September 2005
Volume 46, Issue 9
Free
Retinal Cell Biology  |   September 2005
Genotype–Phenotype Correlation of Mouse Pde6b Mutations
Author Affiliations
  • Alan W. Hart
    From the MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom.
  • Lisa McKie
    From the MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom.
  • Joanne E. Morgan
    From the MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom.
  • Philippe Gautier
    From the MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom.
  • Katrine West
    From the MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom.
  • Ian J. Jackson
    From the MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom.
  • Sally H. Cross
    From the MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom.
Investigative Ophthalmology & Visual Science September 2005, Vol.46, 3443-3450. doi:10.1167/iovs.05-0254
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      Alan W. Hart, Lisa McKie, Joanne E. Morgan, Philippe Gautier, Katrine West, Ian J. Jackson, Sally H. Cross; Genotype–Phenotype Correlation of Mouse Pde6b Mutations. Invest. Ophthalmol. Vis. Sci. 2005;46(9):3443-3450. doi: 10.1167/iovs.05-0254.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To identify the underlying molecular defects causing retinal degeneration in seven N-ethyl-N-nitrosourea (ENU) induced mutant alleles of the Pde6b gene and to analyze the timescale of retinal degeneration in these new models of retinitis pigmentosa.

methods. Conformation sensitive capillary electrophoresis and DNA sequencing were used to identify the mutations in the Pde6b gene. Visual acuity testing was performed with a visual-tracking drum at ages ranging from postnatal day 25 to week 10. Retinal examination was performed with an indirect ophthalmoscope. Animals were killed and eyes were prepared for histologic analysis.

results. Point mutations in the seven new alleles of Pde6b were identified: Three generated premature stop codons, two were missense mutations, and two were splice mutations. The three stop codon mutants and one of the splice mutants had phenotypes indistinguishable from the Pde6b rd1 mouse in rapidity of onset of retinal degeneration, suggesting that they are null alleles. However, the remaining alleles showed slower onset of retinal degeneration, as determined by visual acuity testing, fundus examination, and histology, indicating that they are hypomorphic alleles.

conclusions. These data demonstrate a correlation between genotype and phenotype. Four of the mutants with severe genetic lesions have rapid onset of retinal degeneration, as determined by fundus examination. These mice were indistinguishable from Pde6b rd1 mice, which are effectively blind by 3 weeks of age. In contrast, the milder genetic lesions show a slower progression of the disease and provide the community with models that more closely mimic human retinitis pigmentosa.

