• View in gallery
    Figure 1—

    Diagram depicting allele-specific priming for fGM2 for amplification of the normal allele (A) and mutant allele (B). For both alleles, the mismatched primer (gray) anneals to the noncoding strand to provide arithmetic but not logarithmic amplification.

  • View in gallery
    Figure 2—

    Representative amplification plot of results of a qPCR assay for detection of fGM1 Korat. The DNA from a carrier cat was amplified by use of normal primers (Np) and mutant primers (Mp). No detectable signal was found by use of either primer set for the no template control (NTC) samples. $RN = Change in the relative intensity of fluorescence. Squares = Quadruplicate samples amplified with the Np set. Circles = Quadruplicate samples amplified with the Mp set. Diamonds = Samples from the NTC.

  • View in gallery
    Figure 3—

    Plot of Ct versus well position for detection of fGM1 (A), fGM2 Baker (B), and fGM2 Korat (C) mutations by use of normal (Np) and mutant (Mp) primer sets in qPCR assays conducted on samples obtained from cats with a normal (N; wells 1 to 8), mutant (M; wells 9 to 16), or carrier (C; wells 17 to 24) genotype as well NTC samples (wells 25 to 32). All samples were tested in quadruplicate; amplifications were reproducible with an SD of less than 3% among quadruplicate samples within the same experiment (each set of 4 diamonds).

  • 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.

    • Crossref
    • 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.

    • Crossref
    • 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.

    • Crossref
    • 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.

    • Crossref
    • 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.

    • Crossref
    • 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.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 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.

    • Crossref
    • 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.

    • Crossref
    • 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.

    • Crossref
    • 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. WangScott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn University, AL 36849.

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 PhD
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Bruce F. SmithScott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn University, AL 36849.

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 VMD, PhD

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.

An autosomal recessively inherited defect of the lysosomal hydrolase β-galactosidase, GM1 gangliosidosis results in blockade of the catabolism of GM1 gangliodoside as well as other glycolipids and glycoproteins with the same terminal sugar moiety.1–3 Patients affected with gangliosidosis have progressive neurologic dysfunction and premature death.2–4 The GM1 gangliosidosis in cats was initially described5 in 1971, and it has been considered for use in studying type 2 GM1 gangliosidosis in humans. In addition to neurologic and metabolic abnormalities, GM1 mutant cats have premature thymic involution by 7 months of age when neurologic signs become clinically severe.4 In another study2 conducted by our laboratory group, we determined that GM1 gangliosidosis in Korat and Siamese cats was caused by a single nucleotide substitution (G→C) in the β-galactosidase gene, which resulted in a change of amino acid and loss of hydrolytic activity.

The null variant of GM2 gangliosidosis (ie, Sandhoff disease) has been described in humans and cats.6–9 Deleterious mutations in the gene encoding the β subunit of hexosaminidase affect both isozymes (A and B) of the enzyme, which results in the null variant of GM2 gangliosidosis.10 The null variant of GM2 gangliosidosis has been described in domestic shorthair cats and Korat cats.6,8,9 The fGM2 Korat (ie, Korat mutation) results from deletion of a single base and frame shift, which introduces a premature stop codon in the 5′ end of the coding sequence.8 The fGM2 Baker (ie, Baker mutation) consists of a 25-bp inversion at the extreme 3′ end of the coding sequence, which introduces 3 amino acid substitutions at the carboxyl terminus of the β subunit and a translational stop, which is located 8 amino acids before the expected stop codon.11,a Both mutations cause similar progressive neurologic dysfunction, including tremors, ataxia, dysmetria, nystagmus, paresis, and paralysis.12

Traditionally, diagnosis of the condition and detection of the carrier state for gangliosidosis in cats have been evaluated by selective assay for enzyme activity. This approach is severely limited with regard to sample availability and consistency because enzyme assay requires material that has not been preserved in a fixative.2 Overlapping ranges of enzyme activity for homozygous and heterozygous animals make it difficult to precisely identify carrier animals. Molecular techniques, such as nucleotide sequencing, provide a sensitive and specific approach; however, these are laborious and time-consuming assays.

A qPCR assay combined with allele-specific primers is a relatively novel approach for molecular diagnosis and screening. In the study reported here, qPCR assays were used in combination with fluorescent dye to provide relative quantitation of alleles within cats with 3 types of mutations that resulted in gangliosidosis. These assays monitored the efficiency of PCR amplification on the basis of increasing fluorescence intensity. They may provide a specific and rapid diagnosis and be suitable for large-scale genetic screening.

