Human Molecular Genetics, 2002, Vol. 11, No. 16 1823-1833
© 2002 Oxford University Press
Canine CNGB3 mutations establish cone degeneration as orthologous to the human achromatopsia locus ACHM3
1Center for Canine Genetics and Reproduction, James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA, 2Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, D4-100 Seattle, WA 98109, USA, 3Molecular and Cellular Biology Program, University of Washington, Box 357275, Seattle, WA 98195-7275, USA and 4Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK
Received March 18, 2002; Accepted May 30, 2002
DDBJ/EMBL/GenBank accession nos
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cone degeneration (cd ) is an autosomal recessive canine disease that occurs naturally in the Alaskan Malamute and German Shorthaired Pointer breeds. It is phenotypically similar to human achromatopsia, a heterogeneous autosomal recessive disorder associated with three distinct loci. Both the canine disease and its human counterparts are characterized by day-blindness and absence of retinal cone function in adults. We report linkage of the canine cd locus to marker C29.002 on canine chromosome 29 at recombination fraction
=0.0 with a maximum LOD score of 24.68 in a series of informative outbred pedigrees derived from cd-affected Alaskan Malamutes. Conserved gene order between CFA29 and the long arm of human chromosome 8 argued for homology between the cd locus and the human achromatopsia locus, ACHM3, at 8q21–22. The canine homolog of the cyclic nucleotide-gated channel ß-subunit gene (CNGB3), responsible for the human ACHM3 disease phenotype, was mapped within the zero-recombination interval for the cd locus. A deletion removing all exons of canine CNGB3 was identified in cd-affected Alaskan Malamute-derived dogs. A missense mutation in exon 6 (D262N, nucleotide 784) within a conserved region of the same gene was detected in German Shorthaired Pointers affected with an allelic disorder. Identification of these canine disorders as homologs of human ACHM3 underscores the power of recent developments in canine genomics, and provides a valuable system for exploring disease mechanisms and evaluating potential therapeutic measures in disorders of cone photoreceptors. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Segregation of the domestic dog population into separate breeds, driven by artificial selection for defined phenotypic and behavioral traits, has been marked by the emergence of inherited canine diseases that are often directly comparable to those observed in humans. Mapping disease genes in canine pedigrees is facilitated by the ability to control matings, obtain large sibships and exploit shorter generation times as compared with human families (1–3). Defined breeds of dog, each of which represents a relatively isolated subpopulation within a single species, thus allow identification and mapping of disease loci with relevance to both human and canine health (4).
A spectrum of hereditary retinopathies have been reported in the dog, the most frequent of which form the clinical disease entity termed progressive retinal atrophy (PRA), the canine equivalent of human retinitis pigmentosa (RP). PRA, like RP, is primarily a disease of rod photoreceptors, in which cone function and structure degenerate secondarily. Within this class of diseases, several specific disorders segregate independently in different breeds of dog, including English Mastiffs, Irish Setters, Norwegian Elkhounds, Poodles, Siberian Huskies and others (5–10). Distinct loci primarily responsible for each disorder have been identified 5,11–14). In this study, we extend the list of molecularly defined canine inherited retinopathies by identifying the locus, gene and germline mutations responsible for canine cone degeneration (cd ) in two breeds of dog.
Canine cone degeneration (cd ) was first observed in an inbred strain of Alaskan Malamute dogs in 1960, and is inherited as an autosomal recessive trait (15). Cone-degenerate pups develop day-blindness and photophobia between 8 and 12 weeks postnatal, the age when retinal development is normally completed in dogs. Symptoms are present only in bright light; vision in dim light is normal. Affected dogs remain ophthalmoscopically normal throughout life. Cone function can be detected electroretinographically in very young (3–6-week old) cd-affected pups, but begins to fail at 6–12 weeks of age and is non-recordable in mature cd-affected dogs (16) (G.D. Aguirre and G.M. Acland, unpublished data). Adult cd-affected retinas lack all cones, except for a few displaced cone nuclei (17,18). Rod photoreceptors, however, remain functionally and structurally normal throughout the animals' life.
