EVALUATION OF A COMMERCIAL TEST FOR GENETIC RESISTANCE TO FELINE INFECTIOUS PERITONITIS
Niels C. Pedersen, DVM PhD,
Distinguished Professor Emeritus,
Center for Companion Animal Health,
School of Veterinary Medicine,
University of California, Davis
I would like to summarize the research done in the article by Hsieh and colleagues.1 The researchers reasoned that interferon-gamma was involved in protection against FIP, a reasonable assumption based on earlier research of others. They sequenced the feline interferon-gamma gene (fIFNG) and identified differences between cats (i.e., genetic polymorphisms) at three different positions that they referred to as 401, 408, and 428B. These polymorphisms involved a single nucleotide polymorphism (SNP) at each of these positons. These SNPs were not in parts of the genes that encode the fIFNG protein (i.e., exons), but rather in regions flanking the exons (i.e., introns). Intronic regions are known to contain both nonsense sequences and genetic elements that regulate the level of protein expression by a gene. They then collected DNA from 66 healthy cats and 60 cats with FIP. They found no relationship between SNP differences at positions +401 and +408, but it appeared that there was a relationship with disease status for a SNP at position +428B. Quoting their work – “from all the SNP tested; only fIFNG + 428C/T was found to be significantly associated with the outcome of the infection. At position +428, there was a higher frequency of the CT genotype in asymptomatic control cats (19.5%) than in FIP cats (6.3%), and the data showed a significant correlation with disease resistance (p = 0.03) (Table 2).” It is noteworthy that only cats that were CC or CT were present among the 66 healthy and 60 FIP cats; no cats that were TT were present in both populations. A table listing pertinent findings is given below:
The authors concluded that the difference between 16 healthy cats with CT (19.5% of healthy cats) was significantly different than 4 FIP cats with CT (6.3% of FIP cats). Using an Odds Ratio (OR) they calculated that the odds of CC cats having FIP were 3.6 times greater than cats having CT and concluded that T SNP was dominant to C. However, there were two problems with this conclusion. First, no cats in their study were TT and given the fact that 20 cats in both populations carried the T SNP (20/126=15.8%), the expectation would be that 9% (.158 x .158=.092), or 11 cats among the 126 cats would be TT. This suggests that either TT is an embryonic lethal, or more likely that the study populations were not random representation of all cats. Statistical comparisons are only valid when dealing with populations that are randomly selected and special care needs to be taken when the significance level is close to the minimum of P≤0.05 and the numbers of case and controls is small. The zero value for TT cats in the population also prevented an assessment of whether or not cats containing two copies of the “protective allele” also had significantly more resistance than cats that were CC. One would expect that if T is dominant, and CT is protective, that TT would also be protective. Knowledge of the incidence of cats that are TT at +428 is critical for application of the test, because the only way to breed for CT genotype, if it is the only genotype associated with FIP resistance, is to maintain CC cats in the population. If it can be shown that TT cats show a similar degree of FIP resistance to CT cats, then it is theoretically possible to breed only for the T allele (CT and TT), assuming that the T allele is really related to resistance. Therefore, the value of the test is dependent on the incidence of CC, CT and TT genotypes in the populations where it will be used. It is likely that some breeds will be homozygous for the common allele C, and the test will therefore not apply to them. Although unlikely, some breeds may have a high incidence of T, and again assuming that both CT and TT are protective, it may not be cost effective to test. If the incidence of T is low, positive selection for T carries the risk of inbreeding (see discussion below). The bottom line is that breeders should not pay for this test until they are given the information that is necessary to properly use it in their breeding programs.
