The age of human mutation: genealogical and linkage disequilibrium analysis of the CLN5 mutation in the Finnish population.

Variant late infantile neuronal ceroid lipofuscinosis (vLINCL) is an autosomal recessive progressive encephalopathy of childhood enriched in the western part of Finland, with a local incidence of 1 in 1500. We recently assigned the locus for vLINCL, CLN5, to 13q21.1-q32. In the present study, the haplotype analysis of Finnish CLN5 chromosomes provides evidence that one single mutation causes vLINCL in the Finnish population. Eight microsatellite markers closely linked to the CLN5 gene on chromosome 13q were analyzed, to study identity by descent by shared haplotype analysis. One single haplotype formed by flanking markers D13S160 and D13S162 in strong linkage disequilibrium (P < .0001) was present in 81% of disease-bearing chromosomes. Allele 4 at the marker locus D13S162 was detected in 94% of disease-bearing chromosomes. To evaluate the age of the CLN5 mutation by virtue of its restricted geographical distribution, church records were used to identify the common ancestors for 18 vLINCL families diagnosed in Finland. The pedigrees of the vLINCL ancestors merged on many occasions, which also supports a single founder mutation that obviously happened 20 to 30 generations ago (i.e., approximately 500 years ago) in this isolated population. Linkage disequilibrium was detected with seven markers covering an extended genetic distance of 11 cM, which further supports the young age of the CLN5 mutation. When the results of genealogical and linkage disequilibrium studies were combined, the CLN5 gene was predicted to lie approximately 200 - 400 kb (total range 30 - 1360 kb) from the closest marker D13S162.     

Infantile form of neuronal ceroid lipofuscinosis (CLN1) maps to the short arm of chromosome 1.

The neuronal ceroid lipofuscinoses (CLNs) are one of the most common progressive encephalopathies of childhood in Western countries. They are divided into three main types: infantile, late infantile, and juvenile. The inheritance of all forms is autosomal recessive, and the biochemical background is totally unknown. The infantile type (CLN1) demonstrates the earliest onset of symptoms and the most severe clinical course. CLN1 is enriched in the Finnish population with incidence of 1:20,000, and only about 50 cases have been reported from other parts of the world. We have collected 15 Finnish CLN1 families with one or two diseased children for a linkage analysis with polymorphic probes randomly localized on human chromosomes. After studying 42 polymorphic protein and DNA markers, we found definitive proof of linkage with three different probes on the short arm of chromosome 1, with maximum lod scores of 3.38 at theta = 0.00 (0.00-0.08) for D1S57 (pYNZ2), 3.56 at theta = 0.00 (0.00-0.09) for D1S7 (lambda MS1), and 3.56 at theta = 0.00 (0.00-0.11) for D1S79 (pCMM8). With the assignment of the CLN1 gene, our study demonstrates the power of multiallelic VNTR probes in the search for linkage of a rare recessive disorder using limited family material.     

Batten disease gene, CLN3: linkage disequilibrium mapping in the Finnish population, and analysis of European haplotypes.

The gene for Batten disease (juvenile-onset neuronal ceroid lipofuscinosis, or Spielmeyer-Sjögren disease), CLN3, maps to 16p11.2-12.1. Four microsatellite markers--D16S288, D16S299, D16S298, and SPN--are in strong linkage disequilibrium with CLN3 in 142 families from 16 different countries. These markers span a candidate region of approximately 2.1 cM. CLN3 is most prevalent in northern European populations and is especially enriched in the isolated Finnish population, with an incidence of 1:21,000. Linkage disequilibrium mapping was applied to further refine the localization of CLN3 in 27 Finnish families by using linkage disequilibrium data and information about the population history of Finland to estimate the distance of the closest markers from CLN3. CLN3 is predicted to lie 8.8 kb (range 6.3-13.8 kb) from D16S298 and 165.4 kb (132.4-218.1 kb) from D16S299. Enrichment of allele "6" at D16S298 (on 96% of Finnish and 92% of European CLN3 chromosomes) provides strong evidence that the same major mutation is responsible for Batten disease in Finland as in most other European countries and that it is therefore not a Finnish mutation. Genealogical studies show that Batten disease is widespread throughout the densely populated regions of Finland. The ancestors of two Finnish patients carrying rare alleles "3" and "5" at D16S298 in heterozygous form originate from the southwestern coast of Finland, and these probably represent other foreign mutations. Analysis of the number and distribution of CLN3 haplotypes from 12 European countries provides evidence that more than one mutation has arisen in Europe.     