In humans, a cd-like condition referred to as achromatopsia, total color blindness, day-blindness or rod monochromacy has been extensively described (19–22). At least three susceptibility loci have been mapped: ACHM1 on human chromosome 14 (MIM 603096) (20), ACHM2 on 2q11 (MIM 216900) (19) and ACHM3 on 8q21–q22 (MIM 262300) (21–23). Mutations in CNGA3, the cone-specific cyclic nucleotide-gated channel 
-subunit gene, have been identified as the cause of ACHM2 (24,25), and a rodent knockout of ACHM2 has been developed by targeted disruption of the murine CNGA3 homolog (26). More recently, mutations in the CNGB3 gene (GenBank accession nos AF228520 and AF272900) have been associated with ACHM3 (27,28). Like their canine counterparts, human achromats display day-blindness and a complete lack of cone cell function (29).
At present, cd-affected dogs are the only large animal and naturally occurring model of human achromatopsia. Immuno-cytochemical studies of the cd-affected retina have demonstrated a specific absence or delocalization of two subunits (
8 and ß3) of the canine cone-specific transducin signaling protein from the outer segments of pre-degenerate cone photoreceptors (18,30). Despite this intriguing observation, genes for both subunit proteins have been excluded as the cd gene (30,31). In this study, we report mapping of the canine cd locus to canine chromosome 29 (CFA29), allelism of two forms of cd [in German Shorthaired Pointer (GSP) and Alaskan Malamute-derived (AMD) pedigrees] and co-segregation of CNGB3 mutations with these cd phenotypes. These results strongly support the conclusions that cd is the canine homolog of human ACHM3, and that both the human and canine diseases are caused by mutations in the CNGB3 gene.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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From an initial genome screen of six cd-informative AMD pedigrees, positive linkage was detected between cd and marker C29.002, which is located on CFA29 (32–34). Using genotypes from five additional families and ten informative microsatellite markers in the region surrounding C29.002, a maximum LOD score of 24.68 was observed with marker C29.002 at a recombination fraction
=0.0 (Table 1). The zero recombination interval for cd in these pedigrees is flanked by markers REN86C11 and FH3366 (Table 1).
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To refine map order surrounding cd, data obtained by genotyping eight of the canine reference pedigrees (35) for markers in this region were merged with that from the existing meiotic linkage map (32). The zero recombination interval for cd spans 4.8 cM in the map generated using the canine reference pedigrees (Fig. 1A), and 6.1 cM in the map based on cd-informative pedigrees (Fig. 1B). Marker order in the two meiotic maps is identical. Haplotype analysis confirmed this order, as illustrated in a representative pedigree in Figure 2. No recombination events were observed between cd, C29.002 and FH2385 in the cd pedigrees; thus the order of these markers in the interval REN86C11–FH3366 is ambiguous.
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Because previous radiation hybrid (RH) map data (34) and reciprocal paint data (36–38) established conservation of synteny between CFA29 and human chromosome 8q, the candidacy of CNGB3 was immediately apparent. This gene codes for the ß subunit of the cone cyclic nucleotide-gated channel, and CNGB3 mutations have been identified in human achromats (27,28). A full-length (2349 bp) canine CNGB3 cDNA was cloned, sequenced and aligned with corresponding human and mouse sequences. From conceptual translation of the coding sequence, the canine gene shares 75.7% amino acid identity and 90.5% similarity with the human gene, and slightly lower (71.3%) amino acid identity with the mouse sequence (data not shown). As expected for a functional region, amino acid sequence in the cGMP-binding domain is more highly conserved between species, with canine CNGB3 sharing 89.2% and 88.2% amino acid identity between the human and mouse proteins, respectively.