Retinitis pigmentosa (RP) describes a group of diseases that affect the retina of the eye and are a leading cause of human blindness. 1 Early indicators of the disease are night blindness and impaired visual fields, with a proportion of patients presenting with reduced visual acuity. RP has an incidence of 1 in 3000 to 1 in 5000 and may be inherited as an autosomal dominant (ad), autosomal recessive (ar), X-linked, or simplex/multiplex disease. 1 The identification of mutations in the rhodopsin gene 2 3 4 5 and genes encoding structural proteins, such as peripherin, 6 7 account for 20% to 30% of the adRP cases reported. 8 Defects in photoreceptor phosphodiesterase activity caused by mutations in the β subunit of the rod cGMP-phosphodiesterase (PDE6B) gene have been shown to underlie cases of arRP 9 10 accounting for ∼1% to 2% of all cases of RP. 8  
The product of Pde6b contributes to the heterotetrameric phosphodiesterase complex (PDE, αβγ2), 11 12 which regulates cytoplasmic cGMP levels in rod photoreceptors in response to light. On light stimulation, PDE is activated by removal of the γ-inhibitory subunits, resulting in a decrease in cGMP levels and hyperpolarization of the rod cell. 13 In mice with the retinal degeneration 1 (rd1) mutation elevated cGMP levels persist because of a homozygous null mutation in the Pde6b gene. 14 15 16 17 This results in permanent opening of cGMP-gated cation channels in the membrane of the rod photoreceptors, allowing an excess of extracellular ions to enter the cell, which ultimately leads to cell death by apoptosis. 18 The phenotypic resemblance between patients with arRP due to mutations in PDE6B and the Pde6b rd1 mouse has made this mouse an excellent model of this condition. However, this model (Pde6b rd1 ) presents with rapid photoreceptor degeneration that is apparent at postnatal day 8, and by 3 weeks of age, these animals have lost nearly all of their rod cells. 19 Mouse models that present with slower onset of disease would be useful tools to gain knowledge of and test therapies for this debilitating condition. As part of the MRC Harwell ENU mutagenesis program, 20 21 new models of retinal degeneration were identified. ENU-mutagenized male BALB/cAnN (BALB/c) mice were mated to C3H/HeN (C3H) females, to produce F1 progeny that were screened for eye defects. 21 Because C3H mice are homozygous for the Pde6b rd1 allele, all the F1 mice screened were heterozygous for the recessive Pde6b rd1 allele, which enabled Thaung et al. 21 to identify seven mutant lines that carried new recessive alleles of Pde6b. The phenotype of four of these lines was indistinguishable from that of C3H, and these were named retinal degeneration 1, 1-4 Harwell (Pde6b rd1 -1 -4H ). On fundus examination, the remaining three new mutant lines at 3 weeks of age appeared to have a near-normal retina compared with the degenerated retina observed in age-matched C3H mice. These lines were named atypical retinal degenerations (atrd)1-3 (Pde6b atrd1 -3 ). 
Materials and Methods
Mutation Detection
The 22 exons of the Pde6b gene were screened for mutations by using conformation sensitive capillary electrophoresis (CSCE). Fluorescent (FAM-labeled) PCR products were amplified from genomic DNA with intronic primers (primer sequences are available on request). PCR products were heteroduplexed in 1× PCR buffer by incubating at 96°C for 10 minutes, then cooled by controlled decrease in temperature of 0.5°C every 20 seconds for 24 minutes, followed by a final incubation of 60°C for 30 minutes. CSCE was then performed on a gene analyzer (47-cm capillaries; model 310; Applied Biosystems, Inc. [ABI], Foster City, CA; with Genescan Data Collection and Analysis Software; ABI). Before the products were loaded onto the analyzer, 39 μL dH2O was added to 1 μL of each heteroduplexed sample. The polymer was 5% polymer (cat. no. 401885; Genescan Data Collection and Analysis Software; ABI), 4 M urea (cat. no. U-0631; Sigma-Aldrich) in 1× TTE (20× TTE [1.78 M Tris, 0.57 M taurine, and 10 mM Na2EDTA]; cat. no. EC-871; National Diagnostics, Manville, NJ). The running buffer was also 1× TTE. Run conditions were as follows: injection and run voltages, 15 kV; injection time, 5 seconds; run time, 15 minutes; and run temperature, 32°C. Peaks were compared by eye for shifts indicative of mutations. If a change in peak shape relative to the control was found on analysis of the traces for a sample, and nonfluorescent PCR products were purified with a PCR 96-well filtration system (Multi-screen; Millipore, Bedford, MA) on a robotic platform (2000; Biomek) and sequenced directly by using dye termination cycle sequencing (Big Dye Terminator; ABI). Sequences were analyzed on computer (Sequencher program; Gene Codes, Ann Arbor, MI). 
Reverse Transcription–Polymerase Chain Reaction
RNA for analysis of the Pde6b atrd2 , allele was extracted from enucleated eyes (RNAgents Total RNA Isolation System; Promega, Madison, WI). RT-PCR was performed (Access RT-PCR System; Promega) with a forward primer from exon 10 (5′-GCTGAACACAGACACCTATGAC-3′) and a reverse primer from exon 14 (5′-GATGTCGTGGCACAAGCC-3′). RT-PCR products were analyzed by agarose gel electrophoresis or were cloned into a vector (pGEM-Easy; Promega) using standard procedures. The cloned transcripts—wild-type (67 colonies), Pde6b atrd2/+ (83 colonies), and Pde6b atrd2 (78 colonies)—were analyzed by sequencing. 
Animals
All studies were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and under the guidance of the Medical Research Council in Responsibility in the Use of Animals for Medical Research (July 1993) and UK Home Office Project License 60/3124. The allelic series of Pde6b mutant mice were identified in a genome-wide screening of ENU mutagenized mice, described in detail elsewhere. 21 The mice were maintained on a C3H background. A congenic sighted C3H mouse line was generated by crossing BALB/c with C3H, then backcrossing phenotypically normal (as determined by fundus examination) mice with C3H for 10 generations. Mice of this generation were intercrossed and their progeny typed as described 21 to select animals homozygous for the wild-type Pde6b allele, and these were used for subsequent breeding. 
Histologic Analysis
Animals were killed at various ages ranging, from 3 to 10 weeks, and the eyes were removed and fixed with Davidson solution for up to 48 hours dehydrated and embedded in paraffin wax. Sections were stained with hematoxylin and eosin by use of standard procedures. Images were captured on computer (IPLaboratory; Signal Analysis, Vienna, VA; and Photoshop 7.0 software, Adobe Systems, Mountain View, CA). 
