cells, macrophages, and monocytes. We report a novel CLN8 mutation as a cause for NCL8 in a girl with developmental delay and epilepsy, cerebellar syndrome, visual loss, and progressive cognitive and motor regression. This case, together with an analysis of the available literature, emphasizes the existence of a continuous spectrum of CLN8-associated phenotypes rather than a sharp distinction between them.

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BACKGROUND INFORMATION: The endo-lysosomal system (ELS) comprises a set of membranous organelles responsible for transporting intracellular and extracellular components within cells. Defects in lysosomal proteins usually affect a large variety of processes and underlie many diseases, most of them with a strong neuronal impact. Mutations in the endoplasmic reticulum-resident CLN8 protein cause CLN8 disease. This condition is one of the 14 known neuronal ceroid lipofuscinoses (NCLs), a group of inherited diseases characterised by accumulation of lipofuscin-like pigments within lysosomes. Besides mediating the transport of soluble lysosomal proteins, recent research suggested a role for CLN8 in the transport of vesicles and lipids, and autophagy. However, the consequences of CLN8 deficiency on ELS structure and activity, as well as the potential impact on neuronal development, remain poorly characterised. Therefore, we performed CLN8 knockdown in neuronal and non-neuronal cell models to analyse structural, dynamic and functional changes in the ELS and to assess the impact of CLN8 deficiency on axodendritic development. RESULTS: CLN8 knockdown increased the size of the Golgi apparatus, the number of mobile vesicles and the speed of endo-lysosomes. Using the fluorescent fusion protein mApple-LAMP1-pHluorin, we detected significant lysosomal alkalisation in CLN8-deficient cells. In turn, experiments in primary rat hippocampal neurons showed that CLN8 deficiency decreased the complexity and size of the somatodendritic compartment. CONCLUSIONS: Our results suggest the participation of CLN8 in vesicular distribution, lysosomal pH and normal development of the dendritic tree. We speculate that the defects triggered by CLN8 deficiency on ELS structure and dynamics underlie morphological alterations in neurons, which ultimately lead to the characteristic neurodegeneration observed in this NCL. SIGNIFICANCE: This is, to our knowledge, the first characterisation of the effects of CLN8 dysfunction on the structure and dynamics of the ELS. Moreover, our findings suggest a novel role for CLN8 in somatodendritic development, which may account at least in part for the neuropathological manifestations associated with CLN8 disease.

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CLN8 disease is a rare form of neuronal ceroid lipofuscinosis caused by biallelic mutations in the CLN8 gene, which encodes a transmembrane endoplasmic reticulum protein involved in trafficking of lysosomal enzymes. CLN8 disease patients present with myoclonus, tonic-clonic seizures, and progressive declines in cognitive and motor function, with many cases resulting in premature death early in life. There are currently no treatments that can cure the disease or substantially slow disease progression. Using a mouse model of CLN8 disease, we tested the safety and efficacy of an intracerebroventricularly (i.c.v.) delivered self-complementary adeno-associated virus serotype 9 (scAAV9) gene therapy vector driving expression of human CLN8. A single neonatal injection was safe and well tolerated, resulting in robust transgene expression throughout the CNS from 4 to 24 months, reducing histopathological and behavioral hallmarks of the disease and restoring lifespan from 10 months in untreated animals to beyond 24 months of age in treated animals. While it is unclear whether some of these behavioral improvements relate to preserved visual function, improvements in learning/memory, or other central or peripheral benefits, these results demonstrate, by far, the most successful degree of rescue reported in an animal model of CLN8 disease, and they support further development of gene therapy for this disorder.

