Animal Blood Groups and Biochemical Genetics I4 (1983) 201-21 1 Population studies on the ELA system in American Standardbred and Thoroughbred mares Ernest Bailey Department of Veterinary Science University of Kentucky, Lexington, KY 40546- 0076, USA Received 22 February 1983; accepted 9 June 1983 Key-words: lymphocyte, antigen, ELA, histocompatibility, horse, equine lympho- cyte antigen Summary 336 Standardbred mares and 334 Thoroughbred mares in the vicinity of Lexington, Kentucky, were lymphocyte typed for 11 allelic antigenic specificities of the equine lymphocyte antigen (ELA) system. The Standardbred mares were divided into a population of pacers and a population of trotters. Substantial differences in ELA gene frequencies were found between the 3 groups. When the distribution of anti- gens within populations were compared to Hardy-Weinberg equilibrium expecta- tions, relatively good agreement was found. Introduction All vertebrate species appear to possess a highly polymorphic system of lymphocyte antigens. In mice and human beings the genes for these antigens are involved in im- mune regulation and are closely linked to genes expressing complement compo- nents and immune responsiveness. Therefore, research on the lymphocyte antigen systems in domestic animals may provide discoveries which influence the health, performance and productivity of these animals. The equine lymphocyte antigen (ELA) system was recognized by virtue of re- search conducted in various laboratories (Lazary et al. , 1980; Bailey, 1980a; Motti- roni et al., 1981; Antczak et al., 1982) and subsequent collaborative workshops in- volving 12 laboratories, worldwide, active in ELA research (Bull, 1983; Anony- mous, 1983). At the end of the second ELA workshop, 10 specificities of the ELA system were given the workshop nomenclature W1, W2, W3, W4, W5, W6, W7, W8, W9 and W10 (Bull, 1983; Anonymous, 1983). This is a conservative measure of the varia- tion in the ELA system because individual laboratories recognize still more varia- 201
Transcript
Animal Blood Groups and Biochemical Genetics I4 (1983) 201-21 1
Population studies on the ELA system in American Standardbred and Thoroughbred mares
Ernest Bailey
Department of Veterinary Science University of Kentucky, Lexington, KY 40546- 0076, USA
336 Standardbred mares and 334 Thoroughbred mares in the vicinity of Lexington, Kentucky, were lymphocyte typed for 11 allelic antigenic specificities of the equine lymphocyte antigen (ELA) system. The Standardbred mares were divided into a population of pacers and a population of trotters. Substantial differences in ELA gene frequencies were found between the 3 groups. When the distribution of anti- gens within populations were compared to Hardy-Weinberg equilibrium expecta- tions, relatively good agreement was found.
Introduction
All vertebrate species appear to possess a highly polymorphic system of lymphocyte antigens. In mice and human beings the genes for these antigens are involved in im- mune regulation and are closely linked to genes expressing complement compo- nents and immune responsiveness. Therefore, research on the lymphocyte antigen systems in domestic animals may provide discoveries which influence the health, performance and productivity of these animals.
The equine lymphocyte antigen (ELA) system was recognized by virtue of re- search conducted in various laboratories (Lazary et al. , 1980; Bailey, 1980a; Motti- roni et al., 1981; Antczak et al., 1982) and subsequent collaborative workshops in- volving 12 laboratories, worldwide, active in ELA research (Bull, 1983; Anony- mous, 1983).
At the end of the second ELA workshop, 10 specificities of the ELA system were given the workshop nomenclature W1, W2, W3, W4, W5, W6, W7, W8, W9 and W10 (Bull, 1983; Anonymous, 1983). This is a conservative measure of the varia- tion in the ELA system because individual laboratories recognize still more varia-
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tion (Lazary et al., 1980; Bailey, 1980a; Mottironi et al., 1981; Antczak et al., 1982). However, until two or more laboratories recognize those antigens during a workshop, workshop nomenclature is withheld.
In this report, the distribution of ELA antigens in Standardbred and Thorough- bred mares in Lexington, Kentucky is presented and discussed.
Materials and methods
Standardbred mares (n = 336) and Thoroughbred mares (n = 334) were lympho- cyte-typed and used as the base populations for estimation of gene frequencies. The 336 Standardbred mares were all broodmares residing at two local Standardbred horse farms. The 334 Thoroughbred mares were all brood-mares residing at five lo- cal Thoroughbred farms. In addition, 600 sets of sire-dam-offspring were lympho- cyte typed in order to study the segregation pattern of the genes controlling the lym- phocyte specificities. Many, but not all, of the dams tested for the families were also included in the base population.
