CA2718392A1 - Biomarkers for alzheimer's disease - Google Patents
Biomarkers for alzheimer's disease Download PDFInfo
- Publication number
- CA2718392A1 CA2718392A1 CA2718392A CA2718392A CA2718392A1 CA 2718392 A1 CA2718392 A1 CA 2718392A1 CA 2718392 A CA2718392 A CA 2718392A CA 2718392 A CA2718392 A CA 2718392A CA 2718392 A1 CA2718392 A1 CA 2718392A1
- Authority
- CA
- Canada
- Prior art keywords
- disease
- alzheimer
- genotyping
- group
- individual
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention pertains to the domain of Alzheimer's disease (AD), and provides new genetic markers for this disease, distributed on 17 genes. Some of these markers can also be used for predicting an increased risk of developing an early-onset form of Alzheimer's disease. The invention also pertains to a method for in vitro predicting an increased risk, for an individual, of developing Alzheimer's disease, by SNP genotyping. Kits for performing the method are also part of the invention.
Description
BIOMARKERS FOR ALZHEIMER'S DISEASE
The present invention pertains to the domain of Alzheimer's disease (AD), and provides new genetic markers for this disease, distributed on 17 genes initially selected by transcriptomic analysis because of their over- or under-expression in the cerebral tissue of AD cases compared to normal individuals. Some of these markers can also be used for predicting an increased risk of developing an early-onset form of Alzheimer's disease.
AD is already a major problem of public health in our developed countries. About 5% of the people aged 65 or above are affected with AD and the prevalence rises steeply to 19% after age 75 and to 47% after age 85.
In the brain tissue, two anatomo-pathological damages characterize this pathology:
- an abnormal accumulation of extracellular deposits, mainly constituted by a peptide of 40 to 42 amino acids called amyloid peptide (or A13);
- a neuronal intracellular aggregation of modified proteins form the cytoskeleton under paired helical filaments (PHF).
It is not very probable that a single causal factor is responsible for the development of these two brain damages. AD appears, indeed, as a multifactorial and complex pathology. The appearance and the evolution of this pathology, as those of most of the multifactorial diseases, are conditioned by the interaction of various genetic susceptibility factors and environmental risk factors.
At the clinical level, although difficult to determine, the age of appearance of the first symptoms allows to distinguish the relatively rare early-onset forms, affecting the patient before the age of 65 years, and the frequent late-onset forms arising after 65 years.
Most of the premature forms of AD are hereditary and their transmission follows a mendelian autosomic dominant mode. Although these cases represent less than 2 % of all the forms of AD, the discovery of genes involved in these familial forms allowed understanding better some pathophysiological mechanisms (Campion, Dumanchin et al. 1999).
To date, pathogenic mutations were discovered in the amyloid precursor gene (APP) (protein which metabolism produces the A13 peptide), the presenilin 1 (PSI) and presenilin 2 (PS2) genes. These mutations were associated with a specific increase in the secretion of the A131-42 peptide, the most toxic form of all amyloid peptides (Cruts and Van Broeckhoven 1998).
These modifications in the APP metabolism led to propose a pathophysiological hypothesis based on the concept of the amyloid cascade.
Although the bibliographical data let think of the existence of 4 major genes, only the s4 allele of the apolipoprotein E gene (APOE) is recognized as a genetic determinant of AD. The expression of this allele is associated with an increase from 3 to 5 times of the risk of developing AD (Farrer, Cupples et al. 1997).
Encountered difficulties for characterizing new susceptibility genes in AD are partially related to methodological problems. Indeed, these approaches get organized essentially around two axes: genome-wild linkage or linkage disequilibrium studies (LD) on familial (early-onset) AD and case-control studies in late-onset AD
based on a bibliographical gene selection.
Linkage disequilibrium (LD) studies are very well adapted when looking for pathogenic mutations responsible for early-onset AD forms and presenting a monogenic segregation (Kamboh 2004). However, this approach seems less appropriate when there are several implied genetic factors, for which impact differs from one family to the other, especially because of the implication of environmental factors.
Such LD
studies are made by genomic screening. This technique consists in studying the segregation of genetic markers regularly spaced out along the genome through several generations. This screening allows defining loci of interest susceptible to contain a gene implicated in the pathology.
However, the current studies based on this methodology and using populations suffering from early-onset AD forms lead to define chromosomal regions of interest sometimes extending over more than 60 centimorgan (Roberts, MacLean et al. 1999). These regions contain hundreds of genes and the systematic search for mutations or for pathogenic polymorphisms is consequently a very heavy task.
The use of case-control studies can also seem very effective to bring to light interactions between studied factors (genetic or environmental) and/or restricted effects on the risk of developing the disease (Bertram, McQueen et al. 2007).
However, this approach presents numerous problems as for the selection of the studied genes, for the quality and the size of studied populations and for the functionality of the incriminated polymorphisms. This latter point is indeed crucial because, further to the complete sequencing of the human genome, a considerable number of polymorphisms are now available in databases for this kind of studies.
Hence, despite the intrinsic qualities of each method, the obtained results could not facilitate the identification of new genetic markers of AD.
In this context, the inventors developed filters in order to decrease the number of genes and thus polymorphisms to be studied. Their original approach, described in more details in the experimental part which follows, is based on the hypothesis that new genetic markers of AD could be identified by analysing the differential expression of genes located in chromosomal regions of interest (Bensemain, Hot et al. 2007).
By performing such an analysis on two sub-populations of individuals of Caucasian origin (a French cohort of 1370 individuals and an American cohort of 1700 individuals), the inventors identified single nucleotide polymorphisms (or Tag-SNPs), located in 17 different genes, which are associated with the risk of developing Alzheimer's disease. These genes are indicated in Table 1 below, and the Tag-SNPs are detailed in Table 2.
Number of Association Gene Abbreviation Ref. Seq Chr. associated with age at SNPs onset Myosin X MYO10 NM-012334 5 24 4 Integrin alpha 2 ITGA2 NM 002203 5 3 precursor -Butyrophilin, subfamily BTN3A2 NM 007047 6 2 3, member A2 -Tenascin XB isoform 1 TNXB NM 019105 6 4 Advanced glycosylation AGER NM 001136 6 4 end product-specific -Major histocompatibility HLA-DRA NM019111 6 5 complex, class II, DR
Cyclin D3 CCDN3 NM_001760 6 3 Interleukin 33 /
Nuclear factor from IL-33/NF- NM 033439 9 1 high endothelial HEV -venules FLJ20375 or FLJ20375 AK000382 9 9 Beta-1,4- B4GALT1 NM 001497 9 5 galactosyltransferase 1 -Ciliary neurotrophic CNTFR NM 001842 9 7 2 factor receptor -Carbonic anhydrase IX CA9 NM-00 1216 9 1 precursor Calcium/calmodulin-dependent protein CAMK2G NM_001222 10 7 kinase II gamma Angiopoietin 4 ANGPTL4 NM_009641 20 6 5 Spermine oxidase SMOX NM 175840 20 2 Table 1: list of genes having at least one Tag-SNP associated with the risk of i o developing Alzheimer's disease.
The present invention pertains to the domain of Alzheimer's disease (AD), and provides new genetic markers for this disease, distributed on 17 genes initially selected by transcriptomic analysis because of their over- or under-expression in the cerebral tissue of AD cases compared to normal individuals. Some of these markers can also be used for predicting an increased risk of developing an early-onset form of Alzheimer's disease.
AD is already a major problem of public health in our developed countries. About 5% of the people aged 65 or above are affected with AD and the prevalence rises steeply to 19% after age 75 and to 47% after age 85.
In the brain tissue, two anatomo-pathological damages characterize this pathology:
- an abnormal accumulation of extracellular deposits, mainly constituted by a peptide of 40 to 42 amino acids called amyloid peptide (or A13);
- a neuronal intracellular aggregation of modified proteins form the cytoskeleton under paired helical filaments (PHF).
It is not very probable that a single causal factor is responsible for the development of these two brain damages. AD appears, indeed, as a multifactorial and complex pathology. The appearance and the evolution of this pathology, as those of most of the multifactorial diseases, are conditioned by the interaction of various genetic susceptibility factors and environmental risk factors.
