Ruth F. Itzhaki*, Woan-Ru Lin* and Gordon K. Wilcock+
* Molecular Neurobiology Laboratory, Department of Optometry & Vision Sciences, UMIST, PO Box 88, Manchester M60 1QD
+ Department of Care of the Elderly, Frenchay Hospital, Bristol BS16 1LE
Introduction
The gene for apolipoprotein type E4 (apoE4) is a risk factor for Alzheimer's disease (AD) but it is neither essential nor sufficient to cause the disease. Presumably, environmental agents must be involved also[1]. One factors we have been investigating is herpes simplex virus type 1 (HSV1). Nearly all adults harbour HSV1 [2,3] in the peripheral nervous system (PNS) in a latent state, i.e., the genome is present but no virions nor viral proteins are produced. The virus reactivates periodically, and in certain people causes cold sores.
In this laboratory we have used polymerase chain reaction (PCR) to seek HSV1 DNA in brain [4-6]. We have detected HSV1 in about 78% of brain specimens from 46 AD patients and in 64% of 44 age-matched normals, in the temporal and/or frontal cortices and/or hippocampus, but not in the occipital cortex [5] - a region far less affected in AD than are the former three regions. Reverse PCR assays indicated that the virus is in latent form [6]. We suggested that periodic, mild reactivation of HSV1 occurs in the CNS as a result of immunosuppression [7] or stress, causing greater damage in ADs than in normals because of a difference in host or virus factor (Fig 1).
Aims
1. To examine a possible difference in host factor - the apoE genotype - between those AD patients and age-matched normals respectively HSV1-positive or HSV1-negative in brain.
2. Following these results, to examine a possible difference in apoE genotype between cold sore sufferers and non-sufferers.
Methodology
Subjects for brain studies. Post mortem brain specimens were obtained from 44 normals (25 male, and 19 female, age range 73-93, mean 79), and 46 AD patients (10 male and 36 female, aged 54-96, mean, 79). Brain regions used were temporal and/or frontal cortex and in a few cases, hippocampus. All specimens were coded and PCRs were done blind on the coded DNA products. In HSV1 assays, a 110 bp sequence of the HSV1 TK gene and also a 267 bp sequence of the human HGPRT gene were amplified in the same tube.
Subjects for cold sore studies. . Blood (1-5 ml) was collected for isolating DNA from 33 normals, i.e. people who had never had a cold sore, (15 females and 18 males, age range 20-61, mean 38), and from 40 sufferers (19 females and 21 males, age range 23-63, mean 39). All samples were coded and measurements done on the coded products
ApoE genotype. For determination of the apoE genotype, a 227 bp sequence in the allele was amplified and the product was digested with cfoI (15 u in 55m l for 1h at 37oC, then plus a further 10 u for another 1hr at 37oC.
Results
1. Table 1 lists genotypes and apoE allele numbers for AD patients and normals[8]. The apoE4 allele frequencies of the following groups do not differ significantly: HSV1-negative ADs (10%), HSV1-negative normals (6%) and HSV1-positive normals (4%). Using multiple logistic regression, the apoE4 allele frequency of the HSV1 positive AD group (53%) is significantly different from the above three groups (p<0.0001): the relative risk of AD (apoE4 plus HSV1-positive) versus the latter groups equals 19.0 (95% confidence limits 7.2-49.7) Consistent with this, the apoE4 value of 53% for HSV1-positive AD patients is significantly above the range of values reported for AD patients over-all (34-42%, p<0.01).
2. Table 2 shows that the apoE4 allele frequency of cold sore sufferers is 36% whereas that of non-sufferers is 9% (p<0.0001)[8].
Discussion
Our previous results showed, for the first time, the definite presence of HSV1 DNA in brain of both aged normal people and AD patients. Our present results [8,9] indicate that the risk of developing AD is very high in those people who have at least one apoE4 allele and who have HSV1 DNA in at least one of the brain regions examined, whereas those with either factor alone are not at risk.
The apoE4 allele frequency of cold sore sufferers is significantly greater than that of non-sufferers. This strongly and independently supports our hypothesis that in the CNS, reactivation of the virus is more damaging in apoE4 possessors than in those with the other apoE variants.
Conclusion
1. Our results suggest that HSV1 in brain of possessors of an apoE4 allele is indeed a risk factor in AD - the first environmental factor to be definitely implicated. This is the first case of a neurological disease in which an environmental factor acting with an inherited factor has been identified. Also, the gene for apoE4 is the first genetic factor to be implicated in cold sores.
2. These data point to the therapeutic use of anti-viral agents to prevent viral reactivation in appropriate cases of AD, and ultimately, to the use of a vaccine against the virus in infancy.
References
1. Itzhaki, R.F. (1994) Molec Neurobiol 9, 1-13.
2. Goodpasture, E.W. (1929) Medicine 8, 223-243.
3. Baringer, J.R & Swoveland, P. (1973) N Engl J Med 288, 648-650.
4. Jamieson, G.A., Maitland, N.J., Wilcock, G.K., Craske, J. & Itzhaki, R.F. (1991) J. Med. Virol. 33, 224-227.
5. Jamieson, G.A., Maitland, N.J., Wilcock, G.K., Yates, C.M. & Itzhaki, R.F. (1992) J. Pathol. 167, 365-368.
6. Itzhaki, R.F., Maitland, N.J., Wilcock, G.K., Yates, C.M. & Jamieson, G.A. (1993) in Alzheimer's Disease: Advances in Clinical and Basic Research (Corain, B., Iqbal, K., Nicholini, M., Winblad, B., Wisniewski, H., & Zatta, P., eds.), pp.97-102, Wiley & Sons, Chichester.
7. Saldanha, J., Sutton, R.N.P., Gannicliffe, A., Farragher, B. & Itzhaki, R.F. (1986) J. Neurol. Neurosurg. Psych. 49, 613-619.
8. Itzhaki R.F., Lin W-R., Shang D., Wilcock G.K., Faragher B., Jamieson G.A. (1997) Lancet. 349, 241-244
We thank the Sir Halley Stewart Trust for support of this work, Mettler-Toledo for a donation, Ms J. Graham and Ms S. Matthews for skilled assistance to R.F.I. and G.K.W., respectively, and Drs Brooke and Parker for taking blood samples.
Correspondence to:
Professor Ruth F. Itzhaki
Molecular Neurobiology Laboratory,
Department of Optometry & Vision Sciences,
UMIST,
PO Box 88,
Manchester M60 1QD
United Kingdom
E-mail : Ruth.Itzhaki@umist.ac.uk
Tel: 44-161-200-3879
Fax: 44-161-200-4433
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