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Corresponding author: Monica Aleman, MVZ, PhD, Dipl ACVIM, Department of Medicine and Epidemiology, Tupper Hall 2108, One Shields Avenue, University of California, Davis, CA 95616; e-mail: firstname.lastname@example.org
Background: Anesthetic-induced malignant hyperthermia has been documented in Quarter Horses and is caused by a single-point mutation in the ryanodine receptor 1 gene at nucleotide C7360G generating a R2454G amino acid substitution. An accurate, faster molecular test that is less prone to contamination would facilitate screening for the mutation in horses intended for breeding, in those undergoing surgical procedures, and in those with clinical signs compatible with malignant hyperthermia.
Objective: To report a rapid and accurate method for the detection of the ryanodine receptor 1 C7360G mutation.
Methods: This study included horses with the ryanodine receptor 1 C7360G mutation as detected by gene sequencing. Available genomic and complementary DNA extracted from whole blood, hair or skeletal muscle was used for genetic analysis. Real-time polymerase chain reaction (RT-PCR) melting curve analysis was performed by equine specific primers and 2 hybridization probes (sensor and anchor probes) that contain the site of the mutation. Results from this method were blinded and compared with nucleic acid sequencing for validation.
Results: A rapid genotyping assay with fluorescence resonance energy transfer probes and melting curve analysis was accurate (100% agreement, K= 1) for identification of affected horses. The prevalence of the mutation in a random population of Quarter Horses was 1.3%.
Conclusions and Clinical Importance: Malignant hyperthermia in Quarter Horses can be rapidly and accurately detected by RT-PCR melting curve genotyping with hybridization probes.
Malignant hyperthermia (MH) is a life-threatening disorder of skeletal muscle elicited by halogenated anesthetics, depolarizing muscle relaxants, and stress.1 The genetic basis of MH linked to ryanodine receptor 1 (RyR1) mutations has been reported in humans,2 pigs,3 dogs,4 and horses.5 Nonanesthetic forms of the disease have been documented in humans and pigs.3,6 The authors recently have described fatal and nonfatal clinical manifestations of the disease unassociated with anesthesia in Quarter Horses.7 The presence of a single missense point mutation in the RyR1 gene at different nucleotides has been associated with the disease in horses, pigs, and dogs.3–5 Because of the extended locus heterogeneity of MH susceptibility (RyR1 is 1 of 6 loci) and number of mutations within the RyR1 gene in humans; genetic testing alone is not always recommended.8 Therefore the in vitro contracture test (IVCT) still is considered the gold standard for the diagnosis of MH.8 The IVCT is based on the differential contractile response of normal and MH susceptible skeletal muscle to caffeine and halothane.8 Skeletal muscle from MH susceptible individuals exhibits hypersensitivity to caffeine- and halothane-induced contracture whereas muscle from normal individuals is not hypersensitive to either agent.8
Since the 1st report of the disease, triggered by halothane in the horse, a presumptive diagnosis of MH has been based on clinical manifestations of hypercapnia, acidosis, hyperthermia, profuse sweating, tachycardia, tachypnea, and muscle rigidity triggered by halothane alone or in combination with succinylcholine or nerve stimulation. Few reports of the IVCT for diagnosis of MH in horses have been published.9 The test is not readily available in veterinary medicine because it requires trained personnel, appropriate facilities, standard protocols, and fresh muscle samples.9 Recently, a single missense point mutation (C7360G) at exon 46 of the RyR1 gene, generating R2454G amino acid substitution, was identified in MH susceptible Quarter Horses.5 However, it is uncertain whether or not this mutation is responsible for all cases of MH in horses or if it is specific to the Quarter Horse. Because of the limited availability of the IVCT and presumed presence of a single-point mutation in Quarter Horses, genetic testing is more suitable for the diagnosis of MH. An accurate, faster molecular test that is less prone to contamination than currently available tests would facilitate screening for the mutation in horses intended for breeding, those undergoing surgical procedures, and those with clinical signs compatible with MH.5 Real-time polymerase chain reaction (RT-PCR) melting curve analysis is a method used for the characterization of amplicons associated with microbiologic identification, detection of mutations, and single nucleotide polymorphisms.10 Therefore, the purpose of this study was to report a novel method for the rapid detection of the RyR1 C7360G mutation with RT-PCR melting curve analysis with hybridization probes in MH susceptible horses.
Materials and Methods
This prospective study included 11 Quarter Horses (6 geldings, 2 mares, 2 colts, and 1 stallion) genotyped as MH susceptible (MHRyR1) based on the presence of the RyR1 C7360G mutation as detected by gene sequencing of genomic (gDNA) and complementary (cDNA) DNA from whole blood, hair, or skeletal muscle samples submitted to the Neuromuscular Disease Laboratory (NDL) of the William R. Pritchard Veterinary Medical Teaching Hospital at the University of California at Davis. Both, gDNA and cDNA from 10 healthy Quarter Horses (wild type, WTRyR1; 6 geldings and 4 mares) donated by the NDL were used as controls to validate the genotyping assay. In addition, gDNA from 225 randomly selected Quarter Horses from our research herd and NDL was genotyped with the novel method to investigate the prevalence of the mutation in this population.