At the clinical level, although difficult to determine, the age of appearance of the first symptoms allows to distinguish the relatively rare early-onset forms, affecting the patient before the age of 65 years, and the frequent late-onset forms arising after 65 years.
Most of the premature forms of AD are hereditary and their transmission follows a mendelian autosomic dominant mode. Although these cases represent less than 2 % of all the forms of AD, the discovery of genes involved in these familial forms allowed understanding better some pathophysiological mechanisms (Campion, Dumanchin et al. 1999).
To date, pathogenic mutations were discovered in the amyloid precursor gene (APP) (protein which metabolism produces the A13 peptide), the presenilin 1 (PSI) and presenilin 2 (PS2) genes. These mutations were associated with a specific increase in the secretion of the A131-42 peptide, the most toxic form of all amyloid peptides (Cruts and Van Broeckhoven 1998).
These modifications in the APP metabolism led to propose a pathophysiological hypothesis based on the concept of the amyloid cascade.
Although the bibliographical data let think of the existence of 4 major genes, only the s4 allele of the apolipoprotein E gene (APOE) is recognized as a genetic determinant of AD. The expression of this allele is associated with an increase from 3 to 5 times of the risk of developing AD (Farrer, Cupples et al. 1997).
Encountered difficulties for characterizing new susceptibility genes in AD are partially related to methodological problems. Indeed, these approaches get organized essentially around two axes: genome-wild linkage or linkage disequilibrium studies (LD) on familial (early-onset) AD and case-control studies in late-onset AD
based on a bibliographical gene selection.
Linkage disequilibrium (LD) studies are very well adapted when looking for pathogenic mutations responsible for early-onset AD forms and presenting a monogenic segregation (Kamboh 2004). However, this approach seems less appropriate when there are several implied genetic factors, for which impact differs from one family to the other, especially because of the implication of environmental factors.
Such LD
studies are made by genomic screening. This technique consists in studying the segregation of genetic markers regularly spaced out along the genome through several generations. This screening allows defining loci of interest susceptible to contain a gene implicated in the pathology.
However, the current studies based on this methodology and using populations suffering from early-onset AD forms lead to define chromosomal regions of interest sometimes extending over more than 60 centimorgan (Roberts, MacLean et al. 1999). These regions contain hundreds of genes and the systematic search for mutations or for pathogenic polymorphisms is consequently a very heavy task.
The use of case-control studies can also seem very effective to bring to light interactions between studied factors (genetic or environmental) and/or restricted effects on the risk of developing the disease (Bertram, McQueen et al. 2007).
However, this approach presents numerous problems as for the selection of the studied genes, for the quality and the size of studied populations and for the functionality of the incriminated polymorphisms. This latter point is indeed crucial because, further to the complete sequencing of the human genome, a considerable number of polymorphisms are now available in databases for this kind of studies.
Hence, despite the intrinsic qualities of each method, the obtained results could not facilitate the identification of new genetic markers of AD.
In this context, the inventors developed filters in order to decrease the number of genes and thus polymorphisms to be studied. Their original approach, described in more details in the experimental part which follows, is based on the hypothesis that new genetic markers of AD could be identified by analysing the differential expression of genes located in chromosomal regions of interest (Bensemain, Hot et al. 2007).
By performing such an analysis on two sub-populations of individuals of Caucasian origin (a French cohort of 1370 individuals and an American cohort of 1700 individuals), the inventors identified single nucleotide polymorphisms (or Tag-SNPs), located in 17 different genes, which are associated with the risk of developing Alzheimer's disease. These genes are indicated in Table 1 below, and the Tag-SNPs are detailed in Table 2.
Number of Association Gene Abbreviation Ref. Seq Chr. associated with age at SNPs onset Myosin X MYO10 NM-012334 5 24 4 Integrin alpha 2 ITGA2 NM 002203 5 3 precursor -Butyrophilin, subfamily BTN3A2 NM 007047 6 2 3, member A2 -Tenascin XB isoform 1 TNXB NM 019105 6 4 Advanced glycosylation AGER NM 001136 6 4 end product-specific -Major histocompatibility HLA-DRA NM019111 6 5 complex, class II, DR
Cyclin D3 CCDN3 NM_001760 6 3 Interleukin 33 /
Nuclear factor from IL-33/NF- NM 033439 9 1 high endothelial HEV -venules FLJ20375 or FLJ20375 AK000382 9 9 Beta-1,4- B4GALT1 NM 001497 9 5 galactosyltransferase 1 -Ciliary neurotrophic CNTFR NM 001842 9 7 2 factor receptor -Carbonic anhydrase IX CA9 NM-00 1216 9 1 precursor Calcium/calmodulin-dependent protein CAMK2G NM_001222 10 7 kinase II gamma Angiopoietin 4 ANGPTL4 NM_009641 20 6 5 Spermine oxidase SMOX NM 175840 20 2 Table 1: list of genes having at least one Tag-SNP associated with the risk of i o developing Alzheimer's disease.
12+22 22 Co-SNP Chr. Gene 1/2 versus 11 versus dominant 12+11 model rs153202 5 myosin X C / T 0,005 ns 0,01 rs27430 5 myosin X T / C 0,008 0,005 0,002 rs2401987 5 myosin X A / G 0,05 ns ns rs26315 5 myosin X A / T 0,05 ns ns rs6881621 5 myosin X C / T ns 0,04 ns rs1445946 5 myosin X A / G 0,009 ns 0,05 rs13356962 5 myosin X A / G ns 0,01 ns rs250339 5 myosin X A / G 0,03 ns 0,04 rs 10051929 5 myosin X A/C ns ns 0,03 rs12520877 5 myosin X A / G ns ns 0,04 rs4702173 5 myosin X C / T ns 0,04 ns rs17651023 5 myosin X C / T 0,05 ns ns rs6898592 5 myosin X C / T 0,01 ns ns rs17707609 5 myosin X C / G ns 0,007 ns rs17651165 5 myosin X C / T 0,007 ns ns rs31505 5 myosin X A / G 0,02 ns 0,05 rs17614059 5 myosin X C / G 0,01 ns 0,02 rs 17651266 5 myosin X C / T 0.05 ns 0,05 rs40985 5 myosin X A / G 0,007 ns 0,007 rs716555 5 myosin X C / T Ns 0,02 0,03 rs253315 5 myosin X A/C 0,009 0,02 0,002 rs2560852 5 myosin X C / G 0,002 ns 0,001 rs7710976 5 myosin X C / G 0,008 ns ns rs153709 5 myosin X A/C ns 0,04 ns rs716436 5 ITGA2 C / T ns 0,009 ns rs4865756 5 ITGA2 A / G 0,02 ns 0,03 rs11741738 5 ITGA2 A / G ns 0,02 ns rs9467731 6 BTN3A2 C / T ns 0,01 ns rs3757142 6 BTN3A2 A / G ns 0,009 ns rs3130285 6 TNXB C / T 0,003 ns 0,009 rs3130286 6 TNXB C / T 0,006 ns 0,01 rs185819 6 TNXB T / C ns 0,03 ns rs3130287 6 TNXB C / T ns 0,002 0,004 rs3134943 6 AGER A / G ns 0,005 0,007 rs8365 6 AGER C / G 0,03 ns ns rs3134940 6 AGER A / G 0,04 ns ns rs1800684 6 AGER A / T 0,008 ns 0,01 rs3129876 6 HLA-DRA A / G ns 0,008 0,009 rs3129881 6 HLA-DRA C / T 0,03 ns 0,05 rs9268658 6 HLA-DRA G / A 0.05 0,01 0,008 rs8084 6 HLA-DRA A / C ns 0,009 ns rs7192 6 HLA-DRA G / T 0,004 ns 0,03 rs3116713 6 PHF1 A / G ns ns 0,04 rs442745 6 PHF 1 A / G ns 0,007 0,02 rs3106196 6 PHF 1 G / T ns 0,05 0,03 rs3218114 6 cyclin D3 A / G ns 0,003 0,002 rs9529 6 cyclin D3 A / G 0.05 ns ns rs3218086 6 cyclin D3 A / G ns 0,03 