GENOMICS
11,
744-750
(1991)
Cosegregation of Porcine Malignant Hyperthermia and a Probable Causal Mutation in the Skeletal Muscle Ryanodine Receptor Gene in Backcross Families KINYA OTSU,* VIJAY K. KHANNA,*
ALAN L. Armwm,t
AND DAVID
H. MACLENNAN*,’
*Banting and Best Department of Medicaf Research, University of Toronto, C.H. Best institute, 112 Coliege Street, Toronto, Ontario, CanadaM5G I L6; and tAFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station Roslin, Midiothian EH25 9PS, United Kingdom Received
May
9, 1991;revisedJune
only halothane sensitivity can be measured in the living animal through short, controlled exposure to the anesthetic gas halothane (Eikelenboom and Minkema, 1974). Susceptibility to halothane-induced malignant hyperthermia has been shown to be controlled by a recessive gene at a single autosomal locus (HAL) with both alleles exhibiting incomplete penetrance (Ollivier et al., 1975; Minkema et al., 1977; Smith and Bampton, 1977). Man is also affected by halothaneinduced MH, but the conditions that induce human MH (prolonged halothane exposure coupled with administration of succinylcholine) are sufficiently severe to trigger the reaction in the heterozygote and inheritance is reported to be dominant. Because sensitivity to halothane-induced MH is a recessive trait in pigs, the standard halothane challenge test fails to distinguish between the homozygous resistant animals (N/J/) and the heterozygous carriers (N/n). Progeny testing, which offers the only reliable way of genotyping animals, is both expensive and time-consuming. Therefore, there is a need for identification of the causal mutation and for development of a cheap and accurate test based directly on the mutation that would facilitate the unambiguous identification of all three genotypes-iv/N, N/n, and n/n. In an earlier study, (Fujii et aZ., 1991) we found that the alteration of Cl843 to T1843 in the DNA encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor) was correlated with susceptibility to MH in five breeds of lean, heavily muscled swine and that the mutation was associated with a specific haplotype (HinPI- BanII+ RsaI-) in each of these breeds. These findings led to the potential for development of a DNA-based test for MH in swine. In this study, we have compared results from DNA-based tests for the mutation with results from the indirect halothane challenge test and GP1/
A study of the inheritance of malignant hyperthermia (MH) in the British Landrace breed revealed the same substitution of T for C at nucleotide 1843 in the ryanodine receptor (RYRI) gene that was previously shown to be correlated with MII in five Canadian swine breeds. Cosegregation of the mutation with MII in 338 informative meioses led to a lod score of 101.76 for linkage at 8,, = 0.0. The substitution was also associated with a HinPIBanII+ R8& haplotype in this breed, as in the Ave breeds tested earlier, suggesting its origin in a common founder animal. DNA-based detection of the MH status in 376 MHsusceptible heterozygous (N/n) and homozygous (n/n) pigs was shown to be accurate, eliminating the 5% diagnostic error that is associated with the halothane challenge test and flanking marker haplotyping procedures in current diagnostic use. These results strongly support the view that the substitution of T for C at nucleotide 1843 is the causative mutation in porcine MH and demonstrate the feasibility of rapid, accurate, noninvasive, large-scale testing for porcine MH status using DNA-based tests for the mutation. Q 1991hdd~ POW, IDC.
INTRODUCTION
Sudden, stress-induced deaths, pale soft exudative meat (PSE), and sensitivity to halothane-induced malignant hyperthermia (MH) are manifestations of the porcine stress syndrome (PSS) (for reviews seeHarrison, 1979; Mitchell and Heffron, 1982; Webb et al., 1982; Louis et al., 1990; Archibald, 1991). Deaths arise from uncontrolled skeletal muscle contracture with attendant hypermetabolic and hyperthermic reactions. These reactions are triggered by handling, sexual intercourse, excessive ambient temperature, and a number of chemical agents. Of the three phenotypes, 1 To whom
correspondence
should
OSJJS-7543/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
be addressed. 744
Inc. reserved.
27, 1991
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MALIGNANT
PGD haplotype analysis currently used to diagnose MH in backcrosses between British Landrace heterozygous (HAL N/n) and homozygous MH (HAL n/n) animals. Our study confirms and greatly extends the correlation between inheritance of MH and the alteration of Cl843 to T, lending strong support to the identification of this mutation as causal of porcine MH and setting the stage for the accurate, large-scale diagnosis of porcine MH. MATERIALS
AND
METHODS
Informative pedigrees for testing genetic markers for MH were created by crossing pigs heterozygous for several of the known biochemical polymorphisms linked to the HAL locus with the corresponding homozygotes for MH and the various polymorphisms. The pigs were derived from generations 6 and 7 of the British Landrace selection lines established at the Animal Breeding Research Organization (now IAPGR), Edinburgh. The structure of these selection lines is described by Simpson et al. (1986) and by Webb and Simpson (1986). The multiply heterozygous pigs were generated by crossing specific generation 6 individuals in the halothane-positive (HP) line with specific generation 6 individuals in the halothane-negative (HN) line. The corresponding homozygotes were selected on the basis of their marker genotypes from generations 6 and 7 of the HP line. In the subsequent backcross matings, all heterozygous pigs had the genotype HAL N/n, GPI A/B, PGD A/B and all homozygous pigs had the genotypes HAL n/n, GPI X/X, PGD Y/Y, where X and Y could be either A or B. It was anticipated that these pigs would be heterozygous and homozygous, respectively, for other genetic markers in this region of the pig genome. Of the backcross offspring born in 46 litters in farrowings in late 1987 and Summer 1988, 338 were halothane tested (Webb and Jordan, 1978) and blood was sampled. Washed erythrocytes were phenotyped for glucose-6-phosphate isomerase GPI and 6-phosphogluconate dehydrogenase PGD variants as described by Gahne and Juneja (1985). DNA was prepared from washed white blood cells and stored at 4°C. Ten-microgram aliquots of DNA were dispatched at ambient temperature by courier to Toronto for DNA-based analysis. DNA was analyzed from a total of 376 animals that included 14 heterozygous and 24 homozygous parents and 338 offspring. A 74-bp fragment of the genomic DNA between nucleotides 1811 and 1884 was PCRamplified and subjected to differential oligonucleotide hybridization using probes 17C and 15T, exactly as described previously (Fujii et aZ., 1991). In several cases, the PCR product was digested with a combination of HinPI and HgiAI to confirm the diagnoses