Blood samples for lymphocyte isolation were collected in commercial evacuated tubes containing acid-citrate-dextrose anticoagulant. Lymphocytes were isolated by the thrombin technique of Terasaki & Park (1976). Lymphocytes were tested with reagents developed as described previously (Bailey, 1980a). Most reagents were unabsorbed sera from pregnant, unimmunized mares. Some sera were ab- sorbed with horse spleen cells to produce functionally monospecific reagents. Cal- culations of correlation coefficients were made with a computer program kindly provided by Professor Ray Mickey of the University of California at Los Angeles. In calculating the correlation coefficients, intermediate reactions (25 to 49 % cells killed) were deleted from analysis.
Antigen assignment Several reagents were used to type for each antigen. The agreement between anti- gen assigment and reagent reaction pattern is presented in Table 1. The informa- tion is presented as a correlation coefficient between antigen assignment and each of 2 reagents used. These values were calculated using one set of test plates used to type 98 Standardbred horses and 79 Thoroughbred horses.
Optimally, three high-quality reagents were used for detecting each specificity. A high-quality reagent was one that killed more than 80 % of the cells bearing the corresponding antigen but none of the cells when the antigen was absent. In the ear- ly part of the testing program, only 2 reagents wre available for the specificities W7 and W8. However, the quality of these reagents was high and antigen assignment could be made confidently. For some specificities, such as W5 and W10, reagents were often of lower quality and as many as six reagents were used. Three reagents used in this study and reported here were not produced in this laboratory. Bern 106 and Bern 23 were reagents kindly provided by Dr Sandor Lazary of Bern, Switzer-
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1 In calculating the correlation coefficients, intermediate reactions (score = 4) were deleted from analy- sis.
land; Dav 220 was a reagent kindly provided by Professor Domenico Bernoco of Davis, California. These reagents, plus others, were used to assign the antigens present in each horse's lymphocytes (Table 1). Agreement of antigen assigment with workshop nomenclature was assured by the use, in this test, of at least one re- agent used to identify each antigen during the international workshops on equine lymphocyte antigens.
Estimation of gene frequencies, calculations of Hardy-Weinberg equilibrium dis- tributions and tests for goodness of fit were performed as described by Mattiuz et al. (1970).
Results
Segregation data Segregation data previously have been published demonstrating that W1, W2, W3,
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Table 2. Segregation of W7, W8, W9, W10 and Lex 8 with ELA system markers in Standardbred and Thoroughbred horse families.
a1
w 7 w7 w 7 w7 W8 W8 W8 W8 W8 w 9 . w9 w 9 w9 w9 w10 w10 w10 w10 w10 Lex 8 Lex 8
b‘
w1 w5 W6 w10 w1 w 4 w5 W6 w 1 0 w2 w3 w 5 W6 w10 w 1 w3 w4 w5 W6 W5 w10
Lex 8 W3
Number of parental phenotypes studies
1 1 1 3 3 1 4 3 3 1 1 4 1 2
24 4 12 21 14 7 2 4
Distribution of ‘a’ and ‘b’ in offspring
ab
0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0 0 0 0
a-
21 1 0 2
3 8
28 2 2
0 2 4 0 1
27 2 4 12 6 3 0 2
b-
82 0 0 0 1 0 1 0 0 0 9 0
162 0 1 0 1 0
1 0 3 0 2 0 1 0 1 0
152 0 2 0 8 0 9 0 8 0 4 0 2 0 2 0
1 ‘a’ and ‘b’ represent two antigens present in one parent, neither of which is present in the other parent. The mating type can also be represented as a/b X-1- 2 In these classes, the segregation of alleles deviates from the expected ratio of 1:l (See text for dis- cussion).
W4, W5 and W6 segregated as the product of codominant alleles (Lazary et al., 1980; Bailey, 1980a; Antczak et al., 1982). The results found during this study are consistent with those earlier reports and are not repeated here. The data in Table 2 demonstrate that W7, W8, W9, W10 and Lex 8 also segregated like allelic products of the ELA system. Horses which possessed two of these ELA specificities always transmitted the gene for one or the other but not both of them to their offspring. In the course of this study, no horse was found to possess more than 2 of these 11 spe- cificities.