A mutation in canine CLN5 causes neuronal ceroid lipofuscinosis in Border collie dogs

Neuronal ceroid lipofuscinosis (NCL) is a neurodegenerative disease found in Border collie dogs, humans, and other animals. Disease gene studies in humans and animals provided candidates for the NCL gene in Border collies. A combination of linkage analysis and comparative genomics localized the gene to CFA22 in an area syntenic to HSA13q that contains the CLN5 gene responsible for the Finnish variant of human late infantile NCL. Sequencing of CLN5 revealed a nonsense mutation (Q206X) within exon 4 that correlated with NCL in Border collies. This truncation mutation should result in a protein product of a size similar to that of some mutations identified in human CLN5 and therefore the Border collie may make a good model for human NCL. A simple test was developed to enable screening of the Border collie population for carriers so the disease can be eliminated as a problem in the breed.     

Chromosomal assignment of canine TSC2, PKD1 and CLN3 genes by radiation hybrid- and linkage analyses

The canine tuberous sclerosis 2 (TSC2) gene has been mapped to canine chromosome 6 using a canine whole genome radiation hybrid panel. There is close linkage between canine TSC2 and the polycystic kidney disease 1 gene (PKD1), as has been observed in humans and other mammalian species. The gene responsible for the human juvenile form of neuronal ceroid lipofuscinosis (CLN3), maps close to TSC2 and PKD1 in humans, and is also syntenic in the dog. We further demonstrate linkage to a group of polymorphic markers assigned to canine chromosome 6 (CFA6).     

Neuronal ceroid lipofuscinosis in Devon cattle is caused by a single base duplication (c.662dupG) in the bovine CLN5 gene

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are recessively inherited neurodegenerative disorders that affect humans and other animals, characterised by brain atrophy and the accumulation of lysosome derived fluorescent storage bodies in neurons and most other cells. Common clinical signs include blindness, ataxia, dementia, seizures and premature death. The associated genes for six different human forms have been identified (CLN1, CLN2, CLN3, CLN5, CLN6 and CLN8), and three other human forms suggested (CLNs 4, 7 and 9). A form of NCL in Australian Devon cattle is caused by a single base duplication (c.662dupG) in bovine CLN5. This mutation causes a frame-shift and premature termination (p.Arg221GlyfsX6) which is predicted to result in a severely truncated protein, analogous to disease causing mutations in human Finnish late infantile variant NCL (CLN5), and a simple genetic diagnostic test has been developed. The symptoms and disease course in cattle also matches CLN5. Only one initiation site was found in the bovine gene, equivalent to the third of four possible initiation sites in the human gene. As cattle are anatomically and physiologically similar to humans with a human-like central nervous system and easy to maintain and breed, they provide a valuable alternative model for CLN5 studies.     

Pathogenesis and therapies for infantile neuronal ceroid lipofuscinosis (infantile CLN1 disease)

The neuronal ceroid lipofuscinoses (NCL, Batten disease) are a group of inherited neurodegenerative diseases. Infantile neuronal ceroid lipofuscinosis (INCL, infantile Batten disease, or infantile CLN1 disease) is caused by a deficiency in the soluble lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1) and has the earliest onset and fastest progression of all the NCLs. Several therapeutic strategies including enzyme replacement, gene therapy, stem cell-mediated therapy, and small molecule drugs have resulted in minimal to modest improvements in the murine model of PPT1-deficiency. However, more recent studies using various combinations of these approaches have shown more promising results; in some instances more than doubling the lifespan of PPT1-deficient mice. These combination therapies that target different pathogenic mechanisms may offer the hope of treating this profoundly neurodegenerative disorder. Similar approaches may be useful when treating other forms of NCL caused by deficiencies in soluble lysosomal proteins. Different therapeutic targets will need to be identified and novel strategies developed in order to effectively treat forms of NCL caused by deficiencies in integral membrane proteins such as juvenile neuronal ceroid lipofuscinosis. Finally, the challenge with all of the NCLs will lie in early diagnosis, improving the efficacy of the treatments, and effectively translating them into the clinic. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.     