Visual Acuity Testing
The optokinetic response (OKR) test with the visual-tracking drum has been described elsewhere 22 with the addition in this study of a video camera to record mouse movements (see www.eumorphia.org/ for protocol). Mice were tested at 3 to 4, 6, and 10 weeks of age, with each mouse tested with a stripe width subtending to 2°, 4°, and 8°. 
Funduscopy
After visual acuity testing, mice were administered 1% tropicamide to each eye to dilate their pupils. Fundus examination was performed with a Heine indirect ophthalmoscope and a Volk superfield lens (see www.eumorphia.org/ for protocol). 
Sequence Analysis
ClustalW (version 1.82 with default settings 23 provided by European Bioinformatics Institute, European Molecular Biology Laboratory, Heidelberg, Germany; available at http://www.ebi.ac.uk/clustalw/) was used to generate alignments for protein sequences, which were visualized using Genedoc, version 2.6.002 (http://www.psc.edu/biomed/genedoc/). 
Results
Identifying the Underlying Mutations in the Seven Novel Pde6b Alleles
As part of a screen for ENU-induced eye mutations, seven novel recessive Pde6b alleles were found by Thaung et al. 21 . To identify the underlying mutations in these alleles, genomic DNAs from Pde6b new mutation /Pde6b rd1 heterozygous mice were scanned for sequence variants in the exonic and intron–exon junction regions of the Pde6b gene using CSCE. For comparison, genomic DNAs from BALB/c, C3H, and F1 offspring of a cross between BALB/c and C3H (Pde6b rd1 /+) were also analyzed. In Pde6b rd1 /+ DNA, six peak shifts were found on inspection of the traces. As expected, sequencing demonstrated that one of these was due to the C-to-A base substitution in exon 7 that is found in the rd1 allele. 17 Sequencing revealed that the other five shifts were caused by single-base polymorphisms between BALB/c and C3H in intronic regions. Additional chromatographic peak shifts were found for Pde6b atrd1 , Pde6b atrd2 , Pde6b atrd3 , Pde6b rd1 -2H , and Pde6b rd1 -4H when heterozygous with Pde6b rd1 and subsequent sequencing identified functional single-base substitutions in Pde6b in these alleles (Table 1) . The other two lines (Pde6b rd1 -1H and Pde6b rd1 -3H ) did not show any obvious additional peak shifts; therefore all exons of Pde6b were sequenced to identify the underlying mutation in these two lines. For Pde6b rd1 -1H , a mutation was found in the intron 18 splice donor site (Table 1) ; and, for Pde6b rd1 -3H , a mutation was found in exon 9 (Table 1)
The four alleles that produced phenotypes identical with the null allele, Pde6b rd rd1-1H-4H—had mutations predicted to cause loss-of-function. In three, mutations introducing premature stop codons were present. These were a G-to-A transition in exon 9 (Pde6b rd1 -3H ), a T-to-A transversion in exon 17 (Pde6b rd1 -4H ), and a C-to-T transition in exon 21 (Pde6b rd1 -2H ). In the fourth, Pde6b rd1 -1H , there was a G-to-A transition at position 1 of the intron 18 splice donor site. Because a G residue at this position is absolutely necessary for 5′ splice site function, this mutation would be expected to eliminate correct splicing. In support of this notion, an identical mutation has been found in a patient with arRP. 8 Analysis of the alleles that caused less severe phenotypes revealed missense mutations in two of them, Pde6b atrd1 (in exon 15) and Pde6b atrd3 (in exon 14). Both changed highly conserved residues in the catalytic domain of the Pde6b protein (Fig. 1) . The third less-severe allele, Pde6b atrd2 , had a G -to-A transition at position 5 in the 5′ splice donor site of intron 11. A survey of splice donor site sequences found that a G residue is present at position 5 in 82% of rodent splice donor sites, and it is the most highly conserved residue after the GT at positions 1 and 2. 25 Because atrd2 behaves like a hypomorphic allele, we predicted that this mutation might reduce, but not completely abolish, correct splicing. 
Effect on Splicing of Pde6b by the atrd2 Mutation
We next performed RT-PCR using primers that amplify a 511-bp fragment between exons 10 and 14 of Pde6b, to determine the effect of the Pde6b atrd2 mutation on splicing of the Pde6b nascent transcript. As Pde6b is expressed only in rod photoreceptors that degenerate in the Pde6b mutants, we examined RNA isolated from eyes of animals that were wild type at Pde6b or were heterozygous for either the atrd2 or atrd3 mutation. A DNA product of the expected size, indicating correct splicing, was present in all the samples; but, in addition, the atrd2 /+ sample showed a larger DNA product, suggesting the use of a cryptic splice site in intron 11 in the atrd2 allele (Fig. 2a) . Sequencing of this novel product revealed the inclusion of 83 bases from intron 11 in the transcript, due to skipping of the normal 5′ splice site and the use of a cryptic splice site, with the sequence GTTAGT that has a five-of-six match with the consensus splice site sequence GTRAGT. If this RNA were translated, 54 novel amino acids would be incorporated into the protein before a stop codon in exon 12 (Fig. 2b ; variant 1). 
The phenotype of atrd2 homozygotes suggests that some wild-type transcript must be produced from the mutant allele. To assess this we performed RT-PCR on eye RNA from 2-day-old atrd2 homozygote and from adult wild-type animals. The PCR fragments were cloned and individual colonies sequenced. Three variants in addition to the expected wild-type sequence were found (Fig. 2b) . The major one, variant 1, used the cryptic splice site identified earlier. The other two were rare. Variant 2 skipped exon 11, to produce an in-frame deletion, and variant 3, seen only in wild-type RNA, had a cryptic splice site in exon 10 and resulted in an in-frame 36-bp deletion. The predominant fragment found in Pde6b atrd2 homozygotes was variant 1, accounting for 73.1% (Fig. 2c) . However, 16.7% of the fragments analyzed were correctly spliced. In addition, skipping of exon 11 accounted for 10.3% (variant 2; Fig. 2c ). The wild-type RT-PCR revealed that only 1.5% of the transcripts were from the variant 1 cryptic splice RNA, with the predominant transcript being the expected wild-type product (92.5%; Fig. 2c ). These data show that the Pde6b atrd2 mutation indeed affects splicing, and the predominant transcript, variant 1, yields a truncated and presumably nonfunctional protein. The milder retinal degeneration seen in atrd2 mice compared with rd1, which lack Pde6b protein, must be due to production of a small amount of normal Pde6b. 
