Journal of Heredity Advance Access originally published online on June 15, 2005
Journal of Heredity 2005 96(7):735-738; doi:10.1093/jhered/esi088
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Characterization of Candidate Genes for Neuronal Ceroid Lipofuscinosis in Dog
From the Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559 Hannover, Germany
Address correspondence to C. Drögemüller at the address above, or e-mail: cord.droegemueller{at}tiho-hannover.de.
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Abstract |
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The neuronal ceroid lipofuscinoses (NCL) are a heterogenous group of monogenic autosomal recessive inherited progressive neurodegenerative diseases characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Until today, eight forms of NCL have been classified in humans by clinical criteria, which result from mutations in at least six different genes (TPP1, CLN2, PPT1, CLN5, CLN6, and CLN8). NCL has also been reported in various domestic animal species including cattle, goat, sheep, cat, and certain dog breeds. In this report, the experimental analysis of canine PPT1, CLN5, CLN6, and CLN8 full-length cDNA sequences is described, and the current whole genome sequence assembly was used for gene structure analyses. Characterization of the four canine genes revealed a conserved organization with respect to the human orthologs. In general the gene size in dog is smaller compared to the human sequence due to shorter intron length. Using four individuals of Tibetan terrier with NCL, and a single affected Polish Owczarek Nizinny (PON) dog, we excluded the complete coding region of canine PPT1 and CLN8 and three of four exons of CLN5 and six of seven exons of CLN6 harboring disease-causing mutations.
Hereditary, naturally occurring neuronal ceroid lipofuscinoses (NCLs) have been reported in humans (Goebel and Wisniewski 2004), mice (Messer and Flaherty 1986), and various domestic animal species, including cattle, goat, sheep, cat, and certain dog breeds (Jolly and Walkley 1997). The human NCLs are a heterogenous group of monogenic autosomal recessive inherited progressive neurodegenerative diseases characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells of the body followed by cell degeneration. Grouped together under the eponym of Batten disease, until today eight forms of NCL have been classified in humans (CLN1–8) by clinical criteria, the age of onset and the presence of lysosomal storage material (Goebel and Wisniewski 2004).
Six genes have been identified that cause human NCL (Mole 2004). Two types of NCL are caused by mutations in genes coding for lysosomal enzymes (CLN1, which encodes PPT1, a palmitoyl protein thioesterase; CLN2, which encodes TPP1, a tripeptidyl protein peptidase), two types of NCL are caused by mutations in lysosomal transmembrane encoding genes (CLN3 and CLN5), one type is caused by mutations of the CLN6 gene encoding a membrane protein of the endoplasmatic reticulum, and another type is caused by mutations of the CLN8 gene encoding a protein that recycles between the endoplasmatic reticulum and endoplasmatic reticulum golgi intermediate complex (www.ucl.ac.uk/ncl). The two assigned human genes causing the CLN4 and CLN7 phenotypes have not been identified until now. For the naturally occurring mouse NCL mutant strains mnd and nclf, the causative mutations have been identified in the murine orthologs of CLN8 and CLN6, respectively (Ranta et al. 1999; Gao et al. 2002).
In dogs, autosomal recessive inherited NCL has been described extensively in English setters with similarities to human juvenile NCL (Koppang 1992). After exclusion of causative mutations in the canine TPP1 and CLN3 genes (Shibuya et al. 1998; Katz et al. 2001), recently a single point mutation in the coding region of the canine CLN8 gene has been found in English setter dogs with NCL (Katz et al. 2005). However, the Tibetan terrier dog represents the second canine breed with many reported details concerning the course of disease (Alroy et al. 1992). NCL in Tibetan terriers is inherited in an autosomal recessive pattern that was classified as late-onset disease (Riis et al. 1992). A similar late-onset NCL phenotype has been described in Polish Owczarek Nizinny (PON) dogs (Wrigstad et al. 1995). There is currently no method available for identifying those affected dogs before clinical signs become apparent until after 4–6 years of age. First studies revealed no indication for causative mutations in the canine CLN3 and CLN8 genes after examination of single NCL cases in Tibetan terriers (Shibuya et al. 1998; Katz et al. 2005).
The aim of the present study was to characterize the canine orthologs of further four canine previously unreleased NCL candidate genes (PPT1, CLN5, CLN6, and CLN8) using the current sequence information of the 15 July 2004 dog whole genome shotgun assembly. After experimental full-length cDNA cloning and subsequent genome structure analysis of the canine PPT1, CLN5, CLN6, and CLN8 genes, a mutation analysis was performed to test whether these genes are responsible for the NCL phenotype in Tibetan terrier and PON dogs.