The authors had the greatest problem trying to explain the biologic relevance of the T SNP polymorphism. Most meaningful genetic mutations are in exon (protein coding regions) and not in introns (non-protein coding regions). In order to provide biological relevance, the authors needed to show that the mutation in the intron caused an increase in the production of fIFNG. The normal way to do this would be to isolate the normal gene having the CC SNPs and the alternative gene with the CT SNPs and show that there was a difference in expression levels of fIFNG between the two genes in some sort of test tube (i.e., in-vitro) system. The simplest test would be to identify a large cohort of cats that are CC, CT, or TT and isolate their blood lymphocytes, stimulate them to produce IFNG, and then compare the levels of either specific RNA (by qRT-PCR) or actual protein by ELISA or other assay. If their observations are correct, lymphocytes from cats that are CT or TT should express more fIFNG than cats that are CC. Rather than do this complex experiment, the authors chose to merely measure the levels of fIFNG in the plasma of cats with FIP, reasoning that cats with FIP and the CT SNPs would have higher plasma levels of fIFNG than FIP cats with the CC SNPs. They found that 12/12 FIP cats with the CC SNPs had very low levels of fIFNG, while all three FIP cats with the CT mutation had high levels. The conclusion was that the CT mutation allowed for a higher expression of fIFNG. There were several serious problems with this conclusion. First, it is paradoxical that one would conclude that the CT mutation conferred resistance to FIP, and that this resistance was associated with increased fIFNG expression, but then prove it by using plasma from CT cats that were suffering from FIP. The second problem is that there were only three cats in the CT group and 12 in the CC groups, rendering any statistical comparison moot. Finally, there is not good evidence that cats develop FIP because they are unable to produce sufficient levels of fIFNG. Several studies on FIP have measured cytokine expression in cats with FIP.3 These studies were more apt to show increases in FIFNG in serum of cats with FIP and increased production by blood lymphocytes of cats with FIP upon stimulation. In fact, the general conclusion is that IFNG is stimulated in cats with FIP rather than inhibited.
Up to this point, I am assuming that there is some merit in this research and that further research is warranted. The problem is making the huge leap from a some what tenuous research finding to application in the field. There is already ample evidence that resistance to FIP cannot be attributed to a simple polymorphism in a single gene. Our attempts to find a single gene responsible for FIP resistance in both field studies with Birman cats4 and with random bred cats in our own experimental breeding program5 have failed to identify a single genetic marker for FIP resistance/susceptibility that we would deem significant. In fact, the only genetic risk factor that we could identify for FIP resistance was inbreeding itself.6 Breeding resistant cats to resistant cats did not increase resistance in their offspring, but rather made them even more susceptible. This is exactly what you would expect from a genetic trait that involves many different genes and gene pathways. The more you try to select for a single resistance factor, the more you unintentionally limit the role of multiple genes and gene pathways. Interestingly, the current recommendation is still the best one, i.e., to avoid inbreeding and not to breed cats that have produced FIP kittens. This recommendation is based on the theory that such cats carry a greater proportion of susceptibility factors than cats that do not produce kittens that develop FIP and when bred together will produce kittens with an even greater proportion of risk factors. Based on these findings, selecting for FIP resistance using a single genetic marker is more likely to favor inbreeding and actually increase the incidence of FIP. Finally, it must be remembered that heritability only explains 50% of FIP susceptibility, 7 with the remainder being attributed to epigenetic (genetic changes occurring after birth) and environmental factors.
Although a simple genetic test that will significantly reduce the risk of FIP does not seem likely, there is nonetheless reason to continue to search for genetic explanations for why some cats appear to resist FIP virus infection and others succumb to it, and why some cats develop the wet form of FIP and others the more chronic dry form. These studies should be concentrated in catteries, where FIP exists, pedigrees are known and genetic traits can easily be tracked. However, the cost for such research should not be born at the expense of breeders ordering a test. The normal sequence for the marketing of a genetic test for a disease trait is to validate it before offering it to the public. I do not believe that this test has been adequately researched.
1. Hsieh LE, Chueh LL. Identification and genotyping of feline infectious peritonitis-associated single nucleotide polymorphisms in the feline interferon-γ gene. Vet Res. 2014, 45:57.
2. Pedersen NC. A review of feline infectious peritonitis virus infection: 1963-2008. J Feline Med Surg. 2009, 11(4):225-58.
3. Pedersen NC. An update on feline infectious peritonitis: virology and immunopathogenesis. Vet J. 2014, 201(2):123-32
4. Golovko L, Lyons LA, Liu H, Sørensen A, Wehnert S, Pedersen NC. Genetic susceptibility to feline infectious peritonitis in Birman cats. Virus Res. 2013, 175(1):58-63.
5. Pedersen NC, Liu H, Gandolfi B, Lyons LA. The influence of age and genetics onnatural resistance to experimentally induced feline infectious peritonitis. Vet Immunol Immunopathol. 2014, 162(1-2):33-40.
6. Pedersen NC, et al. Immunity to feline infectious peritonitis virus infection is diminished rather than enhanced by positive selection for a resistant phenotype over three generations.Manuscript in preparation.
7. Foley JE, Pedersen NC: Inheritance of susceptibility of feline infectious peritonitis in purebred catteries. Feline Practice, 1996, 24(1):14-22