Department of Animal Genetics and Institute of Animal Science, Royal VeterinarJi and Agricultural University and National Institute of Animal Science, Copenhagen

Malignant hyperthermia syndrome (MHS) in pigs: Relative risks associated with various genotypes

By E. ANDRESEN, P. JENSEN, P. JONSSON and H . STAUN

Dedicated to Prof. IVAR JOHANSSON

M s . received 1. 2. 80

Introduction

The malignant hyperthermia syndrome (MHS) occurs in susceptible pigs following the administration of various drugs (TOPEL et al. 1975). Halothane inhalation is used routinely for diagnostic purpose (CHRISTIAN 1974; EIKELENBOOM and MINKEMA 1974). A number of studies have shown that close relationships exist between susceptibility to halothane and several production traits, including undesirable traits such as inferior meat quality (TOPEL 1975; EIKELENBOOM et al. 1978; WEBB and JORDAN 1978). Susceptibility to MHS as triggered by halothane is an autosomal, recessive trait (SMITH and BAMPTON 1977). Susceptible pigs possess the genotype Hal" Hal", whereas non-susceptible pigs posses the genotype Hal"'Hal" or Hal" Hal".

In 1976 KASMUSEN and CHRISTIAN discovered an association between MHS susceptibil- ity and H blood types in Yorkshire and Yorkshire-Duroc crossbred pigs. Almost simultaneously JORGENSEN et a[. (1 976) discovered an association between PHI (phos- phohexose isomerase) types and halothane reactivity among pigs of various Landrace breeds. The H and Phi loci are genetically linked with a distance of about 2 centimorgan, and the Hal locus appears to be situated between the two loci, but closer to Phi than to the H locus (ANDRESEN and JENSEN 1977; J~RGENSEN 1978; ANDRESEN 1979). Observed associa- tions within Danish Landrace pigs have been explained by the existence of linkngc disequilibrium (ANDKESEN 1979 b, 1980).

The present study is confined to a sample of Danish Landrace pigs, which have been subjected to H and PHI typing and the halothane test. The purpose of the study is ( 1 ) to estimate the relative risk of contracting MHS for pigs belonging to various genotypic classes, and ( 2 ) to relate the findings to problems pertaining to selection for improvement of production traits.

Theory

The 2 x 2 table is a well known tool for detecting an association between a disease and a prospective disease determinant in case-control studies. For reviews, see for example SCHWABE et al. (1977) or WILLEBERC (1977). For various details, see e.g. BISHOP et al. (1 975).

U.S. Copyright Clearance Center Code Statement: Z. Tierziichrg. Ziichtgsbiol. 97 (1980) 210-216 0 1980 Verlag Paul Parey, Hamburg und Berlin ISSN 0044-3581 /ASTM-Coden: ZTZBAS

0044-3581 /80/9703-0210 $2.50/0

Malignant hyperthermia syndrome ( M H S ) in pigs 21 1

is a measure of the association (cf. CAVALLI-SFORZA and BOOMER 1971), whereas the magnitude of x2 indicates the statistical significance of the association, i.e. whether the r value is significantly different from zero.

In order to illustrate the procedure of estimating relative risk, assume that a, b ,c and d in Table 1 represent the observed number of each category in a random sample of the population. The relative risk (R), which refers to the entire population is the incidence of the disease among animals exposed to (or possessing) the disease determinant divided by the incidence among those not exposed to (or not possessing) the disease determinant. Thus, the

The tetrachoric correlation coefficient, r =

estimate of the relative risk is R = -2- .c=a(c+d). An estimate of R = 1 indicates that a + b ‘ c + d c(a + b)

the effect of an hypothezised disease determinant’ is zero. O n e should also note that k = 1 and the association (r) is 0 if ad = bc. Moreover, the association is 1 if both b and d are zero. Clearly, an association of r = 1 is suggestive of the existence of merely one disease determinant. In this situation the relative risk is not defined. A relevant example is susceptibility to M H S as defined by the halothane reaction assuming full penetrance of the genotype H a p Hal“.

Due to a low a priori probability combined with sampling variation the a or c cell in the 2 x 2 table may have a zero entry. This makes a risk estimation impossible based on the actual observations. A frequent procedure when sampling (random) zeros are encountered is to add one half to each cell in the table (BISHOP et al. 1975). This adjustment is applied for the treatment of Table 2 B in the present paper.

