WO1996025517A9 - Method for detecting a mutation involved in alzheimer's disease and nucleotide sequences for implementing same - Google Patents
Method for detecting a mutation involved in alzheimer's disease and nucleotide sequences for implementing sameInfo
- Publication number
- WO1996025517A9 WO1996025517A9 PCT/FR1996/000263 FR9600263W WO9625517A9 WO 1996025517 A9 WO1996025517 A9 WO 1996025517A9 FR 9600263 W FR9600263 W FR 9600263W WO 9625517 A9 WO9625517 A9 WO 9625517A9
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- WIPO (PCT)
- Prior art keywords
- seq
- cgc
- alzheimer
- mutation
- sequence
- Prior art date
Links
- 230000035772 mutation Effects 0.000 title claims abstract description 28
- 229920001850 Nucleic acid sequence Polymers 0.000 title claims abstract description 19
- 206010001897 Alzheimer's disease Diseases 0.000 title claims abstract description 13
- 238000003752 polymerase chain reaction Methods 0.000 claims abstract description 17
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- 108010060159 Apolipoprotein E4 Proteins 0.000 description 1
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Definitions
- the present invention relates to a method for detecting a new mutation involved in Alzheimer's disease.
- Apolipoprotein E is a 299 amino acid plasma glycoprotein, synthesized primarily by the liver and intestine, but also in most other tissues, including the astrocyte of nervous tissue.
- the normal apo E concentration of the plasma is of the order of 40 mg / l. It is present in lipoproteins chylomicrons, VLDL, ILDL (as well as in the cerebrospinal fluid).
- the sequence of the protein (RALL et al., 1982 J. Biol Chem., 257: 4171-4178), that of the complementary DNA, as well as the structure and sequence of the APO E gene (DAS et al. 1985, J.
- the fourth exon of the gene codes for the domain of the apo E protein, which participates in the binding with the LDL receptor.
- E2 and E4 differ from E3 by base substitutions: in E4, C is in place of T in position 3932, and in E2 is T instead of C in position 4070. This results in the amino acid changes following: Cys (112) instead of Arg for the E4 isoform, and Arg (158) instead of Cys for the E2 isoform.
- apo E isoforms of apo E are classically distinguished by isofocalization of serum proteins. It is currently easier to genotype them by polymerase chain reaction (PCR) based on their DNA sequence variation (Hixson and Vernier, 1990, J. Lipid Res., 31: 545-548), by cleavage. , after amplification, with the restriction enzyme Hha I.
- PCR polymerase chain reaction
- Alzheimer's can manifest in patients apparently as well ⁇ 3 as ⁇ 2.
- the applicant has found a new mutation in the apolipoprotein E gene and has shown that this mutation was associated with a high probability of Alzheimer's disease.
- the Applicant has thus solved the problem set forth above by providing a means of distinguishing patients carrying an additional mutation of Apo E which, together with or independently of ⁇ 4, determines Alzheimer's.
- the subject of the present invention is a method for detecting, in a DNA preparation, a mutation intervening in Alzheimer's disease, characterized in that the presence, in the apolipoprotein E gene, of the presence of one of the following nucleotide sequences SEQ ID N * 1, SEQ ID N * 3 or SEQ ID No. 5: SEQ ID N1: TGC GGC CGC
- SEQ ID N'3 TGC CGC GAC
- SEQ ID NO 5 CGC GAC CGC
- the subject of the present invention is also a method for detecting such a mutation in an RNA preparation, as shown in FIGS. in which case the presence in the RNA corresponding to the apolipoprotein E gene is sought, either of the sequence SEQ ID N * 5, or of the sequences SEQ ID N * 7 and SEQ ID N * 8 respectively corresponding to the sequences SEQ ID N 1 and SEQ ID NO : 3 in which T has been replaced by U.
- the presence of the nucleotide sequence is evidenced by sequencing of DNA, or RNA, in which these sequences are sought.
- the determination of the presence of one of the nucleotide sequences is carried out by specific amplification of the DNA, in which these sequences are sought, that is to say that of the apolipoprotein E gene or part of this gene.
- the present invention further relates to nucleotide sequences comprising from about 20 to about 30 nucleotides, or base pairs, and comprising one of the sequences SEQ ID N * 3 or SEQ ID No. 5 described above.
- sequences and the sequence SEQ ID N * l may advantageously be used as oligo-probes for the specific amplification of the portion of the gene of 1 apolipoprotein E wherein tested for the presence of a mutation.
- the amplification of such a sequence may be carried out using primers not containing one of the sequences SEQ ID N * 1, SEQ ID No. 3 or SEQ ID No. 5.
- the purpose of the process according to the invention is to amplify a fragment of approximately 100 to 400 base pairs, capable of containing the desired mutation. Primers can therefore be chosen on either side of the region to be amplified.
- the specific amplification of the sequence likely to contain a mutation is preferentially carried out by the polymerase chain reaction (PCR) method, and in particular its ARMS (Allele Refractory Mutation System) variant.
- PCR polymerase chain reaction
- ARMS Allele Refractory Mutation System
- the products of the amplification reaction are identified by any method known to those skilled in the art for differentiating two DNA fragments, and in particular by staining with ethidium bromide or by hybridization with oligosondes.
- ARMS of the PCR method comprises the following steps: use of a common primer (C) and two differential primers; one corresponding to the normal allele (B), and the other to the mutated allele (A); the reaction is carried out simultaneously on the one hand between a common primer and a normal primer (AC), and on the other hand between a common primer and a mutated primer (BC).