0,02 rs7044343 9 IL-33 T / C 0.00003 ns 0.00001 rs10757145 9 KIAA1797 A / G ns 0,05 ns rs1332322 9 KIAA1797 A/C ns ns 0,04 rs4978111 9 KIAA1797 A / G 0,02 ns 0,01 rs7860490 9 KIAA1797 G / T ns 0,04 0,05 rs7033259 9 KIAA1797 A / T 0,05 ns ns rs6475473 9 KIAA1797 A/C 0,002 0,02 0,002 rs6475474 9 KIAA1797 C / T ns 0,02 0,01 rs 1081143 8 9 KIAA 1797 C / G ns ns 0,04 rs10964779 9 KIAA1797 A / G 0,03 ns ns rs3780491 9 B4GALT1 C / T ns ns 0,03 rs10511909 9 B4GALT1 C / G 0,01 ns 0,008 rs2774272 9 B4GALT1 A / G ns 0.05 0,02 rs10758194 9 B4GALT1 G / T ns 0,04 0,02 rs1328898 9 B4GALT1 C / T 0,05 ns 0,02 rs2274592 9 CNTFR C / T 0,04 ns 0,03 rs2381164 9 CNTFR C / G 0,008 0,02 0,002 rs10972149 9 CNTFR A / G 0,0008 ns 0,04 rs 10814123 9 CNTFR C / T ns 0,001 0,05 rs10758268 9 CNTFR G / T ns 0,0003 0,001 rs12551429 9 CNTFR C / G ns 0,05 ns rs3763613 9 CNTFR A/C ns 0,03 ns rs3829078 9 CA9 A / G 0,01 ns 0,01 rs6481654 10 KIAA1462 A / G 0,02 ns ns rs9988732 10 KIAA1462 A / G ns 0,003 0,01 rs2488024 10 KIAA1462 G / A 0,006 ns 0,02 rs11000780 10 CAMK2G A / G ns 0,05 ns rs 10824037 10 CAMK2G A / G ns 0,05 ns rs10458656 10 CAMK2G A / G 0,05 ns ns rs2633310 10 CAMK2G G / T 0,02 ns 0,02 rs2675662 10 CAMK2G C / T ns 0,02 0,02 rs2459446 10 CAMK2G C / T 0,01 ns 0,02 rs2688614 10 CAMK2G C / T ns 0,007 0,005 rs6055551 20 ANGPTL4 C / T ns 0,04 ns rs3787566 20 ANGPTL4 C / T ns 0,007 ns rs4816050 20 ANGPTL4 C / T ns 0,002 ns rs13040505 20 ANGPTL4 C / G 0,02 ns 0,01 rs730169 20 ANGPTL4 C / G ns 0,03 0,04 rs574628 20 ANGPTL4 A / G 0,04 ns ns rs1741296 20 SMOX C / T ns 0,004 0,03 rs6076623 20 SMOX G / T 0,01 ns 0,04 Table 2: list of the SNPs showing a significant association with the risk of developing AD based on statistical analyses with recessive, dominant or co-dominant models. P
was adjusted on the age, gender kind and the genetic status of APOE gene. The allele indicated in bold is the more frequent one.
The inventors also identified 16 SNPs which are associated with an increased risk, for an individual, of developing an earlier-onset form of Alzheimer's disease. This means that people having, for at least one of these SNPs, an unfavourable form of said polymorphism, statistically develop Alheimer's disease at least one year earlier than other people developing the disease. These SNPs are located in 4 different genes, as disclosed in Table 3 below.
Gene ANGPTL4 MYO 10 CNTFR KIAA1462 SNPs rs1124740 rs17651266 rs6476454 rs1063205 rs6118095 rs4702179 rs3763615 rs2478839 rs4813852 rs2562343 rs7071535 rs4816051 rs152342 rs2014144 rs6118137 rs9337951 Table 3 : SNPs associated with the age at onset.
Accordingly, the invention pertains to the use of at least one single nucleotide polymorphism (SNP) selected in the group consisting of rs 153202, rs27430, rs2401987, rs26315, rs6881621, rs1445946, rs13356962, rs250339, rsl0051929, rs 12520877, rs4702173, rs 17651023, rs6898592, rs 17707609, rs 17651165, rs31505, rs17614059, rsl7651266, rs40985, rs716555, rs253315, rs2560852, rs7710976, rsl53709, rs716436, rs4865756, rs11741738, rs9467731, rs3757142, rs3130285, rs3130286, rsl85819, rs3130287, rs3134943, rs8365, rs3134940, rs1800684, rs3129876, rs3129881, rs9268658, rs8084, rs7192, rs3116713, rs442745, rs3106196, rs3218114, rs9529, rs3218086, rs7044343, rs10757145, rs1332322, rs4978111, rs7860490, rs7033259, rs6475473, rs6475474, rs10811438, rs10964779, rs3780491, rs10511909, rs2774272, rs10758194, rs1328898, rs2274592, rs2381164, rsl0972149, rs10814123, rs10758268, rs12551429, rs3763613, rs3829078, rs6481654, rs9988732, rs2488024, rsll000780, rs10824037, rs10458656, rs2633310, rs2675662, rs2459446, rs2688614, rs6055551, rs3787566, rs4816050, rs13040505, rs730169, rs574628, rsl741296, rs6076623, rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rs4702179, rs2562343, rsl52342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951, as a genetic marker for determining the genetic predisposition of an individual to Alzheimer's disease. In a preferred embodiment of the invention, a group of 2 to 5, 6 to 10, 11 to 20 or even more than 20 SNPs selected in the above list is used as a genetic predisposition marker.
The present invention also pertains to the use of at least one SNP
selected in the group consisting of rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rs17651266, rs4702179, rs2562343, rs152342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951, as a genetic marker for determining the genetic predisposition of an individual to develop an early-onset form of Alzheimer's disease. Groups of 2, 3, 4, 5, 6 to 10 or even more SNPs selected in the above list can also be used to determine the predisposition of an individual to develop an early-onset form of Alzheimer's disease.
According to a particular embodiment, the present invention relates to a method for in vitro predicting an increased risk, for an individual, of developing Alzheimer's disease, comprising a step of performing a genotyping assay of at least one gene selected in the group consisting of myosin X, integrin alpha 2 precursor, NM 007047, tenascin XB isoform 1, advanced glycosylation end product-specific, major histocompatibility complex, class II, DR, NM_002636, cyclin D3, Interleukin-33, KIAA1797, NM_001497, ciliary neurotrophic factor receptor, carbonic anhydrase IX
precursor, KIAA1462, calcium/calmodulin-dependent protein kinase II gamma, angiopoietin 4 and SMOX, in a biological sample from said individual.
An example of suitable sample for the SNP genotyping is blood. The SNP genotyping assay can be performed using any SNP analysis method known in the art. For example, hybridization methods can be used, with technologies such as macro-or micro-array, or with technologies based on probes which are specific for the changing base of the SNPs of interest (e.g., Tagman probes, Molecular beacons, Scorpion probes etc.).Other methods, such as restriction fragment length polymorphism (RFLP) methods, can also be used, as well as chromatography (dHPLC), capillary electrophoresis sequencing, mass spectrometry, or specific PCR. Various methods, such as SNPplexTM, Amplifluor SNPs Genotyping System and single base extension (SBE) followed by tag-array hybridization and allele-specific primer extension, are commercially available for performing SNP genotyping assays.
In particular, the above method can be performed by genotyping at least one SNP selected in the group consisting of rs153202, rs27430, rs2401987, rs26315, rs6881621, rs1445946, rs13356962, rs250339, rs10051929, rs12520877, rs4702173, rs17651023, rs6898592, rs17707609, rs17651165, rs31505, rs17614059, rs17651266, rs40985, rs716555, rs253315, rs2560852, rs7710976, rs153709, rs716436, rs4865756, rs11741738, rs9467731, rs3757142, rs3130285, rs3130286, rs185819, rs3130287, rs3134943, rs8365, rs3134940, rs1800684, rs3129876, rs3129881, rs9268658, rs8084, rs7192, rs3116713, rs442745, rs3106196, rs3218114, rs9529, rs3218086, rs7044343, rs10757145, rs1332322, rs4978111, rs7860490, rs7033259, rs6475473, rs6475474, rs10811438, rs10964779, rs3780491, rs10511909, rs2774272, rs10758194, rs1328898, rs2274592, rs2381164, rsl0972149, rs10814123, rs10758268, rs12551429, rs3763613, rs3829078, rs6481654, rs9988732, rs2488024, rs11000780, rs10824037, rs10458656, rs2633310, rs2675662, rs2459446, rs2688614, rs6055551, rs3787566, rs4816050, rs13040505, rs730169, rs574628, rs1741296 and rs6076623.