HYPERTHERMIA
(Fujii et al., T for C at breed was some of the subcloning
745
MUTATION
1991). The presence of the substitution of nucleotide 1843 in the British Landrace also confirmed by direct sequencing of PCR products (Gillard et al., 1991) and by for single-strand sequencing. RESULTS
Marker and HAL haplotypes were deduced by inspection of the phenotypes in the backcross litters and from knowledge of the parental and grand-parental genotypes and are summarized for the heterozygous parents and recombinant offspring in Table 1. Offspring numbers tested from crosses between specific individuals ranged from 1 in a litter to 20 spread across two farrowings of a particular sow. Multiple litters, especially for the boars, increased our confidence in the haplotype predictions. Informative offspring per heterozygous parent ranged from 5 sired by boar 2293 to 64 sired by boar 1943. The cosegregation of RYRl genotypes, as determined by DNA-based analysis, and of phenotypes, as determined by halothane testing and confirmed by haplotype analysis, is summarized in Table 2. The halothane challenge test is designed to detect n/n animals (Eikelenboom and Minkema, 1974; Webb and Jordan, 1978). Of the 168 animals reacting in this test, all were confirmed as n/n by the DNA-based test (Table 2). Of the 208 nonreactors, 197 were confirmed as N/n, but 11 were shown to be homozygous (n/n) by the DNA-based test. Analysis of the GPI and PGD haplotypes was consistent with the diagnosis of the 11 discordant animals (5.3%) as n/n nonreactors. The figure of 6.1% of the 179 n/n pigs that were nonreactors (false negatives) lies within the range of incomplete penetrance recorded in other studies (Ollivier et aZ., 1975; Minkema et al., 1977; Smith and Bampton, 1977; Southwood et al., 1988). All of the 11 disagreements between the RYRl genotype and the halothane test alone could be ascribed to incomplete penetrance of the HAL n allele by examining the marker haplotypes. Thus there is a complete correlation between the MH status of 376 animals, as diagnosed through a combination of the halothane challenge test with GPI and PGD haplotyping, and the DNA based test. These data confirm the accuracy of the DNA-based test and strongly support the identity of the Cl843 to T mutation as causal of porcine MH. We have experienced errors in the DNA-based testing of some of the samples transported between our laboratories. Following our initial survey of 376 animals with only oligonucleotide hybridization tests, there was apparent disagreement between the DNA and halothane-based tests for 34 animals, but 8 of these were readily corrected since they resulted from
746
OTSU
ET
TABLE MH Status
and Haplotype
AL.
1 of Heterozygous
Animals Haplotypes”
Haplotypes” Haplotype
Animal
HAL A. Boars
Parental
1943
Recombinant Parental
BC132 2088
Recombinant Parental
BCllO 2115
Recombinant
BC355 BC71 BC359 BC365 BC368 2116
Parental Recombinant
Parental
BC407 BC408 BC90 1944
Parental
2118
Recombinant Parental
BC137 2293
RYRl
GPI
PGD
Number offspring”
of Haplotype
Animal
and offspring C T C/B C :/ii C T C C C/B T c C T C c c C T C T T C T
HAL
RYRl
GPI
PGD
Number of offspring*
B. Sows and offspring A B A
A B AI-4 A/A
B A A B A A B A
38 25
B A
1948
Parental
1950
16 19’
Recombinant
16 18d
Parental
BC37 BC39 BC40 1951
Recombinant Parental
5899 2084
Parental
2085
Parental
2086
Parental
2129
Recombinant Parental
BC243 2131
A BIB A/A A B A/A AI.4 A/A B A A B BIB A B
Parental
26 24d
B A B A
5 3 15 17d
B A
2 3d
C A T B C A T B CfB T BIB C A/A C B T A C /A C B T A C B T A C B T A C A T B C /B C A T B
B A B A A
B A A B A B A B A B A A B A
9 6 5 6d
11 8 4 4 8 2e 2 6’ 4 3 9 8
’ In the first column, N refers to the normal allele and n to the MH allele deduced from information obtained in the halothane challenge test and analysis of marker haplotypes; in the second column, C and T refer to nucleotides at position 1843; in the third and fourth columns, A and B refer to the A and B alleles of GPI and PGD. The regions in which recombinations occurred between RYRl, GPI and PGD are indicated by slashes. Mating types were N/n C/T A/B A/B (boar) X n/n T/T X/X Y/Y (sow), where X and Y could be either A or B in section A and vice versa for section B. * Number of offspring to receive haplotypes, as shown, from their heterozygous parent. Skewing of the results from the 1:l ratio expected is most likely due to the preferential loss of n/n animals prior to testing. ’ Includes two nonreactors that received the haplotype T-B-A from the heterozygous parent. d Includes one nonreactor that received the haplotype T-B-A from the heterozygous parent. ’ Includes one nonreactor that received the haplotype T-A-A from the heterozygous parent. f Includes three nonreactors that received the haplotype T-A-A from the heterozygous parent. trivial transcription or communication problems within and between our laboratories. Samples of the same PCR products from each of these 34 animals were retested using a combination of oligonucleotide probing and HinPI and HgiAI digestion. In addition, samples of the same DNA from these 34 animals were sent from Edinburgh for a second DNA-based diagnosis where necessary. In 11 cases, disagreements were due to incomplete penetrance of the Hal n allele, as described above. In the remaining 15 cases, our initial oligonucleotidebased diagnosis was altered. Our ability to provide an unambiguous reading of hybridization test results using the relatively short oligonucleotides as probes varied slightly from batch to batch, probably due to variation in washing temperature, for which there is little tolerance, and this was responsible, in part, for
initial diagnostic errors. Digestion with a combination of HinPI and HgiAI helped to sort out problems that could not be resolved by repeat oligonucleotide hybridization screening. A more serious problem, however, was an apparent potential for sample-specific error that may have been due to contaminants in specific PCR products. The diagnosis of one difficult sample (BC69) as n/n was ultimately confirmed by direct sequencing of the PCR product and by demonstration of contamination or heterogeneity in the original DNA. Sample BC163 was also shown to be contaminated or heterogeneous. These problems would have been missed if we had not had internal checks on diagnoses or alternative assays to test for the presence of the mutation. Clearly, use of the oligonucleotide probe assay alone provides inadequate diagnosis. HinPI and HgiAI di-
PORCINE
TABLE
MALIGNANT
HYPERTHERMIA
2
Comparison of Halothane Challenge Testing for Phenotype and DNA-Based Analysis for Genotype in the Definition of the MH Status of British Landrace Pigs Phenotype from halothane reaction R NR R NR o Of the eleven all were confirmed
Genotype from DNA-based analysis
Number animals
of % Discordance
168 11” 0 197
n/n n/n N/n N/n halothane nonreactors as n/n by haplotype
(NR) genotyped analysis.