- the mutation can also be simply demonstrated by digestion of the amplification product with an appropriate restriction enzyme, whose mutation (when present) alters the cleavage site: thus, the restriction enzyme Not I (GCGGCCGG ) normally cleaves the sequence once into two restriction fragments of 72 and 172 base pairs; when the mutation is present, a single fragment at 244 base pairs is then obtained.
- an appropriate restriction enzyme whose mutation (when present) alters the cleavage site: thus, the restriction enzyme Not I (GCGGCCGG ) normally cleaves the sequence once into two restriction fragments of 72 and 172 base pairs; when the mutation is present, a single fragment at 244 base pairs is then obtained.
- FIG. 1 is a representation of the DNA sequence of apo E amplified region ranging from the nucleotide sequence of No. 3869 to No. 3958 (upward arrows) (SEQ ID NO 9).
- the corresponding protein sequences range from 90 to 121 (downward arrows) (SEQ ID No. 10).
- the first two sites Hhal (GCGC) are underlined and the third, polymorphic ⁇ 2, ⁇ 3, ⁇ 4, box.
- the four polymorphic sites of the third letter of the codons are indicated.
- the sites corresponding to the new mutation are boxed (GGC - ⁇ Gly / GAC - ⁇ Asp).
- FIGS. 2A to 2C show agarose gels derived from the ARMS PCR genotyping method of the mutation.
- the common primer C is used jointly, on the one hand with the mutant primer A, and on the other hand with the normal primer B.
- the three kinds of genotypes are thus easily identifiable.
- the G-A mutation (3936) is present in eight control individuals, not suffering from Alzheimer's disease at an advanced age: two homozygotes 3-3 (nos 1559 and 145), two heterozygotes 2- 3 (Nos. 1542 and 1597), a 2-4 heterozygote (N'1149), two 3-4 heterozygotes (Nos. 1573 and 1542), and even a 4-4 homozygote ( No. 125).
- the mutation is present 21 times, that is in 50% of the cases.
- One embodiment of the invention uses the DNA sequencing technique, according to which direct sequencing is carried out using the enzyme sequenase (industrial kit), on an Applied Biosystems automatic sequencer apparatus.
- the non-coding strand was sequenced twice for each reaction, using the 5 '-TAAGCTTGGCACGGCTGTCCAAGGA-3' primer.
- Another embodiment of the invention easier to implement than the previous one, consists of to use the PCR technique for mutation detection.
- mutant primer B 3 '-CGGCGGACCACGTCATGGCG-5' (normal primer) are used in the ARMS PCR technique (allele refractory mutation system) with a common primer C located at approximately 300 bp in the adjacent intron.
- AC and BC under the following conditions: 1 ⁇ g of genomic DNA 1 pmol / ⁇ l of each primer, 10 ⁇ mol / ⁇ l of the dNTPs 10% DMSO
- the hybridization temperature is discriminant between the AC and BC reactions.
- the amplifying band observable in agarose gel makes it possible to carry out the genotype as illustrated in FIG.
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
Abstract
Method for detecting, in a DNA preparation, a mutation involved in Alzheimer's disease, by determining the presence, in the apolipoprotein E gene, of one of the following nucleotide sequences, designated as SEQ ID N°1, SEQ ID N°3 and SEQ ID N°5: SEQ ID N°1: TGC GGC CGC; SEQ ID N°3: TGC GAC CGC; SEQ ID N° 5: CGC GAC CGC. The determination is achieved by polymerase chain reaction (PCR) or sequencing techniques.
Description
Procédé de détection d'une mutation intervenant dans la maladie d'Alzheimer et séquences nucléotidiques pour sa mise en oeuvre Method for detecting a mutation intervening in Alzheimer's disease and nucleotide sequences for its implementation
La présente invention a pour objet un procédé de détection d'une nouvelle mutation intervenant dans la maladie d'Alzheimer.The present invention relates to a method for detecting a new mutation involved in Alzheimer's disease.
Elle est en outre relative à des séquences nucléotidiques pour la mise en oeuvre de ce procédé.It is further related to nucleotide sequences for the implementation of this method.
L'apolipoprotéine E (apo E) est une glycoprotéine plasmatique de 299 acides aminés, synthétisée principalement par le foie et l'intestin, mais aussi dans la plupart des autres tissus, notamment par l'astrocyte du tissu nerveux. La concentration normale en apo E du plasma est de l'ordre de 40 mg/1. Elle y est présente dans les lipoprotéines chylomicrons, VLDL, ILDL (ainsi que dans le liquide céphalo-rachidien) . On connaît la séquence de la protéine (RALL et al, 1982 J. Biol. Chem. , 257: 4171-4178), celle de l'ADN complémentaire, ainsi que la structure et la séquence du gène APO E (DAS et al, 1985, J. Biol. Chem., 260: 6240-6247; PAIK et al, 1985 Proc. Natl. Acad. Sci., 82:3445-3449 ). Le quatrième exon du gène code pour le domaine de la protéine apo E, qui participe à la liaison avec le récepteur LDL.Apolipoprotein E (apo E) is a 299 amino acid plasma glycoprotein, synthesized primarily by the liver and intestine, but also in most other tissues, including the astrocyte of nervous tissue. The normal apo E concentration of the plasma is of the order of 40 mg / l. It is present in lipoproteins chylomicrons, VLDL, ILDL (as well as in the cerebrospinal fluid). The sequence of the protein (RALL et al., 1982 J. Biol Chem., 257: 4171-4178), that of the complementary DNA, as well as the structure and sequence of the APO E gene (DAS et al. 1985, J. Biol Chem, 260: 6240-6247, PAIK et al., 1985 Proc Natl Acad Sci 82: 3445-3449). The fourth exon of the gene codes for the domain of the apo E protein, which participates in the binding with the LDL receptor.