Of course, the skilled artisan can chose to genotype a determined group of 2, 3, 4, 5, 6-10, 11-20 or more than 20 SNPs. When performing this method, the skilled artisan can also attribute a weight to each of the genotyped SNPs, in order to obtain a score integrating the results of all SNPs genotypes. Of course, the skilled artisan can also combine the information obtained by SNP genotyping as described above with other information such as, for example, information concerning the e4 allele in said individual.
The present invention also pertains to a method for in vitro predicting an increased risk, for an individual, of developing an earlier-onset form of Alzheimer's disease, comprising a step of performing a genotyping assay of at least one gene selected in the group consisting of myosin X, ciliary neurotrophic factor receptor, KIAA1462 and angiopoietin 4. in particular, this method can be performed by genotyping one, 2, 3, 4, 6 to 10 or even more SNP(s) selected in the group consisting of rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rsl7651266, rs4702179, rs2562343, rsl52342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951.
By "earlier-onset form of Alzheimer's disease" is meant that an individual having a marker as mentioned above will statistically develop Alzheimer's disease at least one year earlier than a person not having this marker.
In a preferred embodiment, the above methods are performed with a sample from a Caucasian individual.
Another aspect of the present invention is a kit for performing a method as above described, comprising means for genotyping at least one SNP
selected in the group consisting of rs153202, rs27430, rs2401987, rs26315, rs6881621, rs1445946, rs13356962, rs250339, rs10051929, rsl2520877, rs4702173, rsl7651023, rs6898592, rs17707609, rs17651165, rs31505, rs17614059, rs17651266, rs40985, rs716555, rs253315, rs2560852, rs7710976, rs153709, rs716436, rs4865756, rs11741738, rs9467731, rs3757142, rs3130285, rs3130286, rs185819, rs3130287, rs3134943, rs8365, rs3134940, rs1800684, rs3129876, rs3129881, rs9268658, rs8084, rs7192, rs3116713, rs442745, rs3106196, rs3218114, rs9529, rs3218086, rs7044343, rs10757145, rs1332322, rs4978111, rs7860490, rs7033259, rs6475473, rs6475474, rs10811438, rs10964779, rs3780491, rs10511909, rs2774272, rs10758194, rs1328898, rs2274592, rs2381164, rs10972149, rs10814123, rs10758268, rs12551429, rs3763613, rs3829078, rs6481654, rs9988732, rs2488024, rs11000780, rs10824037, rs10458656, rs2633310, rs2675662, rs2459446, rs2688614, rs6055551, rs3787566, rs4816050, rs13040505, rs730169, rs574628, rs1741296, rs6076623, rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rs4702179, rs2562343, rs152342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951, and a notice of use mentioning the relevance of said SNP(s) in the prognosis of Alzheimer's disease.
In a preferred embodiment of said kit, the kit comprises means for genotyping at least two, and more preferably at least 5 SNPs selected in the above list.
Non-limitative examples of genotyping means which can be included in a kit according to the invention are oligonucleotides and restriction enzymes specific for said SNP(s). Oligonucleotide(s) included in the kit can be bound to a solid support, for example in a micro- or macro-array. Alternatively or complementarily, oligonucleotide(s) included in the kit can be labelled (for example TagMan probes, molecular beacons, Scorpion probes, etc.). Reagents for performing HPLC, RFLP, capillary electrophoresis, specific PCR (such as SNPplexTM or Amplifluor SNP
genotyping), mass spectrometry etc., can also be included in the kit.
Other characteristics of the invention will also become apparent in the course of the description which follows of the biological assays which have been performed in the framework of the invention and which provide it with the required experimental support, without limiting its scope.
Examples Materials and methods Populations Both analyzed sub-populations were obtained by drawing lots from two case-control studies, a French one (n=1370) and an American one (n=1700).
The characteristics of these two sub-populations are described in table 4 below.
French case- American case-control control sub-population AD controls AD cases controls n 307 238 200 200 Mean age' 74.1 73.1 8.5 77.2 6.1 74.3 5.8 Mean age at 68.6 - 72.0 -% of men 35 43 42 38 Table 4 All samples were from individuals of Caucasian origin. The clinical diagnoses were made according to the criteria NINCDS / ADRDA and DSM-III-R.
Controls were recruited in the same geographical region as individuals affected by AD
(in the North of France or in the East-region of Pennsylvania) and did not present 5 criteria of dementia at the time of the recruitment.
Chip-Genotyping Using HapMap database (http://www.hapmap.org), a set of 1156 polymorphisms was selected on 82 genes. These polymorphisms are considered as being Tag-SNPs, allowing to define the most complete genetic information (frequency 10 of the rare allele > 10 % and r2 < 0.8).
The experiments were realized by a service provider, DNAvision company (Belgium), as described by the supplier (Affymetrix company) by using a GeneChip oven 640 hybridization station and a GeneChip 3000 7G 4C scanner.
Among all analyzed SNPs, only twenty one SNPs (1.8 %) could not be genotyped due to technological problems. All other SNPs were genotyped with a rate of success greater than 90 %. Finally, seventy SNPs (6 %) were not in Hardy-Weinberg equilibrium (p < 0.05), the majority of them weakly differing from this equilibrium (60 %, with a probability included between 0.01 and 0.05). Besides, an important proportion of these SNPs were located inside or near a potential CNV (copy Number Variation).
Statistical Analyses The software SAS 8.02 was used (SAS institute, Cary, NC). The association of the 1156 SNPs with the risk of developing AD was estimated by logistic regression and adjustments on the age, the sex, the presence or the absence of allele e4 and the recruitment centre. Three models were systematically tested:
recessive, co-dominant and dominant.
The model 12+22 versus 11 corresponds to a dominant model (that is at least one copy of an allele is sufficient to induce a modification of the risk).
The model 22 versus 11+12 corresponds to a recessive model (that is two copies of one allele are necessary to induce a modification of the risk) Co-predominant model assume that the more the number of copy is important, the more the risk increase.
Results and conclusions Genes Selection by transcriptomic studies Previous linkage-disequilibrium LD studies allowed determining chromosomal regions susceptible to contain one or several genetic markers of AD.
These regions extend sometimes over more than 95 cM.
From these data was developed within the laboratory a biochip allowing the study of the expression of 2741 ORFs ("Open Reading Frame") contained in these chromosome regions of interest (Lambert, Testa et al. 2003). The level of expression of these ORFs was compared in cerebral tissues resulting from 12 individuals affected by AD and from 12 controls (selected on the quality of extracted total ARN).
107 genes, differentially expressed in AD brains compared to control one, were consequently identified (table 5) (Bensemain, Hot et al. 2007).
Locus Distance in Selected ORFs Differentially expressed cm ORFs Chr. 1 50 393 13 Chr.5 50 174 6 Chr.6 40 535 24 Chr. 9 55 230 12 Chr. 10 95 415 15 Chr. 12 40 306 11 Chr. 20 50 239 9 Chr. 21 58 267 6 Chr. X 25 182 11 Table 5 : number of differentially expressed genes in the various chromosomal regions of interest.
Association of identified genes with the risk of developing AD
To accelerate the analysis of the genetic association of these genes with the risk of developing AD, a genotyping chip with an average flow (Affymetrix) was thus developed. From 82 genes, 1156 Tag-SNPs were selected on the Hapmap site, as mentioned above.
These Tag-SNPs were analyzed on two sub-populations, French (n=545) and American (n=400), obtained by drawing lots from more important populations. Seventy Tag-SNPs were not in Hardy-Weinberg equilibrium in the control combined population (French and American), most of them weakly differing from this balance (60 % between 0.01 < p < 0.05).
Among remainders Tag-SNPs and according to the three statistical models that were used (recessive, co-dominant and dominant), 89 of these Tag-SNPs located in 17 different genes presented at least one polymorphism associated with the risk of developing AD in the combined population and this in at least one of the three used models (p < 0.05). Besides, 4 of these genes also present Tag-SNPs presenting the characteristic to modulate the age of appearance of the pathology. (cf table 6 below and table 2 above).
