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Characterization of canine rod photoreceptor cGMP-gated cation channel α-subunit gene and exclusion of its involvement in the hereditary retinal dystrophy of Swedish Briards
Gene 202 (1997) 115–119
Characterization of canine rod photoreceptor cGMP-gated cation channel a-subunit gene and exclusion of its involvement in the hereditary retinal dystrophy of Swedish Briards Andres Veske a, Sven Erik G. Nilsson b, Andreas Gal a,* a Institut fu¨r Humangenetik, Universita¨ts-Krankenhaus Eppendorf, Butenfeld 42, D-22529 Hamburg, Germany b Department of Ophthalmology, University of Linko¨ping, Linko¨ping, Sweden Received 24 April 1997; accepted 26 July 1997; Received by S. Yokoyama
Abstract The nucleotide sequence of the canine rod photoreceptor cGMP-gated cation channel a-subunit (cCNCG1) cDNA has been determined. The open reading frame consists of 2073 nucleotides, which encode a putative protein of 691 amino acids. In addition, we have established the exon/intron boundaries of the cCNCG1 gene and determined the complete sequence of six introns of a total of eight. The exon/intron organization ( location and length of exons and introns) of the cCNCG1 gene is very similar to that of the human rod photoreceptor cGMP-gated cation channel a-subunit gene. We used single-strand conformation polymorphism analysis to search for potential pathogenic sequence changes in the cCNCG1 gene in a Swedish Briard and Briard–Beagle dog kindred, in which an autosomal recessive retinal dystrophy is segregating, a disease which shows phenotypic similarities to retinitis pigmentosa, a heterogeneous group of hereditary and progressive retinal degeneration in human. In intron 3, we found several DNA polymorphisms, which do not cosegregate with the affected status of the dogs, thus excluding cCNCG1 as a candidate gene for the retinal dystrophy in this strain of Swedish Briards. © 1997 Elsevier Science B.V. Keywords: RACE; RAGE; cCNCG1; Vision
1. Introduction Cyclic nucleotide-gated channels (CNGC ) represent a family of cation-selective ion channels which are activated by direct interaction with cyclic nucleotides (cAMP and cGMP). In photoreceptor cells and olfactory neurons, CNGC play a central role in signal transduction pathways by controlling the flow of Na+ and Ca2+ ions into the cells following activation of a G-protein-mediated signaling cascade which changes the * Corresponding author. Tel.: +49 40 47172120; Fax: +49 40 47175138; e-mail: [email protected] Abbreviations: 5∞-UTR, 5∞-untranslated region; aa, amino acid(s); bp, base pair; cDNA, DNA complementary to RNA; cAMP, cyclic adenosine 3∞, 5∞-monophosphate; cGMP, cyclic guanosine 3∞, 5∞-monophosphate; CNG, cyclic nucleotide-gated; cCNCG1, canine rod cyclic nucleotide-gated cation channel a-subunit gene; kb, kilobase; nt, nucleotide; PCR, polymerase chain reaction; pmol, picomole; RP, retinitis pigmentosa; RT–PCR, reverse transcription–polymerase chain reaction; RACE, rapid amplification of cDNA ends; RAGE, rapid amplification of genomic DNA ends; SSCP, single strand conformation polymorphism. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 4 61 - 7
intracellular levels of cyclic nucleotides (for review see Yau and Chen, 1995; Kaupp, 1995). Recent studies indicate that photoreceptor CNGC consists of two subunits, a ( Kaupp et al., 1989) and b (Chen et al., 1993), that assemble into a hetero-oligomeric complex ( Ko¨rschen et al., 1995) whereas in vitro a-subunits may form functional channels with properties nearly identical to those of the native channel ( Kaupp et al., 1989). Each subunit shares similar characteristic features including six hydrophobic membrane spanning segments ( H1–H6), a cGMP-binding domain near the carboxylterminus, a pore region that plays a major role in ion permeation, and a voltage-sensor-like motif comprising the S4 transmembrane domain (Jan and Jan, 1990; Heginbotham et al., 1992; Bo¨nigk et al., 1993; Henn et al., 1995). Human rod CNGC a-subunit gene consists of 10 exons, of which two are untranslated (Dhallan et al., 1992). While Dhallan and co-workers (1992) found an Alu element in the 5∞-UTR of the transcript, which should form the second exon, Pittler et al. (1992) could not confirm this finding. A unique feature of the gene is the unusually large last exon, which encodes two-thirds of the protein.