6.1 0.0
as n/n,
gestions are definitive for the complete digestion of the N/N and n/n genotypes, respectively, but must be adequately controlled for the partial (50%) digestion seen in detection of the N/n genotype.2 To minimize sample mix-ups, the strictest care must be taken in handling samples and in record keeping and communication. Having confirmed that the alteration of Cl843 to T occurs in the British Landrace breed, it was important to discern whether the mutation was also associated with the HinPI- BanII+ RsaI- haplotype previously associated with the MH phenotype (Fujii et aZ., 1991). We carried out haplotype analysis within the RYRl gene of 12 n/n and 12 N/n British Landrace pigs. In most cases, these were the parental animals used in our study. All 12 n/n animals were homozygous for the H&PIRunII+ RsaI- haplotype, while the heterozygous N/n animals differed only in the HinPI restriction endonuclease site that results from the alteration of Cl643 to T (Table 3). Thus in this strain of Landrace, as in animals from five Canadian breeds afflicted with MH, the HinPIBanII+ RsaIhaplotype is present in all MH susceptible (MHS) individuals, suggesting its origin in a single founder animal. In the human RYRl gene (M. Phillips, J. Fujii, and D. H. MacLennan, unpublished) the restriction endonuclease sites sampled in our study of the porcine RYRl haplotype would cover a span of approximately 145 kb, which, on average in the human genome, would correspond to a recombinant fraction of 0.15%. If these distances were relevant to the porcine gene, they would suggest either that the mutation is relatively recent or that there is strong selection a Footnote added in proof: Further cation and restriction endonuclease et al., in preparation) remove many with the tests described here.
refinements to PCR amplifidigestion techniques (K. 0t.m of the ambiguities associated
747
MUTATION
pressure acting to maintain the linkage disequilibrium that we have observed. By segregation analysis, the HAL locus has been assigned to a well-characterized linkage group on chromosome 6 in pigs (for a review see Archibald and Imlah, 1985). The ordering of the loci in this linkage group, in particular of GPI and HAL, has always been difficult because the frequency of incomplete penetrance of the HAL alleles is similar to the recombination frequency between these loci. Our study of linkage between the RYRl, GPI, and PGD loci has provided us with the opportunity to order these markers on pig chromosome 6. On the basis of crossovers between RYRl, GPI, and PGD (Table l), we can deduce that the order is RYRl-GPI-PGD. The recombination distances between these loci are summarized in Table 4. With this ordering, there is an absence of satisfactory codominantly expressed marker loci to the “left” of the HAL locus. DISCUSSION
The caffeine/halothane contracture test for humans (Kalow et aZ., 1970) and the halothane challenge test for swine (Eikelenboom and Minkema, 1974) have provided diagnoses that are widely used, but are limited in their accuracy. In particular, the halothane challenge test does not discriminate N/n from N/N individuals, it provides a false-positive reaction in up to 5% of N/n animals and it fails to elicit a response from up to 5% of n/n animals. Gahne and Juneja (1985) have shown that a combination of the halothane test and analysis of inheritance of linked biochemical genetic markers allows HAL genotypes to be
TABLE
3
Haplotypes of British Landrace Pigs of Different MH Status Haplotype Genotype n/n N/n
HinPI + -
Ban11
RsaI
+ + +
-
Number of chromosomes 24 12 12
Note. Haplotype analysis refers to the presence or absence, in three different PCR products from the RYRI gene, of a HinPI restriction endonuclease site, present in normal and deleted by the MH mutation at cDNA nucleotide 1643, a Ban11 site about 35 kb downstream in the gene, and an RsaI site about 145 kb downstream in the gene (Ref. (11)). Parental haplotypes tested included all four of the homosygous boars and five of the homozygous sows used in this study plus all seven of the heterozygous boars and five of the heterozygous sows (1946,2084,2065,2129, and 2131) described in Table 1.
748
OTSU
ET
TABLE Estimates Loci RYRl-GPI GPI-PGD RYRl-PGD
of Recombination Number of informative offspring 338 338 338
Distances
between Number of recombinant5 6 10 16
predicted with an accuracy of 90-95% in Swedish Landrace and Yorkshire pigs. DNA-based technology has been used recently in studies of linkage with the MH (HAL) locus of both flanking and candidate genes. DNA clones for the linked markers, GPI and PGD, have been isolated and used to search for DNA polymorphisms linked to the HAL locus (Davies et al., 1987,1988; Archibald, 1989; Brenig et al., 1990a,b; Archibald and Bowden, 1991). While these DNA markers do not give a direct test for the HAL alleles, they have allowed the HAL linkage group to be mapped and oriented on the long arm of porcine chromosome 6 by in situ hybridization (Davies et al., 1988; Yerle et al., 1990). Human RYRl cDNA clones have been used to reveal a number of RFLPs that have been used in linkage analysis in nine human MH pedigrees (MacLennan et aZ., 1990). No recombinants were observed between the RYRl RFLPs and the MH locus in 23 informative meioses, making RYRl a candidate gene for MH. The human RYRl gene was assigned by physical mapping techniques to human chromosome 19q13.1 (MacKenzie et aZ., 1990) and McCarthy et al. (1990) were able to establish linkage of MH to this region of human chromosome 19 using a series of chromosome 19q markers. In a companion study (Gillard et a& 1991), we have shown that substitution of T for Cl840 in human RYRl, corresponding to the porcine substitution studied here, is correlated with MH in a single human family. Thus defects in the RYRl gene are strongly implicated as causal of at least some forms of human MH. The assignment of the porcine RYRl gene to a region in porcine chromosome 6pllq21 that is homologous to the region of human chromosome 19q13.1 that contains RYRl (Harbitz et al., 1990) enhances the likelihood that this gene is responsible for MH in both man and pigs. Establishing tight linkage between the RYRl and MH loci in humans is difficult because of the limited numbers of families that have been tested for MH susceptibility, the lack of complete family diagnoses, and the ambiguities of the diagnosis of human MH using the caffeine halothane contracture test (Rosenberg, 1989; Britt, 1989). The use of the specific N/n X n/n backcross pig pedigrees described here over-
AL.
