Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-09
2001-07-03
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S023500, C536S024100, C536S024330, C536S024310
Reexamination Certificate
active
06255054
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns a method and kit for determining if a subject is at increased or decreased risk of developing Alzheimer's disease. The invention further concerns vectors, transformed cells or transgenic mammals which can be used in a method for the screening of compound capable of modulating the expression of the GluR-5 gene or the activity of the GluR-5 receptor. The compounds selected by the method of the invention, as medicament for the treatment or the prevention of Alzheimer's disease, also form part of the present invention.
BACKGROUND OF THE INVENTION
Alzheimer's disease (AD) is marked by the progressive decline of cognitive functions in affected patients and neuropathological features include brain neuronal loss (See Jellinger, & K. A., Bancher, C. J Neural Trans Suppl 54,77-95 (1998)). The causes of sporadic forms of AD representing more than 90% of all cases are unknown. In the brain glutamate receptors mediate fast excitatory neurotransmission and excitotoxicity, a mechanism proposed at the origin of neuronal death in neurodegenerative disorders including AD (See Choi, D. W. Neuron 1,623-634 (1988)).
While there has been considerable research into the mechanisms underlying Alzheimer's disease, it is desirable to develop methods for determining if a subject is at increased risk of developing Alzheimer's disease, using new markers, alone or together with another already known marker or more. It is also desirable to find new ways to investigate and combat this disorder.
For these reasons, the inventors have investigated the polymorphic variations of the human glutamate (kainate) receptor 5 (GluR-5) gene localized on chromosome 21. A variable number of tetraplet repeats AGAT is present in an intronic region of this gene (Ref. GENBANK Accession Number AP000238) and can be assessed by gene sequencing (See Gregor, P. et al. Hum Genet 94, 565-570 (1994)).
The inventors have provided evidence that the total number of tetraplet repeats AGAT present in an intronic region of this gene is significant of the risk of developing Alzheimer's disease.
SUMMARY OF THE INVENTION
The present invention is directed to a method for determining if a subject is at increased or decreased risk of developing Alzheimer's disease, preferably sporadic forms of Alzheimer's disease, comprising the steps of:
a) collecting a biological sample containing genomic DNA from the subject, preferably isolated from venous blood lymphocytes;
b) determining the tetranucleotide AGAT repeats number present in the intronic polynucleotide of the gene GluR-5 which can be amplified by polymerase chain reaction with the primers having the sequences SEQ ID No: 1 and SEQ ID No: 2on each allele;
c) calculating the total number of tetranucleotide AGAT repeats by adding the tetranucleotide AGAT repeats number determined for each allele in step b);
d) observing whether or not the subject is at increased or decreased risk of developing Alzheimer's disease by observing the total number of tetranucleotide AGAT repeats determined in step c) wherein a total number of AGAT repeats equal or superior to a significant threshold value determined by statistical analysis from a reference population indicates said subject is at increased risk of developing Alzheimer's disease and wherein a total number of AGAT repeats inferior to said significant threshold value indicates said subject is at decreased risk of developing Alzheimer's disease.
In a preferred embodiment, the present invention is directed to a method according to the invention wherein said significant threshold value determined by statistical analysis is 18±1, preferably 18, provided that said subject is known not to be affected by the Down syndrome In a further preferred embodiment, the tetranucleotide AGAT repeats number present in the intronic polynucleotide of the gene GluR-5 on each gene GluR-5 allele of the chromosome 21 is obtained by determining the size of and/or sequencing the amplified products obtained after polymerase chain reaction.
In a second aspect, the present invention relates to a method for determining if a subject is at increased or decreased risk of developing Alzheimer's disease according to the invention, characterized in that said method further comprises a second method for assaying a biological sample from said subject for levels of at least an additional marker associated with the increased risk of developing Alzheimer's disease, the presence of a significant level of said at least one marker allowing to confirm if said subject is at increased risk of developing Alzheimer's disease.
In a particularly preferred embodiment, said second method comprises the following steps of:
(i) detecting the presence or absence of an ApoE4 isoform in a biological sample of said subject by using at least one reagent that specifically detects apolipoprotein E type 4 (ApoE4), wherein said reagent is selected from the group consisting of antibodies that selectively bind ApoE4, and oligonucleotide probes that selectively bind to DNA encoding ApoE4; and
(ii) observing if the presence of ApoE4 is or is not detected with said at least one reagent wherein the presence of ApoE4 confirms said subject is at increased risk of developing Alzheimer's disease.
Said at least additional marker associated in conjunction with the method of the present invention can be also selected from the group of markers consisting of bleomycin hydrolase gene polymorphism, FE65 protein gene polymorphism, polymorphic tetranucleotide ATTT repeat site within the intron 7 of the beta-amyloïd precursor protein gene and the persyn (gamma-synuclein) gene polymorphism.
In a third aspect the present invention concerns cloning and/or expression vector comprising a nucleic acid sequence containing between 7 and 14 tetranucleotide AGAT repeats in the intronic polynucleotide of the gene GluR-5 obtainable from biological sample of a subject at decreased or increased risk of developing Alzheimer's disease by polymerase chain reaction with the primers having the sequences SEQ ID No: 1 and SEQ ID No: 2.
Host cell transformed by said vector or mammal, except man, comprising said transformed cell also form part of the present invention.
In a further aspect the present invention is directed to a method for screening or selecting chemical or biochemical compound capable of modulating the expression or the activity of GluR-5 gene characterized in that it uses a vector, a cell or a mammal according to the invention.
In a further aspect the method of the present invention could be used to select patient who may be treated with compound capable of modulating the expression of the GluR-5 gene or the activity of the GluR-5 receptor, according to a pharmacogenomic selection.
In a further aspect the present invention concerns a kit for determining if a subject is at increased or decreased risk of developing Alzheimer's disease comprising at least one pair of primers capable of amplifying the tetranucleotide AGAT repeats number present in the intronic polynucleotide of the gene GluR-5 which can be amplified by polymerase chain reaction with the primers having the sequences SEQ ID No: 1 and SEQ ID No: 2.
In a preferred embodiment the kit of the present invention comprises in addition instructions for determining that the subject is at increased or decreased risk of developing Alzheimer's disease by:
(i) calculating the total number of tetranucleotide AGAT repeats by adding the tetranucleotide AGAT repeats number determined for each allele with said at least one pair of primers; and
(ii) determining whether or not the subject is at increased or decreased risk of developing Alzheimer's disease by observing if the total number of AGAT repeats is equal or superior to a significant threshold value determined by statistical analysis from a reference population, preferably 18±1, wherein a total number of AGAT repeats equal or superior to said significant threshold value indicates said subje
Baclet Marie-Claire
Hugon Jacques
Arthur Lisa B.
Goldberg Jeanine
Mason, Kolehmainen Rathburn & Wyss
LandOfFree
Polymorphism of the human GluR-5 gene and risk factor for... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Polymorphism of the human GluR-5 gene and risk factor for..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Polymorphism of the human GluR-5 gene and risk factor for... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2459560