Chromosomal Differential Number Number Association Gene Ref. Seq Chr. locations expression b of of with age at studied associated SNPs SNPs onset 16,715,016- +201% 103 MYO10 NM_012334 5 16,989,385 24 4 52,320,913- + 215% 26 ITGA2 NM_002203 5 52,426,365 3 26,473,377-BTN3A2 NM 007047 6 26,486,525 - 45% 13 2 32,116,911- + 100% 31 32,185,131 32,256,724-_ AGER NM 001136 6 32,260,001 - 53% 7 4 32,515,625--HLA-DRA NM 019111 6 32,520,799 - 57% 10 5 33,486,934-_ PHFI NM 002636 6 33,492,187 - 47% 4 3 42,010,650--CCDN3 NM 001760 6 42,017,530 + 157% 9 3 IL-33/NF- 6,231,678-HEV -033439 9 6,247,982 1 20,939,639-FLJ20375 AK000382 9 20,985,947 - 62% 55 9 33,100,642-B4GALT1 NM 001497 9 33,157,231 -43% 9 5 34,541,431- +132 % 16 CNTFR NM_oo 1842 9 34,579,722 7 2 35,663,915-_ CA9 NM001216 9 35,671,152 -50% 11 1 30,343,390- + 262% 18 KIAA 1462 NM-020848 10 30,376,806 3 5 75,242,265-_ CAMK2G NM 001222 10 75,304,344 -50% 10 7 801,604-ANGPTL4 NM-009641 20 844,605 - 65% 42 6 5 4,103,675-_ SMOX NM 175840 20 4,116,352 - 42% 15 2 Table 6 The fact that SNPs different from those associated to the risk of developing AD, are linked to different age of AD onset result from the combination of numerous factors:
= The statistical tools that have been used: logistical decline (qualitative / qualitative) versus analysis of the variance (qualitative /
quantitative) = The statistical models that have been used (recessive, predominant or co-predominant) = The frequency of studied SNPs.
= The variability in the measurement of the age of onset and its heterogeneity between the different individuals = The linkage disequilibrium more or less important between different SNPs Conclusion This integrated approach, based on the study of genes i) located in the chromosomal regions of interest defined by genomic screening and ii) differentially expressed in AD brains compared to control brains, allowed the identification of 17 susceptibility genes for AD. The analysis of separated and/or combined genetic variants of the genes which have been identified as associated with the risk of developing AD
according to the present study will allow developing tools for the diagnosis and prognosis of AD.
REFERENCES
Bensemain, F., D. Hot, et al. (2007). "Evidence for induction of the ornithine transcarbamylase expression in Alzheimer's disease." Mol Psychiatry.
Bertram, L., M. B. McQueen, et al. (2007). "Systematic meta-analyses of Alzheimer disease genetic association studies: the A1zGene database." Nat Genet 39(1): 17-23.
Campion, D., C. Dumanchin, et al. (1999). "Early-onset autosomal dominant Alzheimer disease: prevalence, genetic heterogeneity, and mutation 1o spectrum." Am J Hum Genet 65(3): 664-70.
Cruts, M. and C. Van Broeckhoven (1998). "Molecular genetics of Alzheimer's disease." Ann Med 30(6): 560-5.
Farrer, L. A., L. A. Cupples, et al. (1997). "Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease.
A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium." Jama 278(16): 1349-56.
Kamboh, M. I. (2004). "Molecular genetics of late-onset Alzheimer's disease." Ann Hum Genet 68(Pt 4): 381-404.
Lambert, J. C., E. Testa, et al. (2003). "Relevance and limitations of public databases for microarray design: a critical approach to gene predictions."
Pharmacogenomics J 3(4): 235-41.
Roberts, S. B., C. J. MacLean, et al. (1999). "Replication of linkage studies of complex traits: an examination of variation in location estimates."
Am J Hum Genet 65(3): 876-84.
was adjusted on the age, gender kind and the genetic status of APOE gene. The allele indicated in bold is the more frequent one.
The inventors also identified 16 SNPs which are associated with an increased risk, for an individual, of developing an earlier-onset form of Alzheimer's disease. This means that people having, for at least one of these SNPs, an unfavourable form of said polymorphism, statistically develop Alheimer's disease at least one year earlier than other people developing the disease. These SNPs are located in 4 different genes, as disclosed in Table 3 below.
Gene ANGPTL4 MYO 10 CNTFR KIAA1462 SNPs rs1124740 rs17651266 rs6476454 rs1063205 rs6118095 rs4702179 rs3763615 rs2478839 rs4813852 rs2562343 rs7071535 rs4816051 rs152342 rs2014144 rs6118137 rs9337951 Table 3 : SNPs associated with the age at onset.
Accordingly, the invention pertains to the use of at least one single nucleotide polymorphism (SNP) selected in the group consisting of rs 153202, rs27430, rs2401987, rs26315, rs6881621, rs1445946, rs13356962, rs250339, rsl0051929, rs 12520877, rs4702173, rs 17651023, rs6898592, rs 17707609, rs 17651165, rs31505, rs17614059, rsl7651266, rs40985, rs716555, rs253315, rs2560852, rs7710976, rsl53709, rs716436, rs4865756, rs11741738, rs9467731, rs3757142, rs3130285, rs3130286, rsl85819, rs3130287, rs3134943, rs8365, rs3134940, rs1800684, rs3129876, rs3129881, rs9268658, rs8084, rs7192, rs3116713, rs442745, rs3106196, rs3218114, rs9529, rs3218086, rs7044343, rs10757145, rs1332322, rs4978111, rs7860490, rs7033259, rs6475473, rs6475474, rs10811438, rs10964779, rs3780491, rs10511909, rs2774272, rs10758194, rs1328898, rs2274592, rs2381164, rsl0972149, rs10814123, rs10758268, rs12551429, rs3763613, rs3829078, rs6481654, rs9988732, rs2488024, rsll000780, rs10824037, rs10458656, rs2633310, rs2675662, rs2459446, rs2688614, rs6055551, rs3787566, rs4816050, rs13040505, rs730169, rs574628, rsl741296, rs6076623, rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rs4702179, rs2562343, rsl52342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951, as a genetic marker for determining the genetic predisposition of an individual to Alzheimer's disease. In a preferred embodiment of the invention, a group of 2 to 5, 6 to 10, 11 to 20 or even more than 20 SNPs selected in the above list is used as a genetic predisposition marker.
The present invention also pertains to the use of at least one SNP
selected in the group consisting of rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rs17651266, rs4702179, rs2562343, rs152342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951, as a genetic marker for determining the genetic predisposition of an individual to develop an early-onset form of Alzheimer's disease. Groups of 2, 3, 4, 5, 6 to 10 or even more SNPs selected in the above list can also be used to determine the predisposition of an individual to develop an early-onset form of Alzheimer's disease.
According to a particular embodiment, the present invention relates to a method for in vitro predicting an increased risk, for an individual, of developing Alzheimer's disease, comprising a step of performing a genotyping assay of at least one gene selected in the group consisting of myosin X, integrin alpha 2 precursor, NM 007047, tenascin XB isoform 1, advanced glycosylation end product-specific, major histocompatibility complex, class II, DR, NM_002636, cyclin D3, Interleukin-33, KIAA1797, NM_001497, ciliary neurotrophic factor receptor, carbonic anhydrase IX
precursor, KIAA1462, calcium/calmodulin-dependent protein kinase II gamma, angiopoietin 4 and SMOX, in a biological sample from said individual.
An example of suitable sample for the SNP genotyping is blood. The SNP genotyping assay can be performed using any SNP analysis method known in the art. For example, hybridization methods can be used, with technologies such as macro-or micro-array, or with technologies based on probes which are specific for the changing base of the SNPs of interest (e.g., Tagman probes, Molecular beacons, Scorpion probes etc.).Other methods, such as restriction fragment length polymorphism (RFLP) methods, can also be used, as well as chromatography (dHPLC), capillary electrophoresis sequencing, mass spectrometry, or specific PCR. Various methods, such as SNPplexTM, Amplifluor SNPs Genotyping System and single base extension (SBE) followed by tag-array hybridization and allele-specific primer extension, are commercially available for performing SNP genotyping assays.