Des différences dans la séquence d'ADN de cette région déterminent les trois isoformes communes de l'apo E (E2, E3 et E4). E2 et E4 diffèrent de E3 par des substitutions de bases: dans E4, C se trouve à la place de T en position 3932, et dans E2 se trouve T à la place de C en position 4070. Il en résulte les changements en acides aminés suivants: Cys (112) au lieu de Arg pour l'isoforme E4, et Arg (158) au lieu de Cys pour l'isoforme E2.Differences in the DNA sequence of this region determine the three common isoforms of apo E (E2, E3 and E4). E2 and E4 differ from E3 by base substitutions: in E4, C is in place of T in position 3932, and in E2 is T instead of C in position 4070. This results in the amino acid changes following: Cys (112) instead of Arg for the E4 isoform, and Arg (158) instead of Cys for the E2 isoform.
Ces trois isoformes de l'apo E sont
classiquement distinguées par isofocalisation des protéines du sérum. Il est actuellement plus aisé de les génotyper par amplification en chaîne par polymerase (PCR) sur la base de leur variation de séquence d'ADN (Hixson et Vernier, 1990, J. Lipid Res. , 31:545-548), par coupure, après amplification, avec l'enzyme de restriction Hha I.These three isoforms of apo E are classically distinguished by isofocalization of serum proteins. It is currently easier to genotype them by polymerase chain reaction (PCR) based on their DNA sequence variation (Hixson and Vernier, 1990, J. Lipid Res., 31: 545-548), by cleavage. , after amplification, with the restriction enzyme Hha I.
En 1993, Stritt atter et al. (Proc. Natl. Acad. Sci. USA, 90:1977-1981) ont montré que la fréquence allélique de AP0E-ε4 était de 0,50 + 0,06 dans trente familles de patients atteints par la forme tardive de la maladie d'Alzheimer, alors qu'elle n'était que de 0,16 + 0,03 chez les témoins. Les mêmes chercheurs ont retrouvé par la suite (Saunders et al., 1993, Neurology, 43:1467-1472) la nature élevée de l'association entre APO E-ε4 et la maladie d'Alzheimer pour 176 cas certains établis par autopsie.In 1993, Stritt, et al. (Proc Natl Acad Sci USA, 90: 1977-1981) showed that the allelic frequency of AP0E-ε4 was 0.50 + 0.06 in thirty families of patients with late-onset disease. Alzheimer's, whereas it was only 0.16 + 0.03 in the controls. The same researchers later found (Saunders et al., 1993, Neurology, 43: 1467-1472) the high nature of the association between APO E-ε4 and Alzheimer's disease for 176 cases some established by autopsy.
La même année, Lucotte et al. (Lancet,In the same year, Lucotte et al. (Lancet,
342:1309) ont étendu cette association aux patients atteints par la forme sporadique, qui représente l'essentiel (plus de 95%) des cas d'Alzheimer. Ces résultats ont été confirmés ultérieurement par plus d'une cinquantaine de laboratoires indépendants de par le monde. II ressort donc de l'état de la technique que l'on connaissait déjà des procédés d'analyse génétique permettant de détecter, au niveau de l'ADN de patients, s'ils étaient porteurs d'une mutation du gène Apo E liée à la maladie d'Alzheimer. Cependant, tous les cas d'Alzheimer ne sont pas liés au variant ε4 de l'Apo E, et à l'inverse d'autres342: 1309) have extended this association to patients suffering from the sporadic form, which represents the essential (more than 95%) of Alzheimer's cases. These results were subsequently confirmed by more than fifty independent laboratories around the world. It is thus apparent from the state of the art that genetic analysis methods were already known for detecting, at the level of the DNA of patients, whether they carried a mutation of the Apo E gene linked to Alzheimer's disease. However, not all cases of Alzheimer's are linked to the Apo E variant ε4, and in contrast to others
Alzheimer peuvent se manifester chez des patients apparemment aussi bien ε3 que ε2.Alzheimer's can manifest in patients apparently as well ε3 as ε2.
La demanderesse a trouvé une nouvelle mutation dans le gène de l 'apolipoprotéine E et a montré que
cette mutation était associée avec une forte probabilité à la maladie d'Alzheimer.The applicant has found a new mutation in the apolipoprotein E gene and has shown that this mutation was associated with a high probability of Alzheimer's disease.
La demanderesse a donc résolu le problème exposé ci-dessus en fournissant le moyen de distinguer les patients porteurs d'une mutation supplémentaire de l'Apo E qui, conjointement à ε4 ou indépendamment d'elle, détermine l'Alzheimer.The Applicant has thus solved the problem set forth above by providing a means of distinguishing patients carrying an additional mutation of Apo E which, together with or independently of ε4, determines Alzheimer's.
