Volume 2: Science
2. The Spongiform Encephalopathies - knowledge existing in 1986
Genetic aspects of scrapie
Genes affecting scrapie incubation in mice
Genes affecting natural scrapie in sheep
Interaction between host genes and specific scrapie isolates
Summary

2.80 At the time of the emergence of BSE in cattle in 1986, it was understood that susceptibility to scrapie was controlled by a complex interaction between host genes and the specific scrapie isolate. This understanding had been based on findings from research begun as early as the 1950s. We now describe chronologically the main findings in this area.

2.81 The first suggestion that some breeds of sheep were more susceptible to scrapie than others came from early investigations carried out by Gordon in the 1950s and 1960s. In one study, he inoculated 24 different breeds of sheep with one scrapie isolate. The results, published in 1966, showed that there was a wide range of variation in susceptibility between breeds, ranging from 78 per cent developing disease in the Herdwick sheep sample, to nil in the sample composed of Dorset Down sheep. ¹ In addition, the average incubation periods were different between breeds, in those sheep that did develop disease. The incubation periods ranged from 100 to 690 days.

2.82 This difference in susceptibility was also true for mouse strains. In 1969 Dickinson reported that he had inoculated one scrapie isolate, ME7, into four different mouse strains. ² All mouse strains developed scrapie but the incubation periods differed significantly between strains, ranging from 140 to 280 days.

2.83 Scientists at this time then began to consider why some sheep were resistant to scrapie and what could make one animal more resistant to the disease than another animal. Somehow, the different breeds or strains of mice and sheep were exerting specific controls over the replication or spread of scrapie. The next step, therefore, was to identify the mouse and sheep gene, or genes, that could be regulating scrapie infection.

Genes affecting scrapie incubation in mice

2.84 Dickinson then investigated the role of mouse genes in controlling scrapie incubation periods in mice. These studies consisted of extensive mouse-breeding experiments and scrapie inoculations. The first results, published in 1968, were consistent with length being controlled by one gene. This gene was named sinc for scrapie incubation period. ³

2.85 Dickinson's study also suggested that sinc had a single pair of alleles, s7 and p7. Mice homozygous for the s7 allele (ie, where both alleles are s7) had a relatively short incubation period for a particular scrapie isolate (ME7), while mice homozygous for the p7 allele had a long incubation period. Heterozygous mice (ie, having one s7 and one p7 allele) had an incubation period intermediate between the two homozygotes.

2.86 However, in a later study, when the same mouse strain was inoculated with a different scrapie isolate (22A), the length of the incubation periods was reversed (ie, s7s7 mice had relatively long incubation periods, while p7p7 mice had short incubation periods). ⁴ Moreover, heterozygote mice, s7p7, had longer incubation periods than either of the homozygous mice.

2.87 These findings prompted the hypothesis that the two sinc alleles did not act independently, but that their protein products formed a combined structure essential for scrapie agent replication. These observations formed the basis of the replication site hypothesis (paragraphs 2.62-2.65).

2.88 In 1986 there was no information concerning the function of the sinc gene in a normal animal uninfected with scrapie or its protein product. However, the experimental evidence provided by the isolation of the prion protein (PrP) from scrapie-infected brains suggested that PrP was in fact the transmissible agent. Moreover, the discovery that the PrP gene and the sinc gene were closely linked ⁵ suggested that PrP might be the sinc gene product. ⁶ Since 1986 further research has shown that the sinc and PrP genes are indeed the same. ⁷

2.89 Other genes had also been identified as having a role in controlling scrapie infection. In 1972 Dickinson and Fraser showed that mice with the aberrant form of the dominant hemimelia (Dh) gene had longer incubation periods. ⁸ These mice lack spleens, and the result was thought to be due to this defect, as it had already been shown that the spleen was important in the replication and spread of scrapie (see paragraphs 2.162-2.163). Then, in 1983, another gene, Pid-1 (prion incubation determinant), was shown by Kingsbury to influence the incubation period of scrapie and CJD in mice. ⁹

Genes affecting natural scrapie in sheep

2.90 Evidence for a regulatory gene controlling scrapie incubation periods in sheep has come from a study that started in 1961. This involved the selection and separation of Cheviot sheep into two lines dependent upon the length of the incubation period after inoculation with a complex of scrapie strains. ¹⁰ The two lines were referred to as SIP (short incubation period) when the average incubation period following intracerebral injection was 7 months, or LIP (long incubation period) when the incubation period following intracerebral injection ranged from 18 months through to old age. ¹¹ Using breeding experiments, Dickinson predicted that a single gene controlled the length of these incubation periods in sheep. However, only preliminary results were available in 1986.