CLN3P, the Batten disease protein, localizes to membrane lipid rafts (detergent-resistant membranes)

Juvenile neuronal ceroid lipofuscinosis is an inherited pediatric neurodegenerative disorder, which occurs as a result of mutations in the CLN3 gene that is located on chromosome 16p12.1. The encoded protein, CLN3P, is a putative transmembrane protein with no known function. In this study, we demonstrate that CLN3P resides on membrane lipid raft domains (detergent-resistant membranes) and provide important new data towards possible functions of the protein.     

Mutated genes in juvenile and variant late infantile neuronal ceroid lipofuscinoses encode lysosomal proteins

Positional cloning efforts of genes mutated in Batten disease and in the Finnish type of variant late infantile neuronal ceroid lipofuscinosis resulted in the identification of two novel genes, CLN3 and CLN5, and corresponding gene products that proved to be residents of lysosomes. Although the clinical phenotype of these NCL subtypes differs in the age of onset, average life span and EEG findings, the major component of material accumulating in patients' lysosomes is subunit c of mitochondrial ATPase in both these diseases. The CLN3 and CLN5 genes show ubiquitous expression patterns and are targeted to lysosomes in vitro, but the observed synaptosomal localization of the CLN3 protein in neurons would suggest some cell specificity in targeting and function of these proteins. So far, 31 different mutations of the CLN3 gene have been described in Batten patients, with one deletion of 1.02 kb accounting for 75% of disease alleles worldwide. Four CLN5 mutations are known, with one premature stop representing the major founder mutation in the isolated Finnish population. Functional studies of the yeast homolog of CLN3 and increased pH in patients' lysosomes would suggest an involvement of this protein in lysosomal pH homeostasis. Knock-out mouse models for CLN3 have been produced and the histopathology bears a close resemblance to human counterparts with characteristic lysosomal accumulations. Both CLN3 and CLN5 mouse models will provide experimental tools to resolve the pathological cascade in these neurodegenerative diseases.     

Increased Zinc and Manganese in Parallel with Neurodegeneration,Synaptic Protein Changes and Activation of Akt/GSK3 Signaling in Ovine CLN6 Neuronal Ceroid Lipofuscinosis

Mutations in the CLN6 gene cause a variant late infantile form of neuronal ceroid lipofuscinosis (NCL; Batten disease). CLN6 loss leads to disease clinically characterized by vision impairment, motor and cognitive dysfunction, and seizures. Accumulating evidence suggests that alterations in metal homeostasis and cellular signaling pathways are implicated in several neurodegenerative and developmental disorders, yet little is known about their role in the NCLs. To explore the disease mechanisms of CLN6 NCL, metal concentrations and expression of proteins implicated in cellular signaling pathways were assessed in brain tissue from South Hampshire and Merino CLN6 sheep. Analyses revealed increased zinc and manganese concentrations in affected sheep brain in those regions where neuroinflammation and neurodegeneration first occur. Synaptic proteins, the metal-binding protein metallothionein, and the Akt/GSK3 and ERK/MAPK cellular signaling pathways were also altered. These results demonstrate that altered metal concentrations, synaptic protein changes, and aberrant modulation of cellular signaling pathways are characteristic features in the CLN6 ovine form of NCL.     

Genetic heterogeneity in neuronal ceroid lipofuscinosis (NCL): Evidence that the late-infantile subtype (Jansky-Bielschowsky disease; CLN2) is not an allelic form of the juvenile or infantile subtypes

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in neurons and other cell types. Inheritance is autosomal recessive. Three main childhood subtypes are recognized: infantile (Haltia-Santavuori disease; MIM 256743), late infantile (Jansky-Bielschowsky disease; MIM 204500), and juvenile (Spielmeyer-Sjögren-Vogt, or Batten, disease; MIM 204200). The gene loci for the juvenile (CLN3) and infantile (CLN1) types have been mapped to human chromosomes 16p and 1p, respectively, by linkage analysis. Linkage analysis of 25 families segregating for late-infantile NCL has excluded these regions as the site of this disease locus (CLN2). The three childhood subtypes of NCL therefore arise from mutations at distinct loci.     