La présente invention a pour objet un procédé de détection, dans une préparation d'ADN, d'une mutation intervenant dans la maladie d'Alzheimer caractérisé en ce que l'on détermine la présence, dans le gène de 1 'apolipoprotéine E, d'une des séquences nucléotidiques SEQ ID N* 1, SEQ ID N*3 ou SEQ ID N°5 suivantes: SEQ ID N'1 : TGC GGC CGCThe subject of the present invention is a method for detecting, in a DNA preparation, a mutation intervening in Alzheimer's disease, characterized in that the presence, in the apolipoprotein E gene, of the presence of one of the following nucleotide sequences SEQ ID N * 1, SEQ ID N * 3 or SEQ ID No. 5: SEQ ID N1: TGC GGC CGC
SEQ ID N'3 : TGC GAC CGC SEQ ID N*5: CGC GAC CGC Ces trois séquences correspondent respectivement aux séquences d'acides aminés SEQ ID N°2, SEQ ID N*4 et SEQ ID N*6 suivantes:SEQ ID N'3: TGC CGC GAC SEQ ID NO 5: CGC GAC CGC These three sequences correspond to amino acid sequences SEQ ID NO: 2, SEQ ID No. 4 and SEQ ID No. 6 following:
SEQ ID N'2 : Cys Gly Arg SEQ ID N"4 : Cys Asp Arg SEQ ID N' 6 : Arg Asp Arg La présente invention a aussi pour objet un procédé de détection d'une telle mutation dans une préparation d'ARN, auquel cas on recherche la présence dans l'ARN correspondant au gène de 1 *apolipoprotéine E, soit de la séquence SEQ ID N*5, soit des séquences SEQ ID N*7 et SEQ ID N* 8 correspondant respectivement aux séquences SEQ ID N' 1 et SEQ ID N°3 dans lesquelles T a été remplacé par U.The subject of the present invention is also a method for detecting such a mutation in an RNA preparation, as shown in FIGS. in which case the presence in the RNA corresponding to the apolipoprotein E gene is sought, either of the sequence SEQ ID N * 5, or of the sequences SEQ ID N * 7 and SEQ ID N * 8 respectively corresponding to the sequences SEQ ID N 1 and SEQ ID NO : 3 in which T has been replaced by U.
SEQ ID N'7 : UGC GGC CGC SEQ ID N' 8: UGC GAC CGC Selon un premier mode de mise en oeuvre de l'invention, la présence de la séquence nucléotidique
est mise en évidence par séquençage de l'ADN, ou d'ARN, dans lequel on recherche ces séquences.SEQ ID NO: 8 UGC GGC CGC SEQ ID NO: 8 UGC GAC CGC According to a first embodiment of the invention, the presence of the nucleotide sequence is evidenced by sequencing of DNA, or RNA, in which these sequences are sought.
Des méthodes de séquençage sont bien connues de l'homme du métier et sont en particulier décrites dans MAILLET-BARON "Séquençage des acides nucléiques"Sequencing methods are well known to those skilled in the art and are in particular described in MAILLET-BARON "Sequencing of nucleic acids"
Editions Lavoisier, 1992, Paris auquel on peut se référer pour la mise en oeuvre de telles méthodes.Lavoisier Editions, 1992, Paris to which we can refer for the implementation of such methods.
Selon un second mode de mise en oeuvre de l'invention, la détermination de la présence d'une des séquences nucléotidiques est effectuée par amplification spécifique de l'ADN, dans lequel on recherche ces séquences, c'est-à-dire celui du gène de 1 'apolipoprotéine E ou d'une partie de ce gène.According to a second embodiment of the invention, the determination of the presence of one of the nucleotide sequences is carried out by specific amplification of the DNA, in which these sequences are sought, that is to say that of the apolipoprotein E gene or part of this gene.
Des exemples illustratifs de ces modes de mise en oeuvre sont décrits ci-après.Illustrative examples of these modes of implementation are described below.
La présente invention est en outre relative à des séquences nucléotidiques comprenant d'environ 20 à environ 30 nucléotides, ou paires de bases, et comprenant l'une des séquences SEQ ID N*3 ou SEQ ID N°5 décrites ci-dessus. De telles séquences ainsi que la séquence SEQ ID N*l peuvent être avantageusement utilisées comme oligosondes pour l'amplification spécifique de la partie du gène de 1 'apolipoprotéine E dans laquelle on recherche la présence d'une mutation. On notera néanmoins que l'amplification d'une telle séquence peut être effectuée à l'aide d'amorces ne contenant pas l'une des séquences SEQ ID N* 1, SEQ ID N°3 ou SEQ ID N°5. En effet, le but du procédé selon l'invention est d'amplifier un fragment d'environ 100 à 400 paires de bases, susceptible de contenir la mutation recherchée. Des amorces peuvent donc être choisies de part et d'autre de la région à amplifier.The present invention further relates to nucleotide sequences comprising from about 20 to about 30 nucleotides, or base pairs, and comprising one of the sequences SEQ ID N * 3 or SEQ ID No. 5 described above. Such sequences and the sequence SEQ ID N * l may advantageously be used as oligo-probes for the specific amplification of the portion of the gene of 1 apolipoprotein E wherein tested for the presence of a mutation. It should nevertheless be noted that the amplification of such a sequence may be carried out using primers not containing one of the sequences SEQ ID N * 1, SEQ ID No. 3 or SEQ ID No. 5. Indeed the purpose of the process according to the invention is to amplify a fragment of approximately 100 to 400 base pairs, capable of containing the desired mutation. Primers can therefore be chosen on either side of the region to be amplified.
