J. Anim. Breed. Genet. ISSN 0931-2668
ORIGINAL ARTICLE
BSE-associated polymorphisms in the prion protein gene: an investigation K. Vernerova1, L. Tothova1, A. Mikova1, P. Vodrazka2, B. Simek2, L. Hanusova1 & J. Citek1 1 Faculty of Agriculture, University of South Bohemia, Ceske Budejovice, Czech Republic 2 State Veterinary Institute Jihlava, Jihlava, Czech Republic
Summary
Keywords Cattle; BSE; prion gene; 23 indel; 12 indel. Correspondence J. Citek, Faculty of Agriculture, University of South Bohemia, CZ37005 Ceske Budejovice, Czech Republic. Tel: +420387772591; Fax: +420387772593; E-mail: [email protected] Received: 21 August 2013; accepted: 14 March 2014
The aim of this study was to determine the frequency of the 12-bp and 23-bp indel polymorphisms in the prion protein gene (PRNP) in cattle and to investigate the association between these frequencies and the occurrence of bovine spongiform encephalopathy (BSE). There was no significant difference in the 12-bp indel frequency between the BSE animals and control group. For the 23-bp indel, the BSE animals had a significantly lower + + (insins) genotype frequency and + allele frequency compared with the control animals. The / genotype frequency in the BSE animals was not significantly higher when compared with the control animals. One allele increased the risk of BSE by a factor of 1.55 (i.e. by 55%) for the 12-bp indel and by a factor of 2.10 for the 23-bp indel. When both indels are considered, one allele increased the risk of BSE by a factor of 1.54.
Introduction Over the past two or three decades, bovine spongiform encephalopathy (BSE) has been a cause of disquiet to the public and to the veterinary and biological research communities. At first, slow viruses were thought to be the cause of spongiform encephalopathies (Hadlow 1959; Gajdusek et al. 1966), but more recent findings have shown that prions are the cause. Prions are characterized as unprecedented infectious pathogens that cause several fatal neurodegenerative diseases through an entirely novel mechanism. BSE, scrapie in sheep, and Creutzfeldt-Jakob disease (CJD) in humans are some of the most notable prion diseases (Prusiner 1998), and these diseases are able to circumvent the species barrier (e.g. Casalone et al. 2004). This hypothesis was widely accepted even though it has been convincingly disputed by some scientists (Scholz 2002). More recently, the genetic aspects of these diseases have come into focus. The genomes of the affected species have been analysed, and particular attention © 2014 Blackwell Verlag GmbH
• J. Anim. Breed. Genet. 131 (2014) 403–408
has focused on the prion protein gene (PRNP). Many polymorphic sites have been identified, and their association with BSE symptoms has been investigated. Hills et al. (2001) published the full genomic sequence of the bovine PRNP and reported that there is an insertion/deletion polymorphism (indel) within the promoter region. Clawson et al. (2006) reported 388 polymorphisms within a 25.2 kb genomic region that contains PRNP and identified 19 haplotypes. Murdoch et al. (2010a) investigated whether there is an association between the PRNP haplotypes and BSE using a set of haplotype-tagging single-nucleotide polymorphisms (htSNPs) and two indels. Using this technique, they discovered that there is an association between the PRNP haplotypes and the occurrence of classic BSE. In another study, Murdoch et al. (2010b) identified 27 SNPs in 18 different chromosomes that are associated with the occurrence of BSE. Particular attention has focused on the 23-bp indel polymorphism in the PRNP promoter region, the 12bp polymorphism in intron 1 and the removal of the putative SP1 binding site (Hills et al. 2001; Sander doi:10.1111/jbg.12090
Prion protein gene polymorphisms and BSE