Material and methods

The material includes 289 Danish Landrace pigs belonging to 39 litters from 39 different sows and 20 boars. The pigs are representative for pigs from elite herds during the period 1973-74. They were subjected to blood typing, enzyme typing, and halothane testing at 6-12 weeks of age. In agreement with the original discoveries the relevant disease determinants (genotypes) are [ H”l] and [Phi’ PhiB] as indicated in Table 2 A and B. In addition, the aggregate determinant [Wl, PhiH PhiH] is considered in Table 2 C . The genotype [ H”l] encompasses the genotypes H ” H and W H - , where H - includes all Halleles not producing the H a antigen. The PHI system includes the genotypes PhiA Phi”, Phi” Phi’, and Phi’ Phi’. The statistical methods used are described in the previous section.

Results

The results are indicated in Table 2. MHS susceptibility is positively associated with the disease determinant [Wl] as the correlation coefficient is r = 0.25. The corresponding r values for the comparisons in Table 2 B and C are 0.19 and 0.28, respectively. For the material in Table 2 A the incidence of MHS susceptibility among individuals not possessing the disease determinant is 13 : 182 whereas the incidence is 22 : 107 among those possessing the determinant. Thus, the estimate of the relative risk for the comparison [ W l ] : [ nonH“/ 1 IS

A similar procedure is used for the results based on Table 2 C. Therefore, the estimate of 22 .206 - the relative risk for the comparison [ H a l , PhiHPhi’] : [ non Wl, Phi’Phi’] is R = - - 1 3 . 83 4.2.

212

1)cterminmt

E . Andresen, P. Jensen. P . Jonsson and H . Staun

II,rr.,rr

Prerrnt r \ h x n t

(Tcrt popular ion) (Cuntrt i l Popul.xion) Turd

Table I

X table for the analyses of association (r) and relative risk (R) in a case-control s tudy

ad-bc ' N '"' = (a + c) (d + d) :a + b) (c + d)

r = \/X2/N

- a c a(c + d) R = - T : - = - a + c + d c ( a + b )

Table 2

Summary of case-control s tudy comprising 289 Danish Landrace pigs

Estimates of associations and relative risks are indicated. MHS = malignant hyperthermia. Genotype notations are explained under Materials and methods

A.

[ / i d / ] MHS susceptibility rrr\ent A h n r Total

Prescnt 22 85 107 Absent 13 169 182

Total 35 254 289

w?,i = 14.7: r = 0.25: R = 2.9

B

Present Absent

Total

= 10.4; r = 0.19; R = 18.8

35 0

35

194 60

254

229 60

289

C

Present Absent I 22

13 61

193 83

206

Total 35 254 289 I y&c = 22.7; r = 0.28; R = 4.2

i\lalrgnurzt hypertherrnia syndrome ( M H S ) in pigs 213

Finally, as described in the Theory section the estimate of the relative risk for the A (35 + f ) . (60 + 2 . f )

comparison [ PhrHPhiH] : [non PhLHPhzH1 is R = + + ) , (229 +

. +) = 18.8.

Discussion

The estimated relative risks of MHS susceptibility for two different genotypes, [ H / ] and [Phi’phi’], and their combination [Hl, Phi’Phi’] was found to be 2.9, 18.8, and 4.2, respectively. This corresponds to excess risks of 1.9, 17.8, and 3.2 as compared to the categories not having the genotypes considered. In case-control studies the exact cause of excess risks associated with various disease determinants (factor categories) may be difficult o r impossible to unravel. However, in this situation the task is easy. By definition MHS susceptibility is caused by presence of the genotype Hal“ Hal” in the host itself. Thus, the causal determinant for M H S susccptibility is simply that particular genotype (host determinant). Thereforc, the three determinants studied may be designated as associative disease determinants, i.e. without intrinsic (causal) effect. A detailed explanation for the magnitude of the relative risk values shall not be presented at this place. It may suffice to mention that thc risks are due to an excess of chromosomes possessing a combination of the genes Phi’, Hal”, H within the material studied. Since the three genes belong to three closely linked loci the excess can be explained by the phenomenon of linkage disequilibrium (D 4+ O ) , i.c. a non random combination of genes at linked loci. I o r simplicity one can state that D = 0 (linkage equilibrium) corresponds to r = 0 and R = 1, whereas D +i 0 corresponds to r +i 0 and K # I . The present material combined with additional material has shown that linkage disequilibrium does exist with respect to the three loci within the Danish Landrace breed (ANDRESEN 1979b, 1980).