During the family studies it appeared that three stallions were not passing the genes for each of their ELA antigens to their offspring equally. These deviations led to the distortion noted in Table 2. The cause of these deviations are not known and are being investigated further to determine whether these are chance occur- rences or whether they have an underlying biological cause.
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Table 3. Estimated’ gene frequencies for ELA markers in Thoroughbred and Standardbred mares.
1 Gene frequencies estimated from phenotypic frequencies (pf) as 1-( l-pf)h. * W 9 was found in a Standardbred dam and her offspring subsequent to compiling this data. 3 All mares possessed at least one ELA antigen. Therefore, it was nor possible to estimate the gene fre- quency of a ‘blank’ allele. (See text for discussion).
Gene frequencies Gene frequency data are presented in Table 3. The 336 Standardbred mares were divided into 103 trotters and 233 pacers and treated here as separate breeding pop- ulations. (Trotting and pacing are considered highly heritable traits by Standard- bred breeders. Therefore, the usual practice is to breed pacing mares to pacing stal- lions and trotting mares to trotting stallions. Mares were divided into trotters and pacers depending on whether their sire produced predominantly trotting or pacing offspring.)
Highly significant gene frequency differences were found when comparing the two Standardbred populations with the Thoroughbred population (Table 3). W1 and W4 were entirely absent from Thoroughbred mares but were present in Stand- ardbred mares. W9 and W2 were frequent in Thoroughbred mares but rare in both populations of Standardbred mares.
Although variation between the two populations of Standardbred mares was con- siderable, as might be expected of separate breeding populations, they were not as pronounced as when comparing either of these two breeds to Thoroughbreds.
The sum of gene frequencies for ELA antigens is presented at the bottom of Ta- ble 3. For each population the sum is less than 1.00. Although no horse was found which did not possess at least one of these specificities, segregation studies indicate that a ‘blank’ allele exists, i.e., an allele for which a specificity is not yet detected. This conclusion is based on one stallion and three mares, each possessing a single specificity; half of their offspring inherit the gene for that specificity, while the other
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half do not (data not presented). Therefore, the ELA system does not appear to be a closed system for Thoroughbred and Standardbred horses.
Fit to Hardy- Weinberg equilibrium expectation With the estimated gene frequencies from Table 2, the expected distribution of ELA antigens was determined for each group of horses. (The frequency of the blank allele was considered to be 1.00 minus the sum of frequencies for all specifici- ties, i.e., 0.025 for Thoroughbreds, 0.020 for pacers, and 0.029 for trotters.) Then the expected distribution was compared to the actual distribution as described by Mattiuz et al. (1970). These data are presented in Tables 4 , 5 and 6 for Thorough- bred, pacing Standardbred and trotting Standardbred horses, respectively.
The sum of x* values indicated a good fit to Hardy-Weinberg equilibrium expec- tations for trotting and pacing Standardbred mares. The scores were 3.38 and 6.17,
Table 4. Distribution of ELA markers in 334 Thoroughbred mares and its goodness of fit to Hardy- Weinberg equilibrium expectation.
ELA Number Number 2 phenotype observed expected
w2 w2. w3 w2, w5 w2, w9 W2, Lex 8 w2, X' w3 w3, ws w3, w9 W3, Lex 8 w3, x w5 w5, w9 W5, Lex 8 w5, x w9 w9, Lex 8 w9. x Lex 8 Lex 8. X X or none
I Data for classes with W7, W8, W6 and W10 were combined due to the low number of horses repre- sented in each class; X denotes the presence of one of those antigens. 2 = 13.50 for 15 degrees of freedom; 0.60 > P > 0.50.
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Table 5. Distribution of ELA markers in 233 pacing Standardbred mares and its goodness of fit to Hardy-Weinberg equilibrium expectations.