4 RYRl
and the Marker Recombination 0.018 0.029 0.047
Loci frequency
GPI and PGD 95% Confidence interval 0.004-0.032 0.011-0.047 0.025-0.069
came the difficulties associated with finding sufficient informative families and individuals to allow mapping with an accuracy greater than that provided by human data, while complementary halothane challenge and linked marker haplotyping tests overcame the difficulty in accurate diagnosis that plagues research with humans. In this study, 338 of 338 informative meioses revealed cosegregation of the Cl843 to T mutation in the porcine RYRl gene with the HAL n allele, leading to a lod score for linkage at 8, = 0.0 of 101.75. This study, when added to the correlation that we found with 79 individual pigs, accurately diagnosed for one of three phenotypes--nrll\r, N/n, or n/n (Fujii et al., 1991), to our studies with a human family in which the corresponding mutation correlated with the MH phenotype in a second species (Gillard et al., 1991), and to our finding of a common RYRl haplotype (suggesting a common founder animal) in MH-susceptible individuals in the British Landrace strain and in five different Canadian breeds of pigs (Fujii et al., 1991), provides powerful evidence that the Cl843 to T mutation in RYRl is, indeed, the causal mutation for porcine MH and that the corresponding Cl840 to T mutation in humans is causal of MH in the single family in which it has been found to occur to date. Previous tests designed to diagnose MH, although unable to detect carrier individuals, should have been sufficient, in combination with progeny testing, to facilitate the selective removal of the HAL n allele from pig populations. It is, therefore, surprising that the incidence of the HAL n allele has not already been reduced to insignificant levels. Two factors have militated against the elimination of the HAL n allele. First, the perceived advantages in lean meat production associated with the HAL n allele have resulted in the design of various breeding programs to maximize the number of heterozygous (stress-resistant but lean) pigs in the slaughter generation (Simpson and Webb, 1989). Second, the incidence of the HAL n allele in some breeds or populations, such as the Pietrain, mean that survival of the breed is currently incompatible with selection against HAL n. An important question that can now be addressed properly is whether the MH mutation does, indeed,
PORCINE
MALIGNANT
provide an advantage in lean meat production. While spontaneous firing of skeletal muscle due to hypersensitivity of the MH Ca2+ release channel is a plausible explanation for the leanness and hypertrophy associated with muscle in MH-susceptible pigs, the mapping to the HAL linkage group of the APOE gene or the TGFB-1 gene, with their known effects on myogenesis and adipogenesis (Massague, 1987), and (by extrapolation from the human map) of the LIPE gene (Holm et aZ., 19&J), with its potential effects on body fat content, supports an alternative explanation for some of the body composition phenotypes associated with the HAL locus. Thus APOE, TGF-61, or LIPE genes might be responsible for the differences in muscle and fat ratios, while the RYRl gene might be responsible for MH. This study demonstrates that the noninvasive DNA-based test for the C to T mutation at nucleotide 1843 can be used with speed and accuracy for the diagnosis of the MH status of large numbers of pigs. The DNA-based test can distinguish three genotypes (N/N, N/n, and n/n), in contrast to present tests, eliminating the inaccuracy associated with current tests and resulting in unambiguous definition of the MH status of individual swine. The availability of a rapid, accurate, noninvasive test for MH advances the potential for elimination of the MH gene from pig breeding programs. On the other hand, if the MH gene were found to be beneficial in the breeding of lean heavily muscled animals, its controlled and rational inclusion in breeding programs would be feasible with the advent of an accurate test for the defective gene. The test described here and earlier (Fujii et al., 1991) will allow pig breeders to effect either of these breeding objectives. ACKNOWLEDGMENTS We thank John Bowman, Brian McTeir, Sandra Couperwhite, and Alison Cowper for their excellent technical assistance; the farm statI at Mountmarle for maintaining the animals and performing the halothane tests; Stella de Leon for technical assistance in Toronto; Dr. Peter J. O’Brien for sharing his insights into porcine MH; and Dr. E. Gillard for discussion and assistance in these studies. This work was supported by grants to D.H.M. from the Muscular Dystrophy Association of Canada and the Medical Research Council of Canada and by a grant to A.L.A. at the IAPGR from the U.K. Ministry of Agriculture, Fisheries and Food.
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31. 32.
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34.
ROSENBERG, H. (1989). Testing for malignant hyperthermia. In “Malignant Hyperthermia: Current Concepts” (M. A. Nalda Felipe, S. Gottmann, and H. J. Khambatta, Eds.), pp. 47-52. Normed Verlag, Madrid. SIMPSON, S. P., AND WEBB, A. J. (1989). Growth and carcass performance of British Landrace pigs heterozygous at the halothane locus. Anim. Prod. 49: 503-509. SIMPSON, S. P., WEBB, A. J., AND WILMUT, I. (1986). Performance of British Landrace pigs selected for high and low incidence of halothane sensitivity 1. Reproduction. Anim. Prod. 43: 485-492. SMITH, C., AND BAMPTON, P. R. (1977). Inheritance of reaction to halothane anaesthesia in pigs. Genet. Res. 29: 287292. SOUTHWOOD, 0. I., SIMPSON, S. P., AND WEBB, A. J. (1988). Reaction to halothane anaesthesia among heterozygotes at the halothane locus in British Landrace pigs. Genet.Sel. Evol. 20: 357-366. WEBB, A. J., AND JORDAN, C. H. C. (1978). HaIothane sensitivity as a field test for stress-susceptibility in the pig. Anim. Prod. 26: 157-168. WEBB, A. J., AND SIMPSON, S. P. (1986). Performance of British Landrace pigs selected for high and low incidence of halothane sensitivity. 2. Growth and carcass traits. Anim. Prod. 43: 493-503. WEBB, A. J., CARDEN, A. E., SMITH, C., AND IMLAH, P. (1982). Porcine stress in pig breeding. In “Proceedings of the 2nd World Congress on Genetics Applied to Livestock Production,” Vol. VI, pp. 588-608. YERLE, M., ARCHIBALD, A. L., DALENS, M., AND GELLIN, J. (1990). Localization of PGD and TGFj3-1 to pig chromosome 6q. Anim. Genet. 21: 411-417.