L'amplification spécifique de la séquence susceptible de contenir une mutation est
preferentiellement effectuée par la méthode de réaction de polymérisation en chaîne (PCR) , et en particulier sa variante ARMS ( Allele Refractory Mutation System) . Les produits de la réaction d'amplification sont identifiés par toute méthode connue de l'homme du métier permettant de différencier deux fragments d'ADN, et en particulier par coloration à l'aide du bromure d'éthidium ou par hybridation avec des oligosondes.The specific amplification of the sequence likely to contain a mutation is preferentially carried out by the polymerase chain reaction (PCR) method, and in particular its ARMS (Allele Refractory Mutation System) variant. The products of the amplification reaction are identified by any method known to those skilled in the art for differentiating two DNA fragments, and in particular by staining with ethidium bromide or by hybridization with oligosondes.
Pour la mise en oeuvre de la méthode PCR, l'homme du métier peut consulter de manière avantageuse: LARZUL, " Un procédé de réplication in vitro", Editions Lavoisier, 1993, Paris. On rappelera néanmoins que la variante diteFor the implementation of the PCR method, one skilled in the art can advantageously consult: LARZUL, "An in vitro replication process", Editions Lavoisier, 1993, Paris. It will be recalled however that the so-called variant
ARMS de la méthode PCR comprend les étapes suivantes : utilisation d'une amorce commune (C) et de deux amorces différentielles; l'une correspondant à 1*allele normal (B) , et l'autre à l'allele muté (A); la réaction est effectuée simultanément d'une part entre amorce commune et amorce normale (AC) , et d'autre part entre amorce commune et amorce mutée (BC).ARMS of the PCR method comprises the following steps: use of a common primer (C) and two differential primers; one corresponding to the normal allele (B), and the other to the mutated allele (A); the reaction is carried out simultaneously on the one hand between a common primer and a normal primer (AC), and on the other hand between a common primer and a mutated primer (BC).
La mutation peut aussi être mise simplement en évidence par digestion du produit d'amplification par une enzyme de restriction appropriée, dont la mutation (lorsqu'elle est présente) altère le site de coupure: ainsi, l'enzyme de restriction Not I (GCGGCCGG) coupe normalement la séquence une fois en deux fragments de restriction de 72 et 172 paires de bases; lorsque la mutation est présente, un seul fragment à 244 paires de bases est alors obtenu.The mutation can also be simply demonstrated by digestion of the amplification product with an appropriate restriction enzyme, whose mutation (when present) alters the cleavage site: thus, the restriction enzyme Not I (GCGGCCGG ) normally cleaves the sequence once into two restriction fragments of 72 and 172 base pairs; when the mutation is present, a single fragment at 244 base pairs is then obtained.
La présente invention est illustrée sans être aucunement limitée, par les exemples qui suivent : La figure 1 est une représentation de la
séquence ADN de la région apo E amplifiée, allant de la séquence nucléotidique n' 3869 à la n* 3958 (flèches montantes) (SEQ ID N*9). Les séquences proteiques correspondantes vont de 90 à 121 (flèches descendantes) (SEQ ID N'10). Les deux premiers sites Hhal (GCGC) sont soulignés et le troisième, polymorphe ε2, ε3,ε4, encadré. Les quatres sites polymorphes de la troisième lettre des codons sont indiqués. Les sites correspondants à la nouvelle mutation sont encadrés (GGC —^Gly/GAC —^ Asp) ..The present invention is illustrated without being limited in any way by the following examples: FIG. 1 is a representation of the DNA sequence of apo E amplified region ranging from the nucleotide sequence of No. 3869 to No. 3958 (upward arrows) (SEQ ID NO 9). The corresponding protein sequences range from 90 to 121 (downward arrows) (SEQ ID No. 10). The first two sites Hhal (GCGC) are underlined and the third, polymorphic ε2, ε3, ε4, box. The four polymorphic sites of the third letter of the codons are indicated. The sites corresponding to the new mutation are boxed (GGC - ^ Gly / GAC - ^ Asp).
Les figures 2A à 2C représentent des gels d'agarose issus du procédé de génotypage par PCR ARMS de la mutation. L'amorce commune C est utilisée conjointement, d'une part avec l'amorce mutante A, et d'autre part avec l'amorce normale B. Dans les conditions de température d'hybridation décrites dans le texte, les trois sortes de génotypes: +/+ (une bande BC) , +/m (deux bandes d'égale intensité deux fois moindre) , et m/m (une bande AC) , correspondant respectivement aux figures 2A, 2B et 2C, sont ainsi facilement identifiables. EXEMPLE 1-FIGS. 2A to 2C show agarose gels derived from the ARMS PCR genotyping method of the mutation. The common primer C is used jointly, on the one hand with the mutant primer A, and on the other hand with the normal primer B. Under the hybridization temperature conditions described in the text, the three kinds of genotypes : + / + (a BC band), + / m (two bands of equal intensity twice less), and m / m (an AC band), respectively corresponding to Figures 2A, 2B and 2C, are thus easily identifiable. EXAMPLE 1-
Mise en évidence de la mutation en position 3936 du gène codant pour 1 'apolipoprotéine E. Par séquençage direct du produit PCR obtenu selon Hixson et Vernier (précédemment cités) de la séquence d'ADN du gène de 1 'apolipoprotéine E, correspondant aux positions 3869 à 3955 chez 29 individus différents, sains ou atteints de la maladie d'Alzheimer et génotypes par APOE, trois types de variation ont été mis en évidence (figure 1) .Demonstration of the mutation at position 3936 of the gene coding for apolipoprotein E. By direct sequencing of the PCR product obtained according to Hixson and Vernier (previously mentioned) of the DNA sequence of the apolipoprotein E gene, corresponding to the positions 3869 to 3955 in 29 different individuals, healthy or with Alzheimer's disease and genotypes by APOE, three types of variation were highlighted (Figure 1).