Comparison of the Retinal Phenotype of the Seven New Alleles of Pde6b with that of rd1
We next performed a phenotypic analysis of the new Pde6b mutant alleles to assess the time course of retinal degeneration and the consequence this has on visual function. The rapid loss of photoreceptors observed in Pde6b rd1 -1 -4H homozygotes was indistinguishable from that observed in the Pde6b rd1 mice, therefore we focused our attention on the Pde6b atrd1 -3 mice, because, in the original screening, these appeared to show slower onset of retinal degeneration, as determined by funduscopy. 21  
Visual Acuity and Fundus Examinations
In humans, reduced visual acuity can be an early indicator of RP, and so we used the visual-tracking drum to assess the visual acuity of the Pde6b atrd1 -3 mice and compared these findings with C3H (Pde6b rd1 ) and sighted C3H mice. The sighted C3H mice are a congenic line, C3H.C-Pde6b +, that harbors the wild-type Pde6b gene (from BALB/c) bred into the C3H mouse strain. These mice represent an excellent control for comparison with Pde6b rd1 and Pde6b atrd1 -3 mice, as all mice tested have the same genetic background. Visual acuity testing of the Pde6b rd1 mice at 3.5, 6, and 10 weeks of age confirmed earlier findings that these mice are unable to respond in the drum 22 and are effectively blind from an early age. Sighted C3H mice at similar ages responded in the drum at all stripe widths and at all ages tested (Figs. 3a 3b) . Fundus examination after visual acuity testing revealed the characteristic patchy, pigmented appearance and optic disc pallor in young (3.5-week-old) Pde6b rd1 mice. In contrast, sighted C3H had a normal and healthy fundus throughout the experimental period. 
The Pde6b atrd1 -3 homozygote mice were all tested in the drum for positive head tracking responses. Pde6b atrd3 homozygous mice showed the least response. Only one of six animals responded (data not shown); and, on retesting on subsequent days, this mouse and other Pde6b atrd3 homozygotes failed to show any positive head-tracking movement at any age tested, although the fundus at an early age appeared normal. It is interesting to note that atrd3 was identified in the original screen by its failure to respond in an OKR test. 21  
By contrast, Pde6b atrd1 and Pde6b atrd2 homozygous mice responded well in the drum at 3.5 weeks of age (Figs. 3a 3b)with at least 20% of mice at this age responding to a 2° grating. At 4°, the animals performed much better with 90% of Pde6b atrd1 homozygous (Fig. 3a)and 50% of Pde6b atrd2 homozygous (Fig. 3b)mice responding. As these mice aged, their vision began to deteriorate. Although Pde6b atrd1 homozygotes responded well at 6 weeks of age at both 4° and 8°, by 10 weeks of age, they failed to respond in the drum and so were effectively blind. The Pde6b atrd2 homozygotes responded only at 8° at 6 weeks of age. By 10 weeks of age all mutant Pde6b animals failed to show any head-tracking movements. 
The progressive loss of vision was partially reflected in fundus examinations done immediately after visual acuity testing. At 3.5 weeks of age, all three Pde6b atrd1 -3 mutants had a near normal fundus, despite the absence of head tracking by Pde6b atrd3 mice. Slight speckling of the retina was observed in the Pde6b atrd2 homozygotes, although their optic disc appeared normal in shape and color. Signs of retinal degeneration became apparent as the Pde6b atrd2 mice aged. The optic discs at 6 weeks of age began to discolor, and patches of pigmented cells and whitening of blood vessels could be seen on the retina. However, compared with age-matched Pde6b rd1 mice, all Pde6b atrd1 -3 homozygous mice had considerably less degeneration. Nevertheless, by 10 weeks of age, the Pde6b atrd1 -3 homozygous mice were indistinguishable from Pde6b rd1 mice in retinal degeneration and optic disc pallor. 
We also tested the vision of mice that were compound heterozygous for atrd1 or atrd2 and the null rd1 mutation. Pde6b atrd1/rd1 mice performed less well than their homozygous atrd1 counterparts. None showed a head-tracking response to 2° and only 40% to 4° at 3.5 weeks. At older ages the response of the compound heterozygotes was also poorer than that of homozygotes. Pde6b atrd2/rd1 compound heterozygotes had significantly poorer visual response than Pde6b atrd2 homozygotes and showed no response to 2°, even at 3.5 weeks. Fundus examinations at 3.5 weeks revealed small areas of pigmented cells showing through the retina in both Pde6b atrd1/rd1 and Pde6b atrd2/rd1 mice. This degradation progressed with age; and, by 6 weeks, optic disc discoloration was apparent. 
The compound heterozygous genotypes showed a more rapid loss of vision and accelerated retinal degeneration than did the age-matched mice that were homozygous for the hypomorphic alleles. The hypomorphic alleles showed a dosage effect on both phenotypes, with two copies being less severe than one in combination with the null allele. 
Histology
The genotype-phenotype correlation of mutant allele dosage was even more striking when histologic sections were examined. At 3, 4, 5, and 6 weeks of age Pde6b atrd1 -3 homozygous, Pde6b atrd1 -3/rd1 compound heterozygous, Pde6b rd1 homozygous (only 3 weeks of age), and sighted C3H (only 3 and 6 weeks of age) mice were killed and their eyes removed, fixed, sectioned, and stained with hematoxylin and eosin. The outer nuclear layer (ONL) was counted at these various ages to give a measure of the progression of the retinal degeneration. At 3 weeks of age Pde6b rd1 retinal sections only had a single layer of cells present in their ONL (Figs. 4b 4k)and so older stages were not analyzed. In contrast, at 3 and 6 weeks of age the sighted C3H retina has a full complement of photoreceptor cells (∼12 cell layers; Fig. 4a 4f 4j 4o ). Figure 4shows sections through the retina of different genotypes at 3 and 6 weeks of age, and the cell layer thicknesses of the ONL are shown in Table 2 . Progressive loss of ONL cells was clearly seen in all genotypes; and, in each case, the phenotype of compound heterozygotes with Pde6b rd1 was more severe—in line with the visual-tracking drum data and fundus examination results. The most severe loss of ONL was in Pde6b atrd2 retinas, as homozygous or compound heterozygotes. The mildest cell loss phenotype was in retinas from Pde6b atrd1 mice, which had an ONL only slightly thinner than wild-type mice at 3.5 weeks, but which progressed rapidly over the next 3 weeks. Given their lack of head-tracking response at any age, Pde6b atrd3 mice had an unexpectedly mild ONL cell loss at 3.5 weeks. The ONL at the periphery of retinas from Pde6b atrd1 and Pde6b atrd2 at 6 weeks of age was thicker, with approximately three to four and one to three cell layers, respectively. There was no significant peripheral thickening noted in the Pde6b atrd3 retinas. 