To evaluate CNGB3 as a candidate, multiple pairs of PCR primers were designed to amplify the canine CNGB3 coding region in overlapping fragments using RT–PCR. Successful amplification was achieved using mRNA isolated from both homozygous wild-type and cd-heterozygous retinas, but failed for all samples collected from homozygous affected animals (Fig. 3A and data not shown). Failure to amplify any portion of CNGB3 from the mRNA of cd-affected animals suggests that the entire transcript is missing from cd-affected animals. Northern analysis, using a probe spanning exons 5–7, revealed a prominent 5 kb transcript in wild-type (+/+) dogs, a lower level of the same transcript in heterozygous (cd/+) dogs, and the absence of a detectable transcript in homozygous affected (cd/cd) animals (Fig. 3B).
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To determine if the missing message corresponds to a genomic deletion, Southern analysis of EcoRI-digested DNA from cd/+, cd/cd and +/+ dogs was performed. In lanes containing DNA from cd/+ and +/+ dogs, a 6.5 kb fragment was identified by hybridization with a probe comprising exon 18 and a portion of the 3'-untranslated region (3'-UTR) of the canine CNGB3 gene. In contrast, no appropriately sized fragments were visible in lanes containing DNA from cd/cd affected dogs (Fig. 3C).
A CNGB3-containing BAC clone, 100-O2, was identified by screening an 8.1-fold redundant canine library (39). A 3-fold sequence obtained from a minilibrary prepared from the BAC yielded 19 unordered contigs. BLAST analysis of the sequence data identified multiple regions of high similarity (
70% sequence identity over at least 100 bp) to two human genes, CNGB3 and Copine III (CPNE3, GenBank accession no. NM_003909), that map to the homologous region of human chromosome 8. Of the 19 contigs, 7 contained 15 CNGB3 exons (exons 4–18) and partial adjacent introns. Exon 1, intron 1, exon 2 and exon 3 could be amplified directly from BAC 100-O2 by PCR, demonstrating that although these regions were not represented in the 3x sequence, they are present in BAC 100-O2. Thus, BAC 100-O2 appears to encompass the entire canine CNGB3 gene.
One 20 kb contig from the same BAC contained sequences with very high (>90%) nucleotide identity to exons 8–11; and high nucleotide identity (>75% for lengths greater than 100 bp) to introns 7, 9 and 10 of human CPNE3. This is consistent with the location of CPNE3 and CNGB3 in a contiguous genomic sequence on human chromosome 8.
To place CNGB3 relative to markers on CFA29, a sequence tagged site (STS) corresponding to a microsatellite-containing sequence in canine CNGB3 intron 15, a second STS corresponding to one end of BAC 100-O2, and selected previously placed markers (32) were mapped using RH panel RH08. At LOD 8.0, the RH linkage groups containing TRAMP and CNGB3, respectively, form two distinct groups (Fig. 1C). However, our meiotic linkage maps clearly establish the two RH groups as contiguous on CFA29. Marker order is in complete agreement between the meiotic linkage maps (Fig. 1A and B) and the corresponding RH map (Fig. 1C). CNGB3 and BAC 100-O2 mapped between markers FH2385 and C29.002, and thus were within the cd zero-recombination interval as expected.
Primers for amplification of each CNGB3 exon, three STSs corresponding to a portion of CPNE3 and the two end sequences of BAC 100-O2, amplified appropriately sized products from both homozygous normal and cd-heterozygous dogs. None, however, produced amplification products using genomic DNA from any cd-affected AMD dogs (data not shown). This suggests that the cd-associated genomic deletion removes all 140 kb of genomic DNA represented by BAC 100-O2, including the entire coding region of CNGB3 and at least part of the CPNE3 gene.