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A. Veske et al. / Gene 202 (1997) 115–119
There is a great number of genes whose mutations are causative in a wide range of inherited retinal dystrophies in different species including human. Recently, mutations of the human rod photoreceptor CNGC asubunit gene (CNCG1) have been suggested to be the cause of a form of autosomal recessive retinitis pigmentosa in human (Dryja et al., 1995). In retinopathies of the dog, up to now, the gene encoding the b-subunit of rod photoreceptor cGMP-specific phosphodiesterase has been shown to be mutated in the rod/cone dysplasia of Irish Setter (Suber et al., 1993). It is possible that other forms of canine retinal dystrophies are caused by mutations in the canine equivalents of genes known to cause hereditary retinal disease in other animal species or in human. We have analysed the canine gene encoding CNCG1 and performed segregation analysis on six offspring of a Swedish Briard dog affected by a congenital and slowly progressive retinal dystrophy ( Wrigstad et al., 1994; Narfstro¨m et al., 1994) to obtain genetic evidence for or against the involvement of this gene in the pathogenesis of this type of retinal dystrophy.
2. Experimental and discussion
2.1. Characterization of the cCNCG1 cDNA Total mRNA was extracted and purified from the retina of a cross-bred dog and an affected Briard dog using DynabeadsA Oligo (dT ) (Dynal ) following the 25 manufacturer’s recommendation. Samples of mRNA from wild-type and affected dog retinas were reversely transcribed to first-strand cDNA using SuperScript@ (Gibco BRL, Life Technologies, Gaithersburg, MD, USA) reverse transcriptase and oligoT as synthesis 22 primer. The cDNA pool was used as template for rapid amplification of cDNA ends (RACE) and further PCR reactions (Frohman et al., 1988). Part of the cCNCG1 gene was amplified from canine genomic DNA and sequenced using primers designed for the human analog (data not shown). Based on the canine sequence information obtained we designed primers for further experiments. For 3∞-RACE we used canine ion channel exon 9-specific primer DC1999 (5∞-AAGAGAAGGGAAAGCAAATC-3∞) and oligoT corresponding to the 22 poly A tail. For 5∞-RACE we used Marathon@ cDNA
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Amplification Kit (Clontech, Palo Alto, CA, USA) components following the manufacturer’s instructions. Primers for 5∞-RACE were adaptor-specific (AP1: 5∞-CCATCCTAATACGACTCACTATAGGGC-3∞) and canine exon 9-specific (DC1110: 5∞-AGTGGATAATGATGACGATA-3∞). Amplified PCR products were diluted 1:1000 and used for subsequent nested PCR reactions with adaptor-specific nested primer (AP2: 5∞-ACTCACTATAGGGCTCGAGCGGC-3∞) and genespecific primer (DC863: 5∞-ACCAGCAGTCCTTGTTCTAA-3∞). Instead of Taq polymerase we used highest fidelity Pfu DNA polymerase (Promega, Madison, WI, USA) and touchdown PCR procedure to increase both the specificity and yield of PCR (Don et al., 1991). Cycling conditions for 3∞-RACE were as follows: initial denaturation at 95°C for 3 min, five cycles of denaturation (10 s at 95°C ), annealing (10 s at 56°C ) and elongation (30 s at 72°C ), followed by five cycles of annealing at 54°C (the other parameters were unaltered ), and by 28 cycles of annealing at 52°C and a final elongation step (5 min at 72°C ). For 5∞-RACE touchdown PCR annealing temperatures were for first PCR 56–54–52°C and for second, nested PCR 58–56–54°C (the other parameters were unchanged ). Amplification products were purified by CentriconA100 concentrators (Amicon, Beverly, MA) and both strands of two different unaffected animals were directly sequenced using primers (3.2 pmol ) specific to the amplicons and PRISM@ Ready Reaction Sequencing Kit (Perkin Elmer, Foster City, CA, USA) on an automatic fluorometric DNA sequencer (Applied Biosystems model 373A). Cycle-sequencing reactions were carried out on the GeneAmp PCR System 9600 (Perkin Elmer). 2.2. Analysis of the cCNCG1 cDNA The full-length cDNA identified is 2696 bp long and contains an open reading frame between nt 197 and 2269, encoding a 691 amino acid long protein with a calculated molecular mass of 80.259 kDa. The homology of the cDNA encoding canine CNCG1 is highest with the cow homolog (91.3%) and lowest with the chicken (76.6%). The most obvious differences between the various cGMP-gated channel homologs are in the beginning of the transcript, which contains the amino-terminal 92 amino acids removed post- or cotranslationally and thought to be required for targeting of the channel or for interaction of the channel with other proteins
Fig. 1. Amino acid alignment of canine CNCG1 with that of other vertebrate rod CNGC a-subunits (human, Pittler et al., 1992; cow, Kaupp et al., 1989; mouse, Pittler et al., 1992; rat, Barnstable and Wei, unpublished; and chicken, Bo¨nigk et al., 1993). Amino acid sequences were aligned for maximal homology using Clustal algorithm in the DNASTARA software for Windows. Only differences from canine CNGC a-subunit sequence are indicated. Amino acid residue numbers are given on the right. Conserved CNGC pore-forming region (Heginbotham et al., 1992; Sun et al., 1996) is framed in black, cGMP-binding pocket ( Varnum et al., 1995) is framed by dotted lines, whereas voltage-sensor-like motif S4 (Bo¨nigk et al., 1993) is framed in gray. Putative hydrophobic transmembrane segments ( Kaupp et al., 1989) are consecutively numbered (H1–H6) and their position is indicated by solid lines.
