Volume 2: Science
2. The Spongiform Encephalopathies - knowledge existing in 1986
Prion mutations and polymorphisms in human TSEs ¹

2.172 We conclude our review of knowledge in 1986 by looking forward to further developments concerning the prion protein gene which occurred shortly afterwards and which are central to our understanding of TSEs. The human prion protein gene (PRNP) is located on chromosome 20 and is 16,000 bases in length. The protein coding region itself comprises only 759 base pairs, and this specifies a protein consisting of 253 amino acids. Mutations within the PRNP gene can cause TSEs if they involve a critical region. It is presumed that the amino acid changes which result from these mutations make the protein more susceptible to the conformational change associated with protease resistance. In fact, mutations are the only mechanism at present known to have this result. Theoretically, other mechanisms such as exposure to a toxic chemical might lead to the conversion of PrP^C to PrP^Sc, but so far this has not been demonstrated.

2.173 The identification of the human prion mutations listed in Table 2.2 were all made from 1989 onwards. Mutations occur in the familial forms of CJD, including GSS and FFI, and can sometimes be demonstrated in sporadic cases. They are detected by the analysis of the PRNP gene from DNA extracted from a small sample of blood or tissue. An affected individual passes the disease mutation on average to half his or her offspring, and therefore DNA analysis can be used to identify or exclude the disease in close relatives long before the onset of disease. Point mutations result in the substitution of one amino acid for another. For example, the prion protein gene specifies that proline is the amino acid at codon 102. A point mutation within the gene can change that amino acid from proline to leucine. The shorthand for this mutation is Pro-Leu 102 or P102L. This change is sufficient to cause one form of GSS. ² Another common type of familial mutation results from the insertion of additional amino acids into the protein molecule. Specifically these are insertions of additional octapeptide repeats ³ in the prion protein. Other mutations result in polymorphic variation, ie, a change which is not associated with disease. Over 25 mutations of the PRNP gene are known and these are listed in Table 2.2 below. It is not yet clear how these subtle changes in the prion protein lead to disease.

Table 2.2: List of human mutations

2.174 Mutations that are included in the genome of the germ line cells (sperm and ova) lead to familial disease. However, it is possible that sporadic CJD may be caused by similar PRNP mutations occurring spontaneously in body tissue (somatic) cells. Sufficient PrP^Sc may be generated to lead to disease. As the mutation starts in somatic cells and not germ cells, it is not transmitted to offspring. Somatic mutations are well-known causes of other diseases including cancer. However, it is extremely unlikely that a somatic mutation would be detectable (if it occurs) in CJD, since the mutation may affect only a small number of cells in one tissue, and there is no known method of identifying where these cells may be. In this respect, the origin of sporadic CJD from somatic mutation remains just a very likely hypothesis.

2.175 Some specific familial mutations of the PRNP gene have been associated with more than one clinical pattern of CJD. This has been interpreted as the consequence of other host genes which modify the expression of PrP^Sc. Thus the Asp-Asn 178 mutation is associated with CJD when the host's genotype at codon 129 is valine/valine. ⁴ When the host's genotype at codon 129 is methionine/methionine, the patient has the features of Fatal Familial Insomnia (FFI). Other mutations are affected by the codon 129 genotype, namely Pro-Leu 102 and the 4 octapeptide repeat insertional mutation.

2.176 The first mutation in familial disease was identified in 1989 and was the 144-base-pair insertional mutation in a UK family with CJD. ⁵ This was followed by the discovery of a missense mutation at codon 102 (resulting in a proline-leucine substitution), which was shown to be the cause of GSS. ⁶ The pathogenicity of the codon 102 mutation was demonstrated experimentally in 1990, when transgenic mice over-expressing the prion protein with an analogous mutation were shown to develop spongiform degeneration spontaneously. ⁷ In this study, the mutant gene was introduced into fertilised mouse eggs, with the result that many copies integrated into the mouse genome. However, recent evidence suggests that the observation of spontaneous neurodegeneration in these mice is a result of over-production of the mutant gene (due to the increased number of gene copies), rather than the effect of the mutation itself. Work carried out at the Neuropathogenesis Unit (NPU) in Edinburgh has shown that mice bearing the equivalent P101L mutation, introduced into the PrP gene already present in the mouse genome, did not develop a TSE in over 800 days. ⁸ The mice only developed disease when inoculated with TSE sources, and incubation time was different from that in wild mice. It therefore appears that the P101L mutation in mice is associated with disease susceptibility rather than spontaneous neurodegeneration.

2.177 The most frequent cause of familial CJD is the point mutation at codon 200, which results in a substitution of the amino acid lysine for glutamine. This mutation accounts for more than 70 per cent of the families with hereditary CJD worldwide. It has recently been suggested that these cases may have resulted from a single mutational event, and spread geographically with migration of affected individuals. ⁹

2.178 As well as actually causing disease, genetic factors can affect susceptibility to disease, as described for scrapie in paragraphs 2.90-2.95. In contrast to the mutations involved with disease causation, genetic predisposition is relevant to all types of human TSEs - inherited, sporadic and iatrogenic. By far the most important polymorphism of interest in this respect is that found at codon 129 of the PRNP gene. In the Caucasian population, around 51 per cent are heterozygotes, with around 38 per cent methionine homozygotes and 11 per cent valine homozygotes. Iatrogenic cases resulting from the use of human growth hormone are associated with a significant excess of the valine homozygous genotype, ¹⁰ whereas vCJD cases have all been homozygous for methionine (see Table 2.3). Studies of sporadic cases have found that over 80 per cent are homozygous for either methionine or valine, ¹¹ although a recent classification based on 300 cases has found six differenttypes of sporadic CJD, some of which are heterozygotes. ¹²

Table 2.3: Codon 129 polymorphisms in CJD

2.179 Other polymorphisms of the PRNP gene have been identified, though the full relevance of many of them is as yet not clear. These too are listed in Table 2.2. It remains to be seen whether mutations or polymorphisms outside the PRNP gene affect the resistance or susceptibility to BSE or CJD. As described above (paragraphs 2.73-2.75) another gene (doppel) has recently been identified in humans and mice. This gene is closely linked to the PRNP gene and is similar to all known prion proteins. Findings suggest that the interaction between PrP and doppel may be important in the development of TSEs. Furthermore, it is possible that polymorphisms within doppel may modulate disease phenotype. However, the function of doppel remains to be elucidated.