To confirm the observed deletion, metaphase chromosomal spreads prepared from cultured fibroblasts derived from cd/cd, cd/+ and +/+ animals were probed by fluorescence in situ hybridization (FISH) using BAC 100-O2 as a probe (Fig. 4). In spreads derived from heterozygous and wild-type animals, the BAC probe consistently labeled a specific small chromosome (Fig. 4B, C and H) that can be cytogenetically identified as CFA29 in corresponding DAPI-stained spreads (Fig. 4E, F and J). Both copies of CFA29 were labeled in the wild-type sample (Fig. 4C), but only one copy in the heterozygote (Fig. 4B, G and H). No hybridization was noted in spreads from homozygous affected dogs (Fig. 4A). Although these results are consistent with complete deletion of CNGB3, there was no cytogenetically obvious band missing, suggesting that the maximum size of the deletion is less than 
500 kb to 1 Mb (M. Breen, personal communication).
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A second form of day-blindness was observed to segregate as an autosomal recessive phenotype in an extended German Shorthaired Pointer (GSP) pedigree. Clinical examination of affected individuals within this pedigree established that the phenotype is similar to that of cd-affected dogs from the AMD strain (G. Acland, unpublished data). Electroretinographic testing of one of these dayblind GSPs (dog III-3, Fig. 5B) revealed a total absence of cone-mediated response components, with normal rod-mediated components (data not shown).
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To test whether the GSP form of day-blindness was allelic with cd, a non-affected obligate heterozygous GSP (dog II-3, Fig. 5B) was bred to a cd-affected dog (dog II-2, Fig. 5B) from the AMD strain. When the six resulting pups were tested by electroretinography at 12 weeks postnatal age, one individual (dog III-1, Fig. 5B) exhibited the characteristic absence of recordable cone-mediated responses diagnostic of cd; the other five pups (e.g. dog II-2, Fig. 5B) were electroretinographically normal (data not shown).
Using genomic DNA isolated from four affected GSP dogs, each CNGB3 exon was amplified, sequenced, and scanned for mutations. A single base change (G
A) was detected in each affected GSP sample in exon 6 at nucleotide 784 that would result in altered translation of codon 262 (D262N) (Fig. 5A). No other sequence variants were detected in any sample. However, it should be noted that the intron/exon junctions for the 3' end of exon 2, as well as both ends of exon 3, have not yet been identified. Three of the primers used to amplify exons 2 and 3 (CNGB3-EX2R, CNGB3-EX3F and CNGB3-EX3R) bind, respectively, to the last 20 bp of exon 2, and the first and last 20 bp of exon 3. Thus, the presence of undetected mutations in these regions cannot be formally excluded.
We evaluated the presence of this exon 6 mutation in additional dogs using an allele discrimination test that yields a 181 bp Sau3A1 restriction fragment from animals carrying the missense mutation while the wild-type allele is cleaved to yield a 166 bp fragment (Fig. 5A). This allele discrimination test was undertaken on all eight GSPs known to be affected with day-blindness, and all eight were found to be homozygous for the mutant allele. Similarly, all available obligate heterozygous GSPs were tested, and all were found to be heterozygous for the mutant allele. To test for the specificity of this mutation in GSP-affected dogs, the allele discrimination test was applied to a panel of 101 DNA samples collected from 17 other breeds of dog. The panel included DNA from dogs affected with a variety of retinal disorders that are clinically distinct from cd, as well as dogs that were not obviously affected with any retinal disease. All 101 samples were homozygous for the wild-type allele of CNGB3 exon 6 (data not shown).
To demonstrate that both the GSP missense mutation and the AMD deletion co-segregate with the respective diseases in affected families, the allele discrimination test was applied to a subset of dogs from our experimental pedigrees. The pedigree shown in Fig. 5B includes animals homozygous for the AMD CNGB3 deletion (dogs I-1 and II-2) that yield no exon 6 product, dogs homozygous for the GSP mutant allele that yield only a 181 bp product (dogs III-3 and III-4) and several heterozygous dogs. Dog II-3 is heterozygous for the wild-type and GSP missense mutant alleles, yielding 181 and 166 bp products. Dogs I-2, II-1 and III-2 are heterozygous for the AMD deletion and the wild-type allele; thus, only the normal 166 bp fragment is observed. Finally, dog III-1 is heteroallelic at the CNGB3 locus, carrying both the AMD CNGB3 deletion and, as evidenced by the 181 bp fragment, the GSP missense mutation. The fact that this animal is affected demonstrates that the two diseases are allelic.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Linkage mapping of the canine cone degeneration (cd ) disease locus to microsatellite markers on CFA29, together with the conserved synteny of this canine chromosomal region and the long arm of human chromosome 8, provided the first compelling evidence that cd corresponds to the human ACHM3 locus (MIM 262300). The correspondence of CFA29 to human chromosome 8q, established by reciprocal chromosome paint data (36,37,40) as well as the localization of genes from human chromosome 8q to CFA29 by RH mapping (32–34), firmly establishes the evolutionary relationship between CFA29 and human 8q, and identified the canine CNGB3 gene as an excellent positional candidate for canine cone degeneration.
