Allelic Dropout

A Potential but Rare Source of Error in Genetic Testing.

Karen Downes and Dr Chris Helps.

Allele - “one of a number of alternative forms of the same gene”, from allelomorph (“other form”), which is derived from the Greek allel (“reciprocal” or “each other”)

Accurate genetic testing relies on a robustly designed test; firstly the mutation that causes the specific trait (e.g. a disease or coat colour) needs to be identified (this can take years of research!) and then two primers (short lengths of DNA) that will bind to the cat’s DNA each side of the mutation need to be designed and manufactured. This allows the region of DNA containing the mutation to be amplified by a method called Polymerase Chain Reaction (PCR) and the mutation to be determined.

Cats normally have two copies of each gene, one from each parent, and the failure of a genetic test to detect one of these genes (alleles) is termed allelic dropout. It is rare and occurs when other mutations are present in the cat’s DNA that prevent one or other PCR primer from binding to an allele, which means the allele is not amplified and detected. It is most noticeable in cats that are heterozygous for a genetic mutation (they have two alleles; one that has the mutation and one that does not) where either the normal or mutant allele is not detected by the genetic test. Hence, in rare cases a cat that is heterozygous for a mutation may be reported as either normal or homozygous affected because either the mutant or normal allele is not detected.

For a particular genetic test different laboratories will use different PCR primers that bind to different regions of the gene surrounding the mutation. This could lead to a potential source of error in the reporting of a genetic result for the same cat if one laboratory’s genetic test suffers an allelic dropout. Most genetic testing laboratories will be aware of allelic dropout as a potential source of error and will have taken measures to limit its effect; they should certainly have taken it into account when developing their genetic tests. However, mutations that are rare and have never been previously described obviously cannot be taken into account when designing a genetic test and so may cause erroneous results. An example of just such a situation from our own laboratory, and how we dealt with it, is described below.

We had tested a Bengal cat for the Agouti mutation and the result showed the cat to be melanistic (non-agouti a/a). Whilst this is rare in Bengals, these cats do exist and the result was reported. The breeder quickly contacted us to say that the cat could not be melanistic and sent photographic evidence, she was correct! So we did some further investigation of this cat and found a new mutation that had never previously been described. We also found that the cat was actually a carrier of non-agouti (A/a), which agreed with its photograph. Unfortunately this new mutation prevented one of our PCR primers from binding to the normal Agouti allele (A) and this was the reason why the original result was reported as non-agouti (a/a); we were only detecting the non-agouti allele (a). This is shown in the diagram below. Having found this mutation we were able to redesign our genetic test for Agouti and we re-tested all Bengal cats that had been previously reported as non-agouti (a/a). Interestingly they were all still non-agouti (a/a) with the new test, indicating that very few cats had the new mutation. The new mutation was called Agouti A2, and although it does affect the Agouti protein we have yet to determine whether it has an affect on a Bengal’s coat colour. It should also be noted that we have only found the Agouti A2 mutation in Bengal cats and not in any other breed, hence, although we can’t be certain, it is likely that this mutation entered the Bengal breed from an Asian Leopard Cat ancestor, since Bengals are a hybrid breed. It is also likely that hybrid breeds (Bengal, Savannah, Chausie) may suffer from more cases of allelic dropout since their genetic makeup is more diverse (i.e. they contain various wild feline ancestors) than other domestic cat breeds.

Here at Langford Veterinary Services all our genetic tests undergo rigorous design and validation to minimise the chance of allelic dropout occurring. However, to be sure of never having an allelic dropout we would need to sequence the DNA from thousands of cats, which would be very expensive, very time consuming and probably mean that no genetic test was ever offered to breeders! Even then a very rare mutation may cause an allelic dropout in some cats. Hence, we have to strike a balance between accuracy and what is practical, but this means there is always a chance that a cat could have a very rare mutation that causes a genetic test to give a false result. As the example above demonstrates, we will always work with breeders to investigate cases of possible allelic dropout and to continually improve and update our genetic tests. 

The Agouti A2 mutation causes the Normal Agouti allele (A) to dropout, causing a heterozygous (A/a) cat to be reported as non-agouti (a/a). This problem was solved by moving Primer 1 to a new position.