11,
744-750
(1991)
Cosegregation of Porcine Malignant Hyperthermia and a Probable Causal Mutation in the Skeletal Muscle Ryanodine Receptor Gene in Backcross Families KINYA OTSU,* VIJAY K. KHANNA,*
ALAN L. Armwm,t
AND DAVID
H. MACLENNAN*,’
*Banting and Best Department of Medicaf Research, University of Toronto, C.H. Best institute, 112 Coliege Street, Toronto, Ontario, CanadaM5G I L6; and tAFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station Roslin, Midiothian EH25 9PS, United Kingdom Received
May
9, 1991;revisedJune
only halothane sensitivity can be measured in the living animal through short, controlled exposure to the anesthetic gas halothane (Eikelenboom and Minkema, 1974). Susceptibility to halothane-induced malignant hyperthermia has been shown to be controlled by a recessive gene at a single autosomal locus (HAL) with both alleles exhibiting incomplete penetrance (Ollivier et al., 1975; Minkema et al., 1977; Smith and Bampton, 1977). Man is also affected by halothaneinduced MH, but the conditions that induce human MH (prolonged halothane exposure coupled with administration of succinylcholine) are sufficiently severe to trigger the reaction in the heterozygote and inheritance is reported to be dominant. Because sensitivity to halothane-induced MH is a recessive trait in pigs, the standard halothane challenge test fails to distinguish between the homozygous resistant animals (N/J/) and the heterozygous carriers (N/n). Progeny testing, which offers the only reliable way of genotyping animals, is both expensive and time-consuming. Therefore, there is a need for identification of the causal mutation and for development of a cheap and accurate test based directly on the mutation that would facilitate the unambiguous identification of all three genotypes-iv/N, N/n, and n/n. In an earlier study, (Fujii et aZ., 1991) we found that the alteration of Cl843 to T1843 in the DNA encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor) was correlated with susceptibility to MH in five breeds of lean, heavily muscled swine and that the mutation was associated with a specific haplotype (HinPI- BanII+ RsaI-) in each of these breeds. These findings led to the potential for development of a DNA-based test for MH in swine. In this study, we have compared results from DNA-based tests for the mutation with results from the indirect halothane challenge test and GP1/
A study of the inheritance of malignant hyperthermia (MH) in the British Landrace breed revealed the same substitution of T for C at nucleotide 1843 in the ryanodine receptor (RYRI) gene that was previously shown to be correlated with MII in five Canadian swine breeds. Cosegregation of the mutation with MII in 338 informative meioses led to a lod score of 101.76 for linkage at 8,, = 0.0. The substitution was also associated with a HinPIBanII+ R8& haplotype in this breed, as in the Ave breeds tested earlier, suggesting its origin in a common founder animal. DNA-based detection of the MH status in 376 MHsusceptible heterozygous (N/n) and homozygous (n/n) pigs was shown to be accurate, eliminating the 5% diagnostic error that is associated with the halothane challenge test and flanking marker haplotyping procedures in current diagnostic use. These results strongly support the view that the substitution of T for C at nucleotide 1843 is the causative mutation in porcine MH and demonstrate the feasibility of rapid, accurate, noninvasive, large-scale testing for porcine MH status using DNA-based tests for the mutation. Q 1991hdd~ POW, IDC.
INTRODUCTION
Sudden, stress-induced deaths, pale soft exudative meat (PSE), and sensitivity to halothane-induced malignant hyperthermia (MH) are manifestations of the porcine stress syndrome (PSS) (for reviews seeHarrison, 1979; Mitchell and Heffron, 1982; Webb et al., 1982; Louis et al., 1990; Archibald, 1991). Deaths arise from uncontrolled skeletal muscle contracture with attendant hypermetabolic and hyperthermic reactions. These reactions are triggered by handling, sexual intercourse, excessive ambient temperature, and a number of chemical agents. Of the three phenotypes, 1 To whom
correspondence
should
OSJJS-7543/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
be addressed. 744
Inc. reserved.