Cone degeneration in AMD pedigrees clearly co-segregates with a CFA29 deletion (Fig. 5B). This is further supported by FISH data (Fig. 4), the consistent and specific failure of genomic PCR and RT–PCR to amplify products within the deletion from AMD cd-affected templates, Southern blot data for at least the partial absence of CNGB3 (Fig. 3C), and northern analyses that demonstrate reduction of transcript in heterozygotes and total loss of transcript in affected individuals (Fig. 3B).
Similarly, the day-blindness observed in GSP-derived pedigrees also co-segregates with a CNGB3 mutation—in this case a D262N missense mutation (Fig. 5B). The region of CNGB3 surrounding the D262N missense mutation is well conserved between humans and dogs, and is predicted to encode the second transmembrane domain of the CNGB3 protein. Allelism of the two phenotypes, cd in Alaskan Malamutes and day-blindness in GSPs, as demonstrated by the crossbreeding experiment (Fig. 5B), also supports the conclusion that the identified CNGB3 mutations are causal.
Supportive arguments for this conclusion can also be drawn from comparative genomics. Achromatopsia in humans, which shares its major defining features with these canine phenotypes, has also been associated with mutations in genes coding for subunits of the cone photoreceptor-specific cyclic nucleotide-gated channel, ACHM2 with mutations in the gene coding for the subunit of CNGA3 (24,25), and ACHM3 with mutations in the gene coding for the ß subunit of CNGB3 (27,28). The human achromatopsia locus ACHM3 is thus orthologous to cd in Alaskan Malamutes and day-blindness in GSPs. Furthermore, genetically engineered mice with a knockout mutation of the murine homolog of CNGA3 (26) have a phenotype consistent with ACHM2. Thus, the evidence that defects in the cone photoreceptor-specific cyclic nucleotide-gated channel can be causal for achromatopsia-like phenotypes strongly supports the conclusion that mutations in CNGB3 are causal in both human ACHM3 and the two canine phenotypes reported here.
Although the size of the CFA29 deletion in cd-affected Alaskan Malamutes has not yet been precisely defined, it appears to be at least 140 kb and removes not only CNGB3 but also at least a large part of CPNE3. The corresponding region of human chromosome 8 is relatively gene-poor, but, even so, if the canine deletion were very much larger than the known 140 kb minimum, one would expect a contiguous deletion syndrome, a cytogenetically observable deletion or both. Because there is no cytogenetically obvious band missing, the upper size of the deletion is probably less than 0.5–1 Mb. The absence of any detected phenotype that could be attributed to the disruption of CPNE3 is perhaps unexpected, but the gene is a member of a multigene family, with no specifically identified function (41,42). Presumably, whatever the function of the copine genes, there is sufficient redundancy for the loss of CPNE3 to be inconsequential.
