• 1

    Barker CG, Blakemore WF, Dell A, et al. GM1 gangliosidosis (type I) in a cat. Biochem J 1986;235:151158.

  • 2

    Baker HJ, Smith BF, Martin DR, et al. Molecular diagnosis of the feline gangliosidosis: a model for elimination of inherited disease in pure breeds. In: August J, ed.Consultations in feline internal medicine. Vol 4. Orlando, Fla: WB Saunders Co, 2001;615620.

    • Search Google Scholar
    • Export Citation
  • 3

    Suzuki Y, Sakuraba H, Oshima A. Beta-galactosidase deficiency (beta galactosidosis): GM1 gangliosidosis and Morquio B disease. In:Scriver CR, Beaudet AL, Sly WS, et al, eds.The metabolic and molecular bases of inherited disease. 7th ed. New York: McGraw-Hill Book Co, 1995;27852823.

    • Search Google Scholar
    • Export Citation
  • 4

    Cox NR, Morrison NE, Sartin JL, et al. Alternations in the growth hormone/insulin-like growth factor I pathways in feline GM1 gangliosidosis. Endocrinology 1999;140:56985704.

    • Search Google Scholar
    • Export Citation
  • 5

    Baker HJ, Lindsey JR, McKhann GM, et al. Neuronal GM1 gangliosidosis in a Siamese cat with beta galactosidase deficiency. Science 1971;174:838839.

    • Search Google Scholar
    • Export Citation
  • 6

    Cork LC, Munnell JF, Lorenz MD. The pathology of feline GM2 gangliosidosis. Am J Pathol 1978;90:723734.

  • 7

    De Maria R, Divari S, Bo S, et al. Beta-galactosidase deficiency in a Korat cat: a new form of feline GM1-gangliodosis. Acta Neuropathol 1998;96:307314.

    • Search Google Scholar
    • Export Citation
  • 8

    Muldoon LL, Neuwelt EA, Pagel MA, et al. Characterization of the molecular defect in a feline model for type II GM2-gangliosidosis (Sandhoff disease). Am J Pathol 1994;144:11091118.

    • Search Google Scholar
    • Export Citation
  • 9

    Neuwelt EA, Johnson WG, Blank NK, et al. Characterization of a new model of GM2-gangliosidosis (Sandhoff's disease) in Korat cats. J Clin Invest 1985;76:482490.

    • Search Google Scholar
    • Export Citation
  • 10

    O'Neil DC, Bartholomew WR, Rattazzi MC. Antigenic homology of feline and human beta-hexosaminidase. Biochim Biophys Acta 1979;580:19.

  • 11

    Martin DR, Krum BK, Varadarajan GS, et al. An inversion of 25 base pairs causes feline GM2 gangliosidosis variant. Exp Neurol 2004;187:3037.

    • Search Google Scholar
    • Export Citation
  • 12

    Skelly BJ, Franklin RJM. Recognition and diagnosis of lysosomal storage disease in the cat and dog. J Vet Intern Med 2002;16:133141.

  • 13

    Fujii K, Matsubara Y, Akanuma J, et al. Mutation detection by Taq-Man-allele specific amplification: application to molecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency. Hum Mutat 2000;15:189196.

    • Search Google Scholar
    • Export Citation
  • 14

    Iitia A, Hogdall E, Dahlen P, et al. Detection of mutation DF508 in the cystic fibrosis gene using allelic-specific PCR primers and time-resolved fluorometry. PCR Methods Appl 1992;2:157162.

    • Search Google Scholar
    • Export Citation
  • 15

    Higuchi R, Dollinger G, Walsh PS, et al. Simultaneous amplification of specific DNA sequences. Biotechnology (N Y) 1992;10:413417.

  • 16

    Higuchi R, Fockler C, Dolliner G, et al. Kinetic PCR: real time monitoring of DNA amplification. Biotechnology (N Y) 1993;11:10261030.

  • 17

    Ballerini S, Bellincampi L, Bernardini S, et al. Apolipoprotein E genotyping: a comparative study between restriction endonuclease mapping and allelic discrimination with the lightcycler. Clin Chim Acta 2002;317:7176.

    • Search Google Scholar
    • Export Citation
  • 18

    Belak S, Thoren P. Molecular diagnosis of animal disease: some ex-

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Development of quantitative polymerase chain reaction assays for allelic discrimination of gangliosidoses in cats

Chi-Young J. Wang PhD1 and Bruce F. Smith VMD, PhD2
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  • 1 Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn University, AL 36849.
  • | 2 Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn University, AL 36849.

Abstract

Objective—To develop quantitative PCR (qPCR) assays with allele-specific primers to provide a rapid and accurate diagnostic and screening test for the 3 mutations identified as causes of gangliosidoses in domestic cats.

Sample Population—DNA samples obtained from archived feline blood samples submitted for GM1 and GM2 testing.

Procedures—A qPCR assay was developed for each mutation to monitor the efficiency of PCR amplification. Results were determined on the basis of the fluorescent intensity of DNA staining.

Results—Samples from 60 cats were screened by use of the 3 qPCR assays. Of these, 59 qPCR results agreed with the sequence-derived genotypes. The phenotype (affected) for the other cat agreed with results for the qPCR assay, which indicated that interpretation of the sequence-based result was incorrect.

Conclusions and Clinical Relevance—The qPCR assays offer a sensitive, rapid, and reproducible technique for allelic discrimination without the need for complicated processing steps, such as hybridization or sequencing, after PCR procedures. These assays may prove beneficial for a rapid diagnosis of gangliosidoses in cats and could also provide a means for reliable large-scale screening for the carrier state, thereby accelerating the eradication of these debilitating diseases from feline populations.

Abstract

Objective—To develop quantitative PCR (qPCR) assays with allele-specific primers to provide a rapid and accurate diagnostic and screening test for the 3 mutations identified as causes of gangliosidoses in domestic cats.

Sample Population—DNA samples obtained from archived feline blood samples submitted for GM1 and GM2 testing.

Procedures—A qPCR assay was developed for each mutation to monitor the efficiency of PCR amplification. Results were determined on the basis of the fluorescent intensity of DNA staining.

Results—Samples from 60 cats were screened by use of the 3 qPCR assays. Of these, 59 qPCR results agreed with the sequence-derived genotypes. The phenotype (affected) for the other cat agreed with results for the qPCR assay, which indicated that interpretation of the sequence-based result was incorrect.

Conclusions and Clinical Relevance—The qPCR assays offer a sensitive, rapid, and reproducible technique for allelic discrimination without the need for complicated processing steps, such as hybridization or sequencing, after PCR procedures. These assays may prove beneficial for a rapid diagnosis of gangliosidoses in cats and could also provide a means for reliable large-scale screening for the carrier state, thereby accelerating the eradication of these debilitating diseases from feline populations.

Contributor Notes

Dr. Wangs present address is Department of Life Science, Pingtung University of Science and Technology, Neipu Pingtung, Taiwan.

Supported by the Scott-Ritchey Research Center and a private donor.

The authors thank D. Kennamer and B. Krum for technical assistance with the assays.

Address correspondence to Dr. Smith.