et al. 2004). It has been shown that there is a strong correlation between both of these indel polymorphisms and the occurrence of BSE (Sander et al. 2005; Juling et al. 2006). Msalya et al. (2010) suggested that changes in PRNP expression are linked to these indel polymorphisms and may result in different BSE incubation periods and BSE susceptibility. Another mutation, E211K, has also been reported (Clawson et al. 2008; Heaton et al. 2008; Nicholson et al. 2008). In this study, the allele and genotype frequencies of the 12- and 23-bp insertion/deletion polymorphisms in cattle, including BSE-positive animals, in the Czech Republic were determined. Materials and methods The 23-bp indel polymorphism in the bovine PRNP promoter region and the 12-bp indel polymorphism in intron 1 were analysed in Czech Simmental (n = 25) and Holstein sires (n = 30). Both of these breeds are part of the artificial insemination (AI) programme in the Czech Republic. Czech Red cows and sires (n = 26), an original Czech breed, were also used in this study. BSE-positive cattle (n = 26) were also analysed. The BSE status was determined by the State Veterinary Institute Jihlava using immunohistochemical, histopathological, Western blot and ELISA methods. Genomic DNA was isolated from the sperm, whole blood and muscle tissue using either a commercial kit or a standard chloroform extraction laboratory method. DNA was also isolated from the brains of BSE-positive animals. PCR was performed using the method described in Jeong et al. (2006). The 23-bp indel forward primer sequence was 50 -GTGCCAGCCATGTAAGTG-30 , and the 23-bp indel reverse primer sequence was 50 -TGGACAGGCACAATGGG-30 . The sequences for the 12-bp indel primers were forward 50 -TTACCCTCCTGGTTAGGAG-30 and reverse 50 CTAGATTCCTACACACCAC-30 . The annealing temperature, cycle specifications and reaction mixture were optimized for our laboratory environment. The PCR products were separated on a 3% agarose gel and visualized using ethidium bromide staining and UV light. One of the BSE-positive animals developed clinical symptoms of the disease. The other BSE-positive animals were diagnosed after slaughter and had not shown symptoms of encephalopathy. The analyses in this study were conducted in accordance with the contemporary animal welfare law of the Czech Republic. Neither humans nor animals were injured during this study. 404
K. Vernerova et al.
The frequencies of the alleles, genotypes and diplotypes were calculated. Hardy–Weinberg equilibrium (HWE) was determined using the chi-squared test, and the differences in the frequencies among the breeds were determined using the test of difference in relative frequencies. The effects of an allele on BSE were investigated using logistic regression. This method is useful when the observed outcome is restricted to two values, usually the occurrence or non-occurrence of an outcome event. It predicts the probability of the occurrence as a function of the independent variables. Logistic regression also produces odds ratios (O.R.). The odds of an event occurring is calculated as the probability of the outcome event occurring divided by the probability of the event not occurring. The odds ratio gives the relative amount by which the odds of the outcome increases (O.R. > 1.0) or decreases (O.R. < 1.0) when the value of the predictor value is increased by 1.0 units. Results In total, 81 BSE-negative and 26 BSE-positive animals were analysed for the 12-bp and 23-bp indel regions. The genotype and allele frequencies were compared among the Czech Simmental, Holstein and Czech Red control groups and the BSE-positive animals (Tables 1 and 2). The frequency of the 12-bp indel was largely similar in the various groups. Significant differences, however, were found between the BSE-positive animals and the Holsteins, Simmentals and Reds. Interestingly, there was no significant difference between the all-inclusive breed BSE-positive group and allinclusive breed control group. All of the groups were in Hardy–Weinberg equilibrium except for the Czech Simmentals, and random factors may be responsible for this result. For the 23-bp indel, the BSE-positive group and allinclusive breed control group differed in the insins (+ +) genotype frequency and + allele frequency. These frequencies were significantly lower in the BSE-positive animals compared with the control animals (Table 2). This finding suggests that these two indels may be of different importance. For the 23-bp indel, all of the groups were in Hardy–Weinberg equilibrium. Table 3 (frequencies of the diplotypes) shows that the frequency of the double del genotype ( / ) was not significantly higher in the BSE-positive group compared with the all-inclusive breed control group. The number of animals analysed in this study, however, is too low to draw a definitive conclusion. © 2014 Blackwell Verlag GmbH