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BACKGROUND: Status dystonicus is an underdiagnosed condition, probably due to heterogeneous etiology, presentation and course. Herein, we report the first case of CLN8 disease in the literature presenting with status dystonicus who responded well to pharmacological intervention. CASE: A boy aged five years and three months presented with fever, loss of appetite, intermittent excessive dystonic contractions, opisthotonus with retrocollis, and irritability for three days. His developmental milestones were reported as normal up to the age of three years and six months. At this age, he developed seizures, ataxia, and vision problems. Deterioration in developmental milestones was observed from the age of four. Laboratory tests demonstrated leukocytosis, abnormal renal function, mild metabolic acidosis, elevated creatine kinase and transaminase levels. The brain magnetic resonance imaging demonstrated cerebral and cerebellar atrophy. Homozygous missense mutation of c.709G > A (p.G237R) in the CLN8 gene was revealed. With all these clinical and laboratory findings, he was diagnosed with status dystonicus associated with CLN8 disease. Antibiotherapy, anticonvulsant drugs, and intravenous hydration with alkaline fluids were initiated. Due to irregular breathing, dysphagia, and worsening of dystonic contractions, mechanical ventilation was performed, and baclofen, haloperidol, midazolam infusion and chloral hydrate were administered, respectively. Finally, serum creatine kinase levels decreased, and dystonic contractions improved on the 15th day of hospitalization. CONCLUSION: To the best of our knowledge, our case is the first report describing the status dystonicus in a patient with CLN8 disease. Our report suggested that neuronal ceroid lipofuscinoses should be kept in mind in the etiology of status dystonicus.

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ABSTRACT Neuronal Ceroid Lipofuscinosis (NCL) refers to a group of inherited lysosomal storage disorders characterized by the intracellular accumulation of ceroid-lipofuscin compounds and neurodegeneration. Fourteen genes are currently recognized with disease-causing DNA variants: PPT1/CLN1, TPP1/CLN2, CLN3, DNAJC5/CLN4, CLN5, CLN6, MFSD8/CLN7, CLN8, CTSD/CN10, GRN/CLN11, ATP13A2/CLN12, CTSF/CLN13, KCTD7/CLN14, TBCK/CLN15. In the frame of the Cordoba cohort, we studied N=51 cases. The aim of this paper is the observational and retrospective analysis of the "atypical" phenotypes. PCR-Sanger sequencing and/or massive exome sequencing were used as a screening methodology. One CLN1 subject showed an atypical prolonged (P) phenotype with null PPT1 activity and a heterozygous compound genotype: E5 c.451C>T, p.Arg151*/g.6302T>G (I3 c.363-3T>G). Other 11 CLN2 individuals (except one girl) showed TPP1 activity decreased to around 10% of the minimum value of the reference interval in leukocytes and saliva. The DNA variants E7 c.827A>T, p.Asp276Val and I7 c.887-10A>G were the most prevalent. One CLN8 individual showed an atypical congenital phenotype with a heterozygous combination of DNA variants: E2 c.1A>G, p.?/E3 c.792C>G, p.Asn264Lys. Massive sequencing was installed as a screening methodology for the precision diagnosis of atypical CLN1, CLN2, and CLN8 phenotypes. A genetic/phenotypic local registry is under construction.

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BACKGROUND/OBJECTIVE: Cyp24a1-null mice deficient in 24,25(OH)2D3 display impaired callus formation during the endochondral phase of bone fracture repair. The 24,25(OH)2D3 metabolite acted by binding to the TLC domain containing 3B isoform 2 (TLCD3B2, previously named FAM57B2) effector protein, which then synthesizes lactosylceramide (LacCer). Treatment with 24,25(OH)2D3 or LacCer restored callus size and mechanical properties in Cyp24a1-null mice. METHODS: To assess the safety of these molecules and test their efficacy for bone healing in wild-type, non-genetically modified mice, we treated 12-week-old, osteotomized C57BL/6 female mice with each compound for up to 21 days post-osteotomy. Control cohorts were injected with vehicle. RESULTS: Neither compound was found to exhibit any nephro- nor hepato-toxicity. Calcemia remained stable throughout the experiment and was unaffected by either treatment. Supplementation with 24,25(OH)2D3 increased circulating levels of this metabolite about 8-fold, decreased 1,25(OH)2D3 levels, and significantly increased circulating 1,24,25(OH)3D3 levels, suggesting 1?-hydroxylation of 24,25(OH)2D3. TLCD3B2 was found to be expressed in fracture callus at the surface of unmineralized or pre-mineralized cartilage on day 10 and day 12 post-osteotomy and to progressively recede to become undetectable by day 18. Treatment with 24,25(OH)2D3 or LacCer reduced the number of TLCD3B2-positive cells. Both treatments also significantly increased stiffness and elastic modulus of the healing bone callus. CONCLUSION: Exogenous administration of 24,25(OH)2D3 or LacCer improved the biomechanical properties of repaired bones in wild-type animals without affecting circulating calcium levels or other blood parameters, demonstrating preclinical safety and efficacy. TRANSLATIONAL POTENTIAL: Our data suggest the use of 24R,25-dihydroxyvitamin D3 or lactosylceramide for ameliorating fracture healing in clinical practice.

