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1.7.2.1 Progressive Rod-Cone Degeneration

In the breeds in which these diseases have been well-defined clinically a n d electroretinographically, crossbreeding experiments have demonstrated that they have a

common locus, termed Progressive Rod-Cone Degeneration (prcd) (Aguirre and Acland,

1988, 1989). The phenotype was first investigated in the Miniature (and Toy) Poodle as a recessively inherited condition (Barnett, 1965). The clinical scenario is one of night blindness between 3 and 5 years, at which point ophthalmoscopic signs such as tapetal hyperreflectivity and retinal vascular attenuation are already present. This contrasts with the dysplasias, where

Introduction

atrophy, w ith non-tapetal depigm entation and retin al pigm ent e p ith e lial

hypertrophy/hyperplasia creating pigmented spots between the lighter areas. Most dogs develop posterior subcapsular cataracts which progress to maturity. Blindness is complete by

5-6 years of age (Aguirre et al, 1982; Barnett, 1965b).

Crossbreeding experiments demonstrated that the disease in the poodle is allelic with the forms of PRA seen in English and American cocker spaniels (Aguirre and Acland, 1988) and also in Labrador retrievers (Aguirre and Acland, 1991). The age of onset is considerably more variable than the dysplasias, both within and between breeds which may indicate that there are allelic mutations at the same locus, or that the disease expression is altered in different genetic backgrounds. This is seen particularly in relation to the phenotype in English cocker spaniels where the onset of disease is later, less severe and has a slower and more variable time course. Interestingly, the disease seen in the affected progeny o f spaniel/poodle matings was indistinguishable from that seen in poodles (Aguirre and Acland,

1988).

There is regional, retinal variation in disease severity in prcd heterozygote poodles,

with posterior polar and equatorial regions preferentially affected early on with peripheral spread later in the disease. Leucine and fucose incorporation into opsin are normal in the very early stages of the disease but abnormally slow band displacement, representing a 43- 58% reduction in outer segment renewal rates were found in predegenerate prc<i-affected rods. This is the earliest detectable abnormality and is present by 14.5 weeks, at which time the ERG may, or may not appear normal. This is probably due to phenotypic variability within the breed (or to different anaesthetic regimes known to affect the ERG). As a normal outer segment length is maintained in predegenerate rods, then disc shedding and phagocytosis by the RPE must be concomitantly reduced. Under electron microscopy, the rod, then cone outer segments become irregular, then shortened and disorientated, suggesting ruptures of, and damage to the cell membrane. A critical point appears to be reached where the disorientated rod outer segments appear unable to maintain their length. They then shorten rapidly, generating vesicular profiles in the interphotoreceptor matrix, and progress to

Introduction

al, 1982; Aguirre and O'Brien, 1986). Variation of phenotype with breed exemplified by

English cocker spaniels also occurs at the electon microscopic level, with fewer vesicular profiles present (Aguirre and Acland, 1988).

No abnormalities in cyclic nucleotide metabolism have been detected (Acland et al,

1980). The IRBP was investigated for abnormalities, but its loss only occurs secondary to that

of photoreceptor inner segments (Wiggert et al, 1991). Biochemical investigations then

focussed upon opsin.

Microspectrophotometry and retinal extraction studies on opsin from prcd-affected and normal poodles showed that the absorption spectra were the same (peak absorption at 506nm wavelength) as was the amount of pigment extracted per eye. Apparent changes in

distribution were secondary to the degenerative process (Parkes et al, 1982). Further

investigations of opsin mRNA in situ and immunoreactivity showed that, in intact,

predegenerate cells, opsin transcription and translation is normal. There is a decrease in opsin transcription and loss of immunoreactivity in cytoplasmic domains once degeneration begins, but before cell death, possibly indicating a generalised cellular dysfunction as it coincides

with the 'critical point' after which outer segments rapidly shorten (Huang et al, 1994).

Imaging fundus reflectometry confirmed the presence of a spectrally and functionally normal opsin which decreases in amount in when photoreceptors begin to degenerate (at 1.1 years of age). As well as emphasising the variation in retinal expression of phenotype both within and between animals, this study did reveal an intriguing piece of data. Heterozygotes are clinically normal and have normal ERGs, but pigment levels measured in the central 25® of the retina were lower than control normal dogs (Kemp and Jacobson,

1992). This raises the possibility that although overt disease is inherited in a recessive manner, heterozygotes are not entirely normal. A previous report has documented fluorescein

angiographic abnormalities in poodle prcd heterozygotes with normal ERGs (Koskinen et al,

1985).

Differences in photoreceptor function have also been observed in rds mouse

heterozygotes (Hawkins et al, 1985) whilst mutations in the human rds gene are known to

Introduction

family variation is observed in RP (Berson, 1976), all of which would make prcd a good

model for human inherited retinopathies. Investigation of this disease may therefore benefit from a candidate gene approach at this stage and the possibility that the same gene is involved in GPRA is currently under investigation (Petersen-Jones and Sargan, p e rs o n a l communication).

Subsequent to the discovery of abnormal plasma levels of polyunsaturated fatty acids

in some humans with RP (Converse et al, 1983; Bazan et al, 1986; Anderson et al, 1987),

prcd miniature poodles were also investigated.