Proteolytic cleavage of the disease-related lysosomal membrane glycoprotein CLN7

CLN7 is a polytopic lysosomal membrane glycoprotein of unknown function and is deficient in variant late infantile neuronal ceroid lipofuscinosis. Here we show that full-length CLN7 is proteolytically cleaved twice, once proximal to the used N-glycosylation sites in lumenal loop L9 and once distal to these sites. Cleavage occurs by cysteine proteases in acidic compartments and disruption of lysosomal targeting of CLN7 results in inhibition of proteolytic cleavage. The apparent molecular masses of the CLN7 fragments suggest that both cleavage sites are located within lumenal loop L9. The known disease-causing mutations, p.T294K and p.P412L, localized in lumenal loops L7 and L9, respectively, did not interfere with correct lysosomal targeting of CLN7 but enhanced its proteolytic cleavage in lysosomes. Incubation of cells with selective cysteine protease inhibitors and expression of CLN7 in gene-targeted mouse embryonic fibroblasts revealed that cathepsin L is required for one of the two proteolytic cleavage events. Our findings suggest that CLN7 is inactivated by proteolytic cleavage and that enhanced CLN7 proteolysis caused by missense mutations in selected luminal loops is associated with disease.     

Mutations in a Novel CLN6-Encoded Transmembrane Protein Cause Variant Neuronal Ceroid Lipofuscinosis in Man and Mouse

The CLN6 gene that causes variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), a recessively inherited neurodegenerative disease that features blindness, seizures, and cognitive decline, maps to 15q21-23. We have used multiallele markers spanning this approximately 4-Mb candidate interval to reveal a core haplotype, shared in Costa Rican families with vLINCL but not in a Venezuelan kindred, that highlighted a region likely to contain the CLN6 defect. Systematic comparison of genes from the minimal region uncovered a novel candidate, FLJ20561, that exhibited DNA sequence changes specific to the different disease chromosomes: a G-->T transversion in exon 3, introducing a stop codon on the Costa Rican haplotype, and a codon deletion in exon 5, eliminating a conserved tyrosine residue on the Venezuelan chromosome. Furthermore, sequencing of the murine homologue in the nclf mouse, which manifests recessive NCL-like disease, disclosed a third lesion-an extra base pair in exon 4, producing a frameshift truncation on the nclf chromosome. Thus, the novel approximately 36-kD CLN6-gene product augments an intriguing set of unrelated membrane-spanning proteins, whose deficiency causes NCL in mouse and man.     

Mutation of the glycosylated asparagine residue 286 in human CLN2 protein results in loss of enzymatic activity

Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by the deficiency of the lysosomal tripeptidyl peptidase-I encoded by CLN2. We previously detected in two LINCL patients a homozygous missense mutation, p.Asn286Ser, that affects a potential N-glycosylation site. We introduced the p.Asn286Ser mutation into the wild-type CLN2 cDNA and performed transient expression analysis to determine the effect on the catalytic activity, intracellular targeting, and glycosylation of the CLN2 protein. Expression of mutant p.Asn286Ser CLN2 in HEK293 cells revealed that the mutant was enzymatically inactive. Western blot analysis demonstrated that at steady state the amounts of expressed p.Asn286Ser CLN2 were reduced compared with wild-type expressing cells. The rate of synthesis and the sorting of the newly synthesized p.Asn286Ser CLN2 in the Golgi was not affected compared with wild-type CLN2 protein. The electrophoretic mobility of the immunoprecipitated mutant p.Asn286Ser CLN2 was increased by approximately 2 kDa compared with the wild-type CLN2 protein, whereas deglycosylation led to the generation of polypeptides of the same apparent size. The data suggest that mutant p.Asn286Ser CLN2 lacks one oligosaccharide chain resulting in enzymatic inactivation.     

Defective endoplasmic reticulum-resident membrane protein CLN6 affects lysosomal degradation of endocytosed arylsulfatase A

Variant late infantile neuronal ceroid lipofuscinosis, a lysosomal storage disorder characterized by progressive mental deterioration and blindness, is caused by mutations in a polytopic membrane protein (CLN6) with unknown intracellular localization and function. In this study, transient transfection of BHK21 cells with CLN6 cDNA and immunoblot analysis using peptide-specific CLN6 antibodies demonstrated the expression of a approximately 27-kDa protein that does not undergo proteolytic processing. Cross-linking experiments revealed the presence of CLN6 dimers. Using double immunofluorescence microscopy, epitope-tagged CLN6 was shown to be retained in the endoplasmic reticulum (ER) with no colocalization with the cis-Golgi or lysosomal markers. The translocation into the ER and proper folding were confirmed by the N-linked glycosylation of a mutant CLN6 polypeptide. Pulse-chase labeling of fibroblasts from CLN6 patients and from sheep (OCL6) and mouse (nclf) models of the disease followed by immunoprecipitation of cathepsin D indicated that neither the synthesis, sorting nor the proteolytic processing of this lysosomal enzyme was affected in CLN6-defective cells. However, the degradation of the endocytosed index protein arylsulfatase A was strongly reduced in all of the mutant CLN6 cell lines compared with controls. These data suggest that defects in the ER-resident CLN6 protein lead to lysosomal dysfunctions, which may result in lysosomal accumulation of storage material.     

