|Molecular Vision 2002;
Received 29 November 2001 | Accepted 4 June 2002 | Published 7 June 2002
The canine Phosducin gene: characterization of the exon-intron structure and exclusion as a candidate gene for generalized progressive retinal atrophy in 11 dog breeds
Dekomien, Jörg Thomas Epplen
Molecular Human Genetics, Ruhr University, 44780 Bochum, Germany
Correspondence to: Gabriele Dekomien; Phone: (+49)234 32-25764; FAX: (+49)234 321-4196; email: email@example.com
Purpose: The exon-intron structure of the canine Phosducin (PDC) gene was identified and the gene evaluated as a candidate for generalized progressive retinal atrophy (gPRA) in 20 dog breeds.
Methods: Intronic sequences of the PDC gene were analyzed after amplification using polymerase chain reaction (PCR) and following sequencing from clones isolated from a canine genomic library. Mutation screening was performed by PCR with single strand conformation polymorphism (SSCP) analysis. Conspicuous banding patterns were analyzed via sequence analyses to detect the underlying nucleotide variations.
Results: No polymorphisms were identified after PCR-SSCP analysis within the entire coding region of the PDC gene. A 3 bp deletion in intron intervening sequence (IVS) 3 (position -16 to -18) was observed in 9 breeds, a T->A transversion (position IVS3 -63) in 10 breeds and an A->T transversion (position IVS3 -64) in 2 dog breeds.
Conclusions: PDC was excluded as a candidate gene for autosomal recessively (ar) transmitted gPRA in 11 of the 20 dog breeds investigated.
Generalized progressive retinal atrophy (gPRA) constitutes a group of heterogeneous blinding disorders in a number of pedigree breeds of dogs. The symptoms of the mostly autosomal recessively (ar) transmitted retinal photoreceptor diseases in dog breeds start with night blindness and continue to a restricted visual field ending always with complete blindness [1,2]. The progression rate and the age of onset is variable. In man the homologous inherited disorder is retinitis pigmentosa (RP). RP is genetically heterogeneous and inherited according to either dominant, recessive, X-linked, digenic or maternal modes [3-7]. A number of retina-specific genes encoding proteins involved in the visual transduction cascade in photoreceptors represent candidate genes for this eye disease. gPRA-causing mutations were identified in the β-subunit of the cGMP-specific phosphodiesterase (PDE6B) gene in Irish Setters and Sloughis [8,9] and in the α-subunit of the cGMP-specific phosphodiesterase (PDE6A) gene in Cardigan Welsh Corgis . In the PDC gene, a missense mutation was detected leading potentially to photoreceptor dysplasia (pd), a subform of gPRA in the miniature schnauzer . The RPGR gene is mutated in the X-linked form of PRA (XLPRA) in Samoyede and Siberian huskies . A number of other retinal genes have been investigated and excluded as causes of gPRA in several dog breeds: rhodopsin , RDS/peripherin and ROM-1 [14,15], the α- and γ-subunits of transducin  and PDC [11,17].
PDC modulates the phototransduction cascade by binding to the βγ subunit complexes of transducin (Tdβγ) to form PDC-(Tdβγ) complexes . The cyclic adenosine 3'5'-monophosphate-dependent protein kinase (A-kinase) catalyzes the phosphorylation of PDC. Phosphorylated PDC has lost binding affinity to transducin. In consequence, transducin does not inhibit light-activated rhodopsin [19-21]. PDC was detected only in rod photoreceptors, mainly in the inner segment and the perinuclear region . The mRNA of the canine PDC gene contains an open reading frame of 735 nt coding for 245 amino acids. The PDC nucleotide and amino acid sequences are highly conserved in mammals . Here we report on the identification of intronic sequences and mutation screening of the canine photoreceptor specific PDC gene in 20 different dog breeds.
Blood of 804 dogs from 20 different breeds including 111 gPRA-affected animals (see Table 1 and Table 2) was received from the owners in cooperation with the breeding organizations (Verband für das Deutsche Hundewesen (VDH); Nederlandse Vereniging van Saarlooswolfhonden; Schweizer Kynologische Gesellschaft (SKG)). The blood of most dogs was sent from different regions of Germany. In addition, several Saarloos (Sa), Schapendoes (SD), Sloughis (Sl), and Tibet Terrier (TT) originated from the Netherlands (Sa, SD), Switzerland (Sl, TT), Sweden (Sl), and the USA (Sl). Experienced veterinarians confirmed the gPRA status of affected and unaffected dogs by ophthalmoscopy, which was documented in certificates of the eye examinations.