In particular, the above method can be performed by genotyping at least one SNP selected in the group consisting of rs153202, rs27430, rs2401987, rs26315, rs6881621, rs1445946, rs13356962, rs250339, rs10051929, rs12520877, rs4702173, rs17651023, rs6898592, rs17707609, rs17651165, rs31505, rs17614059, rs17651266, rs40985, rs716555, rs253315, rs2560852, rs7710976, rs153709, rs716436, rs4865756, rs11741738, rs9467731, rs3757142, rs3130285, rs3130286, rs185819, rs3130287, rs3134943, rs8365, rs3134940, rs1800684, rs3129876, rs3129881, rs9268658, rs8084, rs7192, rs3116713, rs442745, rs3106196, rs3218114, rs9529, rs3218086, rs7044343, rs10757145, rs1332322, rs4978111, rs7860490, rs7033259, rs6475473, rs6475474, rs10811438, rs10964779, rs3780491, rs10511909, rs2774272, rs10758194, rs1328898, rs2274592, rs2381164, rsl0972149, rs10814123, rs10758268, rs12551429, rs3763613, rs3829078, rs6481654, rs9988732, rs2488024, rs11000780, rs10824037, rs10458656, rs2633310, rs2675662, rs2459446, rs2688614, rs6055551, rs3787566, rs4816050, rs13040505, rs730169, rs574628, rs1741296 and rs6076623.
Of course, the skilled artisan can chose to genotype a determined group of 2, 3, 4, 5, 6-10, 11-20 or more than 20 SNPs. When performing this method, the skilled artisan can also attribute a weight to each of the genotyped SNPs, in order to obtain a score integrating the results of all SNPs genotypes. Of course, the skilled artisan can also combine the information obtained by SNP genotyping as described above with other information such as, for example, information concerning the e4 allele in said individual.
The present invention also pertains to a method for in vitro predicting an increased risk, for an individual, of developing an earlier-onset form of Alzheimer's disease, comprising a step of performing a genotyping assay of at least one gene selected in the group consisting of myosin X, ciliary neurotrophic factor receptor, KIAA1462 and angiopoietin 4. in particular, this method can be performed by genotyping one, 2, 3, 4, 6 to 10 or even more SNP(s) selected in the group consisting of rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rsl7651266, rs4702179, rs2562343, rsl52342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951.
By "earlier-onset form of Alzheimer's disease" is meant that an individual having a marker as mentioned above will statistically develop Alzheimer's disease at least one year earlier than a person not having this marker.
In a preferred embodiment, the above methods are performed with a sample from a Caucasian individual.
Another aspect of the present invention is a kit for performing a method as above described, comprising means for genotyping at least one SNP
selected in the group consisting of rs153202, rs27430, rs2401987, rs26315, rs6881621, rs1445946, rs13356962, rs250339, rs10051929, rsl2520877, rs4702173, rsl7651023, rs6898592, rs17707609, rs17651165, rs31505, rs17614059, rs17651266, rs40985, rs716555, rs253315, rs2560852, rs7710976, rs153709, rs716436, rs4865756, rs11741738, rs9467731, rs3757142, rs3130285, rs3130286, rs185819, rs3130287, rs3134943, rs8365, rs3134940, rs1800684, rs3129876, rs3129881, rs9268658, rs8084, rs7192, rs3116713, rs442745, rs3106196, rs3218114, rs9529, rs3218086, rs7044343, rs10757145, rs1332322, rs4978111, rs7860490, rs7033259, rs6475473, rs6475474, rs10811438, rs10964779, rs3780491, rs10511909, rs2774272, rs10758194, rs1328898, rs2274592, rs2381164, rs10972149, rs10814123, rs10758268, rs12551429, rs3763613, rs3829078, rs6481654, rs9988732, rs2488024, rs11000780, rs10824037, rs10458656, rs2633310, rs2675662, rs2459446, rs2688614, rs6055551, rs3787566, rs4816050, rs13040505, rs730169, rs574628, rs1741296, rs6076623, rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rs4702179, rs2562343, rs152342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951, and a notice of use mentioning the relevance of said SNP(s) in the prognosis of Alzheimer's disease.
In a preferred embodiment of said kit, the kit comprises means for genotyping at least two, and more preferably at least 5 SNPs selected in the above list.
Non-limitative examples of genotyping means which can be included in a kit according to the invention are oligonucleotides and restriction enzymes specific for said SNP(s). Oligonucleotide(s) included in the kit can be bound to a solid support, for example in a micro- or macro-array. Alternatively or complementarily, oligonucleotide(s) included in the kit can be labelled (for example TagMan probes, molecular beacons, Scorpion probes, etc.). Reagents for performing HPLC, RFLP, capillary electrophoresis, specific PCR (such as SNPplexTM or Amplifluor SNP
genotyping), mass spectrometry etc., can also be included in the kit.
Other characteristics of the invention will also become apparent in the course of the description which follows of the biological assays which have been performed in the framework of the invention and which provide it with the required experimental support, without limiting its scope.
Examples Materials and methods Populations Both analyzed sub-populations were obtained by drawing lots from two case-control studies, a French one (n=1370) and an American one (n=1700).
The characteristics of these two sub-populations are described in table 4 below.
French case- American case-control control sub-population AD controls AD cases controls n 307 238 200 200 Mean age' 74.1 73.1 8.5 77.2 6.1 74.3 5.8 Mean age at 68.6 - 72.0 -% of men 35 43 42 38 Table 4 All samples were from individuals of Caucasian origin. The clinical diagnoses were made according to the criteria NINCDS / ADRDA and DSM-III-R.
Controls were recruited in the same geographical region as individuals affected by AD
(in the North of France or in the East-region of Pennsylvania) and did not present 5 criteria of dementia at the time of the recruitment.
Chip-Genotyping Using HapMap database (http://www.hapmap.org), a set of 1156 polymorphisms was selected on 82 genes. These polymorphisms are considered as being Tag-SNPs, allowing to define the most complete genetic information (frequency 10 of the rare allele > 10 % and r2 < 0.8).
The experiments were realized by a service provider, DNAvision company (Belgium), as described by the supplier (Affymetrix company) by using a GeneChip oven 640 hybridization station and a GeneChip 3000 7G 4C scanner.
Among all analyzed SNPs, only twenty one SNPs (1.8 %) could not be genotyped due to technological problems. All other SNPs were genotyped with a rate of success greater than 90 %. Finally, seventy SNPs (6 %) were not in Hardy-Weinberg equilibrium (p < 0.05), the majority of them weakly differing from this equilibrium (60 %, with a probability included between 0.01 and 0.05). Besides, an important proportion of these SNPs were located inside or near a potential CNV (copy Number Variation).
Statistical Analyses The software SAS 8.02 was used (SAS institute, Cary, NC). The association of the 1156 SNPs with the risk of developing AD was estimated by logistic regression and adjustments on the age, the sex, the presence or the absence of allele e4 and the recruitment centre. Three models were systematically tested:
recessive, co-dominant and dominant.
The model 12+22 versus 11 corresponds to a dominant model (that is at least one copy of an allele is sufficient to induce a modification of the risk).
The model 22 versus 11+12 corresponds to a recessive model (that is two copies of one allele are necessary to induce a modification of the risk) Co-predominant model assume that the more the number of copy is important, the more the risk increase.
Results and conclusions Genes Selection by transcriptomic studies Previous linkage-disequilibrium LD studies allowed determining chromosomal regions susceptible to contain one or several genetic markers of AD.
These regions extend sometimes over more than 95 cM.
From these data was developed within the laboratory a biochip allowing the study of the expression of 2741 ORFs ("Open Reading Frame") contained in these chromosome regions of interest (Lambert, Testa et al. 2003). The level of expression of these ORFs was compared in cerebral tissues resulting from 12 individuals affected by AD and from 12 controls (selected on the quality of extracted total ARN).
107 genes, differentially expressed in AD brains compared to control one, were consequently identified (table 5) (Bensemain, Hot et al. 2007).