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(Molday et al., 1991). The initiation codon at nt 197 is within the sequence AACCATG, which conforms well with the Kozak consensus sequence (CANCATG; Kozak, 1987). All putative structural elements characteristic for the ion channel superfamily and well conserved across evolution are present in cCNCG1 ( Fig. 1). 2.3. Determination of exon/intron structure of the cCNCG1 gene Genomic DNA was isolated from peripheral blood leukocytes of an unaffected dog by conventional methods. Purified DNA was submitted to PCR amplification by overlapping primers designed from canine cDNA sequence and putative flanking intron sequences deduced from the organization of the human CNGC a-subunit gene (Dhallan et al., 1992). Amplification was done by OmniGene Thermal Cycler (Hybaid ) using manual hot start and touchdown PCR. Exon/intron boundaries for introns 1, 3 and 8 were determined by the method of rapid amplification of genomic DNA (RAGE) and Universal GenomeWalker@ Kit (Clontech) according to the manufacturer’s instructions. Sequencing of the large introns was performed by the primer walking method. Purification and sequencing procedures were the same as described in Section 2.1. Results are summarized in Table 1. The cDNA sequence was deposited in the EMBL, GenBank and DDBL Nucleotide Sequence Databases under accession number X99914; intron accession numbers are Y11309, X99913, Y11310, Y10559, Y11372 and Y11311 for introns 2, 3, 5, 6, 7 and 8, respectively. 2.4. SSCP–PCR analysis of cCNCG1 in a Swedish Briard–Beagle kindred with hereditary retinal dystrophy For single-strand conformation polymorphism analysis (Orita et al., 1989) we used at least two different gel conditions to improve mutation detection rate (run at room temperature in 8% polyacrylamide and 5% glycerol or run at 4°C in 6% polyacrylamide and 5% glycerol,
Fig. 2. Detection of the sequence polymorphism 266C/T in cCNCG1 gene intron 3 by restriction enzyme analysis. PCR amplified 284-bp intron 3 fragment was digested with XmnI and separated on 2% agarose gel. The amplicon contains a restriction site for XmnI, which gives rise to restriction fragments of 47 and 237 bp. Nucleotide C at position 266 defines an additional recognition site for XmnI and generates allelic restriction fragments of 65 and 172 bp. Lane 1: homozygous pattern of 266T (unaffected animal ); lane 2: heterozygous sample of 266T/C (affected animal ); lane 3: homozygous pattern of 266C (affected animal ); lane 4: molecular weight marker (pBR322, MspI digested).
for details see Bunge et al., 1996). The size of PCR fragments used for SSCP was 120–380 bp. Due to the high degree of inbreeding, affected animals are expected to be homozygous not only for the diseasecausing mutation but also for other sequence variations in the disease-causing gene. Therefore, a heterozygous pattern for a sequence change excludes the gene in question as the one implicated in the retinal disorder in this strain. As overlapping cCNCG1-transcript amplicons from affected and unaffected animals did not show any difference on SSCP gels the same analysis was also performed for introns. In intron 3, we found three sequence variants, 266C/T, 294T/C and 374T/C (numbers according to the sequence deposited to GenBank). As 266C/T creates an additional XmnI restriction site in genomic DNA, this provides an easy and sequencespecific assay to detect this variant. As affected animals are heterozygous for the 266C/T variant ( Fig. 2), these results exclude the cGMP-gated channel a-subunit gene to be a candidate for the retinal dystrophy in the kinship of Swedish Briards studied here. We have also studied
Table 1 Position and nucleotide sequence of exon/intron boundaries of the cCNCG1 gene Exon
Exon length (bp)
Splice donor site
Intron length (bp)
Position on cDNA
Splice acceptor site
1 2 3 4 5 6 7 8 9
182 121 123 63 42 111 108 107 1415
AAAGgtaagaagcc GCAGgtatctatat AGAGgtgagcagta AGCAgtaagtacaa AGAGgtaagttaga AAGAgtaagtggca CAAGgtatctatat ACAGgtaaatgtgt
n.d. 1295 947 ~4300 89 915 735 1476
182 303 426 489 531 642 750 857
ctctccccagATAT tttaccacagCTCC catttttcagGGAA tcttctttagAGAA ctcttttcagCAAA ttgctgttagGGAG tctcttacagAGCA tctattttagGTTA
Exon sequence is shown in upper-case letters; intron sequence is in lower-case letters. n.d., not determined; position on cDNA refers to the last nucleotide of the exon.