DNA was extracted from peripheral blood according to standard protocols . Parts of the PDC gene were amplified by PCR in a thermocycler (Biometra, Goettingen, Germany) from the inserts of the λ phages in order to identify intronic sequences. Furthermore, genomic DNAs from each affected dog as well as representative healthy dogs and obligatory carriers were screened for mutations. PCRs were performed in 96-well microtiter plates (Thermowell Costar Corning, NY). Each well contained 50 ng DNA in 10 μl reaction volume, 100 mM Tris (pH 8.3), 500 mM KCl, 1 U Taq Polymerase (Genecraft, Münster, Germany), 0.2 mmol of each NTP, 0.4 mM of each primer, and varying concentrations of MgCl2 (see Table 3). For SSCP analysis, 0.06 μl of [α 32P] dCTP (10 mCi/ml) was included in the PCR. Parts of the inserts of the λ phage were amplified with a one step PCR; for annealing temperatures see Table 3. For genomic mutation analysis, a special PCR procedure was applied: initial denaturation step (5 min at 95 °C), 10 initial cycles 1 °C above the annealing temperature (see Table 3), 22-25 cycles of 95 °C (30 s), annealing temperature (30 s), elongation at 72 °C (40 s) and a final elongation step at 72 °C (3 min).
The PDC gene was cloned from a genomic canine λ-DNA library (λFIX II Library; host: E. coli XL1-Blu MRA (P2) Stratagene, La Jolla, Ca, USA) according the Stratagene standard protocol. Recombinant λDNA was fixed to HybondTM-N Nylon membranes (Amersham, Buckinghamshire, UK) and UV-crosslinked (1' 70 mJ/cm2). The library was screened with PCR amplificates of probes from exon 2 corresponding to nucleotide positions 59-143 and from exon 4 positions 296-667 of the mRNA of the canine PDC gene (EMBL accession number CFY17697). These probes were labeled using [α 32P] dATP and the Megaprime Labelling System (Amersham, Buckinghamshire, UK). Hybridizations were performed at 65 °C in 0.5 M sodium phosphate buffer pH 7.2/7% sodium dodecyl sulfate . After hybridization the filters were washed twice for 30 min each in 2X SSC/1% SDS, once for 15 min with 0.2X SSC/1%SDS at 65 °C and for 30 min with 6X SSC at room temperature. The filters were exposed to phosphoimager screens (STORM 860) and evaluated with the programs STORM Scanner Control and Image Quant (Molecular Dynamics). Hybridizing clones were isolated and plaque purified as described . The approximate insert sizes of the different clones were estimated with exon primers via PCR (see conditions described above using about 0.2 ng phage DNA).
Exon/intron boundaries were analyzed by comparison of canine mRNA (, EMBL accession number Y17697) with four genomic sequences of the human retina-specific protein MEKA, i.e., the PDC gene (5'-flanking region ; Exon 1, 2, and 3  EMBL accession numbers L252260, M38058, and M38059, respectively) using the program Blast Search (NCBI). Intronic sizes were estimated by overlapping PCRs including parts of neighboring exons. PCR amplificates were extracted from 0.8% agarose gels using the Easy Pure extraction kit (Biozym, Germany) and sequenced with intron-overlapping primers (see Table 3). Parts of intron 1 of the PDC gene were characterized by nested PCR using the T7 primer sequence of the λ FIX II vector. Long-range PCR using the Elongase enzyme mix (GIBCO BRL, Karlsruhe, Germany) was performed from genomic DNA in order to identify the splice donor site of intron 1 according to the recommendations of the manufacturer. Sequencing reactions from 2-3 clones and conspicuous band patterns of the SSCP analysis were carried out by the dideoxy-chain termination method using the BDT (Perkin-Elmer, Norwalk, CT) according to the manufacturer's instructions. All sequencing reactions were run on an automated DNA sequencer (Applied Biosystems 373 XL, Foster City, USA) and analysed using the ABI PrismTM 373 XL.