Materials and Methods

Isolation of genomic DNA—Blood samples were obtained from cats of known genotype that were part of the GM1 and GM2 gangliosidosis breeding colonies at our facility and from Korat cats tested for GM1 and GM2 as part of the screening program conducted by our facility. Blood samples were also obtained from negative control cats.b,c Fifty microliters of blood mixed with EDTA to prevent coagulation was added to 0.5 mL of Tris-EDTA buffer (10mM Tris HCl [pH, 7.5], 1mM EDTA); the tube was centrifuged at 13,000 X g for 1 minute. Supernatant was decanted, and the pellet was resuspended in 0.5 mL of Tris-EDTA buffer by vortexing, which was followed by centrifugation at 13,000 X g for 1 minute. Supernatant was again decanted. This procedure was repeated twice, and the resulting pellet was then suspended in 100 ML of buffer (1X PCR buffer,d 0.5% Tween 20, and 100 μg of proteinase K/mL). The suspension was digested at 55°C for 1 hour. The digested sample was heated at 95°C for 10 minutes and then stored at −20°C.

For the fGM2 Korat test, blood samples for mutant cats were not readily available. Thus, DNA was extracted from a liver sample obtained from an affected cat.e The DNA was extracted from all samples by use of a commercial kitf in accordance with the manufacturer's instructions.

Primer design and qPCR techniques—A set of PCR primers was designed for each allele at the mutation sites for fGM1 Korat/Siamese, fGM2 Korat, and fGM2 Baker. The sequences, locations of these primers, and PCR conditions were summarized (Appendix). Amplification volume was 25 μL and consisted of 0.2mM each of dATP, dCTP, and dGTP; 0.5mM of dUTP, 0.1 MM of each primer, 2.0mM MgCl2, 0.25 units of uracil N-glycosylase, 0.125 units of Taq polymerase, 1X PCR buffer,g and 2.5 μL of DNA extracted from a blood sample or 0.1 μg of DNA extracted from a liver sample. The reactions were subjected to 30 cycles for the various conditions. The samples were amplified on a 96-well, tube-based qPCR system.h Fluorescence was measured at the annealing and extension steps of all thermal cycles. All fluorescence data were analyzed for relative allele quantification.

Results

Primer design—Target sequences containing mutation sites were amplified in a pairwise manner by use of 2 sets of allele-specific primers in PCR assays. For the fGM1 Korat/Siamese assay, which was designed to detect a single nucleotide substitution at base 1470 of the coding sequence, the coding strand primer was constructed 71 bases upstream from the mutation. The noncoding strand primers were designed with C (to pair with the normal G) or G (to pair with the mutant C) at the extreme 3′ end. A mismatch between the 3′ ends of primers and the templates resulted in negligible amplification.

For the fGM2 Baker assay, which was designed to detect a 25-bp inversion at base 1579 of the coding sequence, the coding strand primer was constructed 45 bases upstream from the mutation. The noncoding strand primer for the normal allele was designed with 19 bases overlapping the normal sequence at the 3′ end of the inversion and 4 bases overlapping the normal sequence at the 3′ end of the inversion. However, the noncoding strand primer for the mutant allele was designed with 16 bases overlapping the 3′ end of the inversion and 4 bases overlapping the normal sequence at the 3′ end of the inversion (Figure 1). These primers could bind the opposite strand at the 5′ end of the inversion of the other genotype, but this did not result in logarithmic amplification.

Figure 1—
Figure 1—

Diagram depicting allele-specific priming for fGM2 for amplification of the normal allele (A) and mutant allele (B). For both alleles, the mismatched primer (gray) anneals to the noncoding strand to provide arithmetic but not logarithmic amplification.

Citation: American Journal of Veterinary Research 68, 3; 10.2460/ajvr.68.3.231

For the fGM2 Korat assay, which was designed to detect a single base deletion at base 151 of the coding sequence, the coding strand primer was constructed 127 bases upstream from the affected site. The noncoding strand primer for the normal allele was designed with the 3′ end having the normal base (G) at position 151. The noncoding strand primer for the mutant allele did not include this base, but it did include the adjacent base to the 3′ side in the noncoding strand (T) to confer allelic specificity. A base (A) was added to the 5′ end of this primer to equalize the annealing temperature for the primer with the annealing temperature for the normal allele.

Amplification and optimization—The DNA was extracted from blood samples obtained from cats known to be homozygous dominant (normal), homozygous recessive (mutant), and heterozygous (carriers) for each of the 3 diseases, with the exception of the fGM2 Korat mutant in which DNA was isolated from a liver sample obtained from an affected cat. Amplification plots were as mentioned previously (Figure 2). A sample from a cat that was a carrier of fGM2 Baker yielded positive results when amplified with the normal or mutant primer sets, and those amplifications were reproducible among quadruplicate samples within the same experiment. No amplification was observed in negative control samples.