E LA phenotype
W1 w1. w 4 "1, W5 w1 , w10 w1, X' w 4 w4. w 5 w4. w10 w4. x w 5 w5 , w10 w5 , x w10 w10. x X or none
Totals
Number observed
15 11 16 34 13
3 8
11 10
4 20 13
31 32
12
233
Number expected
13.3 10.3 14.4 32.8 20.5
3.3 6.5
11.9 9.3
5.9 20.9 13.1
26.8 29.8
11.2
233
X2
0.21 0.05 0.19 0.04 2.74
0.21 0.34 1.03 0.05
0.60 0.04
0.64 0.16
0.06
6.172
I Data for classes with W2, "3. W6. W7 and W8 were combined due to the low number of horses repre- sented in each class: X denotes the presence of one of these antigens. 2 x2 = 6.17 with LO degrees of freedom; 0.90 > P > 0.80.
respectively. When the data for the two populations were pooled and the fit to Hardy-Weinberg equilibrium distribution measured, the x 2 value was 16.68 with 13 degrees of freedom (calculations not shown). This indicates that dividing the Stand- ardbred mares into pacers and trotters was justified and effective.
The sum of x2 values for Thoroughbred mares was not as good, being 13.5. How- ever, it is comparable for the results reported in human populations by Mattiuz et al. (1970). The largest discrepancies were an excess of horses with W3 and Lex 8 as well as a deficiency of mares with W2 and W3.
Discussion
The expected distribution of alleles according to Hardy-Weinberg equilibrium is based on a randomly breeding population in the absence of selection. Therefore, it is pertinent to the discussion to consider the structure of Thoroughbred and Stand- ardbred horse populations. Few stallions are retained as breeding stock while vir- tually all mares are used as broodmares in these two breeds. Matings are planned by breeders who are selecting for racing performance. Intuitively, one could expect the distribution of genetic markers in horse populations to deviate substantially
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Table 6. Distribution of ELA markers in 103 trotting Standardbred mares and its goodness of fit to Hardy-Weinberg equilibrium expectation.
~
ELA Number Number 2 phenotype observed expected
w1 W1, W6 w1, w10 w1, X' W6 W6, W10 W6, X w10 w10, x
11 14 22 12 5 6 6 8
12
14.0 11.9 18.9 13.3 4.0 9.5 6.6 9.2
10.6
0.64 0.37 0.51 0:13 0.23 0.29 0.06 0.16 0.19
X or none 7 5.0 0.80
Totals 103 103 3.382
* Data for classes with W3, W4, W5 and W8 were combined due to the low number of horses represented in each class; X denotes the presence of one of those antigens. 2 2 = 3.38 with 6 degrees of freedom; 0.80 > P > 0.70.
from Hardy-Weinberg equilibrium expectations. However, this test is not consid- ered to be very sensitive for detecting the presence of such forces as selection and inbreeding (Cavalli-Sforza & Bodmer, 1971, p. 58). The fit of data from this study to Hardy-Weinberg equilibrium expectation would appear to support this conclu- sion.
However, the exercise of making these calculations is useful for several pur- poses. First, the selection pressures exerted on these three horse populations have not resulted in large deviations form the expected distribution of specificities. The impact of ELA genes on racing performance, if any, may not be large.
Second, the f values indicate how well the gene frequencies have been esti- mated. When Bernstein's equation is used to estimate gene frequencies, it is as- sumed that the populations fit Hardy-Weinberg equilibrium expectations perfectly. The higher the 2 value, the greater the departure of the estimate from the true val- ue. Clearly, the gene frequency estimates in the two Standardbred populations are better than the estimates in the Thoroughbred population.
Finally, these calculations demonstrate that the trotting and pacing Standard- bred mares represent two different populations. When the data were pooled for all Standardbred mares, and the fit to Hardy-Weinberg equilibrium distribution meas- ured, the 2 value was 16.68. After dividing them into the two populations, the 2 values were 3.38 and 6.17. The reduction of the x* values indicates that our division based on gait was effective and justified in defining the two populations.
Comparable lymphocyte systems in human beings, Rhesus monkeys, chickens,
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Table 7. Comparison of estimated gene frequencies for ELA antigen in Thoroughbred horses reported in Lexington, KY (Lex)'; Davis, California (Dav)Z; Ithaca, New York (Ith)3; and in the First and Second ELA Workshop meetings (WKl)4, (WK2)5.
