|Molecular Vision 2003;
Received 3 March 2003 | Accepted 20 June 2003 | Published 1 July 2003
Genetic, ophthalmic, morphometric and histopathological analysis of the Retinopathy Globe Enlarged (rge) chicken
Chris F. Inglehearn,1
David R. Morrice,2 Douglas H. Lester,3 Graeme W.
Robertson,4 Moin D. Mohamed,1 Ian Simmons,5 Louise M.
Downey,1 Caroline Thaung,6 Leslie R. Bridges,6 Ian R.
Paton,2 Jacqueline Smith,2 Simon Petersen-Jones,7 Paul
M. Hocking,8 David W. Burt2
1Molecular Medicine Unit and 5Eye Department, St. James's University Hospital, University of Leeds, Leeds, UK; Divisions of 2Genomics and Bioinformatics and 4Genetics and Biometry, Roslin Institute, Roslin (Edinburgh), Midlothian, UK; 3University of Abertay, Dundee, UK; 6Academic Unit of Pathology, Leeds General Infirmary, University of Leeds, Leeds, UK; 7Department of Small Animal Clinical Studies, Michigan State University, Veterinary Medical Center, East Lansing, MI
Correspondence to: Dr. David Burt, Division of Genomics and Bioinformatics, Roslin Institute, Roslin (Edinburgh), Midlothian EH25 9PS, UK; Phone: 44 (0) 131 527 4200; FAX: 44 (0) 131 440 0434; email: Dave.Burt@bbsrc.ac.uk
Purpose: To identify the locus responsible for rge (retinopathy globe enlarged) in chickens and further characterise the rge phenotype.
Methods: A colony of chickens carrying the rge mutation was rederived from a single heterozygous animal of the original line. The eyes of blind, heterozygous and normal birds were subjected to ophthalmic, morphometric and histopathological examination to confirm and extend published observations. DNA samples were obtained and subjected to a whole genome linkage search.
Results: From 138 classified backcross progeny, 56 birds were blind and 82 sighted. Heterozygous birds were indistinguishable from wild type, but homozygotes had sluggish or unresponsive pupils, posterior sub-capsular lens opacities and an atrophic pecten. The fundus appeared normal with no significant pigmentary disturbance, but axial length and eye weight were increased. Pathology revealed focal retinal lesions. Linkage analysis placed the rge locus in a small region of chicken chromosome 1.
Conclusions: rge is a severe recessive retinal dystrophy in chickens, with associated globe enlargement. Linkage mapping has highlighted chicken chromosome 1 in a region most probably homologous to human chromosomes 7q31-35, 21q21 or 22q12-21. Candidate disease loci include RP10 (IMPDH1) and uncharacterised Ushers (USH1E) and glaucoma (GLC1F) loci.
Inherited retinal dystrophies are a heterogeneous group of blinding disorders affecting around 1 in 3500 people [1,2]. They account for 11.5% of blindness in the under 65 age group  and are the most common cause of inherited blindness. Retinal dystrophies fall into three main categories. The commonest, retinitis pigmentosa (RP), is primarily a disease of the retinal periphery, causing night blindness and tunnel vision . It can be inherited in dominant, recessive, X-linked, digenic and mitochondrial modes and is also a component of many recessively inherited syndromes. At least 58 different genes have been implicated in RP causation to date. Macular, cone and cone-rod dystrophies result from dominant, recessive and X-linked mutations at a further 26 loci, and cause photophobia and loss of central vision. Leber Congenital Amaurosis (LCA) is the most severe form of retinal dystrophy, leading to severe visual impairment within the first few months of life. Nine loci have been implicated in LCA causation to date. In all, including an assortment of other retinal dystrophies, 137 loci have now been identified (listed at RetNet) at which mutations cause retinal dystrophy, and for 90 of these the genes are known. These include components of the phototransduction cascade and visual cycle, retinal transcription factors, cell adhesion molecules and structural proteins, and several ubiquitously expressed metabolic enzymes and splicing factors [5,6].
Five retinal mutations have been studied in the chicken. These are the retinal dysplasia and degeneration (rdd) phenotype , a retinal degeneration in the Rhode Island Red strain (rd) , blind enlarged globe (beg) , retinopathy globe enlarged (rge)  and a delayed amelanotic strain (DAM) of chicken . Only for the rd mutant, has the gene and mutation been identified. The rd phenotype is caused by a null mutation in the photoreceptor guanylate cyclase (GC1) gene and is thus a model for one form of Leber Congenital Amaurosis in humans .