Figure 2—
Figure 2—

Representative amplification plot of results of a qPCR assay for detection of fGM1 Korat. The DNA from a carrier cat was amplified by use of normal primers (Np) and mutant primers (Mp). No detectable signal was found by use of either primer set for the no template control (NTC) samples. $RN = Change in the relative intensity of fluorescence. Squares = Quadruplicate samples amplified with the Np set. Circles = Quadruplicate samples amplified with the Mp set. Diamonds = Samples from the NTC.

Citation: American Journal of Veterinary Research 68, 3; 10.2460/ajvr.68.3.231

Assays for all 3 mutations were optimized to generate similar plots by adjusting the annealing temperature. When blood samples obtained from cats whose genotype (including homozygous normal, homozygous mutant, and heterozygous carriers) was determined by direct sequencing for the 3 genetic traits (fGM1 Korat/Siamese, fGM2 Baker, and fGM2 Korat) were tested by use of normal and mutant primer sets, results were consistent with those obtained for amplification plots. These data were graphed in plots of the Ct versus well position for each disease (Figure 3). In general, homozygous dominant (normal) or homozygous recessive (mutant) samples had a slightly lower Ct for amplification with normal or mutant primer sets, respectively. Heterozygotes (carrier) had positive amplification for both primer sets with increased Ct values. Failure to amplify was represented by a Ct of 30 cycles.

Figure 3—
Figure 3—

Plot of Ct versus well position for detection of fGM1 (A), fGM2 Baker (B), and fGM2 Korat (C) mutations by use of normal (Np) and mutant (Mp) primer sets in qPCR assays conducted on samples obtained from cats with a normal (N; wells 1 to 8), mutant (M; wells 9 to 16), or carrier (C; wells 17 to 24) genotype as well NTC samples (wells 25 to 32). All samples were tested in quadruplicate; amplifications were reproducible with an SD of less than 3% among quadruplicate samples within the same experiment (each set of 4 diamonds).

Citation: American Journal of Veterinary Research 68, 3; 10.2460/ajvr.68.3.231

Comparison of sequencing and quantitative PCR results with blood samples—Results for samples of the various fGM1 genotypes (normal, 10; mutant, 1; and carrier, 9), as determined by sequencing, were identical with results determined by use of qPCR assay. The same consistency was evident for testing of fGM2 Korat (normal, 17; mutant, 1; and carrier, 2; Table 1). One discrepancy was found for a single fGM2 Baker test, in which results for sequencing (normal, 8; mutant, 7; and carrier, 5) differed from results for the qPCR assay (normal, 8; mutant, 8; and carrier, 4). The cat in question was determined to be a carrier by use of sequencing; however, it died when it was 2 months old as a result of a disease that appeared to be classic GM2 gangliosidosis. A review of the sequencing result indicated that the sequence data had been misinterpreted because of a low signal-to-noise ratio, which was most likely caused by inadequate or contaminated template. Thus, the correct result was obtained in 60 of 60 cats by use of the qPCR assays, which revealed the high specificity of these assays.

Table 1—

Comparison of results* for nucleotide sequencing and qPCR assays on blood samples obtained from cats with a normal, mutant, or carrier genotype.

VariableMutation assayNucleotide sequencingqPCR assay
NormalMutantCarrierNormalMutantCarrier
fGM1 Korat/Siamese10/201/209/2010/201/209/20
fGM2 Baker8/207/205/208/208/204/20
fGM2 Korat17/201/202/2017/201/202/20

Values reported represent No. of samples with a positive result/total No. of samples tested.

Discussion

Lysosomal storage diseases form a fascinating and diverse group of diseases linked by the common pathologic feature of abnormal accumulation of undegraded metabolites within lysosomes. Gangliosidoses have been reported in many species. Diseases in cats and dogs have been identified, but in only a few cases were the molecular bases of the diseases determined. When these diseases are in purebred animals with limited gene pools, the disease may threaten the existence of that breed. A rapid means of genetic testing is needed for management of these animals, both for detection of carriers in purebred animals as well as management of colonies of affected animals.2

The unique characteristics of various mutations, such as inversions, substitutions, and deletions, may require differing approaches or conditions for allelespecific detection, which may pose substantial challenges to achieve optimization. Within this context, a simple DNA diagnostic method must be developed for large-scale screening of clinical samples. The qPCR procedure, which was based on monitoring amplification of alleles by measuring the increase in fluorescence caused by binding of fluorescent dye to double-stranded products, was used in the study reported here to develop 3 new assays. Target DNA was amplified in a pairwise manner with 2 sets of allele-specific primers (a set complementary to the wild-type sequence and the other set complementary to the mutant sequence). Results for each PCR reaction indicated whether the normal allele or mutant allele (or both) were contained in the sample. This technique offers a sensitive and reproducible assay for allelic discrimination that is faster than other PCR-based methods with complicated processing (such as hybridization or sequencing) required after PCR amplification.13,14 Because amplification is performed in a closed tube and requires no analysis after the PCR procedures, contamination with amplicon is avoided.15,16 The entire procedure can be performed in 96-well microtiter plates, and all data are stored on a personal computer during PCR amplification.13 Therefore, the entire procedure is easily automated.