Locus Distance in Selected ORFs Differentially expressed cm ORFs Chr. 1 50 393 13 Chr.5 50 174 6 Chr.6 40 535 24 Chr. 9 55 230 12 Chr. 10 95 415 15 Chr. 12 40 306 11 Chr. 20 50 239 9 Chr. 21 58 267 6 Chr. X 25 182 11 Table 5 : number of differentially expressed genes in the various chromosomal regions of interest.
Association of identified genes with the risk of developing AD
To accelerate the analysis of the genetic association of these genes with the risk of developing AD, a genotyping chip with an average flow (Affymetrix) was thus developed. From 82 genes, 1156 Tag-SNPs were selected on the Hapmap site, as mentioned above.
These Tag-SNPs were analyzed on two sub-populations, French (n=545) and American (n=400), obtained by drawing lots from more important populations. Seventy Tag-SNPs were not in Hardy-Weinberg equilibrium in the control combined population (French and American), most of them weakly differing from this balance (60 % between 0.01 < p < 0.05).
Among remainders Tag-SNPs and according to the three statistical models that were used (recessive, co-dominant and dominant), 89 of these Tag-SNPs located in 17 different genes presented at least one polymorphism associated with the risk of developing AD in the combined population and this in at least one of the three used models (p < 0.05). Besides, 4 of these genes also present Tag-SNPs presenting the characteristic to modulate the age of appearance of the pathology. (cf table 6 below and table 2 above).
Chromosomal Differential Number Number Association Gene Ref. Seq Chr. locations expression b of of with age at studied associated SNPs SNPs onset 16,715,016- +201% 103 MYO10 NM_012334 5 16,989,385 24 4 52,320,913- + 215% 26 ITGA2 NM_002203 5 52,426,365 3 26,473,377-BTN3A2 NM 007047 6 26,486,525 - 45% 13 2 32,116,911- + 100% 31 32,185,131 32,256,724-_ AGER NM 001136 6 32,260,001 - 53% 7 4 32,515,625--HLA-DRA NM 019111 6 32,520,799 - 57% 10 5 33,486,934-_ PHFI NM 002636 6 33,492,187 - 47% 4 3 42,010,650--CCDN3 NM 001760 6 42,017,530 + 157% 9 3 IL-33/NF- 6,231,678-HEV -033439 9 6,247,982 1 20,939,639-FLJ20375 AK000382 9 20,985,947 - 62% 55 9 33,100,642-B4GALT1 NM 001497 9 33,157,231 -43% 9 5 34,541,431- +132 % 16 CNTFR NM_oo 1842 9 34,579,722 7 2 35,663,915-_ CA9 NM001216 9 35,671,152 -50% 11 1 30,343,390- + 262% 18 KIAA 1462 NM-020848 10 30,376,806 3 5 75,242,265-_ CAMK2G NM 001222 10 75,304,344 -50% 10 7 801,604-ANGPTL4 NM-009641 20 844,605 - 65% 42 6 5 4,103,675-_ SMOX NM 175840 20 4,116,352 - 42% 15 2 Table 6 The fact that SNPs different from those associated to the risk of developing AD, are linked to different age of AD onset result from the combination of numerous factors:
= The statistical tools that have been used: logistical decline (qualitative / qualitative) versus analysis of the variance (qualitative /
quantitative) = The statistical models that have been used (recessive, predominant or co-predominant) = The frequency of studied SNPs.
= The variability in the measurement of the age of onset and its heterogeneity between the different individuals = The linkage disequilibrium more or less important between different SNPs Conclusion This integrated approach, based on the study of genes i) located in the chromosomal regions of interest defined by genomic screening and ii) differentially expressed in AD brains compared to control brains, allowed the identification of 17 susceptibility genes for AD. The analysis of separated and/or combined genetic variants of the genes which have been identified as associated with the risk of developing AD
according to the present study will allow developing tools for the diagnosis and prognosis of AD.
REFERENCES
Bensemain, F., D. Hot, et al. (2007). "Evidence for induction of the ornithine transcarbamylase expression in Alzheimer's disease." Mol Psychiatry.
Bertram, L., M. B. McQueen, et al. (2007). "Systematic meta-analyses of Alzheimer disease genetic association studies: the A1zGene database." Nat Genet 39(1): 17-23.
Campion, D., C. Dumanchin, et al. (1999). "Early-onset autosomal dominant Alzheimer disease: prevalence, genetic heterogeneity, and mutation 1o spectrum." Am J Hum Genet 65(3): 664-70.
Cruts, M. and C. Van Broeckhoven (1998). "Molecular genetics of Alzheimer's disease." Ann Med 30(6): 560-5.
Farrer, L. A., L. A. Cupples, et al. (1997). "Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease.
A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium." Jama 278(16): 1349-56.
Kamboh, M. I. (2004). "Molecular genetics of late-onset Alzheimer's disease." Ann Hum Genet 68(Pt 4): 381-404.
Lambert, J. C., E. Testa, et al. (2003). "Relevance and limitations of public databases for microarray design: a critical approach to gene predictions."
Pharmacogenomics J 3(4): 235-41.
Roberts, S. B., C. J. MacLean, et al. (1999). "Replication of linkage studies of complex traits: an examination of variation in location estimates."
Am J Hum Genet 65(3): 876-84.
Claims (10)
1. Use of at least one single nucleotide polymorphism (SNP) selected in the group consisting of rs153202, rs27430, rs2401987, rs26315, rs6881621, rs1445946, rs13356962, rs250339, rs10051929, rs12520877, rs4702173, rs17651023, rs6898592, rs17707609, rs17651165, rs31505, rs17614059, rs17651266, rs40985, rs716555, rs253315, rs2560852, rs7710976, rs153709, rs716436, rs4865756, rs11741738, rs9467731, rs3757142, rs3130285, rs3130286, rs185819, rs3130287, rs3134943, rs8365, rs3134940, rs1800684, rs3129876, rs3129881, rs9268658, rs8084, rs7192, rs3116713, rs442745, rs3106196, rs3218114, rs9529, rs3218086, rs7044343, rs10757145, rs1332322, rs4978111, rs7860490, rs7033259, rs6475473, rs6475474, rs10811438, rs10964779, rs3780491, rs10511909, rs2774272, rs10758194, rs1328898, rs2274592, rs2381164, rs10972149, rs10814123, rs10758268, rs12551429, rs3763613, rs3829078, rs6481654, rs9988732, rs2488024, rs11000780, rs10824037, rs10458656, rs2633310, rs2675662, rs2459446, rs2688614, rs6055551, rs3787566, rs4816050, rs13040505, rs730169, rs574628, rs1741296, rs6076623, rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rs4702179, rs2562343, rs152342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951, as a genetic marker for determining the genetic predisposition of an individual to Alzheimer's disease.
2. The use of claim 1, wherein at least one SNP selected in the group consisting of rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rs17651266, rs4702179, rs2562343, rs152342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951, is used as a genetic marker for determining the genetic predisposition of an individual to develop an earlier-onset form of Alzheimer's disease.
3. A method for in vitro predicting an increased risk, for an individual, of developing Alzheimer's disease, comprising a step of performing a genotyping assay of at least one gene selected in the group consisting of myosin X, integrin alpha 2 precursor, NM007047, tenascin XB isoform 1, advanced glycosylation end product-specific, major histocompatibility complex, class II, DR, NM_002636, cyclin D3, Interleukin-33, KIAA1797, NM_001497, ciliary neurotrophic factor receptor, carbonic anhydrase IX precursor, KIAA1462, calcium/calmodulin-dependent protein kinase II gamma, angiopoietin 4 and SMOX, in a biological sample from said individual.