A. Veske et al. / Gene 202 (1997) 115–119
36 unaffected outbred Briard and foxhound dogs to determine the allele frequency of the 266C/T polymorphism. As expected, several animals were homozygous either for the T or the C allele. The heterozygosity value calculated was H=0.481 with allele frequencies of 0.597 and 0.403 for T and C, respectively.
3. Conclusions (1) Retinal cDNA encoding canine CNCG1 was identifed. Canine and human sequences share 89.4% identity at the nt level and 90.4% identity at the aa level. (2) Exon/intron boundaries of the cCNCG1 gene were determined and six out of eight introns were sequenced completely. Canine and human genes share a similar organization in that both have a large last exon encoding two-thirds of the protein and 8 (in human 9, due to the Alu element) additional small exons (the first of them untranslated) encoding amino terminal part of the protein. The cCNCG1 gene structure is shown in Table 1. (3) Results of segregation analysis by an intronic polymorphism (266C/T ) in the Swedish Briard and Briard–Beagle kindred excluded cCNCG1 as a candidate gene for the retinal dystrophy found in this dog kindred (Fig. 2). Nevertheless, it is possible that mutations in this gene are responsible for other forms of canine retinal dystrophy.
Acknowledgement This study was financially supported by grants from the Deutsche Forschungsgemeinschaft (Ga 210/5-4) and the Swedish Medical Research Council (project 12X-734). This article is based in part on a doctoral study by A.V.
References Bo¨nigk, W., Altenhofen, W., Mu¨ller, F., Dose, A., Illing, M., Molday, R.S., Kaupp, U.B., 1993. Rod and cone photoreceptor cells distinct genes for cGMP-gated channels. Neuron 10, 865–877. Bunge, S., Fuchs, S., Gal, A., 1996. Simple and nonisotopic methods to detect unknown gene mutations in nucleic acids. In: Adolph, K.W. ( Ed.), Methods in Molecular Genetics. Academic Press, Boca Raton, FL, pp. 26–40. Chen, T.-Y., Peng, Y.-W., Dhallan, R.S., Ahamed, B., Reed, R.R., Yau, K.-W., 1993. A new subunit of the cyclic nucleotide-gated cation channel in retinal rods. Nature 362, 764–767. Dhallan, R.S., Macke, J.P., Eddy, R.L., Shows, T.B., Reed, R.R., Yau, K.-W., Nathans, J., 1992. Human rod photoreceptor cGMP-gated channel: amino acid sequence, gene structure, and functional expression. J. Neurosci. 12, 3248–3256.
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Don, R.H., Cox, B.J., Wainwright, B.J., Baker, K., Mattick, J.S., 1991. ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 19, 4008 Dryja, T.P., Finn, J.T., Peng, Y.-W., McGee, T.L., Berson, E.L., Yau, K.-W., 1995. Mutations in the gene encoding the a subunit of the rod cGMP-gated channel in autosomal recessive retinitis pigmentosa. Proc. Natl. Acad. Sci. USA 92, 10177–10181. Frohman, M.A., Dush, M.K., Martin, G.R., 1988. Rapid production of full-length cDNA from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002. Heginbotham, L., Abramson, T., MacKinnon, R., 1992. A functional connection between the pores of distantly related ion channels as revealed by mutant K+ channels. Science 258, 1152–1155. Henn, D.K., Baumann, A., Kaupp, U.B., 1995. Probing of the transmembrane topology of cyclic nucleotide-gated channels by a gene fusion approach. Proc. Natl. Acad. Sci. USA 92, 7425–7429. Jan, L.Y., Jan, Y.N., 1990. A superfamily of ion channels. Nature 345, 672 Kaupp, U.B., 1995. Family of cyclic nucleotide gated ion channels. Curr. Opin. Neurobiol. 5, 434–442. Kaupp, U.B., Niidome, T., Tanabe, T., Terada, S., Bo¨nigk, W., Stu¨hmer, W., Cook, N.J., Kangawa, K., Matsuo, H., Hirose, T., Miyata, T., Numa, S., 1989. Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel. Nature 342, 762–766. Ko¨rschen, H.G., Illing, M., Seifert, R., Sesti, F., Williams, A., Gotzes, S., Colville, C., Mu¨ller, F., Dose, A., Godde, M., Molday, L., Kaupp, U.B., Molday, R.S., 1995. A 240 kDa protein represents the complete b subunit of the cyclic nucleotide-gated channel from rod photoreceptor. Neuron 15, 627–636. Kozak, M., 1987. An analysis of 5∞-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125–8148. Molday, R.S., Molday, L.L., Dose, A., Clark-Lewis, I., Illing, M., Cook, N.J., Eismann, E., Kaupp, U.B., 1991. The cGMP-gated channel of the rod photoreceptor cell. Characterization and orientation of the amino terminus. J. Biol. Chem. 266, 21917–21922. Narfstro¨m, K., Wrigstad, A., Ekesten, B., Nilsson, S.E.G., 1994. Hereditary retinal dystrophy in the Briard dog: clinical and hereditary characteristics. Vet. Comp. Ophthalmol. 4, 85–92. Orita, M., Suzuki, Y., Sekiya, T., Hayashi, K., 1989. A rapid and sensitive detection of point mutations and genetic polymorphism using polymerase chain reaction. Genomics 5, 874–879. Pittler, S.J., Lee, A.K., Altherr, M.R., Howard, T.A., Seldin, M.F., Hurwitz, R.L., Wasmuth, J.J., Baehr, W., 1992. Primary structure and chromosomal localization of human and mouse photoreceptor cGMP-gated cation channel. J. Biol. Chem. 267, 6257–6262. Suber, M.L., Pittler, S.J., Qin, N., Wright, G.C., Holcombe, V., Lee, R.H., Craft, C.M., Lolley, R.N., Baehr, W., Hurwitz, R.L., 1993. Irish setter dogs affected with rod/cone dysplasia contain a nonsense mutation in the rod cGMP phosphodiesterase b-subunit gene. Proc. Natl. Acad. Sci. USA 90, 9968–9972. Sun, Z.-P., Akabas, M.H., Goulding, E.H., Karlin, A., Siegelbaum, S.A., 1996. Exposure of residues in the cyclic nucleotide-gated channel pore: P region structure and function in gating. Neuron 16, 141–149. Varnum, M.D., Black, K.D., Zagotta, W.N., 1995. Molecular mechanism for ligand discrimination of cyclic nucleotide-gated channels. Neuron 15, 619–625. Wrigstad, A., Narfstro¨m, K., Nilsson, S.E.G., 1994. Slowly progressive changes of the retina and retinal pigment epithelium in briard dogs with hereditary retinal dystrophy. Documenta Ophthalmol. 87, 337–354. Yau, K.-W., Chen, T.-Y., 1995. Cyclic nucleotide-gated channels. In: North, R.A. ( Ed.), Ligand- and Voltage-gated channels. CRC Press, Boca Raton, FL, pp. 307–335.
Characterization of canine rod photoreceptor cGMP-gated cation channel a-subunit gene and exclusion of its involvement in the hereditary retinal dystrophy of Swedish Briards Andres Veske a, Sven Erik G. Nilsson b, Andreas Gal a,* a Institut fu¨r Humangenetik, Universita¨ts-Krankenhaus Eppendorf, Butenfeld 42, D-22529 Hamburg, Germany b Department of Ophthalmology, University of Linko¨ping, Linko¨ping, Sweden Received 24 April 1997; accepted 26 July 1997; Received by S. Yokoyama
Abstract The nucleotide sequence of the canine rod photoreceptor cGMP-gated cation channel a-subunit (cCNCG1) cDNA has been determined. The open reading frame consists of 2073 nucleotides, which encode a putative protein of 691 amino acids. In addition, we have established the exon/intron boundaries of the cCNCG1 gene and determined the complete sequence of six introns of a total of eight. The exon/intron organization ( location and length of exons and introns) of the cCNCG1 gene is very similar to that of the human rod photoreceptor cGMP-gated cation channel a-subunit gene. We used single-strand conformation polymorphism analysis to search for potential pathogenic sequence changes in the cCNCG1 gene in a Swedish Briard and Briard–Beagle dog kindred, in which an autosomal recessive retinal dystrophy is segregating, a disease which shows phenotypic similarities to retinitis pigmentosa, a heterogeneous group of hereditary and progressive retinal degeneration in human. In intron 3, we found several DNA polymorphisms, which do not cosegregate with the affected status of the dogs, thus excluding cCNCG1 as a candidate gene for the retinal dystrophy in this strain of Swedish Briards. © 1997 Elsevier Science B.V. Keywords: RACE; RAGE; cCNCG1; Vision