• J. Anim. Breed. Genet. 131 (2014) 403–408
Prion protein gene polymorphisms and BSE
K. Vernerova et al.
Table 1 Genotype and allele frequencies of insertion/deletion 12 Genotypes Breed
Frequency
++
+
HWE
Allele +
Czech Simmental
Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected
9 0.360 0.212 11 0.367 0.401 9 0.346 0.403 29 0.358 0.336 6 0.231 0.213
5 0.200ACb 0.497 16 0.533A 0.465 15 0.577C 0.464 36 0.444 0.487 12 0.462b 0.497
11 0.440BD 0.292 3 0.100Ba 0.135 2 0.077Dc 0.133 16 0.198 0.176 8 0.308ac 0.289
35.581**
0.460
2.189ns
0.633
5.898ns
0.635
0.799ns
0.580
0.523ns
0.462
Holstein
Czech Red
Control group total
BSE positive
+, insertion; , deletion; BSE, bovine spongiform encephalopathy. HWE, Hardy–Weinberg equilibrium evaluated by chi-squared test. ns non significant; **significant at p < 0.01; A values in one column with the same letter differ at p < 0.01 (capital letter), or at p < 0.05 (small letter). Table 2 Genotype and allele frequencies of insertion/deletion 23 Genotypes Breed Czech Simmental
Holstein
Czech red
Control group total
BSE positive
Frequency Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected
++ 4 0.160 0.176 9 0.300b 0.303 6 0.231 0.289 19 0.235a 0.256 2 0.077ab 0.120
+ 13 0.520 0.487 15 0.500 0.495 16 0.615 0.497 44 0.543 0.500 14 0.538 0.453
8 0.320 0.336 6 0.200 0.203 4 0.154 0.213 18 0.222 0.244 10 0.385 0.428
Allele +
HWE 0.445
ns
0.420
0.012ns
0.550b
5.600ns
0.538c
0.740ns
0.506a
3.568ns
0.346abc
+, insertion; , deletion; BSE, bovine spongiform encephalopathy. HWE, Hardy–Weinberg equilibrium evaluated by chi-squared test. ns non significant. a values in one column with the same letter differ at p < 0.05.
The results from the logistic regression analysis are shown in Table 4. In this analysis, the exposure variable allele was considered continuous, and the risk of BSE with an increasing number of alleles was determined. This approach assumed that the relationship was linear on a logit scale. For the 12-bp indel, the risk of BSE increased by a factor of 1.55 (i.e. by © 2014 Blackwell Verlag GmbH
• J. Anim. Breed. Genet. 131 (2014) 403–408
55%) for every one allele increase. For the 23-bp indel, the odds ratio was as high as 2.10. When both indels are taken into account, the risk of BSE increased by a factor of 1.54 for every one allele increase. For the 12-bp indel analysis, however, the confidence interval included 1, which indicates that the reliability of this result is lower. In the other 405
Prion protein gene polymorphisms and BSE
K. Vernerova et al.
Table 3 Frequencies of 12/23 diplotypes Diplotypes Breed
Frequency
++/++
++/+
++/
+/++
+/+
+/
/++
/+
/
Czech Simmental
Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected
2 0.080 0.037 7 0.233 0.122 5 0.192 0.116 14 0.173 0.086 2 0.077 0.026
5 0.200 0.130 3 0.100 0.198 4 0.154 0.200 12 0.148 0.168 2 0.077 0.097
2 0.080 0.071 1 0.033 0.081 0 0.000 0.086 3 0.037 0.082 2 0.077 0.091
0 0.000 0.087 1 0.033 0.141 0 0.000 0.134 1 0.012 0.125 0 0 0.060
4 0.160 0.242 11 0.367 0.230 12 0.462 0.231 27 0.333 0.244 9 0.346 0.225
1 0.040 0.167 4 0.133 0.094 3 0.115 0.099 8 0.099 0.119 3 0.115 0.213
2 0.080 0.051 1 0.033 0.041 1 0.038 0.038 4 0.049 0.045 0 0 0.035
4 0.160 0.142 1 0.033 0.067 0 0.000 0.066 5 0.062 0.088 3 0.115 0.131
5 0.200 0.098 1 0.033 0.027 1 0.038 0.028 7 0.086 0.043 5 0.192 0.124
Holstein
Czech Red
Control group total
BSE positive
+, insertion; , deletion; BSE, bovine spongiform encephalopathy. All groups were not in Hardy–Weinberg equilibrium, but with regard to the low number of diplotypes, the results are not shown. The differences between control group total and BSE positive were tested; the significance was not found.