Other allelic discrimination systems (such as allele-specific amplifications or molecular beacons) are based on qPCR techniques.13 Such methods require numerous efficient probes with distinguishable fluorescent reporter dyes in each reaction, and synthesis of fluorescent-labeled probes is always a major cost factor.17,18 Additionally, those assays require probes with comparable annealing efficiencies, and primers must be created within strict temperature constraints. Several attempts to use those assays by our laboratory group have failed because of the high contents of the GC bases and the unique arrangement of the sequences surrounding these particular mutations. The qPCR procedure described here provided the flexibility to design primer pairs that successfully distinguished among the 3 types of mutation.

The fluorescent dye system used in the study reported here was an inexpensive and practical choice for qPCR assay. With allele-specific primers, the dye can be used to create DNA-based screening assays for various genetic diseases. These assays are highly stringent, inexpensive, nonradioactive, and nontoxic and can be quickly performed, and they do not require electrophoresis and can be automated. They allow rapid and accurate diagnosis of gangliosidoses and also provide for reliable identification of carrier animals, which should accelerate eradication of these debilitating diseases from purebred cat populations.2

We believe that we were able to effectively adapt an assay approach for 3 separate mutations that cause gangliosidosis in cats. Furthermore, we believe these assays can be used in a screening program for Korat cats.

ABBREVIATIONS

qPCR

Quantitative PCR

Ct

Threshold cycle

a.

Baker HJ, Smith BF, Martin DR, et al. The molecular bases of feline GM1 and GM2 gangliosidoses (abstr). Feline Pract Suppl 1999;1:11.

b.

Provided by Dr. Henry J. Baker, Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn University, Ala.

c.

Provided by Dr. Douglas R. Martin, Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn University, Ala.

d.

PCR core kit, Applied Biosystems, Foster City, Calif.

e.

Provided by Dr. Leslie Muldoon, University of Oregon Health Science Center, Department of Neurology, School of Medicine, Oregon Health and Science University, Portland, Ore.

f.

DNAeasy tissue kit, Qiagen, Valencia, Calif.

g.

Sybr Green PCR kit, Applied Biosystems, Foster City, Calif.

h.

7700 sequence detector system, Applied Biosystems, Foster City, Calif.

References

  • 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.

    • Crossref
    • 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.

    • Crossref
    • 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.

    • Crossref
    • 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.

    • Crossref
    • 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.

    • Crossref
    • 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.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 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.

    • Crossref
    • 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.

    • Crossref
    • 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.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18

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

Appendix

Appendix

Oligonucleotide sequences and PCR conditions for primers used to detect 3 mutations that cause gangliosidoses in cats.

Mutation assayNormal primersMutant primersPCR conditions (30 cycles)
fGM1Korat/Siamese*Forward: 5′-GGAGTCCTGGAGCGAAG-3′ Nucleotides 1382–1398Forward: 5′-GGAGTCCTGGAGCGAAG-3′ Nucleotides 1382–139895°C for 30 seconds, 54°C for 30 seconds, and 72°C for 60 seconds
Reverse: 5′-GTATCTG CCATAGTTCACAG-3′ Nucleotides 1489–1470 
fGM2 BakerForward: 5′-AACAGACTGACAGTTCA-3′ Nucleotides 1517–1533Forward: 5′-AACAGACTGACAGTTCA-3′ Nucleotides 1517–153395°C for 30 seconds, 66°C for 30 seconds, and 72°C for 60 seconds
Reverse: 5′-TTTTGTATTCATAGTCACAATAT-3′ Nucleotides 1607-1585Reverse: 5′-TTTTTACTGGATATTGTGAC-3′ Nucleotides 1607-1588
fGM2 KoratForward: 5′-CGCGACCGGCCATGAGG-3′ Nucleotides 7-23Forward: 5′-AGTTGTCGCGGGAGAGG-3′ Nucleotides 7-2395°C for 30 seconds, 68°C for 30 seconds, and 72°C for 60 seconds
Reverse: 5′-AGTTGTCGCGGGAGAGG-3′ Nucleotides 167-151Reverse: 5′-AAGTTGTCGCGGGAGAGT-3′ Nucleotides 168-151

Nucleotide numbering was based on the feline Bgal sequence (GenBank accession No. AF006749).

Nucleotide numbering was based on the Korat hexosaminidase B sequence (GenBank accession No. S70340)

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.