1. Introduction Cyclic nucleotide-gated channels (CNGC ) represent a family of cation-selective ion channels which are activated by direct interaction with cyclic nucleotides (cAMP and cGMP). In photoreceptor cells and olfactory neurons, CNGC play a central role in signal transduction pathways by controlling the flow of Na+ and Ca2+ ions into the cells following activation of a G-protein-mediated signaling cascade which changes the * Corresponding author. Tel.: +49 40 47172120; Fax: +49 40 47175138; e-mail: [email protected] Abbreviations: 5∞-UTR, 5∞-untranslated region; aa, amino acid(s); bp, base pair; cDNA, DNA complementary to RNA; cAMP, cyclic adenosine 3∞, 5∞-monophosphate; cGMP, cyclic guanosine 3∞, 5∞-monophosphate; CNG, cyclic nucleotide-gated; cCNCG1, canine rod cyclic nucleotide-gated cation channel a-subunit gene; kb, kilobase; nt, nucleotide; PCR, polymerase chain reaction; pmol, picomole; RP, retinitis pigmentosa; RT–PCR, reverse transcription–polymerase chain reaction; RACE, rapid amplification of cDNA ends; RAGE, rapid amplification of genomic DNA ends; SSCP, single strand conformation polymorphism. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 4 61 - 7
intracellular levels of cyclic nucleotides (for review see Yau and Chen, 1995; Kaupp, 1995). Recent studies indicate that photoreceptor CNGC consists of two subunits, a ( Kaupp et al., 1989) and b (Chen et al., 1993), that assemble into a hetero-oligomeric complex ( Ko¨rschen et al., 1995) whereas in vitro a-subunits may form functional channels with properties nearly identical to those of the native channel ( Kaupp et al., 1989). Each subunit shares similar characteristic features including six hydrophobic membrane spanning segments ( H1–H6), a cGMP-binding domain near the carboxylterminus, a pore region that plays a major role in ion permeation, and a voltage-sensor-like motif comprising the S4 transmembrane domain (Jan and Jan, 1990; Heginbotham et al., 1992; Bo¨nigk et al., 1993; Henn et al., 1995). Human rod CNGC a-subunit gene consists of 10 exons, of which two are untranslated (Dhallan et al., 1992). While Dhallan and co-workers (1992) found an Alu element in the 5∞-UTR of the transcript, which should form the second exon, Pittler et al. (1992) could not confirm this finding. A unique feature of the gene is the unusually large last exon, which encodes two-thirds of the protein.
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A. Veske et al. / Gene 202 (1997) 115–119
A. Veske et al. / Gene 202 (1997) 115–119
There is a great number of genes whose mutations are causative in a wide range of inherited retinal dystrophies in different species including human. Recently, mutations of the human rod photoreceptor CNGC asubunit gene (CNCG1) have been suggested to be the cause of a form of autosomal recessive retinitis pigmentosa in human (Dryja et al., 1995). In retinopathies of the dog, up to now, the gene encoding the b-subunit of rod photoreceptor cGMP-specific phosphodiesterase has been shown to be mutated in the rod/cone dysplasia of Irish Setter (Suber et al., 1993). It is possible that other forms of canine retinal dystrophies are caused by mutations in the canine equivalents of genes known to cause hereditary retinal disease in other animal species or in human. We have analysed the canine gene encoding CNCG1 and performed segregation analysis on six offspring of a Swedish Briard dog affected by a congenital and slowly progressive retinal dystrophy ( Wrigstad et al., 1994; Narfstro¨m et al., 1994) to obtain genetic evidence for or against the involvement of this gene in the pathogenesis of this type of retinal dystrophy.