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Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative disorders caused by mutations in fourteen distinct ceroid lipofuscinoses, neuronal (CLN) genes described with various severe symptoms such as seizures, visual failure, motor decline, and progressive cognitive deterioration. The current research represents novel CLN5 (c.741G > A) and CLN8 (c.565delT) mutations in two different Iranian families with late-infantile NCL (LINCL) and their relatives by using whole-exome sequencing (WES). The first family had a 10-year-old male with consanguineous parents and severe NCL symptoms, including motor clumsiness, telangiectasia, and cerebellar atrophy. The second family with a child who suffered from nystagmus rotation, motor difficulties, and seizure was a 5-year-old male with consanguineous parent. WES of probands 1 and 2 revealed homozygotic mutations in exon 4 of CLN5 (c.741G > A, p.W247X) and deletion in exon 3 (c.565delT, p.F189fs) of CLN8, respectively. Both patients' parents were heterozygous for these alterations. In concordance with previous studies, our results indicate that pathogenic mutations in CLN genes, especially CLN5 and 8, are a main cause of LINCL; these results also suggest that LINCL is not a regionally or nationally dependent disorder and can occur in any ethnic group despite the fact that some populations may be more at risk. Consequently, CLN gene screening for patients with typical signs of LINCL is recommended.

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The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN8 deficiency causes a subtype of NCL, referred to as CLN8 disease. CLN8 is an ER resident protein with unknown function; however, a role in ceramide metabolism has been suggested. In this report, we identified PP2A and its biological inhibitor I2PP2A as interacting proteins of CLN8. PP2A is one of the major serine/threonine phosphatases in cells and governs a wide range of signaling pathways by dephosphorylating critical signaling molecules. We showed that the phosphorylation levels of several substrates of PP2A, namely Akt, S6 kinase, and GSK3ß, were decreased in CLN8 disease patient fibroblasts. This reduction can be reversed by inhibiting PP2A phosphatase activity with cantharidin, suggesting a higher PP2A activity in CLN8-deficient cells. Since ceramides are known to bind and influence the activity of PP2A and I2PP2A, we further examined whether ceramide levels in the CLN8-deficient cells were changed. Interestingly, the ceramide levels were reduced by 60% in CLN8 disease patient cells compared to controls. Furthermore, we observed that the conversion of ER-localized NBD-C6-ceramide to glucosylceramide and sphingomyelin in the Golgi apparatus was not affected in CLN8-deficient cells, indicating transport of ceramides from ER to the Golgi apparatus was normal. A model of how CLN8 along with ceramides affects I2PP2A and PP2A binding and activities is proposed.

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BACKGROUND: Neuronal ceroid lipofuscinoses (NCLs) are one of the most frequent childhood-onset neurodegenerative pathologies characterized by seizures, progressive cognitive decline, motor impairment and loss of vision. For the past two decades, more than 430 variants in 13 candidate genes have been identified in the affected patients. Most of the variants were almost exclusively reported in Western patients, and very little clinical and genetic information was available for Chinese patients. CASE PRESENTATION: We report a Chinese boy whose clinical phenotypes were suspected to be NCL, including intractable epilepsy, cognitive and motor decline and progressive vision loss. Using targeted next-generation sequencing, two novel null variants in CLN8 (c.298C > T, p.Gln100Ter; c.551G > A, p.Trp184Ter) were detected in this patient in trans model. These two variants were interpreted as pathogenic according to the variant guidelines of the American College of Medical Genetics and Genomics. CONCLUSIONS: This is the first case report of NCL due to CLN8 variants in China. Our findings expand the variant diversity of CLN8 and demonstrate the tremendous diagnosis value of targeted next-generation sequencing for pediatric NCLs.