2.91 As with the situation in mice, the findings were found to be specific for this scrapie complex but not for the sheep breed, since they were repeated in Herdwick sheep. ¹²

2.92 Epidemiological evidence had also suggested that natural scrapie was controlled by a single gene. This evidence came from the study of the incidence of natural scrapie over 20 years in sheep flocks and from a breeding programme testing the theory of genetic control. ¹³

2.93 Later, these initial predictions were shown to be correct and a gene, sip, was identified which was found to control the incubation period of scrapie. ¹⁴ As with the sinc gene in mice, evidence also became available to suggest that the PrP gene and sip were identical. ¹⁵

2.94 Since 1986 a more complex picture of the effect of sheep genetics on resistance and susceptibility to scrapie has emerged. It has been documented, for example, that within a closed flock of sheep in which scrapie is endemic, PrP gene polymorphisms at codons 136, 154 and 171 are disease-linked, and are associated with susceptibility and resistance to infections and with differences in incubation period. ¹⁶

2.95 Suffolk sheep with valine (V), arginine (R) and glutamine (Q) at codons 136, 154 and 171 respectively are most susceptible to scrapie, while the ARR variants (A is shorthand for alanine) at these codons confer greatest resistance. The ARQ pattern of variants is also associated with susceptibility in Suffolk sheep, but not in Cheviot sheep, in which the same pattern confers resistance. The explanation for these polymorphisms is fundamental to efforts to breed scrapie-resistant flocks.

Interaction between host genes and specific scrapie isolates

2.96 Another interesting finding from this early research was that different results were seen with the various scrapie isolates in the same mouse strain (see paragraphs 2.85-2.86). This finding implied that disease progression was dependent not only on the host genes (eg, sinc), but also upon the interaction between the host genes and the specific scrapie isolate.

2.97 Some scrapie isolates were extremely stable in their properties, when passaged through different mouse strains. For example, ME7 scrapie isolate had similar incubation periods and lesion profiles whether passaged repeatedly through C57BL mice or VM mice. ¹⁷ Other scrapie isolates were known to be entirely stable in their properties when passaged through a particular mouse strain, but when subsequently passaged into another mouse strain, their properties changed. For example, 22A scrapie isolate passaged through VM mice had significantly different properties to 22A scrapie isolate passaged through C57BL mice.

2.98 In 1986 this change in properties was thought to be the result of a mutation of the scrapie agent in the new host. However, such thinking was based on the underlying assumption that the scrapie agent carried nucleic acid that could be mutated. ¹⁸ Later experimental evidence does not support this premise for the change in properties of the scrapie agent, which is now thought to be a result of conformational changes. One mechanism for the change in properties might be the formation of a hybrid prion molecule produced by a combination of two scrapie strains resulting from the presence in the inoculation of a mixture of strains. The hybrid prion molecule might then convert the host prion molecule into a new conformation, recognised as a new strain of the scrapie agent.

2.99 In summary, by 1986 and the emergence of BSE in cattle, experimental evidence had indicated that scrapie disease progression was regulated by a complicated interaction between the host genes (eg, sinc in mice) and the specific scrapie isolate.

Summary

2.100 Many years of research before 1986 on scrapie and the transmission of scrapie strains to experimental mice had led to the identification in both mice and sheep of genetic factors which were associated with susceptibility and resistance to infection. In both sheep and mice the factors appeared to be controlled by alleles at a single genetic locus. It was confirmed later that the gene involved in both species was the prion protein gene. The main conclusion of the work on genetics was that transmission of the infection, the incubation period, and the pattern of disease (phenotype) all depended on interactions between the particular strain of infecting agent and the host's genotype. These findings raised the question whether phenotypic variation in CJD was the result of the same interactions that similar findings were. We shall see expected from investigation of animals affected with BSE. In the event, it transpired that only one strain of infective agent was implicated in BSE and that the phenotype was similar in all breeds of cattle. Consequently, genetic susceptibility factors seem unimportant in this particular species.