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Materials and Methods |
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The canine PPT1, CLN5, CLN6, and CLN8 genes were localized on contigs of the current dog genome assembly (Boxer genome assembly 1.0) by cross-species BLAST (basic local alignment search tool) searches with the corresponding human reference mRNA sequences (NM_000310 [GenBank] , NM_006493 [GenBank] , NM_017882 [GenBank] , NM_018941 [GenBank] ). For PPT1, CLN6, and CLN8, further BLAST searches with experimentally obtained canine gene-specifc cDNA sequences (as will be described) were performed against the canine whole genome shotgun sequences at NCBI Trace Archive to try a closure or an extension of previously identified contigs by a chromosome walking strategy.
The isolation of full length cDNAs was achieved by a modified rapid amplification of cDNA ends (RACE) protocol. The human mRNA sequences were used to identify putative exons on the canine genomic sequence, which were used for RACE primer design. Total RNA from dog lung of a normal female Beagle (Biocat, Heidelberg, Germany) was used for the overlapping 5'- and 3'-RACE polymerase chain reaction (PCR) products with the FirstChoice RNA ligase-mediated (RLM)-RACE kit (Ambion Europe, Huntingdon, UK) according to the instructions of the manufacturer. After cloning and sequencing of full-length canine cDNAs, the exact canine genomic structure was determined using the mRNA-to-genomic alignment program Spidey (www.ncbi.nlm.nih.gov/ieb/research/ostell/spidey/index.html).
To evaluate the four canine genes as candidates for NCL, exons with flanking regions were PCR-amplified from genomic DNA of six dogs and subsequently directly sequenced (primer sequences and PCR conditions available on request). Four female NCL positive Tibetan terrier dogs (8, 9, 10, 14 years of age), a single NCL affected 4.5-year-old PON dog, and a single clinical unsuspicious 16-year-old male Tibetan terrier dog have been sampled. The phenotypes of the affected animals have been confirmed by histopathological examination of brain and retina.
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Results and Discussion |
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In general the characterization of the transcript and genomic sequences of canine PPT1, CLN5, CLN6, and CLN8 genes revealed a conserved organization with respect to the human orthologs. The canine exon/intron boundaries conform perfectly to the GT/AG rule, and the gene size in dog is smaller compared to the human sequence due to shorter intron length. Four supplementary tables containing genome structure information of canine PPT1, CLN5, CLN6, and CLN8 are available as supplementary data online.
The RACE protocol generated a full-length PPT1 cDNA sequence of 2,021 bp containing an open reading frame of 921 bp encoding a protein of 306 amino acids (AJ875416 [GenBank] ). The coding sequence of canine PPT1 displays 91% and 83% similarity to the human and murine PPT1 gene, respectively. The canine PPT1 gene was localized on two separate contigs (AAEX01015066.1, AAEX01015067.1) of the current dog genome assembly. The genomic sequences of these two contigs were extended for another 6 bp using an overlapping single dog whole genome shotgun sequence read from the trace archive (Trace Archive identifier TI279943580). Thus, a contiguous canine genomic sequence of 29,994 bp harboring the complete PPT1 gene of 25.9 kb with nine exons could be assembled.
The full-length transcript of canine CLN5 contained 1,922 bp with an open reading frame of 1,053 bp encoding a protein of 350 amino acids (AJ875417 [GenBank] ). This is in accordance to the prediction of canine CLN5 mRNA sequence derived by automated computational analysis (accession number XM_542617). A protein alignment with the longer human and murine polypeptides consisting of 407 and 486 amino acids, respectively, revealed large deviations at the N-terminus. Human CLN5 gets translated in more than one polypeptide form starting at amino acid positions 1, 30, 50, and 62 (Vesa et al. 2002) and after the first putative transmembrane domain, consisting of amino acids 76–91, the canine CLN5 protein shows quite high identities to the orthologous human and murine protein, respectively. The 8.4-kb encompassing canine CLN5 gene was located in a single genomic contig (AAEX01010623.1) harboring all four exons that are present in the 3.5-kb smaller human CLN5 gene.
The cloned canine CLN6 cDNA sequence of 2,352 bp contains an open reading frame of 939 bp encoding a protein of 312 amino acids (AJ875418 [GenBank] ). The coding sequence of canine CLN6 displays 89% identity to the human gene, and the deduced protein shows 90% identity to the orthologous human protein of 311 amino acids. The canine CLN6 gene was localized on a single genomic contig (AAEX01008667.1) harboring exon 2 to 7 of the ortholog human CLN6 gene. As the first exon of the canine CLN6 gene was not contained on this contig, the genomic sequence was extended for another 910 bp using three dog whole genome shotgun sequences from the trace archive (identifier TI 311672021, TI 290038500, TI 256281130). The total size of the canine CLN6 gene of 16.6 kb is about 7 kb smaller than the corresponding human gene.