Table 4 Parameter estimates and odds ratios of a logistic regression model for the effect of allele on the bovine spongiform encephalopathy status Effect of allele del
Intercept
Coefficient estimate
Standard error
p
Odds ratio
95% confidence interval
indel 12 indel 23 indel 12 and 23
1.56 1.99 2.04
0.44 0.74 0.43
0.31 0.36 0.21
0.156 0.039 0.038
1.55 2.10 1.54
0.85; 2.85 1.04; 4.23 1.02; 2.31
analyses, the confidence interval did not include 1, and the results are considered dependable. Because the sample size is
ORIGINAL ARTICLE
BSE-associated polymorphisms in the prion protein gene: an investigation K. Vernerova1, L. Tothova1, A. Mikova1, P. Vodrazka2, B. Simek2, L. Hanusova1 & J. Citek1 1 Faculty of Agriculture, University of South Bohemia, Ceske Budejovice, Czech Republic 2 State Veterinary Institute Jihlava, Jihlava, Czech Republic
Summary
Keywords Cattle; BSE; prion gene; 23 indel; 12 indel. Correspondence J. Citek, Faculty of Agriculture, University of South Bohemia, CZ37005 Ceske Budejovice, Czech Republic. Tel: +420387772591; Fax: +420387772593; E-mail: [email protected] Received: 21 August 2013; accepted: 14 March 2014
The aim of this study was to determine the frequency of the 12-bp and 23-bp indel polymorphisms in the prion protein gene (PRNP) in cattle and to investigate the association between these frequencies and the occurrence of bovine spongiform encephalopathy (BSE). There was no significant difference in the 12-bp indel frequency between the BSE animals and control group. For the 23-bp indel, the BSE animals had a significantly lower + + (insins) genotype frequency and + allele frequency compared with the control animals. The / genotype frequency in the BSE animals was not significantly higher when compared with the control animals. One allele increased the risk of BSE by a factor of 1.55 (i.e. by 55%) for the 12-bp indel and by a factor of 2.10 for the 23-bp indel. When both indels are considered, one allele increased the risk of BSE by a factor of 1.54.
Introduction Over the past two or three decades, bovine spongiform encephalopathy (BSE) has been a cause of disquiet to the public and to the veterinary and biological research communities. At first, slow viruses were thought to be the cause of spongiform encephalopathies (Hadlow 1959; Gajdusek et al. 1966), but more recent findings have shown that prions are the cause. Prions are characterized as unprecedented infectious pathogens that cause several fatal neurodegenerative diseases through an entirely novel mechanism. BSE, scrapie in sheep, and Creutzfeldt-Jakob disease (CJD) in humans are some of the most notable prion diseases (Prusiner 1998), and these diseases are able to circumvent the species barrier (e.g. Casalone et al. 2004). This hypothesis was widely accepted even though it has been convincingly disputed by some scientists (Scholz 2002). More recently, the genetic aspects of these diseases have come into focus. The genomes of the affected species have been analysed, and particular attention © 2014 Blackwell Verlag GmbH
• J. Anim. Breed. Genet. 131 (2014) 403–408
has focused on the prion protein gene (PRNP). Many polymorphic sites have been identified, and their association with BSE symptoms has been investigated. Hills et al. (2001) published the full genomic sequence of the bovine PRNP and reported that there is an insertion/deletion polymorphism (indel) within the promoter region. Clawson et al. (2006) reported 388 polymorphisms within a 25.2 kb genomic region that contains PRNP and identified 19 haplotypes. Murdoch et al. (2010a) investigated whether there is an association between the PRNP haplotypes and BSE using a set of haplotype-tagging single-nucleotide polymorphisms (htSNPs) and two indels. Using this technique, they discovered that there is an association between the PRNP haplotypes and the occurrence of classic BSE. In another study, Murdoch et al. (2010b) identified 27 SNPs in 18 different chromosomes that are associated with the occurrence of BSE. Particular attention has focused on the 23-bp indel polymorphism in the PRNP promoter region, the 12bp polymorphism in intron 1 and the removal of the putative SP1 binding site (Hills et al. 2001; Sander doi:10.1111/jbg.12090
Prion protein gene polymorphisms and BSE