Discussion
We describe seven new alleles of mouse Pde6b. Four of these have a phenotype similar to the well-described Pde6b rd1 mouse. The three nonsense mutations (Arg799X, Trp378X, and Tyr689X) and one mutation that alters the canonical GT in the 5′ splice donor site in intron 18 to AT (Table 1)predicts severe disruptions of the Pde6B protein that would compromise its function. The latter of these mutations has been found in a patient with autosomal recessive RP, with the second contributing allele causing a missense mutation. 8 These mutant alleles are all predicted to be null alleles, as they are indistinguishable from Pde6b rd1 in retinal degeneration. 
In contrast, the Pde6b atrd1 -3 mice present with a slower onset of rod cell degradation. Two have missense mutations (His620Gln and Asn606Ser) and the third a 5′ splice site mutation (IVS11+5G→A). The milder phenotypes caused by these mutations suggest that some residual function of the Pde6b protein remains that may help prevent the toxic buildup of cGMP levels that precedes cell death in the Pde6b rd1 mouse. 26 27 The relative severity of phenotypes of the Pde6b atrd1 -3 mutants indicates that the Pde6b atrd1 mutation is the least detrimental to protein function, because homozygotes present with the slowest onset of retinal degeneration, as demonstrated by their good visual acuity, near normal fundus, and histology at 3 weeks of age. The atrd1 mutation is located within the putative catalytic domain of Pde6b and causes a histidine-to-glutamine substitution. A lineup of the phosphodiesterase proteins (Fig. 1)from various species shows that in other phosphodiesterases, a tyrosine is found in place of histidine at this position, indicating that histidine is not necessarily essential for function. The only other missense mutation, Pde6b atrd3 is Asn606Ser, situated nearby in the putative catalytic domain, and is more detrimental to protein function based on the phenotype analysis. Even at 3 weeks of age when the extent of degeneration, as judged by the number of photoreceptor nuclei, is relatively mild Pde6b atrd3 mice do not appear to be able to see (Figs. 3 4) . The affected asparagine residue is highly conserved in all known mammalian phosphodiesterases (Fig. 1) , and the phenotypic consequence of the substitution indicates the importance of this residue in protein function. Similar correlations between missense mutations and severity of RP phenotype have been reported in patients with mutations in the rhodopsin gene. 28 29 The Cys110Tyr mutation (intradiscal) shows a late-onset and milder phenotype, 28 whereas the Arg135Leu, which affect the transmembrane domain of the protein, appears to present with a more severe form of RP. 29  
The atrd2 mutation results in a defect in splicing of the Pde6b transcript and a consequent reduction in normal mRNA. Analysis of the relative amount of splice forms indicates that Pde6b atrd2 homozygotes have approximately 17% the normal level of functional transcript and presumably an equivalent amount of functional protein. The photoreceptor cells appear to be very sensitive to the level of Pde6b. It is notable that this decrease to about one fifth of normal has a fairly severe consequence for the photoreceptor cells, and a decrease to one tenth of normal (when compound heterozygous with Pde6b rd1 ) results in a histologic phenotype almost as severe as the loss-of-function mutation. We can use these data to suggest what the impact of the His620Gln atrd1 missense mutation is on Pde6b function. As the atrd1 phenotype is somewhat less severe than atrd2, the protein function must be impaired by less than one fifth. Furthermore, as rd1 heterozygotes (with 50% normal Pdeb6) are phenotypically normal, then the Pde6b protein translated from the atrd1 allele must have between 20% and 50% normal function. The impact on protein function of the Asn606Ser mutation in atrd3 is less readily assessable. The absence of visual response suggests that it is a more severe mutation than atrd2. However, histology indicates that the photoreceptor cells are degenerating more slowly. It is possible that the presence of Pde6b protein in the PDE complex (albeit with very much reduced activity) spares the cells from rapid degeneration, even though they function poorly. 
The effect on splicing of the atrd2 mutation is interesting. The most common outcome of mutation of the conserved +5G in the 5′ splice donor site is skipping of the preceding exon. Three spliced forms were found on analysis of atrd2 transcripts; and, although one of these skipped exon 11, it was the least frequent variant. Most had a cryptic splice site in intron 11, but approximately 17% of transcripts are normally spliced and had the mutant splice site. It is surprising that the mutant site was still used, because of the high conservation of G at the +5 position. However, the mutant splice site has a Senapathy score of 68.8 (of a possible 100), which is high for mutant splice sites (average, 65.6). The cryptic splice site that is activated in the atrd2 mutant has a Senapathy score of 77.2, which is slightly higher than the average of 72.4 found for cryptic splice sites. 30 The functionality of this site is indicated by the finding that variant 1 is formed at a low level (1.5%) from the wild-type gene. 
Conclusions
We describe three new models of retinal degeneration—Pde6b atrd1 , Pde6b atrd2 and Pde6b atrd3 —that show slower onset of disease than does Pde6b rd1 . A clear correlation of genotype to phenotype can explain their disease progression. Pde6b atrd1 mice show the slowest onset of disease, caused by a missense mutation in a conserved residue that lies just outside a PDE domain. Pde6b atrd3 , the next slowest onset of retinal degeneration in this allelic series, is another missense mutation in a highly conserved amino acid that lies within a PDE domain and homozygous animals with this mutation appear to be blind from an early age. Finally, Pde6b atrd2 is a splice-site mutant displaying the next slowest onset of disease. This mutation generates transcripts that when translated will reduce levels of wild-type protein. The models of RP that we have described will be useful in future studies and may provide clues to therapeutic intervention in this debilitating group of retinal dystrophies. 
 