Antigen designation Lex Ith Dav
w 1 w2 w 3 w 4 w5 W6 w7 W8 w9 w10 Lex 8
Ith08 E l2 CaOl E5 Ith 05 Ith 07 Ca03 E9 C a M E l Ith 011
0.00 0.00 0.16 0.16 0.15 0.18 0.00 0.00 0.17 0.18 0.02 0.01 NR 0.01 NR 0.01 NR 0.19 NR 0.03 NR NR
I This report; 2Bailey (1980b); 'Antctak et al. (1982); 'Bull (1983); 5Anonymous (1983); 6NR = not re- ported. 7 Antigens designated by Lex 8 and E3 are probably homologous, however, this has not been definitely established.
mice, swine, dogs and some other species include several closely linked loci for lym- phocyte antigens. There has been some indication for multiple linked loci in horses (Lazary et al., 1980; Bailey, 1980a). However, conclusive evidence has not yet been presented.
The difficulty of finding multiple loci may be due to inbreeding and short breed histories resulting in linkage disequilibrium. Linkage disequilibrium has been re- ported between the ELA system and the closely !inked A blood group system (Bai- ley, 1983). If two different antigens usually occur together and segregate together, then it will be difficult to distinguish between them without very well defined re- agents and genetic recombination. If the antigens in linkage disequilibrium are im- portant only as markers of a haplotype, then the importance of distinguishing be- tween them is not very great.
In previous reports on Standardbred horses, the data for trotters and pacers has been pooled. Without knowing the proportion of trotters and pacers in those stud- ies, it is not possible to compare this data with the results of other studies. However, we can compare our data for Thoroughbred horses with the data of others. In Table 6 , the estimated gene frequencies of ELA-W1 to W10 are compared with the data of Bailey (1980a), Antczak et al. (1982) and the First and Second Workshops on Equine Lymphocyte Antigens (Bull, 1983; Anonymous, 1983.) Note that 41 of the Thoroughbred horses typed for the first workshop were typed in Lexington and are represented in the data of this report as well.
There is relatively good agreement between the reports. The largest discrepancy occurs with the distribution of W5. The estimated gene frequency reported by
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Antczak et al. (1982) is less than half that reported here. (The data reported by Bai- ley (1980b) are comparable to that of this study. The 1980 and 1983 data refer to dif- ferent reagents and different horses, from a different laboratory.) The workshop values, both years, have been intermediate between the results of this report and Antczak et al. (1982). It may be that there are regional differences in the frequency of W5. Why this difference is so great for W5 but not for other antigens is unclear. Another possibility for the discrepancy between the Ithaca laboratory and this study may be differences in antigen assignment for W5. However, limited cell ex- changes and reagent exchanges between our laboratory and the Ithaca laboratory indicated agreement on antigen assignment for W5 (unpublished data). Therefore, the differences may be due to the population of horses sampled in each location.
The impetus for such studies comes from the observations in human beings that certain diseases are associated more frequently with certain HLA antigens (Daus- set & Svejgaard, 1977). Lazary et al. (1982) reported associations for an equine lymphocyte specificity and several diseases in the horse. (This particular specificity was not a product of the ELA system and was designated Ely-1.1.)
The information in this report has important ramifications for studies of diseases associated with ELA antigens. The wide variation between Thoroughbred and Standardbred horse breeds indicates that keeping separate data for these two breeds is important. Failure to do so might result in spurious associations. For ex- ample, the disease ‘equine ataxia’, commonly referred to as ‘wobbles’, occurs pre- dominantly in Thoroughbred horses and less frequently in Standardbred horses. A genetic basis is suspected for the disease (Dimock, 1950). If association of wobbles with ELA antigens were investigated in a pool of Thoroughbred and Standardbred horses, it is certain that W2 and W9 would be positively correlated with the disease, while W1 and W4 would be negatively associated. Irrespective of any actual rela- tion for ELA antigens and wobbles, Thoroughbred horses are more likely to have wobbles, W2 and W9 than Standardbred horses.
If, on the other hand, disease associations are investigated within a breed, posi- tive correlations could be very significant. The distribution of the ELA antigens re- ported in this study is close to that based on chance and random expectation. Con- sequently, deviations will be significant.
Acknowledgements
The author is grateful to Ms. Pamela Henney for excellent technical assistance, to local horsemen for providing blood samples for this research, and to Professors Clyde Stormont and Domenico Bernoco for critically reading the manuscript and making many helpful suggestions.
This work is in connection with a project of the Kentucky Agricultural Experi- ment Station and is published as paper No 83-4-23 with the approval of the Director of the Station.
210 Animal Blood Groups and Biochemicnl Genetics 14 (1983)
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