The cd locus is the eighth canine locus to be identified that determines inheritance of a retinal degenerative disease (5,9–14, 43–45). Recent progress in identifying such loci reflects advances in canine and comparative genomics, combined with the opportunity to exploit advantages conferred by canine population structure, including breed-associated reductions in locus heterogeneity and the availability of large, multigenerational families. Identification of the mutations associated with cd, and establishment of homology between cd and ACHM3, establishes cd as a validated animal model of human ACHM3. These dogs thus offer a unique opportunity to evaluate potential gene therapies for disorders caused by cone-specific genes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Pedigrees and sample preparation
The cd strain of dogs is maintained under sponsorship of NEI/NIH (Grant EY06855; Models of Hereditary Retinal Degeneration). This colony was derived from the Alaskan Malamutes in which the disease was originally described (15–17,46), but the phenotype is currently maintained on a mixed-breed background. For the current study, multiple three-generation subsets of the pedigree of this colony were selected in which the backcross or F2 generations were informative for segregation of the cd phenotype. The initial genome screen used six such three-generation pedigrees. To confirm and refine the results of the initial screen, five additional families from the same colony were genotyped for markers in the identified linkage group. In total, the 11 families comprized 135 individuals with 88 informative progeny. Markers were also typed on a set of eight reference families currently in use for construction of the canine meiotic linkage map (32,35). Genomic DNA was prepared from citrated whole blood or splenic tissue using standard methods (47).
All GSPs studied were privately owned pets, and were made available by their owners to one of the authors (G.M.A.) for clinical examination, collection of blood for research studies and, in one case, for breeding purposes. All procedures involving animals were performed in adherence to the ARVO Resolution for the Use of Animals in Ophthalmic and Vision Research.
Diagnostic methods
Diagnosis of cd phenotype in all dogs was established by electroretinography. Electroretinograms (ERGs) were recorded from halothane-anesthetized dogs as previously described (7,12). Assignment of cd phenotype (non-affected/affected) was based on evaluation of ERG waveforms and amplitudes by criteria previously established (16).
Microsatellite typing and linkage analysis
Microsatellite markers used in this study were developed as previously described or drawn from existing datasets (32,33,48,49). Genotypes were determined by PCR amplification of genomic DNA with a -32P end-labeled forward primer, as previously described (35,49). Primer sequences and PCR conditions can be found on our website htpp://www.fhcrc.org/science/dog_genome/dog.html. Linkage between the cone-degeneration locus and other markers was determined using the best-twopoints function of the MultiMap software package (50), and markers were ordered by multipoint analyses. Data generated using the canine reference families were merged with that from the most recently published version of the canine meiotic linkage map to determine final marker order (32). Maps were constructed with framework markers ordered with odds greater than 1000 : 1 and all remaining markers ordered at odds greater than 10 : 1 using MultiMap (50).
Radiation hybrid analysis
Primers CNGB3 K9ln15-F and CNGB3 K9ln15-R, designed to amplify a microsatellite-containing sequence in canine CNGB3 intron 15, and primers 100-02-TJ-1 and 100-02-TJ-2, designed to amplify an STS from the end of BAC 100-O2, were used to map CNGB3 and BAC 100-O2, respectively. Additional markers were selected from those assigned to CFA29 on canine linkage and RH maps (32,33,49). A 3000 rad canine–hamster radiation hybrid panel RH08 was purchased from Research Genetics (Huntsville, AL). Markers were genotyped on the panel using standard protocols (33,34). Framework markers were ordered with odds greater than 1000 : 1; the remaining markers were all ordered at odds greater than 10 : 1 using MultiMap (50). Sequences for PCR primers and associated amplification conditions are available on the websites htpp://www-recomgen.univ-rennes1.fr/doggy.html and http://www.fhcrc.org/science/dog_genome/dog.html.
Cloning of canine CNGB3 cDNA
The canine CNGB3 gene was obtained by screening a canine retinal cDNA library (Stratagene, La Jolla, CA) using a PCR-based method described previously (51). Primer sequences and PCR conditions can be found on the websites http://www.fhcrc.org/science/dog_genome/dog.html.