Genotyping by Melting Curve Analysis with the LightCycler
Primers were designed from previously published ryanodine receptor 1 exons 45–46 (GenBank AY375484) to work with both cDNA and gDNA.5 Forward and reverse primers were MH-F 5′-CTTCTACGCTGCCTTGA-3′ and MH-R 5′-CAGAGGGAGGCTGATGA-3′, respectively. Because the RYR1 gene mutation in the horse is in close proximity to the start of exon 46,5 it prevented the construction of the forward and reverse primers within the same exon. Therefore, the forward primer was designed within the adjacent exon 45. Hybridization probes were designed and synthesized by TIB MolBiol LLC,a and comprised a sensor probe (MH-SP, 5′-GCTCTGAGGATCCGAGCCATCC-3′) and anchor probe (MH-AP, 5′-GCGCTCCCTCGTGCCCCT-3′). The sensor probe was labeled at its 3′ end with fluorescein isothiocyanate, had a calculated melting temperature of 63.7 °C, and contained the mutation site at its center. Because it was determined from a previous study that approximately 10% of Quarter Horses may have a polymorphic site at nucleotide 7,362 (C or T) that neither generates an amino acid substitution nor confers MH susceptibility,5 the sensor probe also contained a mismatch (A instead of C or T) at nucleotide 7,362. Furthermore, a mismatch at that location interrupted the high GC-rich region of the probe and improved the resolution of the site of interest. The anchor probe was labeled at its 5′ end with LightCycler-Red 640, and phosphorylated at its 3′ end to prevent polymerase extension. This probe had a calculated melting temperature of 68.9 °C, and was located 1 nucleotide downstream of the detection probe (sensor probe). Primers, hybridization probes, and polymorphic sites are shown in Figure 1.
Reactions were performed in triplicates in 96-well plates by with a LightCycler 480 system.b A reaction volume of 20 μL was used as follows: water, 2 μL of sample cDNA or gDNA, 0.5 μL of each primer (0.5 μM), 0.3–0.4 μL of each HybProbea (0.15 and 0.20 μM for cDNA and gDNA, respectively), 0.2 μL uracyl glycosylasec (0.2 U) and 4 μL LightCycler 480 genotyping masterc containing a modified hot-start Taq DNA polymerase, reaction buffer and dNTP mix. Final concentrations of MgCl2 in the reaction were 3.0 and 4.0 mM for cDNA and gDNA, respectively. Reaction conditions were 37 °C for 10 minutes initially, preincubation at 95 °C for 5 minutes, followed by 39 cycles of 95 °C for 10 seconds for denaturation, 54 °C for 10 seconds for annealing, and 72 °C for 6 seconds (17 seconds for gDNA) for extension. After completion of the amplification process, the double-stranded PCR product was denatured at 95 °C for 1 minute, cooled to 40 °C for an additional minute to achieve maximum hybridization of the probes, and then slowly heated to 85 °C. During this process, declining fluorescence was continually monitored and melting curves were constructed and converted to melting peaks by plotting the negative derivative of the fluorescence with respect to temperature (−dF/dT).
Genomic and complementary DNA samples from WTRyR1 and MHRyR1 were blinded and subjected to PCR amplification and melting curve analysis with the primers and hybridization probes described previously. Agreement between amplicon sequencing (gold standard, method 1) and melting curve analysis (novel method, method 2) was evaluated by means of the kappa (K) statistic (K < 0.4 indicates poor agreement, and K > 0.75 excellent agreement).
All 11 MH susceptible horses were heterozygous for the RyR1 C7360G mutation. Three (1.3%) of the 225 randomly selected Quarter Horses also were heterozygous for the mutation as confirmed by sequencing.
Genotyping by Melting Curve Analysis with the LightCycler
The PCR primers were designed to work with cDNA and gDNA, and resulted in 151 and 503 base pairs amplicons, respectively. The sensor probe was designed specifically to detect WTRyR1 and MHRyR1 (homozygous and heterozygous). A clear single temperature melting peak was present in WTRyR1 at 68.1 ± 0.4 °C (mean ± SD) and 69.1 ± 0.4 °C for cDNA (Fig 2) and gDNA, respectively. Samples heterozygous for the MHRyR1 mutation indicated 2 melting peaks at 61.6 ± 0.4 and 68.7 ± 0.3 °C for cDNA (Fig 2); and at 62.8 ± 0.4 and 69.4 ± 0.3 °C for gDNA. The difference between the 2 melting peaks for heterozygous samples was 7.0 ± 0.1 and 6.7 ± 0.4 °C for cDNA and gDNA, respectively. In the event of a sample homozygous for MHRyR1, there would be a single melting peak in the low 60's °C for both cDNA and gDNA. The presence of C7362T polymorphism did not alter the temperature of the melting peaks for both WTRyR1 and MHRyR1 samples, and did not generate additional melting peaks. The C7362T polymorphism was identified by gene sequencing in 1 horse from each group.
