Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Abstract

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.

A New Approach for Accurate Identification of Allele State Based on Real-Time PCR for Biomedical Tasks

“…We checked the reproducibility of the obtained resultsof the diagnostic system for the detection of the PKLR wild-type allele (Table 6). Based on the data obtained, one can see that the ranges of efficiency values for nonspecific reaction and reaction on a heterozygous matrix do not overlap with each other.The minimum ΔEff (between the worst result in a heterozygote and the best in a nonspecific reaction) was 3.69, which allows accurate genotyping of the presented samples.Comparing the difference in efficiency between specific and nonspecific reactions in our system, and in the system, which uses MGB probes in multiplex mode [1], one can see that in the second case, the nonspecific reaction is not effectively inhibited. In particular, its significant growth is observed on the mutant allele.…”

“…Genotyping of a single nucleotide polymorphism (SNP) is an urgent and common task for genetic mapping of complex traits and, in particular, for the diagnosis of hereditary diseases [1,2,3,4]. The most popular methods for solving this type of problem are methods based on the polymerase chain reaction due to the simplicity of the analysis and relatively low price.A fairly large number of suchlike techniques have been developed [6], for example, allele-specific PCR [3,8], oligonucleotide ligation [8], and others.…”

Successful treatment of pyruvate kinase deficiency and inflammatory bowel disease comorbid with exocrine pancreatic insufficiency and hypocobalaminaemia in a cat

“…PK deficiency is an inherited glycolytic enzyme disorder of RBCs caused by a mutation in the PKLR gene, which is predominant in Abyssinian, Somali, Bengal and Singapura cats in the United States, Europe, Australia and Japan. 1,[8][9][10] Because PK deficiency is transferred via autosomal recessive inheritance, affected cats show severe haemolytic anaemia, whereas carriers rarely show clinical signs. 1 The clinical course of haemolytic anaemia in cats with PK deficiency is chronic and progressive, but it is often misdiagnosed as other haemolytic anaemias with aetiologies, including immune-mediated diseases or RBC-infectious organisms.…”

“…PK deficiency is an inherited glycolytic enzyme disorder of RBCs caused by a mutation in the PKLR gene, which is predominant in Abyssinian, Somali, Bengal and Singapura cats in the United States, Europe, Australia and Japan 1,8–10 . Because PK deficiency is transferred via autosomal recessive inheritance, affected cats show severe haemolytic anaemia, whereas carriers rarely show clinical signs 1 .…”

A simple SNP genotyping method reveals extreme invasions of non-native haplotypes in pale chub Opsariichthys platypus, a common cyprinid fish in Japan

“…[ 24 , 33 , 34 ]) Moreover, other studies have successfully applied this technique for distinguishing closely-related organisms or genotypes in various study fields (e.g. [ 35 – 37 ]). Therefore, the SNP genotyping assay that is able to accurately distinguish between the EJ and WJ haplotypes of pale chub would be highly suitable for large-scale survey for monitoring the presence of non-native haplotypes in the eastern Japan.…”