RNA isolation, RT–PCR and northern blot
Retinas were dissected from canine eyes enucleated immediately postmortem, placed in sterile RNase-free tubes, frozen by immersion in liquid nitrogen, and stored at -70°C until utilized for RNA extraction. Total RNA was isolated from retinas of cd/+, cd/cd or +/+ dogs using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) following manufacturer's protocol. RT–PCR reactions utilized 2 µg of total RNA isolated from cd/+, cd/cd or +/+ dogs, reverse-transcribed with oligo (dT) primers and the GeneAmp RNA PCR kit (Perkin Elmer, Foster City, CA). The partial CNGB3 cDNA was amplified using primers CNGB3RT-EX5F and CNGB3RT-EX7R following the manufacturer's protocol. Primer sequences can be found on the websites http://www.fhcrc.org/science/dog_genome/dog.html.
For northern blot analysis, total RNA (20 µg/lane) was electrophoresed and transferred utilizing standard protocols (52). Hybridization was done with ExpressHyb Hybridization Solution (Clontech, Palo Alto, CA) according to the manufacturer's protocol. The membrane was hybridized with a partial CNGB3 cDNA probe generated by RT–PCR using primers CNGB3RT-EX5F and CNGB3RT-EX7R as described above. This probe was stripped from the filter and the membrane rehybridized with canine ß-actin cDNA probe (GenBank accession no. AF021873). Probes were radio-labeled with [-32P]dCTP (New England Nuclear) using the RadPrime DNA Labeling System (Invitrogen Life Tech-nologies, Carlsbad, CA).
BAC clone isolation
Primer sequences and detailed methods can be found on the websites http://www.fhcrc.org/science/dog_genome/dog.html. In brief, an arrayed canine 8.1-fold BAC library (39) was probed with a partial CNGB3 cDNA probe generated by RT–PCR using primers CNGB3RT-EX5F and CNGB3RT-EX7R. A CNGB3-positive BAC clone (100-O2) was isolated, purified by alkaline lysis, NotI-digested and size-separated on a FIGE mapper (BioRad, Hercules, CA) to determine the size of the BAC 100-O2 insert (140 kb). STS markers were developed to each BAC end sequence. The presence of both BAC end sequences in genomic DNA from cd/+, cd/cd and +/+ animals was assayed by PCR and agarose gel electrophoresis.
‘Shotgun’ sequencing of the BAC 100-O2 insert to 3x was undertaken by The Institute for Genomic Research (Rockville, MD), and resulted in 19 unordered contigs. Exons 1–3 and their intron/exon boundaries were not present in the shotgun library generated from BAC 100-02, and instead were amplified from BAC 100-O2 by PCR using primers that anneal to coding sequence prior to sequencing.
Primers CPNE3-f1 and CPNE3-r1 (http://www.fhcrc.org/science/dog_genome/dog.html) were also designed to amplify a 215 bp STS corresponding to a fragment of BAC 100-O2 that represents a highly conserved non-coding region in the 3'-UTR (exon 12) of the canine homolog of CPNE3 (Copine 3, MIM 604207).
Fluorescence in situ hybridization (FISH) labeling and chromosome 29 painting of chromosomal spreads
Canine metaphase spreads were prepared from primary fibroblast cultures derived from cd/cd, cd/+ and +/+ dogs using standard procedures (53). Chromosomes were identified cytogenetically based on diaminidino-2-phenylindole (DAPI)-banding patterns, as described previously (37), according to established karyotypes (54,55) and nomenclature (32).
The CFA29 paint, made by degenerate oligonucleotide-primed–PCR (DOP–PCR) amplification of flow-sorted chromosomes, had previously been characterized by its homology with the paracentric region of Red fox (Vulpes vulpes) chromosome 13q (37). One microgram of BAC 100-O2 DNA was labeled with Biotin-16-dUTP (Roche, Indianapolis, IN) using a nick translation kit (Roche, Indianapolis, IN), and was further digested using DNaseI (Sigma, St Louis, MO) to an optimum size range (200–500 bp). One microgram of unlabeled carrier DNA, produced by sonicating canine liver genomic DNA, was added to 100 ng of labeled probe for each hybridization. Probes were hybridized to canine metaphase spreads as previously described (37). Images were captured using the CytoVision system (Applied Imaging, Santa Clara, CA).