Chez les 4 on retrouve le remplacement de T par C (3932), aboutissant au niveau protéique à la substitution de Cys par Arg (112). Quatre polymorphismes, correspondant à des
remplacements portant sur la troisième base des codons, et neutres du point de vue protéique: G par T (3877), G par A (3886), G par T (3889), et G par T (3946) sont mis en évidence. Enfin et surtout, il a été montré, chez la plupart des malades Alzheimer ε4 une substitution G par A en position 3936, devant déterminer au niveau protéique la substitution Gly par Asp en position 113. Cette dernière substitution, déduite de celle détectée au niveau de l'ADN, est susceptible d'avoir d'importantes conséquences sur la structure de l'APOE.In the 4 we find the replacement of T by C (3932), resulting in the protein level to the substitution of Cys by Arg (112). Four polymorphisms, corresponding to replacements on the third base of the codons, and protein neutral: G by T (3877), G by A (3886), G by T (3889), and G by T (3946) are highlighted. Finally, and most importantly, in most Alzheimer patients ε4, a G-substitution at position 3936 has been shown to determine at the protein level the Gly substitution by Asp in position 113. This latter substitution, deduced from that detected at the level of DNA, is likely to have important consequences on the structure of APOE.
Parmi les 29 individus séquences, la mutation G par A (3936) est présente chez huit individus témoins, non atteints de la maladie d'Alzheimer à un âge avancé: deux homozygotes 3-3 (Nos 1559 et 145), deux hétérozygotes 2-3 (Nos 1542 et 1597), un hétérozygote 2-4 (N'1149), deux hétérozygotes 3-4 (Nos 1573 et 1542), et même un homozygote 4-4 (N° 125).Of the 29 individuals sequenced, the G-A mutation (3936) is present in eight control individuals, not suffering from Alzheimer's disease at an advanced age: two homozygotes 3-3 (nos 1559 and 145), two heterozygotes 2- 3 (Nos. 1542 and 1597), a 2-4 heterozygote (N'1149), two 3-4 heterozygotes (Nos. 1573 and 1542), and even a 4-4 homozygote ( No. 125).
Elle est par contre présente à l'état homozygote chez cinq malades Alzheimer probables 4-4 (Nos 65, 100, 203, 214 et 265) et un malade Alzheimer certain 3-3 (n*81). Elle est aussi présente à l'état homozygote chez un individu à risque primaire 3-3 (n*207), et à l'état hétérozygote chez quatre autres individus à risque primaire 3-3 (Nos 159, 160 , 206 et 209). Elle est également présente à l'état hétérozygote chez deux autres malades Alzheimer probables 3-4 (Nos 233 et 236) et un autre malade Alzheimer probable 3-3 (n° 235). Elle est cependant absente chez sept autres malades Alzheimer probables: quatre 3-3 (Nos 215, 226, 231 et 232), un 2-3 (n" 229), et deux 3-4 (Nos 213 et 227).It is against this in the homozygous state in five probable Alzheimer patients 4-4 (Nos 65, 100, 203, 214 and 265) and some sick Alzheimer 3-3 (No. 81). It is also present in the homozygous state in an individual at primary risk 3-3 (n * 207), and in the heterozygous state in four other individuals at primary risk 3-3 (nos 159, 160, 206 and 209) . It is also present in the heterozygous state in two other patients probable Alzheimer 3-4 (Nos 233 and 236) and another patient probable Alzheimer 3-3 (No. 235). However, it is absent in seven other probable Alzheimer's patients: four 3-3 (Nos. 215, 226, 231 and 232), one 2-3 (No. 229), and two 3-4 (Nos. 213 and 227).
Au total, chez vingt et un malades Alzheimer probables, certains, ou à risque primaire (42 allèles), la mutation est présente 21 fois, soit dans
50% des cas .In total, in twenty-one probable Alzheimer patients, some, or at primary risk (42 alleles), the mutation is present 21 times, that is in 50% of the cases.
Dix huit fois sur vingt et une ( 86% des cas), elle est associée à au moins un allele ε4.Eighteen times out of twenty-one (86% of cases), it is associated with at least one ε4 allele.
L'association observée est explicable en termes de déséquilibre de liaison entre deux sites mutés correspondant à des codons contigus.The observed association is explicable in terms of linkage disequilibrium between two mutated sites corresponding to contiguous codons.
Le microséquençage après digestion peptidique de l'apo E chez un malade Alzheimer probable homozygote 4-4 et présentant à l'état homozygote la mutation G par A (3936) montre que le résidu Asp (113) est effectivement présent.Microsequencing after peptide digestion of apo E in a probable 4-4 homozygous Alzheimer's patient with homozygous G-A mutation (3936) shows that the Asp residue (113) is indeed present.