Table 1.
 
Mutations Identified in the Mouse Pde6b Gene
Table 1.
 
Mutations Identified in the Mouse Pde6b Gene
Mutant Allele Mutation Location DNA Change Protein Change
rd1-1H Splice Intron 18 IVS18 + 1G>A
rd1-2H Nonsense Exon 21 C2395T Arg799X
rd1-3H Nonsense Exon 9 G1133A Trp378X
rd1-4H Nonsense Exon 17 T2067A Tyr689X
atrd1 Missense Exon 15 T1860A His620Gln
atrd2 Splice Intron 11 IVS11 + 5G>A
atrd3 Missense Exon 14 A1817G Asn606Ser
Figure 1.
 
Multiple alignment of phosphodiesterase proteins. Sequences used in the multiple alignment have the following RefSeq accession number (www.ncbi.nlm.nih.gov/locuslink/refseq/ NCBI, Bethesda, MD): mm_pde6b: NP_032832; hs_pde6b: NP_000274; cf_pde6b: NP_001002934; gg_pde6b: XP_424876; rn_pde6b: XP_214126; mm_pde2a: NP_001008548; mm_pde10a: NP_035996; and mm_pde6c: NP_291092. In addition to these, three protein predictions were obtained on the Ensembl Web site (www.ensembl.org/ 24 ), version 28: mm_pde11a orthologue: Ensembl protein ID: ENSMUSP00000043862; xt_pde6b orthologue: Ensembl protein ID: ENSXETP00000039066; tn_pde6b orthologue: Ensembl protein ID: GSTENT00017355001. The highly conserved domains of Pde6b are highlighted with a line above the conserved sequences. Arrow: the position of the atrd1 and atrd3 mutations; parentheses: the amino acid position and change. To illustrate the differences between atrd1 and atrd3, other phosphodiesterase proteins are included, showing that the atrd1 amino acid is less well conserved than that of the atrd3 residue. mm: Mus musculus, mouse; hs: Homo sapiens, human; cf.: Canis familiaris, dog; gg: Gallus gallus, chicken; rn: Rattus norvegicus, rat; xt: Xenopus tropicalis, frog; tn: Tetraodon nigroviridis, pufferfish.
Figure 1.
 
Multiple alignment of phosphodiesterase proteins. Sequences used in the multiple alignment have the following RefSeq accession number (www.ncbi.nlm.nih.gov/locuslink/refseq/ NCBI, Bethesda, MD): mm_pde6b: NP_032832; hs_pde6b: NP_000274; cf_pde6b: NP_001002934; gg_pde6b: XP_424876; rn_pde6b: XP_214126; mm_pde2a: NP_001008548; mm_pde10a: NP_035996; and mm_pde6c: NP_291092. In addition to these, three protein predictions were obtained on the Ensembl Web site (www.ensembl.org/ 24 ), version 28: mm_pde11a orthologue: Ensembl protein ID: ENSMUSP00000043862; xt_pde6b orthologue: Ensembl protein ID: ENSXETP00000039066; tn_pde6b orthologue: Ensembl protein ID: GSTENT00017355001. The highly conserved domains of Pde6b are highlighted with a line above the conserved sequences. Arrow: the position of the atrd1 and atrd3 mutations; parentheses: the amino acid position and change. To illustrate the differences between atrd1 and atrd3, other phosphodiesterase proteins are included, showing that the atrd1 amino acid is less well conserved than that of the atrd3 residue. mm: Mus musculus, mouse; hs: Homo sapiens, human; cf.: Canis familiaris, dog; gg: Gallus gallus, chicken; rn: Rattus norvegicus, rat; xt: Xenopus tropicalis, frog; tn: Tetraodon nigroviridis, pufferfish.
Figure 2.
 
RT-PCR and splice variants between exons 10 and 14 in mouse Pde6b. An RT-PCR DNA product of the expected size (511bp, a) can be seen in all samples—wild type (wt), Pde6b atrd2/+, and Pde6b atrd3/+—however, an additional band corresponding to approximately 600 bp (PhiX174, 603-bp marker) was observed in the Pde6b atrd2/+ track. The 511-bp fragment corresponds to the expected WT transcript (b) and the larger band (594 bp) on sequencing corresponded to the variant 1 transcript represented in (b) as a cartoon. Colony PCR and sequencing identified an additional two variants, which are also represented schematically in (b) as variants 2 and 3. (c) Results obtained from the three separate genotypes analyzed, revealing that the predominant transcript in wild-type mice is the expected WT variant. In contrast, the predominant transcript found in Pde6b atrd2/atrd2 mice is variant 1. The number of colonies analyzed: wt, n = 67; Pde6b atrd2/+, n = 83; and Pde6b atrd2/atrd2 , n = 78.
Figure 2.
 
RT-PCR and splice variants between exons 10 and 14 in mouse Pde6b. An RT-PCR DNA product of the expected size (511bp, a) can be seen in all samples—wild type (wt), Pde6b atrd2/+, and Pde6b atrd3/+—however, an additional band corresponding to approximately 600 bp (PhiX174, 603-bp marker) was observed in the Pde6b atrd2/+ track. The 511-bp fragment corresponds to the expected WT transcript (b) and the larger band (594 bp) on sequencing corresponded to the variant 1 transcript represented in (b) as a cartoon. Colony PCR and sequencing identified an additional two variants, which are also represented schematically in (b) as variants 2 and 3. (c) Results obtained from the three separate genotypes analyzed, revealing that the predominant transcript in wild-type mice is the expected WT variant. In contrast, the predominant transcript found in Pde6b atrd2/atrd2 mice is variant 1. The number of colonies analyzed: wt, n = 67; Pde6b atrd2/+, n = 83; and Pde6b atrd2/atrd2 , n = 78.
Figure 3.
 
Visual acuity test. Sighted Pde6b +, Pde6b atrd1/rd1 and Pde6b atrd1 (a) and (along with Pde6b +), Pde6b atrd2rd1 , and Pde6b atrd2 (b), all maintained on a C3H background, were tested in the visual-tracking drum with gratings of 2°, 4°, and 8°. Each mouse was placed in the drum on a stationary elevated platform in photopic conditions. The mouse was filmed with a video camera throughout the duration of the test, and the footage was analyzed at the end of each testing session. The x-axis shows the age of the mice at each test, and the y-axis denotes the percentage of mice scoring a positive head-tracking movement (n = 4–10).
Figure 3.
 