4. The method of claim 3, wherein said genotyping assay is performed by genotyping at least one SNP selected in the group consisting of rs153202, rs27430, rs2401987, rs26315, rs6881621, rs1445946, rs13356962, rs250339, rs10051929, rs12520877, rs4702173, rs17651023, rs6898592, rs17707609, rs17651165, rs31505, rs17614059, rs17651266, rs40985, rs716555, rs253315, rs2560852, rs7710976, rs153709, rs716436, rs4865756, rs11741738, rs9467731, rs3757142, rs3130285, rs3130286, rs185819, rs3130287, rs3134943, rs8365, rs3134940, rs1800684, rs3129876, rs3129881, rs9268658, rs8084, rs7192, rs3116713, rs442745, rs3106196, rs3218114, rs9529, rs3218086, rs7044343, rs10757145, rs1332322, rs4978111, rs7860490, rs7033259, rs6475473, rs6475474, rs10811438, rs10964779, rs3780491, rs10511909, rs2774272, rs10758194, rs1328898, rs2274592, rs2381164, rs10972149, rs10814123, rs10758268, rs12551429, rs3763613, rs3829078, rs6481654, rs9988732, rs2488024, rs11000780, rs10824037, rs10458656, rs2633310, rs2675662, rs2459446, rs2688614, rs6055551, rs3787566, rs4816050, rs13040505, rs730169, rs574628, rs1741296 and rs6076623.
5. The method of claim 3, for in vitro predicting an increased risk, for an individual, of developing an earlier-onset form of Alzheimer's disease, comprising a step of performing a genotyping assay of at least one gene selected in the group consisting of myosin X, ciliary neurotrophic factor receptor, KIAA1462 and angiopoietin 4.
6. The method of claim 5, wherein said genotyping assay is performed by genotyping at least one SNP selected in the group consisting of rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rs17651266, rs4702179, rs2562343, rs152342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951.
7. The method according to any of claims 1 to 6, wherein said individual is Caucasian.
8. A kit for performing a method according to any of claims 1 to 7, comprising means for genotyping at least one SNP selected in the group consisting of rs153202, rs27430, rs2401987, rs26315, rs6881621, rs1445946, rs13356962, rs250339, rs10051929, rs12520877, rs4702173, rs17651023, rs6898592, rs17707609, rsl 7651165, rs31505, rs17614059, rs17651266, rs40985, rs716555, rs253315, rs2560852, rs7710976, rs153709, rs716436, rs4865756, rs11741738, rs9467731, rs3757142, rs3130285, rs3130286, rs185819, rs3130287, rs3134943, rs8365, rs3134940, rs1800684, rs3129876, rs3129881, rs9268658, rs8084, rs7192, rs3116713, rs442745, rs3106196, rs3218114, rs9529, rs3218086, rs7044343, rs10757145, rs1332322, rs4978111, rs7860490, rs7033259, rs6475473, rs6475474, rs10811438, rs10964779, rs3780491, rs10511909, rs2774272, rs10758194, rs1328898, rs2274592, rs2381164, rs10972149, rs10814123, rs10758268, rs12551429, rs3763613, rs3829078, rs6481654, rs9988732, rs2488024, rs11000780, rs10824037, rs10458656, rs2633310, rs2675662, rs2459446, rs2688614, rs6055551, rs3787566, rs4816050, rs13040505, rs730169, rs574628, rs1741296, rs6076623, rs1124740, rs6118095, rs4813852, rs4816051, rs6118137, rs4702179, rs2562343, rs152342, rs6476454, rs3763615, rs1063205, rs2478839, rs7071535, rs2014144 and rs9337951 and a notice of use mentioning the relevance of said SNP(s) in the prognosis of Alzheimer's disease.
9. The kit of claim 8, wherein said genotyping means comprise at least one oligonucleotide.
10. The kit of claim 9, wherein said oligonucleotide is bound to a solid support.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/IB2008/002161 WO2009112882A1 (en) | 2008-03-14 | 2008-03-14 | Snp biomarkers for alzheimer ' s disease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2718392A1 true CA2718392A1 (en) | 2009-09-17 |
Family
ID=39990030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2718392A Abandoned CA2718392A1 (en) | 2008-03-14 | 2008-03-14 | Biomarkers for alzheimer's disease |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2718392A1 (en) |
| WO (1) | WO2009112882A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609618A (en) * | 2019-01-08 | 2019-04-12 | 青岛大学 | A kind of kit detecting pathological myopia and its application method and purposes |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4286517A3 (en) | 2013-04-04 | 2024-03-13 | President and Fellows of Harvard College | Therapeutic uses of genome editing with crispr/cas systems |
| WO2015037681A1 (en) * | 2013-09-11 | 2015-03-19 | 国立大学法人京都大学 | Test method for evaluating the risk of anti-thyroid drug-induced agranulocytosis, and evaluation kit |
| EP3077533B1 (en) * | 2013-12-06 | 2019-04-24 | Life & Brain GmbH | Methods for establishing a clinical prognosis of diseases associated with the formation of aggregates of abeta1-42 |
| IL302341A (en) | 2015-03-27 | 2023-06-01 | Harvard College | Modified t cells and methods of making and using the same |
| US11725232B2 (en) * | 2016-10-31 | 2023-08-15 | The Hong Kong University Of Science And Technology | Compositions, methods and kits for detection of genetic variants for alzheimer's disease |
| WO2018195129A1 (en) | 2017-04-17 | 2018-10-25 | University Of Maryland, College Park | Embryonic cell cultures and methods of using the same |
-
2008
- 2008-03-14 CA CA2718392A patent/CA2718392A1/en not_active Abandoned
- 2008-03-14 WO PCT/IB2008/002161 patent/WO2009112882A1/en active Application Filing
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609618A (en) * | 2019-01-08 | 2019-04-12 | 青岛大学 | A kind of kit detecting pathological myopia and its application method and purposes |
| CN109609618B (en) * | 2019-01-08 | 2021-11-26 | 青岛大学 | Kit for detecting pathological myopia and use method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2009112882A1 (en) | 2009-09-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6338530B2 (en) | Methods for treating, diagnosing, and monitoring Alzheimer's disease | |
| Macé et al. | ABCA2 is a strong genetic risk factor for early-onset Alzheimer's disease | |
| Brouwers et al. | Alzheimer risk associated with a copy number variation in the complement receptor 1 increasing C3b/C4b binding sites | |
| Takei et al. | Genetic association study on in and around the APOE in late-onset Alzheimer disease in Japanese | |
| Andersen et al. | Clinical genetics of amyotrophic lateral sclerosis: what do we really know? | |
| Heuser et al. | Variants of the genes encoding AQP4 and Kir4. 1 are associated with subgroups of patients with temporal lobe epilepsy | |
| Zheng et al. | Association of the carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase gene with schizophrenia in the Chinese Han population | |
| US8080371B2 (en) | Markers for addiction | |
| Pastor et al. | Further extension of the H1 haplotype associated with progressive supranuclear palsy | |
| US20050196794A1 (en) | Use of haplotypes and SNPs in lipid-relevant genes for the analysis and diagnosis of cardiovascular disease | |
| Shibata et al. | Association studies of cholesterol metabolism genes (CH25H, ABCA1 and CH24H) in Alzheimer's disease | |
| CA2718392A1 (en) | Biomarkers for alzheimer's disease | |
| JP2008509674A (en) | Compositions and methods for determining and predicting treatment responses for depression and anxiety | |
| Luedecking-Zimmer et al. | Investigation of oxidized LDL-receptor 1 (OLR1) as the candidate gene for Alzheimer's disease on chromosome 12 | |
| Taguchi et al. | Identification of hippocampus‐related candidate genes for Alzheimer's disease | |
| Horiuchi et al. | A polymorphism in the PDLIM5 gene associated with gene expression and schizophrenia | |
| Alvarez et al. | Mitochondrial transcription factor A (TFAM) gene variation and risk of late-onset Alzheimer's disease | |
| Zou et al. | Screening of VCP mutations in Chinese amyotrophic lateral sclerosis patients | |
| US20130266937A1 (en) | Compositions and methods for diagnosing and treating macular degeneration | |
| Ozturk et al. | Three SNPs in the GSTO1, GSTO2 and PRSS11 genes on chromosome 10 are not associated with age-at-onset of Alzheimer's disease | |
| Wood et al. | Association between neprilysin polymorphisms and sporadic Alzheimer's disease | |
| Lämsä et al. | Genetic study evaluating LDLR polymorphisms and Alzheimer's disease | |
| US20130324431A1 (en) | Olfactory receptor copy number association with age at onset of alzheimer's disease | |
| Li et al. | Positive association between PDLIM5 and schizophrenia in the Chinese Han population | |
| Yu et al. | Genetic association of rs11610206 SNP on chromosome 12q13 with late-onset Alzheimer's disease in a Han Chinese population |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |