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Health Co-ordinators Statement re Aguirre Study on Cord 1 PRA (March 2013) - Health News page

STATEMENT RE WITHDRAWAL OF CORD1 PRA DNA TEST BY LABOKLIN LABORATORY

In August 2012, the Laboklin Laboratory announced that it would no longer be offering the Cord1 PRA DNA test, other than for research purposes. Its decision was based on a study of early onset PRA in Cord1 genetically Affected Dachshunds, which was carried out by researchers at the University of Pennsylvania and published in July 2012. Immediately following the Laboklin announcement, the ESS Health Co-ordinators contacted the Animal Health Trust for clarification of the findings of this study and whether there were any implications which might alter the advice that should be offered to ESS breeders and owners.

Dr. Cathryn Mellersh of The Animal Health Trust and Dr. David Sargan of Cambridge University issued a statement in August 2012, which can be read in full here. The situation can be summarised as follows:

• The research study only investigated early onset PRA in Dachshunds, so strictly speaking the conclusions related only to Dachshunds, although it could be assumed that a similar situation applies in ESSs. (The major stumbling block with research specifically using ESSs is the serious lack of suitable samples.)
• The study adds nothing to what has been known since 2009, which is that there is almost certainly a second (as yet unidentified), modifying gene mutation that needs to be present for the development of early onset PRA in Cord1 genetically Affected dogs. Without this second mutation, Cord1 PRA is more likely to be late onset.
• Although some dogs without the second modifying gene may never clinically develop Cord1 PRA during their lifetimes, some of them will. AHT research using ERG (electroretinography) testing showed that Cord1 genetically Affected dogs that were thought to be clinically normal, do indeed have reduced retinal function which can't be detected when using the standard clinical eye test.
• In Dachshunds, although the second mutation hasn't yet been precisely identified, they do know in which region of the genome it lies, and we await news of any further developments on that front.

The clear advice from the AHT was that the Cord1 DNA test for ESSs remains valid and valuable, and that Affected and Carrier dogs should only be mated to Clear dogs. A decision was made not to issue a statement to ESS owners last August, as the advice on using the ESS Cord1 PRA DNA test has not changed. However, as this issue has recently been highlighted on various ESS internet discussion forums, it is hoped this statement will help clarify the situation for everyone.

The Cord1 PRA DNA test is available from the Animal Health Trust and remains an ESS requirement under the KC Assured Breeder Scheme.

Louise Scott & Lesley Bloomfield
UK English Springer Spaniel Clubs Joint Health Co-ordinators

1st March 2013

Statement from The Animal Health Trust/Cambridge University
Re University of Pennsylvania Research Paper on
Early Onset Cord1 PRA (Published July 2012)