CNGB3 exon scanning in GSPs
Genomic DNA isolated from affected GSPs was scanned for CNGB3 mutations. Primer sequences and PCR conditions can be found on the websites http://www.fhcrc.org/science/dog_genome/dog.html. For exons 1 and 4–18, primers were designed to amplify each exon plus about 50 bp of flanking intronic sequence. For exon 2, CNGB3-EX2R anneals to the last 20 bp of the exon. Similarly, CNGB3-EX3F anneals to the first 20 bp and CNGB3-EX3R to the last 20 bp of exon 3.
Exon 6 allele discrimination test
A restriction enzyme assay was developed to test for the presence of the GA change identified in GSPs. Exon 6 of CNGB3 was amplified from genomic DNA using primers CNGB3-EX6F and CNGB3-EX6T. Primer sequences and PCR conditions can be found on the websites http://www.fhcrc.org/science/dog_genome/dog.html. Ten microliters of the 258 bp PCR product were digested with a Sau3AI enzyme in a total volume of 20 µl for 2 h, and the products were separated by electrophoresis. Digestion with Sau3AI of the wild-type DNA carrying the ‘G’ allele at nucleotide 784 in exon 6 results in 25, 77 and 166 bp products following gel electrophoresis. DNA from animals carrying the missense GA mutation at nucleotide 784 could not be cut at the Sau3AI restriction site created by CNGB3-EX6T, and consequently produced only two products of 77 and 181 bp following digestion.
Southern blot analysis
DNA was extracted from whole blood according to standard methods (47), and was digested to completion with EcoRI. Following quantification by spectrophotometry, 20 µg DNA was electrophoresed and transferred to nylon membrane, and was probed as previously described using standard 50% formamide blocking and hybridization buffers (56,57). Probe template was amplified by PCR from wild-type canine genomic DNA using primers CNGB3 Ex18-F, CNGB3 3'-UTR-R, CRYBA2 probe-F and CRYBA2 probe-R (http://www.fhcrc.org/science/dog_genome/dog.html), and was gel-purified using the QiaEx II Gel Extraction kit (Qiagen, Valencia, CA). Probe was labeled by random hexamer-primed labeling with [-32P]dCTP (Amersham Pharmacia Biotech, Piscataway, NJ) according to standard methods (56). Bands were visualized on a Typhoon 8600 phosphoimager (Amersham Pharmacia Biotech, Piscataway, NJ).
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge excellent technical support from Gerri Antonini, Amanda Nickle, Susan Nitroy, Sue Pearce-Kelling, Brian Miller and the staff at the Retinal Disease Studies Facility, and the invaluable graphical assistance from Keith Watamura and David Prober. We thank Nate Sutter and Kenine Comstock for helpful discussions and technical assistance, as well as Joel Malek, Grace Pai, Tamara Feldblyum, Claire Fraser and the Random Sequencing team from The Institute for Genome Research DNA Sequencing Core Facility for BAC sequencing. Additional helpful discussions with Dr Oliver Sacks are also gratefully acknowledged. J.K.L. was supported by PHS National Research Grant T32 GM07270. This work was funded by grants from the National Eye Institute (EY06855 to G.M.A. and EY13132 to G.D.A.), the Foundation Fighting Blindness to G.M.A. and G.D.A. The Morris Animal Foundation/The Seeing Eye Inc. and the Van Sloun Fund for Canine Genetic Research to G.D.A., the Pet Plan Charitable Trust to D.R.S. and the American Kennel Club Canine Health Foundation to E.A.O.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 6072565684; Fax: +1 6072565689; Email: gma2{at}cornell.edu (GMA): Tel: +1 2266676979; Fax: +1 6072565689; Email: eostrand@fred.fhcrc.org(EAO).
AF490511 (canine CNGB3), BH760135 (BAC 100-O2, SP6 end sequence) and BH760136 (BAC 100-O2, T7 end sequence) The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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