Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-16
2002-05-21
Clark, Deborah J. R. (Department: 1633)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S091200, C536S024300, C536S024310
Reexamination Certificate
active
06391553
ABSTRACT:
The present invention relates to a method for diagnosing Alzheimer's disease.
Alzheimer's disease is a neurodegenerative dementia characterized by a loss of cortical neurons associated with &bgr;-amyloid plaques, of neurofibrillary tangles and, in most cases, an amyloid angiopathy. It is strongly suspected that there is a genetic influence in the aetiology of Alzheimer's disease (WO 94/01772).
This genetic component has been brought to the fore over many years by indirect observations which suggest that the disease is inherited in an autosomal dominant fashion with an age-dependent penetrance in order to explain certain familial forms of Alzheimer's disease. Recent molecular genetic studies have enabled putative genes for Alzheimer's disease to be isolated by looking for chromosome-specific polymorphic genetic markers (Bird et al., 1989, Neurobiology of Aging 10, 432-434).
Four chromosomal localizations have been described as being involved: three on chromosomes 1, 14 and 21 in the early onset familial forms (age at onset under 60 years), and one on chromosome 19 in the late onset familial and sporadic forms. Two linkage studies have suggested that the chromosomal region 19q13.2 was associated with late onset familial forms of Alzheimer's disease (Pericak-Vance et al, Am. J. Hum. Genet. (1991), 48, 1034-1050). Within this chromosomal region, the group of genes for apolipoproteins (APO) E-CI-CI′-CII is a candidate zone. Among the products of these genes, apolipoprotein E (APOE) is involved especially in the nervous system APOE is present in the senile plaques and possesses a binding affinity for the peptide A&bgr;. APOE is characterized by three major alleles &egr;2, &egr;3, &egr;4. Strittmatter et al. (Proc. Natl. Acad. Sci. (1993) 90, 177-181) have described an increased frequency of the &egr;4 allele of the APOE gene in the late onset familial forms of Alzheimer's disease. This observation has been confirmed for the familial forms (Corder et al., Science (1993), 261, 921-923) and the sporadic forms of Alzheimer's disease (Corder et al., Science (1993), 261, 921-923; Saunders et al., Neurology (1993), 13, 1467-1472).
FR-2 716 894 describes a method which makes it possible to prognosticate, for a given disease, the risks of developing Alzheimer's disease with respect to the general population. This method is based on the detection of the &egr;4 alleles of the APOE gene, of the short alleles of the marker D19S178 and of the long alleles of the APO CII gene, all localized on chromosome 19.
Several hypotheses make it possible to explain this phenomenon.
Recent studies by the inventors, who are the authors of the present invention, now confirm the hypothesis of the existence of at least one other functional mutation in the 19q13.2 region.
Indeed, in addition to a functional effect specific to the polymorphism of apolipoprotein E, the studies by the inventors show differences in levels of expression which are significant in sick subjects compared with healthy controls, which indicates that one or more mutations in the regulatory regions of the APOE gene are involved in the onset of Alzheimer's disease. Furthermore, relative differences in expression are found in the controls.
The inventors have more particularly identified a new polymorphism in the region of the promoter of the gene encoding the apolipoprotein E protein found in a potential binding site for the Th1/E47cs transcription factors.
For the determination of this polymorphism, the sequence described by Paik et al. (1985, Proc. Natl. Aca. Sci., vol. 82, p. 3447) will be taken as reference.
This polymorphism has been called by the inventors Th1/E47cs for Th1/E47cs consensus, since it is situated in a consensus sequence for binding of the Th1/E47cs transcription factor.
The mutation identified is characterized by a Thymine to Guanine substitution (G→T) in the sequence:
GGGTGTCTGT(or G)ATTACTGGG,
G being the most frequent allele in the normal population.
The alleles corresponding to this polymorphism are called hereinafter T (when the base is Thymine) or G (when the base is Guanine).
The determination of the allele can be carried out after PCR amplification of the DNA region comprising this polymorphism:
either by creating in one of the PCR primers a cleavage site for a restriction enzyme not existing in individuals carrying one of the alleles,
or by a hybridization technique using oligonucleotide probes specific for the alleles.
The inventors have studied the influence of the Th1/E47cs polymorphism on the expression of the alleles of the APOE gene and demonstrated an increase in the risk of developing Alzheimer's disease associated with the T allele of Th1/E47cs, specific to this allele and not due to the &egr;4 allele.
Furthermore, the Th1/E47cs polymorphism modulates the risk associated with the &egr;4 allele in individuals with the &egr;3/&egr;4 genotype, the individuals with the GT genotype having an increased risk of developing Alzheimer's disease compared with the individuals homozygous for the Th1/E47cs polymorphism. Reinforcing this observation, the authors have considered that the combination of the T allele of Th1/E47cs with the &egr;4 allele on the same chromosome corresponds to the most unfavourable combination.
The subject of the invention is thus a method for diagnosing Alzheimer's disease, comprising the identification of one or more mutations in the genomic DNA region for regulating the expression of the apolipoprotein E gene, inducing a modification of the expression of the apolipoprotein E gene relative to a control population or a modification of the relative expression of the alleles of the apolipoprotein E gene.
For the purposes of the present invention, diagnosis is understood to mean the confirmation of a mutation in the regulatory region of the APOE gene in a patient whose clinical picture signals a symptomatology which may be attributed to Alzheimer's disease, or alternatively an increased probability in subjects of developing Alzheimer's disease relative to the population as a whole, the increase in probability being statistically significant.
The chromosomal DNA region for regulating the APOE gene is broadly defined as being the chromosomal region 19q13.2 other than the region encoding apolipoprotein E (Human Molecular Genetics, 1994, vol. 3, No. 4, 569-574).
Advantageously, the chromosomal DNA region in which one or more mutations are identified is situated between the marker D19S178 and the APOCII gene, and comprises more particularly the introns and the flanking regions of the APOE gene, extending over a distance of 5 kb upstream and downstream of the APOE gene.
The subject of the invention is more particularly a method for diagnosing Alzheimer's disease comprising the identification of at least one mutation in the promoter of the APOE gene, situated at 186 bases from the TATA box of this gene, the mutation consisting more particularly of the replacement T→G in the sequence defined above.
More particularly, the method consists in testing for one or more mutations in the region of the promoter of the gene encoding apolipoprotein E, existing in particular in a potential binding site for the Th1/E47 transcription factors.
The subject of the invention is also a method for diagnosing Alzheimer's disease comprising a determination of the genotype of apolipoprotein E and the test for a mutation of the type described above in the regulatory region of the APOE gene.
In accordance with this diagnostic method, the presence of at least one &egr;4 allele of apolipoprotein E conjointly with the existence of a mutation in the regulatory region of the APOE gene, in particular the mutation defined above inducing a modification of the expression of the APOE gene or a relative difference in expression of the alleles of apolipoprotein E where appropriate, will range towards the diagnosis of Alzheimer's disease in patients whose clinical picture presents a symptomatology which can be attributed to Alzheimer&apo
Amouyel Philippe
Chartier-Harlin Marie-Christine
Lambert Jean-Charles
Chen Shin-Lin
Clark Deborah J. R.
Institut Pasteur de Lille
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