Batten disease (Spielmeyer-Vogt disease, juvenile onset neuronal ceroid-lipofuscinosis) gene (CLN3) maps to human chromosome 16

The ceroid-lipofuscinoses are a group of inherited neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in neurons and other cell types. The underlying biochemical defect is unknown. Batten disease (Spielmeyer-Vogt disease, juvenile onset neuronal ceroid-lipofuscinosis) displays autosomal recessive inheritance. Genetic linkage studies were undertaken to determine the chromosomal location of the Batten disease mutation (CLN3). Following identification of linkage to the haptoglobin locus, linkage analysis has been carried out in 42 families by using DNA markers for loci on the long arm of human chromosome 16. The maximal lod score between Batten disease and the locus D16S148 calculated for combined sexes is 6.05 at a recombination fraction theta = 0.00. Multilocus analysis using five loci indicated the most likely order to be HP-D16S151-D16S150-CLN3-D16S148-D16S147. The maximal location score for CLN3 was 48 (equivalent to a lod score of 10.4) in that interval within this fixed marker map.     

Molecular screening of Batten disease: identification of a missense mutation (E295K) in the CLN3 gene

Batten disease, the juvenile form of neuronal ceroid lipofuscinosis, is a prevalent neuron degenerative disorder of childhood. A 1.02-kb genomic deletion in the Batten disease gene CLN3 has been determined to be a common mutation. We developed a PCR method to screen for this deletion and tested 43 Batten disease probands. We found 36% (31/86) of Batten disease chromosomes did not carry the 1.02-kb deletion. Of the three heterozygotes for the 1.02-kb deletion, a novel G-to-A missense mutation at nucleotide 1020 of the CLN3 cDNA sequence was found on two of the non-1.02-kb deletion chromosomes. The missense mutation resulted in a substitution of glutamic acid (E) by lysine (K) at position 295 (E295 K). The E295 K mutation causes a change in predicted local protein conformation. This glutamic acid is a highly conserved acidic amino acid, being present in human, mouse, dog and yeast, which suggests it may play an important role in the function of the Batten disease protein. Received: 12 May 1997 / Accepted: 21 August 1997     

Novel interactions of CLN5 support molecular networking between Neuronal Ceroid Lipofuscinosis proteins

Background  

Neuronal ceroid lipofuscinoses (NCLs) comprise at least eight genetically characterized neurodegenerative disorders of childhood. Despite of genetic heterogeneity, the high similarity of clinical symptoms and pathology of different NCL disorders suggest cooperation between different NCL proteins and common mechanisms of pathogenesis. Here, we have studied molecular interactions between NCL proteins, concentrating specifically on the interactions of CLN5, the protein underlying the Finnish variant late infantile form of NCL (vLINCL_Fin).     

The Visual Assignment of Genes by Fiber-Fish: BTF3 Protein Homologue Gene (BTF3) and a Novel Pseudogene of Human RNA Helicase A (DDX9P) on 13q22

Chromosome 13 is one of the poorly mapped human chromosomes. As an example, only two cloned genes have been assigned to bands 13q22–q31. Our characterization of the critical region for the variant form of late infantile neuronal ceroid lipofuscinosis (vLINCL, locus definition CLN5) disease region on 13q22 resulted in the identification of the sequences encoding the BTF3 protein homologue gene (HGMW-approved symbol BTF3) and a novel pseudogene for RNA Helicase A (HGMW-approved symbol DDX9P). Precise visual assignment to the physical clones covering this region and the positional relationships of these genes were achieved by the use of tyramine enhancement of Fiber-FISH hybridization signals, demonstrating the power of this technique in efficient positioning of coding regions on the physical maps.