27, 1991
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MALIGNANT
PGD haplotype analysis currently used to diagnose MH in backcrosses between British Landrace heterozygous (HAL N/n) and homozygous MH (HAL n/n) animals. Our study confirms and greatly extends the correlation between inheritance of MH and the alteration of Cl843 to T, lending strong support to the identification of this mutation as causal of porcine MH and setting the stage for the accurate, large-scale diagnosis of porcine MH. MATERIALS
AND
METHODS
Informative pedigrees for testing genetic markers for MH were created by crossing pigs heterozygous for several of the known biochemical polymorphisms linked to the HAL locus with the corresponding homozygotes for MH and the various polymorphisms. The pigs were derived from generations 6 and 7 of the British Landrace selection lines established at the Animal Breeding Research Organization (now IAPGR), Edinburgh. The structure of these selection lines is described by Simpson et al. (1986) and by Webb and Simpson (1986). The multiply heterozygous pigs were generated by crossing specific generation 6 individuals in the halothane-positive (HP) line with specific generation 6 individuals in the halothane-negative (HN) line. The corresponding homozygotes were selected on the basis of their marker genotypes from generations 6 and 7 of the HP line. In the subsequent backcross matings, all heterozygous pigs had the genotype HAL N/n, GPI A/B, PGD A/B and all homozygous pigs had the genotypes HAL n/n, GPI X/X, PGD Y/Y, where X and Y could be either A or B. It was anticipated that these pigs would be heterozygous and homozygous, respectively, for other genetic markers in this region of the pig genome. Of the backcross offspring born in 46 litters in farrowings in late 1987 and Summer 1988, 338 were halothane tested (Webb and Jordan, 1978) and blood was sampled. Washed erythrocytes were phenotyped for glucose-6-phosphate isomerase GPI and 6-phosphogluconate dehydrogenase PGD variants as described by Gahne and Juneja (1985). DNA was prepared from washed white blood cells and stored at 4°C. Ten-microgram aliquots of DNA were dispatched at ambient temperature by courier to Toronto for DNA-based analysis. DNA was analyzed from a total of 376 animals that included 14 heterozygous and 24 homozygous parents and 338 offspring. A 74-bp fragment of the genomic DNA between nucleotides 1811 and 1884 was PCRamplified and subjected to differential oligonucleotide hybridization using probes 17C and 15T, exactly as described previously (Fujii et aZ., 1991). In several cases, the PCR product was digested with a combination of HinPI and HgiAI to confirm the diagnoses
HYPERTHERMIA
(Fujii et al., T for C at breed was some of the subcloning
745
MUTATION
1991). The presence of the substitution of nucleotide 1843 in the British Landrace also confirmed by direct sequencing of PCR products (Gillard et al., 1991) and by for single-strand sequencing. RESULTS
Marker and HAL haplotypes were deduced by inspection of the phenotypes in the backcross litters and from knowledge of the parental and grand-parental genotypes and are summarized for the heterozygous parents and recombinant offspring in Table 1. Offspring numbers tested from crosses between specific individuals ranged from 1 in a litter to 20 spread across two farrowings of a particular sow. Multiple litters, especially for the boars, increased our confidence in the haplotype predictions. Informative offspring per heterozygous parent ranged from 5 sired by boar 2293 to 64 sired by boar 1943. The cosegregation of RYRl genotypes, as determined by DNA-based analysis, and of phenotypes, as determined by halothane testing and confirmed by haplotype analysis, is summarized in Table 2. The halothane challenge test is designed to detect n/n animals (Eikelenboom and Minkema, 1974; Webb and Jordan, 1978). Of the 168 animals reacting in this test, all were confirmed as n/n by the DNA-based test (Table 2). Of the 208 nonreactors, 197 were confirmed as N/n, but 11 were shown to be homozygous (n/n) by the DNA-based test. Analysis of the GPI and PGD haplotypes was consistent with the diagnosis of the 11 discordant animals (5.3%) as n/n nonreactors. The figure of 6.1% of the 179 n/n pigs that were nonreactors (false negatives) lies within the range of incomplete penetrance recorded in other studies (Ollivier et aZ., 1975; Minkema et al., 1977; Smith and Bampton, 1977; Southwood et al., 1988). All of the 11 disagreements between the RYRl genotype and the halothane test alone could be ascribed to incomplete penetrance of the HAL n allele by examining the marker haplotypes. Thus there is a complete correlation between the MH status of 376 animals, as diagnosed through a combination of the halothane challenge test with GPI and PGD haplotyping, and the DNA based test. These data confirm the accuracy of the DNA-based test and strongly support the identity of the Cl843 to T mutation as causal of porcine MH. We have experienced errors in the DNA-based testing of some of the samples transported between our laboratories. Following our initial survey of 376 animals with only oligonucleotide hybridization tests, there was apparent disagreement between the DNA and halothane-based tests for 34 animals, but 8 of these were readily corrected since they resulted from
746
OTSU
ET
TABLE MH Status
and Haplotype
AL.
1 of Heterozygous
Animals Haplotypes”
Haplotypes” Haplotype
Animal
HAL A. Boars
Parental
1943
Recombinant Parental
BC132 2088
Recombinant Parental
BCllO 2115
Recombinant
BC355 BC71 BC359 BC365 BC368 2116
Parental Recombinant
Parental
BC407 BC408 BC90 1944
Parental
2118
Recombinant Parental
BC137 2293
RYRl
GPI
PGD
Number offspring”
of Haplotype
Animal
and offspring C T C/B C :/ii C T C C C/B T c C T C c c C T C T T C T
HAL
RYRl
GPI
PGD
Number of offspring*
B. Sows and offspring A B A
A B AI-4 A/A
B A A B A A B A
38 25
B A
1948
Parental
1950
16 19’
Recombinant
16 18d
Parental
BC37 BC39 BC40 1951
Recombinant Parental
5899 2084
Parental
2085
Parental
2086
Parental
2129
Recombinant Parental
BC243 2131
A BIB A/A A B A/A AI.4 A/A B A A B BIB A B
Parental
26 24d
B A B A
5 3 15 17d
B A
2 3d
C A T B C A T B CfB T BIB C A/A C B T A C /A C B T A C B T A C B T A C A T B C /B C A T B
B A B A A
B A A B A B A B A B A A B A
9 6 5 6d
11 8 4 4 8 2e 2 6’ 4 3 9 8