A paper has recently been published by a team of researchers, led by Prof Gus Aguirre, at the School of Veterinary Medicine, University of Pennsylvania entitled EXCLUSION OF RPGRIP1 INS44 FROM PRIMARY CAUSAL ASSOCIATION WITH EARLY ONSET CONE-ROD DYSTROPHY IN DOGS. The term RPGRIP1 INS44 refers to the mutation in the gene called RPGRIP1 that was identified in 2006, by researchers from the Animal Health Trust (AHT), in a colony of miniature longhaired dachshunds (MLHDs) with early-onset cone-rod dystrophy (also known as cord1). Mutations in the RPGRIP1 gene are known to cause inherited retinal degeneration in other species, including humans and mice.
Since the initial publication in 2006 Cathryn Mellersh's team at the AHT, in collaboration with David Sargan and Keiko Miyadera from Cambridge University, has become increasingly aware of phenotypic variation and genotype-phenotype discordance between some dogs that are homozygous (i.e. carry two copies) for the RPGRIP1 INS44 mutation (referred to as ins/ins dogs in this document) and it is now very well recognised that not all ins/ins dogs develop early-onset retinal degeneration. Data supporting this observation have been published formally in three separate, peer-reviewed publications [1-3] and shared informally with the Dachshund community on many occasions.
Two of the publications [1, 2] include the observation that ins/ins dogs usually have absent or reduced cone photoreceptors function, even if their retinas don't appear grossly abnormal. The third publication [3] describes very strong evidence for a second region of the genome that acts as a modifier, determining the age at which dogs develop cone-rod dystrophy and it now seems clear that dogs require two copies of both mutations to develop early-onset retinal degeneration. We now know that all the dogs in the AHT colony were homozygous for the modifying mutation, meaning our original findings point unequivocally to a mutation in the RPGRIP1 region that is involved with the development of cone-rod dysplasia and although we now know a second mutation is also involved the existence of a mutation in the RPGRIP1 region is beyond doubt. (Note: the precise mutation in the second region has yet to be formally identified but dogs carrying two copies can be identified by virtue of the fact they carry two identical stretches of DNA in this region. Work is on-going to identify the second mutation).
The recent paper from the Aguirre lab makes many of the same observations that have already been made, regarding genotype-phenotype discordance and abnormal cone function in ins/ins dogs, and these data, although consistent with previous published reports, are not novel findings. The Aguirre lab, like us, find that in young dogs (ERG tested at up to 1.8 years old) only a proportion of animals show loss of retinal function, and like us, they show this as loss of function of the cone cells. Some additional, new, observations have been made, however, including some aspects of abnormal retinal histopathology in ins/ins dogs which are consistent with the fact that the retinas of ins/ins dogs are abnormal. The Aguirre lab has not tested the functional effect of the mutation in older MLHD (above 5 years), where we have found slower degenerative effects even in those animals not affected by the mutation at a young age.
The Aguirre lab has undertaken some simple experiments to investigate whether the RPGRIP1 protein is made normally in ins/ins dogs. These experiments have revealed abnormalities in the RPGRIP1 RNA consistent with loss of the 5' end of the protein but have not revealed loss of normal amounts of the remainder of the protein, leading the researchers to conclude that it is not the RPGRIP1 INS44 mutation that is responsible for the abnormalities observed, but more likely a different mutation located nearby. Our own results also suggest that at least part of the normal protein is likely to be produced. But in our opinion, the experiments to investigate the function of the partial RPGRIP1 protein are not exhaustive, and do not prove unequivocally that the RPGRIP1 protein is normal in ins/ins dogs. In addition, despite a thorough investigation of the RPGRIP1 gene, the Aguirre team have not been able to identify an alternative mutation that explains the observed abnormalities any better than the RPGRIP1 INS44.
Conclusion
We agree with many of the conclusions made by the Aguirre lab, but not all. We agree that the RPGRIP INS44 mutation is not solely responsible for early-onset cone-rod dystrophy in MLHDs and indeed we have already published that an additional mutation acts to modify the age of onset of retinal degeneration in MLHDs. We do not agree, however, that sufficient evidence has been presented to conclude that the RPGRIP1 INS44 mutation is not involved with cone-rod dystrophy in this breed and we note that the Aguirre paper does not investigate the effects of the mutation on later onset PRA in the dachshund, showing only that a proportion of dachshunds genotyped as ins/ins did not develop an early onset functional disease. We note that in this investigation the majority of ins/ins animals did show retinal and ERG abnormalities whilst the controls did not. Until more conclusive evidence of non-involvement of RPGRIP1 INS44 in cone-rod dystrophy is presented we will continue to recommend that Dachshund breeders avoid breeding dogs that are homozygous for this mutation.
Summary
Data presented in the recent paper from the Aguirre lab are consistent with many previously published observations from other research teams and the following points summarise our current understanding of cone-rod dysplasia in miniature longhaired dachshunds;
• Dogs that are homozygous for the RPGRIP1 mutation (ins/ins) will not necessarily develop retinal degeneration (RD) at an early age, although some will.
• Ins/ins dogs usually have abnormal or absent cone photoreceptor function although the degree of loss depends on age of testing and is later in some dogs than others. They may also have abnormal retinal histopathology.
• There is an additional mutation, in a second gene, that influences the age of onset of retinal degeneration and homozygosity at both the RPGRIP1 locus and the newly mapped second region is necessary for a dog to develop early-onset RD.
• The involvement of the RPGRIP1 locus is definitive, based on the perfect association with cord1 in the AHT research colony, although we now know an additional mutation is involved, in a second region of the genome, that determines the age of onset of RD.

References
1. Miyadera, K., et al., Phenotypic variation and genotype-phenotype discordance in canine cone-rod dystrophy with an RPGRIP1 mutation. Mol Vis, 2009. 15: p. 2287-305.
2. Busse, C., et al., Ophthalmic and cone derived electrodiagnostic findings in outbred Miniature Long-haired Dachshunds homozygous for a RPGRIP1 mutation. Veterinary Ophthalmology, 2011. 14(3): p. 146-52.
3. Miyadera, K., et al., Genome-wide association study in RPGRIP1 (-/-) dogs identifies a modifier locus that determines the onset of retinal degeneration. Mamm Genome, 2011. 23(1-2): p. 212-23.