Retinopathy globe enlarged (rge) is an autosomal recessive ocular disease phenotype that was seen in around 0.2% of birds in several commercial UK chicken flocks in the early 1980s. The initial report  and a more detailed follow-up paper  stated that affected birds could be distinguished by their behaviour from as early as 3 weeks, though effects were more marked at 8 weeks. They exhibited a poor pupillary light response and cataract formation was common in older birds. Ophthalmoscopic examination revealed a relatively normal fundus but choroidal vasculature was more prominent than in controls. In addition, a number of discrete white linear streaks were noted extending peripherally from the pecten. Histological examination revealed focal retinal lesions with a more general reduction in numbers of photoreceptors in some birds. Electroretinograms in two 3-month old homozygous birds were of abnormally low amplitude. No name was given to the line in these initial reports, and globe enlargement was not described. However on the basis of subsequent observations, the phenotype in these birds has been named "retinopathy globe enlarged" (rge).
We have created a breeding colony from a single heterozygous rge female, in order to extend the original observations on the rge phenotype and carry out genetic studies. In this way we aim to determine whether blindness in these birds is a model for one of the many forms of human inherited blindness.
The rge line is maintained at the Roslin Institute as a small pedigree population derived from a single heterozygous female that was obtained by SPJ from the original stock. This female was mated to a single male White Leghorn and male and female progeny were inter-mated. The rge line was rederived from homozygous (blind) birds. The birds were subsequently divided into 8 single male-female families that are reproduced annually to control inbreeding . All matings are by artificial insemination. The rge line is maintained and the crosses produced under a Home Office project licence and all husbandry procedures and experimental techniques were conducted under appropriate project and personal licences in a manner consistent with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research
Classification of blind/sighted birds
The visual function of birds was assessed using three behavioural criteria. Firstly, each bird was gently removed from its home cage and allowed to grip the metal food trough in front of the cage. Sighted birds balance easily and promptly turn and re-enter the cage. Blind birds cannot balance easily, do not re-enter the cage and may stumble off the perch. Secondly, the lateral side of the head was moved towards a solid object such as a part of the cage structure. Sighted birds keep their eyes at a distance from the object whereas blind birds do not take avoiding action. If any doubt remained, the bird was placed on a concrete floor and its activity was monitored. Sighted birds walk purposefully and may try to escape, in marked contrast to blind birds, which may not move at all even under gentle pressure. Two people performed each backcross assessment on at least two occasions, at 12-15 and again at 18-20 weeks of age, because of variation in the age at onset of blindness.
Ophthalmic and morphometric analyses
Homozygous, heterozygous and control birds from two age groups, of 6-9 months and approximately 3 years, were subjected to an ophthalmic examination. This was preceded by instillation of the local anaesthetic Amethocaine (1%) in each eye. Intraocular pressures were measured with the Tono-Pen XL (TM) tonometer. This measures the pressure required to flatten the cornea, but given the loss of corneal curvature in homozygous rge birds, as shown in Figure 1, this measurement must be interpreted with caution. Axial lengths were measured with the BVI Axis II A-scanner. Corneal diameters were measured, followed by direct and indirect non-mydriatic fundus examination. After death, homozygous and heterozygous birds were subject to an orbital exenteration. The excess adnexal and orbital tissue were cleaned off the globe, and photographs were taken to allow observation of gross morphology. Individual eyes were weighed to evaluate overall volume. Eyes were then fixed in 10% buffered formalin and retained for subsequent pathological examination.
After fixation, eyes were processed by dehydration in a series of increasing concentrations of alcohol, embedded in paraffin wax, cut into 4 μm sections and stained with haematoxylin and eosin.
Samples of fresh blood were collected at 10-14 weeks of age by superficial venepuncture of a wing vein. DNA extraction was performed using the single step DNAzol method (Gibco BRL). A total of 150 microsatellite markers were selected from an initial set of 249  on the basis of their optimization for polymorphism from pools of the genotypes of the grandparent chickens. These markers, covering all the autosomal linkage groups and the sex chromosomes except chromosome 16, were typed on 138 F2 offspring. PCR reactions for all microsatellite markers were performed separately in a total reaction volume of 15 μl. Reactions contained 15 ng genomic DNA, 1.5 mM MgCl2, 10 mM KCl, 10 mM Tris.HCl pH 8.3, 0.1% Triton X-100, 0.01% gelatin, 200 μM dNTP, 0.5 Unit of FastStart Taq DNA polymerase (Roche) and 5 pmol of each primer. PCR was performed with a hot start of 15 min at 95 °C, then 35 cycles of 15 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C, followed by a final step of 30 min at 60 °C. One μl of each reaction was pooled in 100 μl dH2O with the others in the set. One μl of this was added to 100 nl of the GENESCAN-350 ROX internal size standard and 20 μl HiDi formamide (Applied Biosystems, UK). Reliable and polymorphic markers were selected and organised into compatible marker sets based on the fragment size and dye colour of the PCR product. The PCR products from the same animal were diluted, pooled and run on the ABI 3700 automated sequencer. Fragment sizes were calculated relative to the ROX-350 internal size standard by using GeneScan 3.5.1 DNA fragment analysis and Genotyper 2.5 software (Applied Biosystems, UK).