Visual acuity test. Sighted Pde6b +, Pde6b atrd1/rd1 and Pde6b atrd1 (a) and (along with Pde6b +), Pde6b atrd2rd1 , and Pde6b atrd2 (b), all maintained on a C3H background, were tested in the visual-tracking drum with gratings of 2°, 4°, and 8°. Each mouse was placed in the drum on a stationary elevated platform in photopic conditions. The mouse was filmed with a video camera throughout the duration of the test, and the footage was analyzed at the end of each testing session. The x-axis shows the age of the mice at each test, and the y-axis denotes the percentage of mice scoring a positive head-tracking movement (n = 4–10).
Figure 4.
 
Hematoxylin and eosin–stained sections from Pde6b atrd1-3 mouse retinas. Retinal images illustrating photoreceptor degeneration (ONL) at 3 (ae, jn) and 6 (fi, or) weeks of age. All animals were maintained on a C3H background. Pde6b + (a, f, j, o), Pde6b rd1 (b, k), Pde6b atrd1 (c, g), Pde6b atrd1/rd1 (l, p), Pde6b atrd2 (d, h), Pde6b atrd2/rd1 (m, q), Pde6b atrd3 (e, i), Pde6b atrd3/rd1 (n, r). n = 3. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.
Figure 4.
 
Hematoxylin and eosin–stained sections from Pde6b atrd1-3 mouse retinas. Retinal images illustrating photoreceptor degeneration (ONL) at 3 (ae, jn) and 6 (fi, or) weeks of age. All animals were maintained on a C3H background. Pde6b + (a, f, j, o), Pde6b rd1 (b, k), Pde6b atrd1 (c, g), Pde6b atrd1/rd1 (l, p), Pde6b atrd2 (d, h), Pde6b atrd2/rd1 (m, q), Pde6b atrd3 (e, i), Pde6b atrd3/rd1 (n, r). n = 3. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.
Table 2.
 
Measurement of the Thickness of the Outer Nuclear Layer by Number of Cell Layers
Table 2.
 
Measurement of the Thickness of the Outer Nuclear Layer by Number of Cell Layers
Pde6b rd1 Pde6b + Pde6b atrd1 Pde6b atrd1/rd1 Pde6b atrd2 Pde6b atrd2/rd1 Pde6b atrd3 Pde6b atrd3/rd1
Cell layers (ONL) at 3 weeks 1 12–15 9–10 4–5 4–5 1–2 8–9 2–3
Cell layers (ONL) at 6 weeks 1 12–15 2–3 1 1–2 1 1–2 1
The authors thank Helen Davies for advice on the CSCE technique. 
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Figure 1.
 
Multiple alignment of phosphodiesterase proteins. Sequences used in the multiple alignment have the following RefSeq accession number (www.ncbi.nlm.nih.gov/locuslink/refseq/ NCBI, Bethesda, MD): mm_pde6b: NP_032832; hs_pde6b: NP_000274; cf_pde6b: NP_001002934; gg_pde6b: XP_424876; rn_pde6b: XP_214126; mm_pde2a: NP_001008548; mm_pde10a: NP_035996; and mm_pde6c: NP_291092. In addition to these, three protein predictions were obtained on the Ensembl Web site (www.ensembl.org/ 24 ), version 28: mm_pde11a orthologue: Ensembl protein ID: ENSMUSP00000043862; xt_pde6b orthologue: Ensembl protein ID: ENSXETP00000039066; tn_pde6b orthologue: Ensembl protein ID: GSTENT00017355001. The highly conserved domains of Pde6b are highlighted with a line above the conserved sequences. Arrow: the position of the atrd1 and atrd3 mutations; parentheses: the amino acid position and change. To illustrate the differences between atrd1 and atrd3, other phosphodiesterase proteins are included, showing that the atrd1 amino acid is less well conserved than that of the atrd3 residue. mm: Mus musculus, mouse; hs: Homo sapiens, human; cf.: Canis familiaris, dog; gg: Gallus gallus, chicken; rn: Rattus norvegicus, rat; xt: Xenopus tropicalis, frog; tn: Tetraodon nigroviridis, pufferfish.
Figure 1.
 
Multiple alignment of phosphodiesterase proteins. Sequences used in the multiple alignment have the following RefSeq accession number (www.ncbi.nlm.nih.gov/locuslink/refseq/ NCBI, Bethesda, MD): mm_pde6b: NP_032832; hs_pde6b: NP_000274; cf_pde6b: NP_001002934; gg_pde6b: XP_424876; rn_pde6b: XP_214126; mm_pde2a: NP_001008548; mm_pde10a: NP_035996; and mm_pde6c: NP_291092. In addition to these, three protein predictions were obtained on the Ensembl Web site (www.ensembl.org/ 24 ), version 28: mm_pde11a orthologue: Ensembl protein ID: ENSMUSP00000043862; xt_pde6b orthologue: Ensembl protein ID: ENSXETP00000039066; tn_pde6b orthologue: Ensembl protein ID: GSTENT00017355001. The highly conserved domains of Pde6b are highlighted with a line above the conserved sequences. Arrow: the position of the atrd1 and atrd3 mutations; parentheses: the amino acid position and change. To illustrate the differences between atrd1 and atrd3, other phosphodiesterase proteins are included, showing that the atrd1 amino acid is less well conserved than that of the atrd3 residue. mm: Mus musculus, mouse; hs: Homo sapiens, human; cf.: Canis familiaris, dog; gg: Gallus gallus, chicken; rn: Rattus norvegicus, rat; xt: Xenopus tropicalis, frog; tn: Tetraodon nigroviridis, pufferfish.
Figure 2.
 