In this context it is of interest that the zero value in the lower left cell i n Table 2 B must be regarded as a sampling, or random, zero rather than afixed or structural, zero. This is due to the fact that the genotype Hal” Hal” has been observed in combination with both genotypes Phi“ Phi’’ and Phi” Phi’ in Pietrain and Dutch Landrace pigs (ANSAY and OLLIVJER 1978; GUERIN et al. 1978; WATANABE et al. 1978). This proves the existence of the [Phi”, Hal”] haplotype (gametic type), which so far has not been detected within the Danish Landrace breed. Moreover, the observations indicate that the Hal and Phz loci are two separate loci between which recombination can occur. Thus, the missing genotype within the Danish Landrace breed seems entirely due to the relatively small number of individuals tested for both systems.

The sampling zero just referred to requires an adjustment prior to estimating the relative risk. This problem was adequately solved in the present study for the material in Table 2 Bas described in the Theory section. However, an additional problem remains to be solved. The k values were calculated as if the individuals comprised unrelated population material. However, the material comprised exclusively pigs representing groups of full sibs and haLf sibs. This inclusion of related individuals in the sample is not expected to bias the estimate K but is expected to influence the standard error. The cumbersome task of applying the maximum likelihood method for estimating the standard error has not been applied in this study. However, it may suffice to mention that additional material on record corroborates the present estimates of relative risks. Thus in the combined material the statistical significance of k * I is beyond any doubt.

Measures of associations and estimates of relative risks are important because ultimately they may be helpful in revealing genetical and biochemical paths in disease ethiology. Quite often, however, observed associations and estimates of relative risks are of immediate practical use. For example among pigs of the Danish Landrace breed observed associations have been exploited before the genetic details were known. Thus, JENSEN et al. (1976) and

214 E . Andrrsen, P. Jenscn, P. Jonsson and H. Staun

BARTON et al. (1977) have observed statistical significant associations between genotypes of the H and Phi systems and meat quality, in particular presence or absence of PSE (pale, soft, exudative) meat. The two systems have therefore been used as genetic markers in selection for improved meat quality. N o w it is clear that the potential importance of the H and Phi loci in selection is caused by linkage disequilibrium with respect to the Hal locus. Thus, they serve as marker loci for the Hal locus and/or other loci of importance for meat quality and o ther productive traits. However, within the Danish Landrace breed the extra genetic gain is small by using these marker genes. This is partly due to the fact that the frequencies of the relevant gametes are far from being optimal, and partly due to high heritabilities of important components of meat quality (cf. ANDKESEN ENSEN EN 1978; ANDKESEN et al. I ~ ~ ~ ; ] E N S E N

and ANDRESEN I979a, b). T h e use of relative risk estimates as guides in prevention of disease requires special

precautions. If, for example, a disease determinant is a property of the host itself, in casu a specific genotype, then the success of a selection scheme depends on the frequency of the genotype and the strength of association rather than the relative risk. This is illustrated by the present results summatised in Table 2 . The relative risk for a pig being M H S susceptible is high (R = 18.8), if it possesses the genotype PhiHPhi”, whereas the strength ofassociation is relatively low ( r = 0.19). However, removal of this disease determinant would imply that

7 9 % (g. 100) of the breeding animals should be culled. Meanwhile, a considerable

reduction in M H S susceptibility would be obtained by culling 2 9 % (g 100) of the

pigs, i.e. those possessing the aggregate disease determinant [ H l , Phi” Phi”]. Although the halothane test is applied as an aid to selection within various breeds thetest

is deficient as it does not reveal heterozygosity among the tested individuals, and the testing procedure may be endowed with health hazards (COHEN et al. 1979; FOKSTER and BUTLER 1979). Thus, a knowledge of associations and relative risks in connection with disease determinants, in cusu genetic markers for halothane reactivity and thereby for production traits, is important when using such markers as aids to selection (cf. SMITH 1967).

Summary

A sample of 289 pigs of the Danish Landrace breed w a s subjected to blood typing (/f system), enzvine typ ing (Phi system), and halothane testing. A total of 35 pigs were halothane susceptible and, therefore, by dcfinition susceptible to the malignant hvperthermia syndrome (MHS). A positive association was observed between IMHS susceptibility and (I) presence of the blood group antigen H a (r = 0.25), (2) presence of the gcnotypc Phi” Phi” ( r = 0.19), and (3) presence of both H a and Phi” Phi” (r = 0.28). T h e corres onding estimates of relative risks were 2.9, 18.8, and 4.2, respectively. These I-isk valucs are c a u s e I b y linkage disequilibrium between :he Hal locus for halothatie reactivity .ind the H and Phi loci, T h e usefulness and limitations of relative risk estimates are discussed with regard to selection against M H S susceptibility And associated production traits.