All pedigree, marker genotype and trait data were recorded in resChick, a generic resource database  maintained at Roslin, then imported into the multipoint linkage analysis programme Crimap. Linkage analysis was carried out in three phases. (1) Two point analysis was carried out to determine whether there was significant linkage between markers. (2) Build analysis determined the most likely order of the markers and position of the rge region. (3) A fixed analysis was done, where the rge locus was moved along the length of chromosome 1 to determine its most likely position. Information on the genetic markers is listed in the the chicken database.
To map the rge gene, three homozygous (blind) males were crossed with three female White Leghorns. The female progeny from one of these families were reared and mated to another rge homozygous male to produce the backcross generation. A total of 138 progeny were classified, of which 56 were blind and 82 were sighted. The ratio of blind to sighted individuals was 0.41:0.59 and was similar in all three families. This proportion is significantly different from the 1:1 ratio expected under a model of simple recessive inheritance (χ2 (1 degree of freedom) 4.9, p<0.05). Mortality in this cross was low and was not observed after the first few weeks of life, long before the detection of birds with compromised sight. It is therefore possible that the rge mutation affects embryonic survival or is linked to another a gene that has a pleiotropic effect on survival.
Ophthalmic examinations were carried out on 10 blind birds aged 6-9 months, 16 heterozygous sighted carriers and 8 wild type controls, with approximately half of each sex in each group. In addition 4 homozygous male birds over 3 years of age were examined. No differences were observed between heterozygotes and controls. Homozygous rge birds aged 6-9 months consistently had pupils that were sluggish or non-reactive to light stimulus, posterior sub-capsular lens opacities and an atrophic pecten, often associated with a ring of retinal atrophy at the base of the pecten. The Pecten is a comb-like, highly vascularised structure which projects from the optic nerve head into the vitreous. The fundus in most birds looked normal with no evidence of pathologic intraretinal pigmentation, though in several the choroid looked pale and attenuated. Three year old birds had absent pupillary response and dense cataracts, precluding posterior segment examination. However in one the pecten and fundus were visible. Once again the pecten was atrophic but the fundus looked relatively normal, with only a few pigment flecks. The white linear streaks seen by Curtis and co-workers in some birds in their earlier report on rge were not observed.
Intraocular pressure (IOP), axial length (AL), corneal diameter (CD) and eye weight were determined for ten homozygous, 12 heterozygous and eight normal birds in the age range 6-10 months. In addition eye weights were determined at hatch for three rge homozygotes and five wild type birds. Preliminary analyses showed no differences between the right and left eyes for any of these measurements, so the mean values of both eyes were analysed in a linear model with terms for sex and sight. The model for the mean weight of the two eyes also included a linear term for the bird's body weight. Mean measurements for IOP, AL, CD and eye weight for each sex and for blind (rge/rge) and sighted (rge/+) birds in the 6-10 month age range are given in Table 1. Similar comparisons between heterozygous and control birds revealed no significant differences.
There was no significant difference in IOP between sexes or between blind and sighted birds, as measured by the tonopen. However the loss of corneal curvature in affected birds could alter the reading obtained with this instrument, so an increase in ocular pressure cannot be ruled out. Corneal diameters were not significantly altered, but axial length was greater and eye weight was increased, in blind compared with sighted birds. This is demonstrated in Figure 1, which compares the eye of a blind homozygote with that of a sighted heterozygote. The blind eye is enlarged in both the anterior/posterior and equatorial planes, and shows a marked loss of the corneal curvature normally characteristic of an avian eye . Eye weights at hatch were not significantly different (data not shown).
In 5 eyes of 3 homozygous rge birds aged 9-14 months, the only abnormality found was a cellular focus in the ganglion cell layer in one bird. However in 3 of 4 rge homozygous birds aged 4 years (5 out of 7 eyes), focal retinal lesions were seen. These comprised atrophy and gliosis of the inner retinal layers internal to the outer plexiform layer. In some places the retina was atrophic and replaced by collagenous tissue, and in others the adjacent choroid and sclera were attenuated and contained an outpouching of the atrophic retina. The fourth bird showed disorganisation, atrophy, fibrosis and ossification of the entire retina of both eyes. The focal lesions observed in some birds are almost certainly the histological correlates of the white streaks described by Curtis et al.  in birds of up to 15 weeks of age. An example is shown in Figure 2. These were not seen in ophthalmic observations made during this study, but this may reflect the fact that the birds examined herein are older than those seen by Curtis and co-workers and the lesions, as they have matured, have become less visible to the naked eye. No similar abnormalities were found in the eyes of 3 sighted heterozygous rge birds (2 at hatch and one at one year), or in the eyes of one Brown Leghorn and one White Leghorn control, both at 4 months of age. Because of lead time and cost implications, it was not possible to examine a 4 year old control bird.