In particular, plasma docosahexaenoic acid (DHA) is reduced in humans, in affected

poodles (Anderson et al, 1991a) and also in Abyssinian cats with autosomal recessive PRA

(Anderson et al, 1991b). DHA is a 22:6n-3 essential fatty acid (FA) which comprises

approximately 50% of the FAs in the rod outer segment. In the affected poodles, plasma levels of some precursor fatty acids are elevated, whereas no discrepancy is observed between

normal and rcd-1 affected Irish setters (Anderson et al, 1991a). Other workers did, however

find abnormalities in arachidonic acid (20:4n-6) and DHA in affected setters (Bazan et al,

1985; Birkle et al, 1985), so the specificity of this phenomenon in the poodle is unclear.

Initial studies implicated a A4-desaturase enzyme present in the liver as a candidate gene, since neither normal nor affected retinae appeared to convert intravitreally injected precursor linolenic acid (18:3n-3) and were therefore assumed to be dependent upon

circulating DHA released from the liver (Wetzel et al, 1990). However, in vivo studies of rat

liver function have implicated a different pathway for the conversion of 22:5n-3 to DHA

which does not involve that desaturase (Voss et al, 1991). Therefore, this candidate appears to

have been ruled out.

The most recent work has demonstrated the presence of DHA pathway precursors in

the RPE (Wang and Anderson, unpublished observations cited by Alvarez et al, 1994),

although only 22;5n-3 is found in any amount in the poodle retina. The retina in normal and

prcd affected poodles is able to convert 22:5n-3 to 22;6n-3 but slightly less was converted in

Introduction

The biological consequences of this ability of the retina to convert 22:5n-3 to 22:6n- 3 requires further clarification but results indicate that the same enzyme system may be operating in the retina as in the liver, since identical intermediates are present in both tissues. This does not rule out the effects of a mutation in a liver-specific enzyme which forms the major contribution to transporting the DHA necessary to maintain the rod outer segments

from the liver. Indeed, prcd poodles challenged with high levels of dietary DHA have levels in

the liver which are higher than controls (Alvarez et al, 1994). However, the fact that decreased

plasma DHA is seen in prcd, arguably in the Irish setter rcrf-7and in RP conditions known to

have different genetic loci (X-linked RP, adRP for example), makes it appear more likely that this is a secondary effect of photoreceptor dysfunction rather than a primary cause.

The overall picture for prcd is that it seems to be a good model for human RP, with

temporal, structural and biochemical parallels, but with few good biochemical clues which would point towards particular candidate genes, as yet. Additionally, this form of PRA is also very common in the breeds in which it is found, possibly due to the fact that even affected dogs may not be detected until after they have produced offspring. Continuing research to identify the genetic locus for this disease is therefore, very important.

1.7.2.2 GPRA in Tibetan terriers

This disease is reported to be widespread in Europe (Curtis et al, 1991) and a small

breeding colony is maintained at the A HT in Newmarket, Suffolk. There is considerable variation in the onset of clinical signs, even within a single litter but night blindness generally presents between 12 and 18 months of age, concom itant with peripheral tapetal hyperreflectivity and granularity. Within a few months, vascular attenuation and mottled depigmentation of the non-tapetal fundus have commenced, with optic nerve atrophy then following, over a variable time course of up to 3 years . Posterior cortical cataracts develop

between 4 and 6 years of age (Millichamp et al, 1988).

ERG abnormalities at 10 months show a reduction in rod and cone responses but later, reflect rod loss, followed by cones. However, isolated, perfused retinas from 9 week-old affected dogs indicate that transduction kinetics are abnormal at this stage (Millichamp,

Introduction

1985). At this time histology shows disorganised rod and cone outer segments with abnormally long lamellae and vesicular profiles in the interphotoreceptor matrix. Degeneration of outer segments which follows is almost complete by 8 months of age

(Millichamp et al, 1988).

No candidate genes have yet been implicated. Biochemically, opsin concentrations measured by microspectrophotometry are normal in 9-10 week old predegenerate affected

dogs (Millichamp et al, 1988), whilst fundus reflectometry shows a decline in a normal opsin

which is secondary to outer segment loss (Curtis and Kemp, 1988). The phototransduction abnormalities implied by the ERG of isolated retinae do not result in elevation of cGM P (Millichamp, 1985). It is not known whether or not this disease is allelic with any of the other PRAs, but the early ERG and morphological changes may indicate a gene which influences the late stages of photoreceptor maturation.

1.7.2.3 GPRA in Miniature longhaired dachshunds

This condition has only been recently described in the literature and a breeding colony is maintained at the A HT. Preliminary observations indicate an autosomal recessive

trait (Curtis et al, 1991), the locus of which has not been tested in relation to any of the

known PRAs. Ophthalmoscopic signs are similar to prcd and the Tibetan terrier but the

temporal distribution differs. The onset is relatively early, at 6-12 months with the ERG remaining relatively unaffected during the first few weeks of life, in contrast to the dysplasias. Indeed, the ERG shows a normal trace at 10 weeks, but is markedly reduced by 9 months. Histologically, there is selective rod outer segment attenuation and irregularity by 10.5 weeks with gross degeneration and cone involvement detectable by 25 weeks (Curtis and Barnett,

1993).