Un mode de mise en oeuvre de l'invention utilise la technique de séquençage d'ADN, selon laquelle on réalise un séquençage direct par utilisation de l'enzyme séquenase (kit industriel), sur un appareil séquenceur automatique Applied Biosystems. Le brin non-codant a été séquence deux fois pour chaque réaction, par utilisation de l'amorce 5 '-TAAGCTTGGCACGGCTGTCCAAGGA-3 ' Un autre mode de mise en oeuvre de l'invention, plus facile à mettre en oeuvre que le précédent, consiste à utiliser la technique PCR pour la détection de la mutation.One embodiment of the invention uses the DNA sequencing technique, according to which direct sequencing is carried out using the enzyme sequenase (industrial kit), on an Applied Biosystems automatic sequencer apparatus. The non-coding strand was sequenced twice for each reaction, using the 5 '-TAAGCTTGGCACGGCTGTCCAAGGA-3' primer. Another embodiment of the invention, easier to implement than the previous one, consists of to use the PCR technique for mutation detection.
Deux amorces différentielles: A: 3'-TGGCGGACCACGTCATGGCG-5'Two differential primers: A: 3'-TGGCGGACCACGTCATGGCG-5 '
(amorce mutante) B: 3 '-CGGCGGACCACGTCATGGCG-5 ' (amorce normale) sont utilisées en technique PCR ARMS (allele refractory mutation System) avec une amorce commune C située à environ 300 pb dans l'intron adjacent. Pour chaque échantillon sont réalisées deux PCR simultanées: AC et BC dans les conditions suivantes: 1 μg d'ADN génomique 1 pmol/μl de chaque amorce, lOμmol/μl des dNTP
10% DMSO(mutant primer) B: 3 '-CGGCGGACCACGTCATGGCG-5' (normal primer) are used in the ARMS PCR technique (allele refractory mutation system) with a common primer C located at approximately 300 bp in the adjacent intron. For each sample, two simultaneous PCRs are performed: AC and BC under the following conditions: 1 μg of genomic DNA 1 pmol / μl of each primer, 10 μmol / μl of the dNTPs 10% DMSO
0,025 U/μl de Taq polymerase0.025 U / μl of Taq polymerase
dans un volume de 50 μl de tampon (Tris-HCl, 10 mM, pH=8,3; 50 mM KC1; 1,5 mM MgCl2) la réaction est de 30 cycles 95*C 1 minutein a volume of 50 .mu.l of buffer (Tris-HCl 10 mM, pH = 8.3; 50 mM KC1; 1.5 mM MgCl 2) the reaction is 30 cycles 95 ° C 1 min
(dénaturation)(denaturation)
64"C 1 minute (hybridation)64 "C 1 minute (hybridization)
70'C 2 minutes70'C 2 minutes
(élongation) .(elongation).
La température d'hybridation est discriminante entre les réactions AC et BC. La bande d'amplifiât observable en gel d'agarose permet de réaliser le génotype comme illustré à la figure 2.
The hybridization temperature is discriminant between the AC and BC reactions. The amplifying band observable in agarose gel makes it possible to carry out the genotype as illustrated in FIG.
LISTE DE SEQUENCESSEQUENCE LIST
(1) INFORMATIONS GENERALES:(1) GENERAL INFORMATION:
(i) DEPOSANT:(i) DEPOSITOR:
(A) NOM: LABORATOIRES EUROBIO(A) NAME: EUROBIO LABORATORIES
(B) RUE: 7, Avenue de Scandinavie(B) STREET: 7, Scandinavia Avenue
(C) VILLE: LES ULIS(C) CITY: THE ULIS
(E) PAYS: FRANCE(E) COUNTRY: FRANCE
(F) CODE POSTAL: 91953(F) POSTAL CODE: 91953
(G) TELEPHONE: 69 07 94 77 (H) TELECOPIE: 69 07 95 34 (I) TELEX: 681 425 F(G) TELEPHONE: 69 07 94 77 (H) TELEPHONE: 69 07 95 34 (I) TELEX: 681 425 F
(ii) TITRE DE L' INVENTION: PROCEDE DE DETECTION D'UNE MUTATION INTERVENANT DANS LA MALADIE D'ALZHEIMER ET SEQUENCES NUCLEOTIDIQUES POUR SA MISE EN OEUVRE(ii) TITLE OF THE INVENTION: METHOD FOR DETECTING ALZHEIMER DISEASE MUTATION AND NUCLEOTIDE SEQUENCES FOR ITS IMPLEMENTATION
(iii) NOMBRE DE SEQUENCES: 10(iii) NUMBER OF SEQUENCES: 10
(iv) FORME DECHIFFRABLE PAR ORDINATEUR:(iv) COMPUTER-DEPENDABLE FORM:
(A) TYPE DE SUPPORT: Floppy disk(A) SUPPORT TYPE: Floppy disk
(B) ORDINATEUR: IBM PC compatible(B) COMPUTER: IBM PC compatible
(C) SYSTEME D" EXPLOITATION: PC-DOS/MS-DOS(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) LOGICIEL: Patentin Release #1.0, Version #1.30 (OEB)(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPO)
(vi) DONNEES DE LA DEMANDE ANTERIEURE:(vi) DATA FROM THE PRIOR APPLICATION:
(A) NUMERO DE LA DEMANDE: FR 95 01 884(A) APPLICATION NUMBER: FR 95 01 884
(B) DATE DE DEPOT: 17-FEB-1995(B) DEPOSIT DATE: 17-FEB-1995
(2) INFORMATIONS POUR LA SEQ ID NO: 1:(2) INFORMATION FOR SEQ ID NO: 1:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 9 paires de bases(A) LENGTH: 9 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: double(C) NUMBER OF BRINS: double
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN (génomique)(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETIQUE: NON(iii) Hypothesis: No
(vi) ORIGINE:(vi) ORIGIN:
(A) ORGANISME: Homo sapiens(A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 1; TGCGGCCGC