’ In the first column, N refers to the normal allele and n to the MH allele deduced from information obtained in the halothane challenge test and analysis of marker haplotypes; in the second column, C and T refer to nucleotides at position 1843; in the third and fourth columns, A and B refer to the A and B alleles of GPI and PGD. The regions in which recombinations occurred between RYRl, GPI and PGD are indicated by slashes. Mating types were N/n C/T A/B A/B (boar) X n/n T/T X/X Y/Y (sow), where X and Y could be either A or B in section A and vice versa for section B. * Number of offspring to receive haplotypes, as shown, from their heterozygous parent. Skewing of the results from the 1:l ratio expected is most likely due to the preferential loss of n/n animals prior to testing. ’ Includes two nonreactors that received the haplotype T-B-A from the heterozygous parent. d Includes one nonreactor that received the haplotype T-B-A from the heterozygous parent. ’ Includes one nonreactor that received the haplotype T-A-A from the heterozygous parent. f Includes three nonreactors that received the haplotype T-A-A from the heterozygous parent. trivial transcription or communication problems within and between our laboratories. Samples of the same PCR products from each of these 34 animals were retested using a combination of oligonucleotide probing and HinPI and HgiAI digestion. In addition, samples of the same DNA from these 34 animals were sent from Edinburgh for a second DNA-based diagnosis where necessary. In 11 cases, disagreements were due to incomplete penetrance of the Hal n allele, as described above. In the remaining 15 cases, our initial oligonucleotidebased diagnosis was altered. Our ability to provide an unambiguous reading of hybridization test results using the relatively short oligonucleotides as probes varied slightly from batch to batch, probably due to variation in washing temperature, for which there is little tolerance, and this was responsible, in part, for
initial diagnostic errors. Digestion with a combination of HinPI and HgiAI helped to sort out problems that could not be resolved by repeat oligonucleotide hybridization screening. A more serious problem, however, was an apparent potential for sample-specific error that may have been due to contaminants in specific PCR products. The diagnosis of one difficult sample (BC69) as n/n was ultimately confirmed by direct sequencing of the PCR product and by demonstration of contamination or heterogeneity in the original DNA. Sample BC163 was also shown to be contaminated or heterogeneous. These problems would have been missed if we had not had internal checks on diagnoses or alternative assays to test for the presence of the mutation. Clearly, use of the oligonucleotide probe assay alone provides inadequate diagnosis. HinPI and HgiAI di-
PORCINE
TABLE
MALIGNANT
HYPERTHERMIA
2
Comparison of Halothane Challenge Testing for Phenotype and DNA-Based Analysis for Genotype in the Definition of the MH Status of British Landrace Pigs Phenotype from halothane reaction R NR R NR o Of the eleven all were confirmed
Genotype from DNA-based analysis
Number animals
of % Discordance
168 11” 0 197
n/n n/n N/n N/n halothane nonreactors as n/n by haplotype
(NR) genotyped analysis.
6.1 0.0
as n/n,
gestions are definitive for the complete digestion of the N/N and n/n genotypes, respectively, but must be adequately controlled for the partial (50%) digestion seen in detection of the N/n genotype.2 To minimize sample mix-ups, the strictest care must be taken in handling samples and in record keeping and communication. Having confirmed that the alteration of Cl843 to T occurs in the British Landrace breed, it was important to discern whether the mutation was also associated with the HinPI- BanII+ RsaI- haplotype previously associated with the MH phenotype (Fujii et aZ., 1991). We carried out haplotype analysis within the RYRl gene of 12 n/n and 12 N/n British Landrace pigs. In most cases, these were the parental animals used in our study. All 12 n/n animals were homozygous for the H&PIRunII+ RsaI- haplotype, while the heterozygous N/n animals differed only in the HinPI restriction endonuclease site that results from the alteration of Cl643 to T (Table 3). Thus in this strain of Landrace, as in animals from five Canadian breeds afflicted with MH, the HinPIBanII+ RsaIhaplotype is present in all MH susceptible (MHS) individuals, suggesting its origin in a single founder animal. In the human RYRl gene (M. Phillips, J. Fujii, and D. H. MacLennan, unpublished) the restriction endonuclease sites sampled in our study of the porcine RYRl haplotype would cover a span of approximately 145 kb, which, on average in the human genome, would correspond to a recombinant fraction of 0.15%. If these distances were relevant to the porcine gene, they would suggest either that the mutation is relatively recent or that there is strong selection a Footnote added in proof: Further cation and restriction endonuclease et al., in preparation) remove many with the tests described here.
refinements to PCR amplifidigestion techniques (K. 0t.m of the ambiguities associated
747
MUTATION
pressure acting to maintain the linkage disequilibrium that we have observed. By segregation analysis, the HAL locus has been assigned to a well-characterized linkage group on chromosome 6 in pigs (for a review see Archibald and Imlah, 1985). The ordering of the loci in this linkage group, in particular of GPI and HAL, has always been difficult because the frequency of incomplete penetrance of the HAL alleles is similar to the recombination frequency between these loci. Our study of linkage between the RYRl, GPI, and PGD loci has provided us with the opportunity to order these markers on pig chromosome 6. On the basis of crossovers between RYRl, GPI, and PGD (Table l), we can deduce that the order is RYRl-GPI-PGD. The recombination distances between these loci are summarized in Table 4. With this ordering, there is an absence of satisfactory codominantly expressed marker loci to the “left” of the HAL locus. DISCUSSION
The caffeine/halothane contracture test for humans (Kalow et aZ., 1970) and the halothane challenge test for swine (Eikelenboom and Minkema, 1974) have provided diagnoses that are widely used, but are limited in their accuracy. In particular, the halothane challenge test does not discriminate N/n from N/N individuals, it provides a false-positive reaction in up to 5% of N/n animals and it fails to elicit a response from up to 5% of n/n animals. Gahne and Juneja (1985) have shown that a combination of the halothane test and analysis of inheritance of linked biochemical genetic markers allows HAL genotypes to be
TABLE
3
Haplotypes of British Landrace Pigs of Different MH Status Haplotype Genotype n/n N/n
HinPI + -
Ban11
RsaI
+ + +
-
Number of chromosomes 24 12 12
Note. Haplotype analysis refers to the presence or absence, in three different PCR products from the RYRI gene, of a HinPI restriction endonuclease site, present in normal and deleted by the MH mutation at cDNA nucleotide 1643, a Ban11 site about 35 kb downstream in the gene, and an RsaI site about 145 kb downstream in the gene (Ref. (11)). Parental haplotypes tested included all four of the homosygous boars and five of the homozygous sows used in this study plus all seven of the heterozygous boars and five of the heterozygous sows (1946,2084,2065,2129, and 2131) described in Table 1.