2. Experimental and discussion
2.1. Characterization of the cCNCG1 cDNA Total mRNA was extracted and purified from the retina of a cross-bred dog and an affected Briard dog using DynabeadsA Oligo (dT ) (Dynal ) following the 25 manufacturer’s recommendation. Samples of mRNA from wild-type and affected dog retinas were reversely transcribed to first-strand cDNA using SuperScript@ (Gibco BRL, Life Technologies, Gaithersburg, MD, USA) reverse transcriptase and oligoT as synthesis 22 primer. The cDNA pool was used as template for rapid amplification of cDNA ends (RACE) and further PCR reactions (Frohman et al., 1988). Part of the cCNCG1 gene was amplified from canine genomic DNA and sequenced using primers designed for the human analog (data not shown). Based on the canine sequence information obtained we designed primers for further experiments. For 3∞-RACE we used canine ion channel exon 9-specific primer DC1999 (5∞-AAGAGAAGGGAAAGCAAATC-3∞) and oligoT corresponding to the 22 poly A tail. For 5∞-RACE we used Marathon@ cDNA
117
Amplification Kit (Clontech, Palo Alto, CA, USA) components following the manufacturer’s instructions. Primers for 5∞-RACE were adaptor-specific (AP1: 5∞-CCATCCTAATACGACTCACTATAGGGC-3∞) and canine exon 9-specific (DC1110: 5∞-AGTGGATAATGATGACGATA-3∞). Amplified PCR products were diluted 1:1000 and used for subsequent nested PCR reactions with adaptor-specific nested primer (AP2: 5∞-ACTCACTATAGGGCTCGAGCGGC-3∞) and genespecific primer (DC863: 5∞-ACCAGCAGTCCTTGTTCTAA-3∞). Instead of Taq polymerase we used highest fidelity Pfu DNA polymerase (Promega, Madison, WI, USA) and touchdown PCR procedure to increase both the specificity and yield of PCR (Don et al., 1991). Cycling conditions for 3∞-RACE were as follows: initial denaturation at 95°C for 3 min, five cycles of denaturation (10 s at 95°C ), annealing (10 s at 56°C ) and elongation (30 s at 72°C ), followed by five cycles of annealing at 54°C (the other parameters were unaltered ), and by 28 cycles of annealing at 52°C and a final elongation step (5 min at 72°C ). For 5∞-RACE touchdown PCR annealing temperatures were for first PCR 56–54–52°C and for second, nested PCR 58–56–54°C (the other parameters were unchanged ). Amplification products were purified by CentriconA100 concentrators (Amicon, Beverly, MA) and both strands of two different unaffected animals were directly sequenced using primers (3.2 pmol ) specific to the amplicons and PRISM@ Ready Reaction Sequencing Kit (Perkin Elmer, Foster City, CA, USA) on an automatic fluorometric DNA sequencer (Applied Biosystems model 373A). Cycle-sequencing reactions were carried out on the GeneAmp PCR System 9600 (Perkin Elmer). 2.2. Analysis of the cCNCG1 cDNA The full-length cDNA identified is 2696 bp long and contains an open reading frame between nt 197 and 2269, encoding a 691 amino acid long protein with a calculated molecular mass of 80.259 kDa. The homology of the cDNA encoding canine CNCG1 is highest with the cow homolog (91.3%) and lowest with the chicken (76.6%). The most obvious differences between the various cGMP-gated channel homologs are in the beginning of the transcript, which contains the amino-terminal 92 amino acids removed post- or cotranslationally and thought to be required for targeting of the channel or for interaction of the channel with other proteins
Fig. 1. Amino acid alignment of canine CNCG1 with that of other vertebrate rod CNGC a-subunits (human, Pittler et al., 1992; cow, Kaupp et al., 1989; mouse, Pittler et al., 1992; rat, Barnstable and Wei, unpublished; and chicken, Bo¨nigk et al., 1993). Amino acid sequences were aligned for maximal homology using Clustal algorithm in the DNASTARA software for Windows. Only differences from canine CNGC a-subunit sequence are indicated. Amino acid residue numbers are given on the right. Conserved CNGC pore-forming region (Heginbotham et al., 1992; Sun et al., 1996) is framed in black, cGMP-binding pocket ( Varnum et al., 1995) is framed by dotted lines, whereas voltage-sensor-like motif S4 (Bo¨nigk et al., 1993) is framed in gray. Putative hydrophobic transmembrane segments ( Kaupp et al., 1989) are consecutively numbered (H1–H6) and their position is indicated by solid lines.