Markers were mapped to chromosome 1 and their positions supported at a level of 1000:1 likelihood, in agreement with the consensus chicken genetic linkage map [15,18]. Positive two point and multipoint LOD scores were obtained for markers on chicken chromosome 1 in order to determine the position of the rge locus (Table 2 and Table 3). A graph of log(relative likelihood) against genetic distance for the region of chicken chromosome 1 between ADL0314 and MCW0112 is shown in Figure 3, with the area where the rge locus is most likely to lie marked within two LOD scores in either direction (243±6 cM), which represents 99% confidence limits . This distance could therefore, represent 100-200 genes .
We report the rederivation and analysis of a line of chickens first described in the late 1980s, with a poorly characterised ocular disease. The observations and procedures described herein confirm that the rge mutation causes autosomal recessive blindness in birds carrying it. At 10 weeks, homozygotes are blind on behavioural testing. 6-9 month old rge birds have atrophy of the pecten, a sluggish pupillary response, posterior sub-capsular lens opacities and in some cases a pale and attenuated choroid. Fundus appearance is relatively unchanged however. Histopathological examination revealed focal lesions similar to those in previous reports. Heterozygous rge carriers have normal vision at up to ten months of age, though this does not exclude the possibility of a late onset dominant effect. The globe enlargement observed in this study was not reported in earlier descriptions of the rge phenotype, probably because previous reports only examined birds up to 15 weeks of age. Globe enlargement is seen in chickens in association with a variety of visual stimulus deprivations . However it is not a universal finding, since it is absent from two other forms of chicken retinal degeneration, rd  and rdd . The conservation of gene order between the chicken and human genomes is similar to that between humans and mice, in spite of the much greater evolutionary separation . We can therefore use comparative mapping to predict both candidate disease loci and candidate genes by comparison with the human genome. The homologous region of chicken chromosome 1 does not lie in a large region of conserved synteny with the human genome. Instead, various smaller sections of the human genome show homology to this region, including parts of chromosomes 12p13, 22q13, 7q35 and 21q22. When these areas were examined, four possible genes/loci were highlighted by virtue of their location and their involvement in human inherited eye disease. TIMP3 maps to human chromosome 22q12-13  and IMPDH1 to 7q31.3 [24,25]. Both these genes are known to be involved in human retinal disease: mutations in TIMP3 cause Sorsby Fundus Dystrophy (SFD) and IMPDH1 was recently implicated in the RP10 form of dominant Retinitis pigmentosa. The dominant glaucoma locus GLC1F mapping to human chromosome 7q35-36  and the Usher type 1 locus USH1E located at 21q21 , are also candidates for the human equivalent of the rge phenotype.
The TIMP3 gene was analysed in normal and affected birds, but no sequence differences were observed. Furthermore, the TIMP3 gene was found to map well outside the region of 99% confidence for linkage. IMPDH1 catalyses the rate limiting step in guanine nucleotide biosynthesis and has recently been identified as the gene underlying RP10 , a classical form of dominant retinitis pigmentosa. In order to test IMPDH1 as a possible candidate site for this mutation, we attempted to map the gene using sequence from a chicken EST that showed homology to human IMPDH1. From this, the gene appeared to map to chicken chromosome 12. On further inspection of the sequence, it was realized that the IMPDH2 paralogue had in fact been mapped. IMPDH1 sequence has not been found in chicken and it has been suggested that there may, in fact, only be the one IMPDH gene in chicken . However, this remains to be clarified.
IMPDH1, USH1E and GLC1F are therefore the most likely candidates based on position, since these genes/loci are in regions homologous to the area of chicken chromosome 1 which shows the highest confidence values for the presence of the rge locus. However it is also possible that this chicken mutant represents a new and as yet unreported disease phenotype. Structural and physiological differences between humans and birds make it difficult to draw direct comparisons between the rge mutant and human diseases such as retinal degenerations or glaucoma. Further genetic studies should determine whether, like the rd chicken, rge is a model for a known human phenotype, or whether it could be used to identify a new candidate gene for involvement in human eye disease.
This work was supported by the Wellcome Trust (Grant Numbers 057288 and 035535) and by the Biotechnology and Biological Science Research Council (BBSRC).
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