748
OTSU
ET
TABLE Estimates Loci RYRl-GPI GPI-PGD RYRl-PGD
of Recombination Number of informative offspring 338 338 338
Distances
between Number of recombinant5 6 10 16
predicted with an accuracy of 90-95% in Swedish Landrace and Yorkshire pigs. DNA-based technology has been used recently in studies of linkage with the MH (HAL) locus of both flanking and candidate genes. DNA clones for the linked markers, GPI and PGD, have been isolated and used to search for DNA polymorphisms linked to the HAL locus (Davies et al., 1987,1988; Archibald, 1989; Brenig et al., 1990a,b; Archibald and Bowden, 1991). While these DNA markers do not give a direct test for the HAL alleles, they have allowed the HAL linkage group to be mapped and oriented on the long arm of porcine chromosome 6 by in situ hybridization (Davies et al., 1988; Yerle et al., 1990). Human RYRl cDNA clones have been used to reveal a number of RFLPs that have been used in linkage analysis in nine human MH pedigrees (MacLennan et aZ., 1990). No recombinants were observed between the RYRl RFLPs and the MH locus in 23 informative meioses, making RYRl a candidate gene for MH. The human RYRl gene was assigned by physical mapping techniques to human chromosome 19q13.1 (MacKenzie et aZ., 1990) and McCarthy et al. (1990) were able to establish linkage of MH to this region of human chromosome 19 using a series of chromosome 19q markers. In a companion study (Gillard et a& 1991), we have shown that substitution of T for Cl840 in human RYRl, corresponding to the porcine substitution studied here, is correlated with MH in a single human family. Thus defects in the RYRl gene are strongly implicated as causal of at least some forms of human MH. The assignment of the porcine RYRl gene to a region in porcine chromosome 6pllq21 that is homologous to the region of human chromosome 19q13.1 that contains RYRl (Harbitz et al., 1990) enhances the likelihood that this gene is responsible for MH in both man and pigs. Establishing tight linkage between the RYRl and MH loci in humans is difficult because of the limited numbers of families that have been tested for MH susceptibility, the lack of complete family diagnoses, and the ambiguities of the diagnosis of human MH using the caffeine halothane contracture test (Rosenberg, 1989; Britt, 1989). The use of the specific N/n X n/n backcross pig pedigrees described here over-
AL.
4 RYRl
and the Marker Recombination 0.018 0.029 0.047
Loci frequency
GPI and PGD 95% Confidence interval 0.004-0.032 0.011-0.047 0.025-0.069
came the difficulties associated with finding sufficient informative families and individuals to allow mapping with an accuracy greater than that provided by human data, while complementary halothane challenge and linked marker haplotyping tests overcame the difficulty in accurate diagnosis that plagues research with humans. In this study, 338 of 338 informative meioses revealed cosegregation of the Cl843 to T mutation in the porcine RYRl gene with the HAL n allele, leading to a lod score for linkage at 8, = 0.0 of 101.75. This study, when added to the correlation that we found with 79 individual pigs, accurately diagnosed for one of three phenotypes--nrll\r, N/n, or n/n (Fujii et al., 1991), to our studies with a human family in which the corresponding mutation correlated with the MH phenotype in a second species (Gillard et al., 1991), and to our finding of a common RYRl haplotype (suggesting a common founder animal) in MH-susceptible individuals in the British Landrace strain and in five different Canadian breeds of pigs (Fujii et al., 1991), provides powerful evidence that the Cl843 to T mutation in RYRl is, indeed, the causal mutation for porcine MH and that the corresponding Cl840 to T mutation in humans is causal of MH in the single family in which it has been found to occur to date. Previous tests designed to diagnose MH, although unable to detect carrier individuals, should have been sufficient, in combination with progeny testing, to facilitate the selective removal of the HAL n allele from pig populations. It is, therefore, surprising that the incidence of the HAL n allele has not already been reduced to insignificant levels. Two factors have militated against the elimination of the HAL n allele. First, the perceived advantages in lean meat production associated with the HAL n allele have resulted in the design of various breeding programs to maximize the number of heterozygous (stress-resistant but lean) pigs in the slaughter generation (Simpson and Webb, 1989). Second, the incidence of the HAL n allele in some breeds or populations, such as the Pietrain, mean that survival of the breed is currently incompatible with selection against HAL n. An important question that can now be addressed properly is whether the MH mutation does, indeed,
PORCINE
MALIGNANT
provide an advantage in lean meat production. While spontaneous firing of skeletal muscle due to hypersensitivity of the MH Ca2+ release channel is a plausible explanation for the leanness and hypertrophy associated with muscle in MH-susceptible pigs, the mapping to the HAL linkage group of the APOE gene or the TGFB-1 gene, with their known effects on myogenesis and adipogenesis (Massague, 1987), and (by extrapolation from the human map) of the LIPE gene (Holm et aZ., 19&J), with its potential effects on body fat content, supports an alternative explanation for some of the body composition phenotypes associated with the HAL locus. Thus APOE, TGF-61, or LIPE genes might be responsible for the differences in muscle and fat ratios, while the RYRl gene might be responsible for MH. This study demonstrates that the noninvasive DNA-based test for the C to T mutation at nucleotide 1843 can be used with speed and accuracy for the diagnosis of the MH status of large numbers of pigs. The DNA-based test can distinguish three genotypes (N/N, N/n, and n/n), in contrast to present tests, eliminating the inaccuracy associated with current tests and resulting in unambiguous definition of the MH status of individual swine. The availability of a rapid, accurate, noninvasive test for MH advances the potential for elimination of the MH gene from pig breeding programs. On the other hand, if the MH gene were found to be beneficial in the breeding of lean heavily muscled animals, its controlled and rational inclusion in breeding programs would be feasible with the advent of an accurate test for the defective gene. The test described here and earlier (Fujii et al., 1991) will allow pig breeders to effect either of these breeding objectives. ACKNOWLEDGMENTS We thank John Bowman, Brian McTeir, Sandra Couperwhite, and Alison Cowper for their excellent technical assistance; the farm statI at Mountmarle for maintaining the animals and performing the halothane tests; Stella de Leon for technical assistance in Toronto; Dr. Peter J. O’Brien for sharing his insights into porcine MH; and Dr. E. Gillard for discussion and assistance in these studies. This work was supported by grants to D.H.M. from the Muscular Dystrophy Association of Canada and the Medical Research Council of Canada and by a grant to A.L.A. at the IAPGR from the U.K. Ministry of Agriculture, Fisheries and Food.
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HYPERTHERMIA
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MUTATION
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