et al. 2004). It has been shown that there is a strong correlation between both of these indel polymorphisms and the occurrence of BSE (Sander et al. 2005; Juling et al. 2006). Msalya et al. (2010) suggested that changes in PRNP expression are linked to these indel polymorphisms and may result in different BSE incubation periods and BSE susceptibility. Another mutation, E211K, has also been reported (Clawson et al. 2008; Heaton et al. 2008; Nicholson et al. 2008). In this study, the allele and genotype frequencies of the 12- and 23-bp insertion/deletion polymorphisms in cattle, including BSE-positive animals, in the Czech Republic were determined. Materials and methods The 23-bp indel polymorphism in the bovine PRNP promoter region and the 12-bp indel polymorphism in intron 1 were analysed in Czech Simmental (n = 25) and Holstein sires (n = 30). Both of these breeds are part of the artificial insemination (AI) programme in the Czech Republic. Czech Red cows and sires (n = 26), an original Czech breed, were also used in this study. BSE-positive cattle (n = 26) were also analysed. The BSE status was determined by the State Veterinary Institute Jihlava using immunohistochemical, histopathological, Western blot and ELISA methods. Genomic DNA was isolated from the sperm, whole blood and muscle tissue using either a commercial kit or a standard chloroform extraction laboratory method. DNA was also isolated from the brains of BSE-positive animals. PCR was performed using the method described in Jeong et al. (2006). The 23-bp indel forward primer sequence was 50 -GTGCCAGCCATGTAAGTG-30 , and the 23-bp indel reverse primer sequence was 50 -TGGACAGGCACAATGGG-30 . The sequences for the 12-bp indel primers were forward 50 -TTACCCTCCTGGTTAGGAG-30 and reverse 50 CTAGATTCCTACACACCAC-30 . The annealing temperature, cycle specifications and reaction mixture were optimized for our laboratory environment. The PCR products were separated on a 3% agarose gel and visualized using ethidium bromide staining and UV light. One of the BSE-positive animals developed clinical symptoms of the disease. The other BSE-positive animals were diagnosed after slaughter and had not shown symptoms of encephalopathy. The analyses in this study were conducted in accordance with the contemporary animal welfare law of the Czech Republic. Neither humans nor animals were injured during this study. 404
K. Vernerova et al.
The frequencies of the alleles, genotypes and diplotypes were calculated. Hardy–Weinberg equilibrium (HWE) was determined using the chi-squared test, and the differences in the frequencies among the breeds were determined using the test of difference in relative frequencies. The effects of an allele on BSE were investigated using logistic regression. This method is useful when the observed outcome is restricted to two values, usually the occurrence or non-occurrence of an outcome event. It predicts the probability of the occurrence as a function of the independent variables. Logistic regression also produces odds ratios (O.R.). The odds of an event occurring is calculated as the probability of the outcome event occurring divided by the probability of the event not occurring. The odds ratio gives the relative amount by which the odds of the outcome increases (O.R. > 1.0) or decreases (O.R. < 1.0) when the value of the predictor value is increased by 1.0 units. Results In total, 81 BSE-negative and 26 BSE-positive animals were analysed for the 12-bp and 23-bp indel regions. The genotype and allele frequencies were compared among the Czech Simmental, Holstein and Czech Red control groups and the BSE-positive animals (Tables 1 and 2). The frequency of the 12-bp indel was largely similar in the various groups. Significant differences, however, were found between the BSE-positive animals and the Holsteins, Simmentals and Reds. Interestingly, there was no significant difference between the all-inclusive breed BSE-positive group and allinclusive breed control group. All of the groups were in Hardy–Weinberg equilibrium except for the Czech Simmentals, and random factors may be responsible for this result. For the 23-bp indel, the BSE-positive group and allinclusive breed control group differed in the insins (+ +) genotype frequency and + allele frequency. These frequencies were significantly lower in the BSE-positive animals compared with the control animals (Table 2). This finding suggests that these two indels may be of different importance. For the 23-bp indel, all of the groups were in Hardy–Weinberg equilibrium. Table 3 (frequencies of the diplotypes) shows that the frequency of the double del genotype ( / ) was not significantly higher in the BSE-positive group compared with the all-inclusive breed control group. The number of animals analysed in this study, however, is too low to draw a definitive conclusion. © 2014 Blackwell Verlag GmbH