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The Neuronal Ceroid Lipofuscinoses (NCLs, Batten disease) are a group of inherited neurodegenerative disorders with variable age of onset, characterized by the lysosomal accumulation of autofluorescent ceroid lipopigments. The endoplasmic reticulum (ER) is a critical organelle for normal cell function. Alteration of ER homeostasis leads to accumulation of misfolded protein in the ER and to activation of the unfolded protein response. ER stress and the UPR have recently been linked to the NCLs. In this review, we will discuss the evidence for UPR activation in the NCLs, and address its connection to disease pathogenesis. Further understanding of ER-stress response involvement in the NCLs may encourage development of novel therapeutical agents targeting these pathogenic pathways.

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BACKGROUND: The presence of deletions can complicate genetic diagnosis of autosomal recessive disease. METHOD: The DNA of patients was analyzed in a diagnostic setting. RESULTS: We present three unrelated patients each carrying deletions that encompass the 37 kb CLN8 gene and discuss their phenotype. Two of the cases were hemizygous for a mutant allele - their deletions unmasked a mutation in CLN8 on the other chromosome. CONCLUSION: Microarray analysis is recommended in any patient suspected of NCL who is apparently homozygous for a mutation that is not present in one of the parents or when the family has no known consanguinity.

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Neuronal ceroid lipofuscinosis (NCL), one of the most common neurodegenerative childhood-onset disorders, is characterized by autosomal-recessive inheritance, epileptic seizures, progressive psychomotor deterioration, visual impairment, and premature death. Based on the country of origin of the patients, the clinical features/courses, and the molecular genetics background of the disorder, 14 distinct NCL subtypes have been described to date. CLN8 mutation was first identified in Finnish patients, and the condition was named Northern Epilepsy (NE); however, the severe phenotype of the CLN8 gene was subsequently found outside Finland and named 'variant late-infantile' NCL. In this study, five patients and their six healthy relatives from a large Turkish consanguineous family were enrolled. The study involved detailed clinical, radiological and molecular genetic evaluations. Whole-exome sequencing and homozygosity mapping revealed a novel homozygous CLN8 mutation, c.677T>C (p.Leu226Pro). We defined NE cases in Turkey, caused by a novel mutation in CLN8. WES can be an important diagnostic method in rare cases with atypical courses.

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Neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage diseases that have been described in a variety of dog breeds, where they are caused by different mutations in different genes. However, the causative gene defect in the breed Alpenländische Dachsbracke remained unknown so far. Here we present two confirmed cases of NCL in Alpenländische Dachsbracke dogs from different litters of the same sire with a different dam harboring the same underlying novel mutation in the CLN8 gene. Case 1, a 2-year-old male Alpenländische Dachsbracke was presented with neurological signs including disorientation, character changes including anxiety states and aggressiveness, sudden blindness and reduction of food intake. Magnetic resonance imaging (MRI) scans showed cerebral atrophy with dilation of all cerebral ventricles, thinning of the intermediate mass of the thalamus and widening of the cerebral sulci. Postmortem examination of the central nervous system (CNS) showed neuronal loss in the cerebral cortex, cerebellum and spinal cord with massive intracellular deposits of ceroid pigment. Additional ceroid-lipofuscin deposits were observed in the enteric nervous system and in macrophages within spleen, lymph nodes and lung. Ultrastructural analyses confirmed NCL with the presence of osmiophilic membrane bounded lamellar-like structures. Case 2, a 1,5-year old female Alpenländische Dachsbracke was presented with progressive generalized forebrain disease including mental changes such as fearful reactions to various kinds of external stimuli and disorientation. The dog also displayed seizures, absence of menace reactions and negative cotton-ball test with normal pupillary light reactions. The clinical and post mortem examination yielded similar results in the brain as in Case 1. Whole genome sequencing of Case 1 and PCR results of both cases revealed a homozygous deletion encompassing the entire CLN8 gene as the most likely causative mutation for the NCL form observed in both cases. The deletion follows recessive inheritance since the dam and a healthy male littermate of Case 1 were tested as heterozygous carriers. This is the first detailed description of CLN8 gene associated NCL in Alpenländische Dachsbracke dogs and thus provides a novel canine CLN8 model for this lysosomal storage disease. The presence of ceroid lipofuscin in extracerebral tissues may help to confirm the diagnosis of NCL in vivo, especially in new dog breeds where the underlying mutation is not known.