The canine CLN8 cDNA contains an open reading frame of 867 bp encoding a protein of 288 amino acids (AJ875419 [GenBank] ). The translation start codon was assigned based on the homology to the human ortholog because there are two more additional in-frame ATG codons within the 5'-UTR. A first ATG codon at cDNA position 21 and a second ATG codon at position 77 with short open reading frames of 60 and 99 bp, respectively. Both translated amino acid sequences showed no homology to any known mammalian protein. Therefore, the canine CLN8 gene seems to be another example for the rapidly growing family of genes with unused ATG translation initiation codons in the 5'-UTR (Peri and Pandey 2001). The canine CLN8 protein shows 89% and 84% identity to the orthologous human and murine protein, respectively. The canine CLN8 gene was localized on a single genomic contig (AAEX01055130.1) harboring exon 2 to 3 of the ortholog human CLN8 gene. As the first untranslated exon of the canine CLN8 gene was not contained on this contig, a single nonoverlapping dog whole genome shotgun sequence read from the trace archive (identifier TI264711558) could be identified. Unfortunately, no further sequence reads could be found by the BLAST-based chromosome walking strategy to close the sequence gap of intron 1. Therefore, the canine CLN8 gene spans at least 18.4 kb and is highly conserved in comparison to the human gene, where the protein initiation codon is also found in exon 2. Our experimental annotation probably indicates errors in the first automatic computer assisted gene annotation of canine chromosome 37 where CLN8 has been annotated with five exons (accession number NW_139916).
All coding exons with flanking intronic regions of PPT1 and CLN8 could be amplified from the examined six dogs and the sequences were compared to the Boxer genome assembly 1.0. Because no sequence variation was found for PPT1 and CLN8, these two genes can be excluded as candidates for the NCL phenotype in Tibetan terrier and PON dogs. Except for the first exons, the remainder of the exons with flanking intronic regions of CLN5 and CLN6 could be amplified from the examined six dogs. For CLN6 a single nucleotide polymorphisms (SNP) was found at nucleotide 27 of exon 4, the healthy Tibetan terrier dog was heterozygous T/G and the five NCL affected dogs were homozygous T/T. Interestingly, the Boxer sequence showed a G at this position, but the observed polymorphism did not alter the predicted amino acid sequence of leucine CLN6 codon 109. Our data thus indicate that this polymorphism in the canine CLN6 gene can be excluded as candidate mutation for the NCL phenotype in the Tibetan terrier and PON dogs. The amplification of CLN5 exon 1 and CLN6 exon 1 failed due to the strong GC content of the regions, respectively. Therefore, a final exclusion of these genes is not possible yet, although the sequence comparison of the analyzed exons encoding 85% of CLN5 and 91% of CLN6, respectively, revealed no polymorphism.
The present study revealed no functional mutations of the complete coding region of PPT1 and CLN8. Except the first exons of CLN5 and CLN6, no functional polymorphisms could be found in the remaining exons of CLN5 and CLN6 as well. In addition to the previously reported exclusion of canine TPP1, CLN3, and CLN8 in Tibetan terrier (Shibuya et al. 1998; Katz et al. 2001, 2005), the PPT1 and CLN8 genes, and probably CLN5 and CLN6 as well, could be excluded as candidate genes for NCL in Tibetan terrier and PON dogs. Perhaps the still-unknown human CLN4 gene assigned to adult NCL might be a better candidate gene for the late-onset NCL in Tibetan terrier and PON dogs. Probably there are still more than the six known human NCL genes, as indicated by findings in sheep where a point mutation in the cathepsin D (CTSD) gene causes a congenital lysosomal storage disease with profound neurodegeneration that share features with human NCL (Tyynela et al. 2000) and chloride channel 3 (CLCN3) knockout mice showing phenotypes similar to human NCL (Yoshikawa et al. 2002). Ultimately, if the candidate gene approach did not reveal the causative gene in Tibetan terrier and PON dogs it might be indicated to perform a genome-wide linkage scan using NCL segregating families to map the canine chromosome region harboring the deleterious gene.
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Supplementary Data |
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Four supplementary tables are available at Journal of Heredity online (www.jhered.oxfordjournals.org).