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(Molday et al., 1991). The initiation codon at nt 197 is within the sequence AACCATG, which conforms well with the Kozak consensus sequence (CANCATG; Kozak, 1987). All putative structural elements characteristic for the ion channel superfamily and well conserved across evolution are present in cCNCG1 ( Fig. 1). 2.3. Determination of exon/intron structure of the cCNCG1 gene Genomic DNA was isolated from peripheral blood leukocytes of an unaffected dog by conventional methods. Purified DNA was submitted to PCR amplification by overlapping primers designed from canine cDNA sequence and putative flanking intron sequences deduced from the organization of the human CNGC a-subunit gene (Dhallan et al., 1992). Amplification was done by OmniGene Thermal Cycler (Hybaid ) using manual hot start and touchdown PCR. Exon/intron boundaries for introns 1, 3 and 8 were determined by the method of rapid amplification of genomic DNA (RAGE) and Universal GenomeWalker@ Kit (Clontech) according to the manufacturer’s instructions. Sequencing of the large introns was performed by the primer walking method. Purification and sequencing procedures were the same as described in Section 2.1. Results are summarized in Table 1. The cDNA sequence was deposited in the EMBL, GenBank and DDBL Nucleotide Sequence Databases under accession number X99914; intron accession numbers are Y11309, X99913, Y11310, Y10559, Y11372 and Y11311 for introns 2, 3, 5, 6, 7 and 8, respectively. 2.4. SSCP–PCR analysis of cCNCG1 in a Swedish Briard–Beagle kindred with hereditary retinal dystrophy For single-strand conformation polymorphism analysis (Orita et al., 1989) we used at least two different gel conditions to improve mutation detection rate (run at room temperature in 8% polyacrylamide and 5% glycerol or run at 4°C in 6% polyacrylamide and 5% glycerol,
Fig. 2. Detection of the sequence polymorphism 266C/T in cCNCG1 gene intron 3 by restriction enzyme analysis. PCR amplified 284-bp intron 3 fragment was digested with XmnI and separated on 2% agarose gel. The amplicon contains a restriction site for XmnI, which gives rise to restriction fragments of 47 and 237 bp. Nucleotide C at position 266 defines an additional recognition site for XmnI and generates allelic restriction fragments of 65 and 172 bp. Lane 1: homozygous pattern of 266T (unaffected animal ); lane 2: heterozygous sample of 266T/C (affected animal ); lane 3: homozygous pattern of 266C (affected animal ); lane 4: molecular weight marker (pBR322, MspI digested).
for details see Bunge et al., 1996). The size of PCR fragments used for SSCP was 120–380 bp. Due to the high degree of inbreeding, affected animals are expected to be homozygous not only for the diseasecausing mutation but also for other sequence variations in the disease-causing gene. Therefore, a heterozygous pattern for a sequence change excludes the gene in question as the one implicated in the retinal disorder in this strain. As overlapping cCNCG1-transcript amplicons from affected and unaffected animals did not show any difference on SSCP gels the same analysis was also performed for introns. In intron 3, we found three sequence variants, 266C/T, 294T/C and 374T/C (numbers according to the sequence deposited to GenBank). As 266C/T creates an additional XmnI restriction site in genomic DNA, this provides an easy and sequencespecific assay to detect this variant. As affected animals are heterozygous for the 266C/T variant ( Fig. 2), these results exclude the cGMP-gated channel a-subunit gene to be a candidate for the retinal dystrophy in the kinship of Swedish Briards studied here. We have also studied
Table 1 Position and nucleotide sequence of exon/intron boundaries of the cCNCG1 gene Exon
Exon length (bp)
Splice donor site
Intron length (bp)
Position on cDNA
Splice acceptor site
1 2 3 4 5 6 7 8 9
182 121 123 63 42 111 108 107 1415
AAAGgtaagaagcc GCAGgtatctatat AGAGgtgagcagta AGCAgtaagtacaa AGAGgtaagttaga AAGAgtaagtggca CAAGgtatctatat ACAGgtaaatgtgt
n.d. 1295 947 ~4300 89 915 735 1476
182 303 426 489 531 642 750 857
ctctccccagATAT tttaccacagCTCC catttttcagGGAA tcttctttagAGAA ctcttttcagCAAA ttgctgttagGGAG tctcttacagAGCA tctattttagGTTA
Exon sequence is shown in upper-case letters; intron sequence is in lower-case letters. n.d., not determined; position on cDNA refers to the last nucleotide of the exon.
A. Veske et al. / Gene 202 (1997) 115–119
36 unaffected outbred Briard and foxhound dogs to determine the allele frequency of the 266C/T polymorphism. As expected, several animals were homozygous either for the T or the C allele. The heterozygosity value calculated was H=0.481 with allele frequencies of 0.597 and 0.403 for T and C, respectively.
3. Conclusions (1) Retinal cDNA encoding canine CNCG1 was identifed. Canine and human sequences share 89.4% identity at the nt level and 90.4% identity at the aa level. (2) Exon/intron boundaries of the cCNCG1 gene were determined and six out of eight introns were sequenced completely. Canine and human genes share a similar organization in that both have a large last exon encoding two-thirds of the protein and 8 (in human 9, due to the Alu element) additional small exons (the first of them untranslated) encoding amino terminal part of the protein. The cCNCG1 gene structure is shown in Table 1. (3) Results of segregation analysis by an intronic polymorphism (266C/T ) in the Swedish Briard and Briard–Beagle kindred excluded cCNCG1 as a candidate gene for the retinal dystrophy found in this dog kindred (Fig. 2). Nevertheless, it is possible that mutations in this gene are responsible for other forms of canine retinal dystrophy.
Acknowledgement This study was financially supported by grants from the Deutsche Forschungsgemeinschaft (Ga 210/5-4) and the Swedish Medical Research Council (project 12X-734). This article is based in part on a doctoral study by A.V.
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