Acknowledgements

Thanks are due to Mrs. I N G E R HVID.I.FEI.DT CHRISTENSEN, Dept. of Animal Genetics and Mr. E. BACH, National Insti tute of Animal Science fo r excellent technical assistance.

Resume

Syndrome d’hyperthermie marigne (SHM) des p O r C 3 : risque relati fdes g‘:~iofypes v a r k s

U n khan t i l l on d e 289 porcs de la Landrace danoise a et6 typisi. a u point d e vuc des groupcs sanguins (systkme H), enzymes ( s y s t h e P H I ) et test6 quant 1 la scnsibiliti d’halothane. U n total d e 35 porcs i taient sensibles a u halothane et de 11, par dtfinit ion, riceptifs pour le syndr6me d’hyperrherniie

Malignant hyperthermia syndrome (MHS) in pzgs 215

maliyne (SHM). Une association positive a etc observCe entre la receptivite S H M et ( I ) , prksence de I’antigkne H a du groupe sanguin, ( r = 0.25), (2), prcsence du gcnotype Phi” Phi” ( r = 0.19), et ( 3 ) , pri.sence, a u s s i bien de H,’, ainsi que de Phi” Phi’ ( r = 0.28).Les estimations evaluees du risque relatif ktaient 2.9, 18.8 et 1.2 . Ces valeurs de risque sont causees par un d6sequilibre de linkage entre le locus Hal pour la rbactivite d’halothmc et Ies loci H et Phi. L’utiliti. des estimations des risques relatifs est discut ie en vue de stlectionncr contre la receptiviti. S H M et Ics caractkres de production ass0cii.s.

Zusammenfassung

h/alignc~s Hyperthermie Syridrom (AIHS) in Schweinen: Relatives Risiko verschiedcrier Genotypen

Eine Stichprobe voti 289 Schweinen der dinischen Landrasse wurde hinsichtlich Blutgruppen ( H - System), Enzyme (PHI-System) typisiert und auf Halothanempfindlichkeit gepruft. 35 Schweine waren halothancnipfindlich u n d daher per definitionen enipfanglich fur das Maligne Hyperthermie Syndrom. Positive Assoziation konntc bcobachtet werden zwischen MHS-Empfinglichkeit iind 1 .): Anwesenheit des Blutgruppenantigens H a (r = 0.25), 2.): Vorhandensein des Genotyps Phi” Phi” ( r =

nheit, sowohl von H,’, \vie auch Phi” Phi” ( r = 0.28). Berechnete Schitzungen des relativen Kisikos waren 2.9, 18.8 u n d 4.2. Diese Kisikowerte s ind durch Kopplungsungleichgewiclit zwischen detn Hal-Lokus fiir t lalott ianreakti\~itat und den H und PHI-Loci verursacht. Die Verwend- barkeit der relati\.cn Kisikoschatzungen wird in bezug auf Selektion gegen MHS-Empfanglichkcit u n d damit verbundene Produktionsmerkniale diskutiert.

Resumen

Sitrdronie tie hiperternzia maligna ( S H h f ) en certios: riesgos r e l a t i w s tie ttiferentes genotrpos

Una muestra de 289 cerdos rle la Landrace danesa fue sometida a detcrniinacion del grupo sanguinco (sistema H ) , del grupo cnzinil t ico (sistema Phi) asi como al test de halothane. U n total de 35 cerdos result6 susceptible al halothane y, en consecuencia, susceptible al sindroine de hipertermia maligna (SHM) . Se observo u n a asociacion positiva entre la susceptibilidad al halothane y : I ) la presencia del antigeno H a dcl grupo sanguine0 (r = 0.25), 2 ) la presencia del genotipo Phi” Phi” ( r = 0.19), 3 ) la presencia tanto de Ha como de Phi” Phi” ( r = 0.28). Las cstimativas corrcspondientes de 10s riesgos relativos fueron de 2.9, 18.8 y 4.2 rcspectivamente. Estos valores de riesgo son causados por una concxion desequilibrada entre el locus Hal para reactividad de halothane y 10s loci H y Phi. L,a utilidad y las limitaciones de estimativas del riesgo relativo son discutidos teniendo en vista una selecci6n contra la susceptibilidad al S H M y ciertas caracteristicas de produccion asociadas con ella.

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Authors'addrrss: T h e Royal Veterinary and Agriculturc University, Department of Animal Genetics, Biilowsvej 13, DK-1870 Copenhagen V