• J. Anim. Breed. Genet. 131 (2014) 403–408
Prion protein gene polymorphisms and BSE
K. Vernerova et al.
Table 1 Genotype and allele frequencies of insertion/deletion 12 Genotypes Breed
Frequency
++
+
HWE
Allele +
Czech Simmental
Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected
9 0.360 0.212 11 0.367 0.401 9 0.346 0.403 29 0.358 0.336 6 0.231 0.213
5 0.200ACb 0.497 16 0.533A 0.465 15 0.577C 0.464 36 0.444 0.487 12 0.462b 0.497
11 0.440BD 0.292 3 0.100Ba 0.135 2 0.077Dc 0.133 16 0.198 0.176 8 0.308ac 0.289
35.581**
0.460
2.189ns
0.633
5.898ns
0.635
0.799ns
0.580
0.523ns
0.462
Holstein
Czech Red
Control group total
BSE positive
+, insertion; , deletion; BSE, bovine spongiform encephalopathy. HWE, Hardy–Weinberg equilibrium evaluated by chi-squared test. ns non significant; **significant at p < 0.01; A values in one column with the same letter differ at p < 0.01 (capital letter), or at p < 0.05 (small letter). Table 2 Genotype and allele frequencies of insertion/deletion 23 Genotypes Breed Czech Simmental
Holstein
Czech red
Control group total
BSE positive
Frequency Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected
++ 4 0.160 0.176 9 0.300b 0.303 6 0.231 0.289 19 0.235a 0.256 2 0.077ab 0.120
+ 13 0.520 0.487 15 0.500 0.495 16 0.615 0.497 44 0.543 0.500 14 0.538 0.453
8 0.320 0.336 6 0.200 0.203 4 0.154 0.213 18 0.222 0.244 10 0.385 0.428
Allele +
HWE 0.445
ns
0.420
0.012ns
0.550b
5.600ns
0.538c
0.740ns
0.506a
3.568ns
0.346abc
+, insertion; , deletion; BSE, bovine spongiform encephalopathy. HWE, Hardy–Weinberg equilibrium evaluated by chi-squared test. ns non significant. a values in one column with the same letter differ at p < 0.05.
The results from the logistic regression analysis are shown in Table 4. In this analysis, the exposure variable allele was considered continuous, and the risk of BSE with an increasing number of alleles was determined. This approach assumed that the relationship was linear on a logit scale. For the 12-bp indel, the risk of BSE increased by a factor of 1.55 (i.e. by © 2014 Blackwell Verlag GmbH
• J. Anim. Breed. Genet. 131 (2014) 403–408
55%) for every one allele increase. For the 23-bp indel, the odds ratio was as high as 2.10. When both indels are taken into account, the risk of BSE increased by a factor of 1.54 for every one allele increase. For the 12-bp indel analysis, however, the confidence interval included 1, which indicates that the reliability of this result is lower. In the other 405
Prion protein gene polymorphisms and BSE
K. Vernerova et al.
Table 3 Frequencies of 12/23 diplotypes Diplotypes Breed
Frequency
++/++
++/+
++/
+/++
+/+
+/
/++
/+
/
Czech Simmental
Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected Absolute Relative Expected
2 0.080 0.037 7 0.233 0.122 5 0.192 0.116 14 0.173 0.086 2 0.077 0.026
5 0.200 0.130 3 0.100 0.198 4 0.154 0.200 12 0.148 0.168 2 0.077 0.097
2 0.080 0.071 1 0.033 0.081 0 0.000 0.086 3 0.037 0.082 2 0.077 0.091
0 0.000 0.087 1 0.033 0.141 0 0.000 0.134 1 0.012 0.125 0 0 0.060
4 0.160 0.242 11 0.367 0.230 12 0.462 0.231 27 0.333 0.244 9 0.346 0.225
1 0.040 0.167 4 0.133 0.094 3 0.115 0.099 8 0.099 0.119 3 0.115 0.213
2 0.080 0.051 1 0.033 0.041 1 0.038 0.038 4 0.049 0.045 0 0 0.035
4 0.160 0.142 1 0.033 0.067 0 0.000 0.066 5 0.062 0.088 3 0.115 0.131
5 0.200 0.098 1 0.033 0.027 1 0.038 0.028 7 0.086 0.043 5 0.192 0.124
Holstein
Czech Red
Control group total
BSE positive
+, insertion; , deletion; BSE, bovine spongiform encephalopathy. All groups were not in Hardy–Weinberg equilibrium, but with regard to the low number of diplotypes, the results are not shown. The differences between control group total and BSE positive were tested; the significance was not found.
Table 4 Parameter estimates and odds ratios of a logistic regression model for the effect of allele on the bovine spongiform encephalopathy status Effect of allele del
Intercept
Coefficient estimate
Standard error
p
Odds ratio
95% confidence interval
indel 12 indel 23 indel 12 and 23
1.56 1.99 2.04
0.44 0.74 0.43
0.31 0.36 0.21
0.156 0.039 0.038
1.55 2.10 1.54
0.85; 2.85 1.04; 4.23 1.02; 2.31
analyses, the confidence interval did not include 1, and the results are considered dependable. Because the sample size is