After blinded melting curve analysis, samples were identified and results compared with sequenced amplicons. The overall agreement between the 2 methods using cDNA and gDNA for WTRyR1 and MHRyR1 in this study was 100% (K of 1.0).
This study described a novel rapid and accurate detection method for the RyR1 mutation in Quarter Horses with MH susceptibility that was less prone to contamination. Based on the results from this novel method, there were no MH homozygous horses in the present study, which was in agreement with the results from gene sequencing.
Three different techniques have been described for the detection of the RyR1 mutation in exon 46 in MH susceptible horses.5 The 1st technique involved the use of qualitative PCR with primers that amplified exon 46 in its entire length (121 base pairs)5 followed by sequencing, a process that involved several days. The 2nd technique comprised amplification of exon 46 followed by digestion of the amplicon with the restriction enzyme BamHI that recognizes and digests the GGATCC sequence, present in WTRyR1 horses, and generated 2 PCR products as seen on an agarose gel.5 In MHRyR1 heterozygous horses, 3 bands were observed. Because the sequence GGATCG in the mutated RyR1 gene is not recognized and therefore not digested by BamHI, a homozygous horse would have 1 band. The 3rd method used 2 sets of sequence-specific primers in the forward direction; 1 containing the wild-type nucleotide C located at its 3′ end, and a different forward primer with the mutated nucleotide G at its 3′ end.5 The reverse primer was the same for both amplifications. Primer C amplified the wild-type and heterozygous mutated sequence whereas primer G exclusively amplified the mutated sequence.5 These processes required a few hours (not including gDNA or cDNA extraction), and involved several post-PCR steps that could have increased the risk of end-product contamination, sample tracking errors, and incomplete enzyme digestion.
In this report, we described a highly specific RT-PCR technique that can be performed in less than 1 hour and give immediate results for the detection of WTRyR1 and MHRyR1 horses (heterozygous and homozygous). The principle of the technique is based on fluorescence resonance energy transfer (FRET). During FRET, a donor fluorophore (fluorescein) in the sensor probe, which is excited by a light-emitting diode light source, and transfers its energy to an acceptor fluorophore (LightCycler-Red 640) in the anchor probe only when positioned in close proximity to a donor. The acceptor fluorophore emits light of a longer wavelength, which can be detected by the thermocycler. The technique involves a pair of primers (forward and reverse) and a pair of probes (donor and acceptor) designed to bind close to each other (1–5 bases; 4–25 Å molecular distance) during DNA amplification. The melting temperatures of the probes should be 5–10 °C higher than those of the primers. For mutation analysis, the sensor probe is designed to cover the target polymorphic site near the center of the probe. In addition, the sensor probe must have a lower melting temperature than the anchor probe to guarantee that it will melt first. Any base mismatch will make the probe unstable and therefore will have a strong impact on the melting temperature. During melting curve analysis, the temperature slowly increases and if there is a perfect match, the probe will melt away at a higher temperature. However, if there is a mismatch (mutation) with the sensor probe, melting will occur at a lower temperature as shown in Figure 2. This rapid detection method is accurate, not affected by known polymorphisms of Quarter Horses, does not require post-PCR steps (eg, gel preparation and extraction, enzymatic digestion, sequencing), and works with both cDNA and gDNA. In the event of the presence of an additional polymorphism in the region of the sensor probe, different melting curves and temperatures are expected.
In conclusion, genotyping by melting curve analysis with hybridization probes is a rapid and accurate detection method for the RyR1 C7360G mutation that works on both cDNA and gDNA. This novel technique can be used to screen horses intended for breeding, before surgical intervention that may require inhalation anesthesia, or in horses with clinical signs compatible with both anesthetic and nonanesthetic forms of the disease. Furthermore, Quarter Horses with sudden death, exertional and nonexertional rhabdomyolysis, colic-like episodes, muscle rigidity, stiffness, intermittent hyperthermia, sweating, and subclinical increases in serum muscle enzyme activities should be tested if other causes have been ruled out. Lastly, the prevalence of the described mutation in Quarter Horses appears to be higher than previously thought. However, additional studies in a larger Quarter Horse population are needed. Several factors such as lack of recognition, information, triggering events, and testing may contribute to the apparent low prevalence of the disease.
aTIB Molbiol LLC, Adelphia, NJ
bRoche Diagnostics Corporation, Roche Applied Science, Indianapolis, IN
cInvitrogen Corporation, Carlsbad, CA
This project was funded by the Comparative Gastroenterology Laboratory of the University of California at Davis. Genetic testing is available at the Neuromuscular Disease Laboratory at the William R. Pritchard Veterinary Medical Teaching Hospital, One Shields Avenue, University of California, Davis CA 95616.