Positions of intronic primers which were used for mutation screening were created after DNA sequence analysis of the genomic PDC clones (see Table 3). SSCP samples were treated as described [16,28]. PCR products were digested dependent on the lengths of the fragments  with different restriction enzymes (exon 2 BsuRI, exon 3 MnlI, exon 4 part 1 MboII, and exon 4 part 2 MnlI/RsaI). The PCRs (3 μl) were denatured with 7 μl of loading buffer (95% deionized formamide 10 mM NaOH, 20 mM EDTA, 0.06% (w/v) xylene cyanol, and 0.06% (w/v) bromophenol blue). The samples were heated to 95 °C for 5 min and snap cooled on ice. 3 μl aliquots of the single-stranded fragments were separated through two sets of 6% polyacrylamide (acrylamide/bisacrylamide: 19/1) gels, one set containing 10% glycerol, another containing 5% glycerol and 1 M urea. Gels were run with 1X TBE buffer at 50-55 W for 4-6 h at 4 °C. All gels were dried and subjected to autoradiography over night. Selected DNA samples with band shifts evidenced by SSCP electrophoresis were purified and cycle sequenced as described above.
Results & Discussion
The screening of the genomic library with probes of exons 2 and 4 led to the isolation of four genomic DNA clones, each containing some different parts of the PDC gene. Yet exon 1 was lacking in these clones. Comparisons with genomic DNA of man (EMBL accession number AL162722) show an intron 1 size of more than 75000 base pairs (bp). Therefore, the canine intron 1 insert may well exceed clonable sizes in l-phages. Using long-range PCR of intron 1 a 14 kb fragment was identified. Sequencing revealed the 3' part of exon 1 and partial intronic sequences (EMBL accession number AJ429100). The sequencing data indicate that the canine PDC gene has four exons. Exon 1 (69 bp) and 24 bp of exon 2 comprise the 5' untranslated region (UTR) of the PDC gene. The coding region contains 738 bp: exon 2 85 bp, exon 3 152 bp and exon 4 525 bp and additional 409 bp in the 3' UTR (see Figure 1). Comparing the canine with the human PDC gene, in exon 4 three additional bases lead to an extra amino acid in man compared to the 245 amino acids in dogs. Otherwise, the interspecific homology amounts to 90% at the amino acid level. Introns 2 and 3 are smaller in dogs (intron 2: human 2820 bp, dog about 1900 bp; intron 3: man 1947 bp, dog about 1500 bp). The canine splice donor and acceptor sites are in agreement with the GT/AG rule (see Table 4). The human PDC gene maps to chromosome 1q24.3-31.1. On the basis of reciprocal chromosome painting , the canine PDC gene is, therefore, predicted to map to CFA 7, the homologous chromosomal region in dogs.
Furthermore, the PDC gene was examined here for polymorphisms in 20 breeds, including all gPRA-affected dogs and selected normal and obligate gPRA carriers by PCR-SSCP analysis of exons 2, 3 and 4, parts of the three introns, as well as the UTRs. In the coding region of the PDC gene, no polymorphism was detected. However, in eleven breeds of dogs, differences were detected in intron 3 of the PDC gene: a TCT deletion in positions -16 to -18 (see Figure 2A) in BDP, Co, D, ECS, EM, IRS, LR, MP, Sl, TM and TT as well as a T/A transversion in position -63 in BDP, Co, D, ECS, GR, IRS, LR, MP, Sl TM and TT, as well as an A/T nucleotide exchange in position -64 in EM and Sa (see Figure 2B; for abbreviations see Table 1). The polymorphism in position IVS3 -64 was never observed in linkage with the TCT deletion. Allele frequencies of the 3 polymorphisms in the different breeds are consistent with the expectations from Hardy Weinberg equilibrium (Table 2). The R82G missense mutation described in exon 4 of the PDC gene in miniature schnauzers  was not identified in any of the dog breeds examined. Two 3' UTR polymorphisms described in Miniature poodles and Irish wolfhounds  were also not detected here.
The new polymorphisms specified above were identified in heterozygous state in some gPRA-affected dogs in 11 of the 20 breeds. Therefore, these sequence variations can be used as intragenic markers lacking segregation with ar gPRA. The breeding history, small population size and gPRA abundance in the investigated breeds point to few meiotic events in which intragenic recombinations could have occured between an unidentified mutation in the PDC locus in gPRA dogs and the investigated polymorphism .
Given ar transmission, our typing results suggest that the sequence variations in the PDC gene are not causative for gPRA in the following 11 dog breeds: Co, D, ECS, EM, GR, IRS, MP, Sa, Sl, TM, and TT.
We thank the owners of the dogs for blood samples, the veterinarians of the Dortmunder Ophthalmologenkreis (DOK) for the ophthalmologic investigations of the dogs, and the support of different breeding clubs. These studies were supported in part by the Gesellschaft für kynologische Forschung, Bonn (Germany).
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