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Neuronal ceroid lipofuscinoses (NCLs) are clinically and genetically heterogeneous neurodegenerative lysosomal diseases. Fourteen distinct NCL subtypes (CLN1-CLN14) are known, and they are caused by mutations in different genes. CLN8 was first identified in Finnish patients, and the phenotype was subsequently found in Turkish, Italian, and Pakistani patients. We report a 6-year-old Japanese boy with NCL with a novel missense mutation in CLN8. At the age of 3years, he manifested frequent drop seizures, and then progressively developed motor difficulties with an ataxic gait, myoclonus, left conjugate deviation, and rotational nystagmus. At age 5, he developed profound visual difficulty and dysphagia, and he has now lost his mobility. A bone marrow examination at age 5 showed sea-blue histiocytes. An electroretinogram was non-recordable. No giant somatosensory evoked potentials were found. Brain magnetic resonance imaging revealed bilateral diffuse hyperintensities in the white matter around the lateral ventricles and cerebellar and pontine atrophy on T2-weighted images. In a lysosomal enzyme study, the palmitoyl-protein-thioesterase and pepinase activity was within normal limits. Whole-exome sequencing revealed a homozygous CLN8 mutation: c.620T>G (p.L207R). His parents were both heterozygous for this mutation. To our knowledge, this is the first report of a CLN8 mutation in late infantile NCL in Japan.

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The neuronal ceroid lipofuscinoses (NCLs) are hereditary neurodegenerative diseases characterized by seizures and progressive cognitive decline, motor impairment, and vision loss accompanied by accumulation of autofluorescent lysosomal storage bodies in the central nervous system and elsewhere in the body. Mutations in at least 14 genes underlie the various forms of NCL. One of these genes, CLN8, encodes an intrinsic membrane protein of unknown function that appears to be localized primarily to the endoplasmic reticulum. Most CLN8 mutations in people result in a form of NCL with a late infantile onset and relatively rapid progression. A mixed breed dog with Australian Shepherd and Blue Heeler ancestry developed neurological signs characteristic of NCL starting at about 8months of age. The signs became progressively worse and the dog was euthanized at 21months of age due to seizures of increasing frequency and severity. Postmortem examination of the brain and retinas identified massive accumulations of intracellular autofluorescent inclusions characteristic of the NCLs. Whole genome sequencing of DNA from this dog identified a CLN8:c.585G>A transition that predicts a CLN8:p.Trp195* nonsense mutation. This mutation appears to be rare in both ancestral breeds. All of our 133 archived DNA samples from Blue Heelers, and 1481 of our 1488 archived Australian Shepherd DNA samples tested homozygous for the reference CLN8:c.585G allele. Four of the Australian Shepherd samples tested heterozygous and 3 tested homozygous for the mutant CLN8:c.585A allele. All 3 dogs homozygous for the A allele exhibited clinical signs of NCL and in 2 of them NCL was confirmed by postmortem evaluation of brain tissue. The occurrence of confirmed NCL in 3 of 4 CLN8:c.585A homozygous dogs, plus the occurrence of clinical signs consistent with NCL in the fourth homozygote strongly suggests that this rare truncating mutation causes NCL. Identification of this NCL-causing mutation provides the opportunity for identifying dogs that can be used to establish a canine model for the CLN8 disease (also known as late infantile variant or late infantile CLN8 disease).

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