RT-PCR and splice variants between exons 10 and 14 in mouse Pde6b. An RT-PCR DNA product of the expected size (511bp, a) can be seen in all samples—wild type (wt), Pde6b atrd2/+, and Pde6b atrd3/+—however, an additional band corresponding to approximately 600 bp (PhiX174, 603-bp marker) was observed in the Pde6b atrd2/+ track. The 511-bp fragment corresponds to the expected WT transcript (b) and the larger band (594 bp) on sequencing corresponded to the variant 1 transcript represented in (b) as a cartoon. Colony PCR and sequencing identified an additional two variants, which are also represented schematically in (b) as variants 2 and 3. (c) Results obtained from the three separate genotypes analyzed, revealing that the predominant transcript in wild-type mice is the expected WT variant. In contrast, the predominant transcript found in Pde6b atrd2/atrd2 mice is variant 1. The number of colonies analyzed: wt, n = 67; Pde6b atrd2/+, n = 83; and Pde6b atrd2/atrd2 , n = 78.
Figure 2.
 
RT-PCR and splice variants between exons 10 and 14 in mouse Pde6b. An RT-PCR DNA product of the expected size (511bp, a) can be seen in all samples—wild type (wt), Pde6b atrd2/+, and Pde6b atrd3/+—however, an additional band corresponding to approximately 600 bp (PhiX174, 603-bp marker) was observed in the Pde6b atrd2/+ track. The 511-bp fragment corresponds to the expected WT transcript (b) and the larger band (594 bp) on sequencing corresponded to the variant 1 transcript represented in (b) as a cartoon. Colony PCR and sequencing identified an additional two variants, which are also represented schematically in (b) as variants 2 and 3. (c) Results obtained from the three separate genotypes analyzed, revealing that the predominant transcript in wild-type mice is the expected WT variant. In contrast, the predominant transcript found in Pde6b atrd2/atrd2 mice is variant 1. The number of colonies analyzed: wt, n = 67; Pde6b atrd2/+, n = 83; and Pde6b atrd2/atrd2 , n = 78.
Figure 3.
 
Visual acuity test. Sighted Pde6b +, Pde6b atrd1/rd1 and Pde6b atrd1 (a) and (along with Pde6b +), Pde6b atrd2rd1 , and Pde6b atrd2 (b), all maintained on a C3H background, were tested in the visual-tracking drum with gratings of 2°, 4°, and 8°. Each mouse was placed in the drum on a stationary elevated platform in photopic conditions. The mouse was filmed with a video camera throughout the duration of the test, and the footage was analyzed at the end of each testing session. The x-axis shows the age of the mice at each test, and the y-axis denotes the percentage of mice scoring a positive head-tracking movement (n = 4–10).
Figure 3.
 
Visual acuity test. Sighted Pde6b +, Pde6b atrd1/rd1 and Pde6b atrd1 (a) and (along with Pde6b +), Pde6b atrd2rd1 , and Pde6b atrd2 (b), all maintained on a C3H background, were tested in the visual-tracking drum with gratings of 2°, 4°, and 8°. Each mouse was placed in the drum on a stationary elevated platform in photopic conditions. The mouse was filmed with a video camera throughout the duration of the test, and the footage was analyzed at the end of each testing session. The x-axis shows the age of the mice at each test, and the y-axis denotes the percentage of mice scoring a positive head-tracking movement (n = 4–10).
Figure 4.
 
Hematoxylin and eosin–stained sections from Pde6b atrd1-3 mouse retinas. Retinal images illustrating photoreceptor degeneration (ONL) at 3 (ae, jn) and 6 (fi, or) weeks of age. All animals were maintained on a C3H background. Pde6b + (a, f, j, o), Pde6b rd1 (b, k), Pde6b atrd1 (c, g), Pde6b atrd1/rd1 (l, p), Pde6b atrd2 (d, h), Pde6b atrd2/rd1 (m, q), Pde6b atrd3 (e, i), Pde6b atrd3/rd1 (n, r). n = 3. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.
Figure 4.
 
Hematoxylin and eosin–stained sections from Pde6b atrd1-3 mouse retinas. Retinal images illustrating photoreceptor degeneration (ONL) at 3 (ae, jn) and 6 (fi, or) weeks of age. All animals were maintained on a C3H background. Pde6b + (a, f, j, o), Pde6b rd1 (b, k), Pde6b atrd1 (c, g), Pde6b atrd1/rd1 (l, p), Pde6b atrd2 (d, h), Pde6b atrd2/rd1 (m, q), Pde6b atrd3 (e, i), Pde6b atrd3/rd1 (n, r). n = 3. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.
Table 1.
 
Mutations Identified in the Mouse Pde6b Gene
Table 1.
 
Mutations Identified in the Mouse Pde6b Gene
Mutant Allele Mutation Location DNA Change Protein Change
rd1-1H Splice Intron 18 IVS18 + 1G>A
rd1-2H Nonsense Exon 21 C2395T Arg799X
rd1-3H Nonsense Exon 9 G1133A Trp378X
rd1-4H Nonsense Exon 17 T2067A Tyr689X
atrd1 Missense Exon 15 T1860A His620Gln
atrd2 Splice Intron 11 IVS11 + 5G>A
atrd3 Missense Exon 14 A1817G Asn606Ser
Table 2.
 
Measurement of the Thickness of the Outer Nuclear Layer by Number of Cell Layers
Table 2.
 
Measurement of the Thickness of the Outer Nuclear Layer by Number of Cell Layers
Pde6b rd1 Pde6b + Pde6b atrd1 Pde6b atrd1/rd1 Pde6b atrd2 Pde6b atrd2/rd1 Pde6b atrd3 Pde6b atrd3/rd1
Cell layers (ONL) at 3 weeks 1 12–15 9–10 4–5 4–5 1–2 8–9 2–3
Cell layers (ONL) at 6 weeks 1 12–15 2–3 1 1–2 1 1–2 1
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