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Acknowledgments |
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This paper was delivered at the 2nd International Conference on the "Advances in Canine and Feline Genomics: Comparative Genome Anatomy and Genetic Disease," Universiteit Utrecht, Utrecht, The Netherlands, October 14–16, 2004.
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Corresponding Editor: Elaine Ostrander
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Alroy J, Schelling SH, Thalhammer JG, Raghavan SS, Natowicz MR, Prence EM, and Orgad U, 1992. Adult onset lysosomal storage disease in a Tibetan terrier: clinical, morphological and biochemical studies. Acta Neuropathol (Berl) 84:658–663.[Medline]
Gao H, Boustany RM, Espinola JA, Cotman SL, Srinidhi L, Antonellis KA, Gillis T, Qin X, Liu S, Donahue LR, and others, 2002. Mutations in a novel CLN6-encoded transmembrane protein cause variant neuronal ceroid lipofuscinosis in man and mouse. Am J Hum Genet 70:324–35.[CrossRef][Web of Science][Medline]
Goebel HH and Wisniewski KE, 2004. Current state of clinical and morphological features in human NCL. Brain Pathol 14:61–69.[Web of Science][Medline]
Jolly RD and Walkley SU, 1997. Lysosomal storage diseases of animals: an essay in comparative pathology. Vet Pathol 34:527–548.[Abstract]
Katz ML, Shibuya H, and Johnson GS, 2001. Animal models for the ceroid lipofuscinoses. Adv Genet 45:183–203.[Medline]
Katz ML, Khan S, Awano T, Shahid SA, Siakotos AN, and Johnson GS, 2005. A mutation in the CLN8 gene in English Setter dogs with neuronal ceroid-lipofuscinosis. Biochem Biophys Res Commun 327:541–547.[CrossRef][Web of Science][Medline]
Koppang N, 1992. English setter model and juvenile ceroid-lipofuscinosis in man. Am J Med Genet 42:599–604.[CrossRef][Web of Science][Medline]
Messer A and Flaherty L, 1986. Autosomal dominance in a late-onset motor neuron disease in the mouse. J Neurogenet 3:345–355.[Web of Science][Medline]
Mole S, 2004. The genetic spectrum of human neuronal ceroid-lipofuscinoses. Brain Pathol 14:70–76.[Web of Science][Medline]
Peri S and Pandey A, 2001. A reassessment of the translation initiation codon in vertebrates. Trends Genet 17:685–687.[CrossRef][Web of Science][Medline]
Ranta S, Zhang Y, Ross B, Lonka L, Takkunen E, Messer A, Sharp J, Wheeler R, Kusumi K, Mole S, and others, 1999. The neuronal ceroid lipofuscinoses in human EPMR and mnd mutant mice are associated with mutations in CLN8. Nat Genet 23:233–236.[CrossRef][Web of Science][Medline]
Riis RC, Cummings JF, Loew ER, and de Lahunta A, 1992. Tibetan terrier model of canine ceroid lipofuscinosis. Am J Med Genet 42:615–21.[CrossRef][Web of Science][Medline]
Shibuya H, Liu PC, Katz ML, Siakotos AN, Nonneman DJ, and Johnson GS, 1998. Coding sequence and exon/intron organization of the canine CLN3 (Batten disease) gene and its exclusion as the locus for ceroid-lipofuscinosis in English setter dogs. J Neurosci Res 52:268–275.[CrossRef][Web of Science][Medline]
Tyynela J, Sohar I, Sleat DE, Gin RM, Donnelly RJ, Baumann M, Haltia M, and Lobel P, 2000. A mutation in the ovine cathepsin D gene causes a congenital lysosomal storage disease with profound neurodegeneration. EMBO J 19:2786–2792.[CrossRef][Web of Science][Medline]
Vesa J, Chin MH, Oelgeschlager K, Isosomppi J, DellAngelica EC, Jalanko A, and Peltonen L, 2002. Neuronal ceroid lipofuscinoses are connected at molecular level: interaction of CLN5 protein with CLN2 and CLN3. Mol Biol Cell 13:2410–2420.
Wrigstad A, Nilsson SE, Dubielzig R, and Narfstrom K, 1995. Neuronal ceroid lipofuscinosis in the Polish Owczarek Nizinny (PON) dog. A retinal study. Doc Ophthalmol 91:33–47.[CrossRef][Web of Science][Medline]
Yoshikawa M, Uchida S, Ezaki J, Rai T, Hayama A, Kobayashi K, Kida Y, Noda M, Koike M, Uchiyama Y, and others, 2002. CLC-3 deficiency leads to phenotypes similar to human neuronal ceroid lipofuscinosis. Genes Cells 7:597–605.[Abstract]
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