(2) INFORMATIONS POUR LA SEQ ID NO: 2:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1; TGCGGCCGC (2) INFORMATION FOR SEQ ID NO: 2:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 3 acides aminés(A) LENGTH: 3 amino acids
(B) TYPE: acide aminé(B) TYPE: amino acid
(C) NOMBRE DE BRINS:(C) NUMBER OF STRANDS:
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide(ii) TYPE OF MOLECULE: peptide
(iii) HYPOTHETIQUE: NON(iii) Hypothesis: No
(vi) ORIGINE:(vi) ORIGIN:
(A) ORGANISME: Homo sapiens(A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 2:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
Cys Gly ArgCys Gly Arg
11
(2) INFORMATIONS POUR LA SEQ ID NO: 3:(2) INFORMATION FOR SEQ ID NO: 3:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 9 paires de bases(A) LENGTH: 9 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: double(C) NUMBER OF BRINS: double
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN (génomique)(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETIQUE: NON(iii) Hypothesis: No
(vi) ORIGINE:(vi) ORIGIN:
(A) ORGANISME: Homo sapiens(A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 3; TGCGACCGC (2) INFORMATIONS POUR LA SEQ ID NO: 4:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3; TGCGACCGC (2) INFORMATION FOR SEQ ID NO: 4:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 3 acides aminés(A) LENGTH: 3 amino acids
(B) TYPE: acide aminé(B) TYPE: amino acid
(C) NOMBRE DE BRINS:(C) NUMBER OF STRANDS:
(D) CONFIGURATION: linéaire
(ii) TYPE DE MOLECULE: ADN (génomique)(D) CONFIGURATION: linear (ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETIQUE: NON(iii) Hypothesis: No
(vi) ORIGINE:(vi) ORIGIN:
(A) ORGANISME: Homo sapiens(A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 4:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
Cys Asp Arg 1Cys Asp Arg 1
(2) INFORMATIONS POUR LA SEQ ID NO: 5:(2) INFORMATION FOR SEQ ID NO: 5:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 9 paires de bases(A) LENGTH: 9 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: double(C) NUMBER OF BRINS: double
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN (génomique)(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETIQUE: NON(iii) Hypothesis: No
(vi) ORIGINE:(vi) ORIGIN:
(A) ORGANISME: Homo sapiens(A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 5: CGCGACCGC (2) INFORMATIONS POUR LA SEQ ID NO: 6:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: CGCGACCGC (2) INFORMATION FOR SEQ ID NO: 6:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 3 acides aminés(A) LENGTH: 3 amino acids
(B) TYPE: acide aminé(B) TYPE: amino acid
(C) NOMBRE DE BRINS:(C) NUMBER OF STRANDS:
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide(ii) TYPE OF MOLECULE: peptide
(iii) HYPOTHETIQUE: NON(iii) Hypothesis: No
( vi ) ORIGINE :(vi) ORIGIN:
(A) ORGANISME : Homo sapiens
( i) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 6:(A) ORGANIZATION: Homo sapiens (i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
Arg Asp Arg 1Arg Asp Arg 1
(2) INFORMATIONS POUR LA SEQ ID NO: 7:(2) INFORMATION FOR SEQ ID NO: 7:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 9 paires de bases(A) LENGTH: 9 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ARNm(ii) MOLECULE TYPE: mRNA
(iii) HYPOTHETIQUE: NON(iii) Hypothesis: No
(vi) ORIGINE:(vi) ORIGIN:
(A) ORGANISME: Homo sapiens(A) ORGANIZATION: Homo sapiens
( i) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 7: UGCGGCCGC (2) INFORMATIONS POUR LA SEQ ID NO: 8:(i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: UGCGGCCGC (2) INFORMATION FOR SEQ ID NO: 8:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 9 paires de bases(A) LENGTH: 9 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ARNm(ii) MOLECULE TYPE: mRNA
(iii) HYPOTHETIQUE: NON(iii) Hypothesis: No
(vi) ORIGINE:(vi) ORIGIN:
(A) ORGANISME: Homo sapiens(A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 8: UGCGACCGC (2) INFORMATIONS POUR LA SEQ ID NO: 9:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: UGCGACCGC (2) INFORMATION FOR SEQ ID NO: 9:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 90 paires de bases(A) LENGTH: 90 base pairs
(B) TYPE: nucléotide
(C) NOMBRE DE BRINS: simple(B) TYPE: nucleotide (C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADN (génomique)(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETIQUE: NON(iii) Hypothesis: No
(vi) ORIGINE:(vi) ORIGIN:
(A) ORGANISME: Homo sapiens(A) ORGANIZATION: Homo sapiens
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: allele(A) NAME / KEY: allele
(B) EMPLACEMENT:replace(9, "t")(B) LOCATION: replace (9, "t")
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: allele(A) NAME / KEY: allele
(B) EMPLACEMENT:replace(18, "a")(B) LOCATION: replace (18, "a")
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: allele(A) NAME / KEY: allele
(B) EMPLACEMENT:replace(21, "t")(B) LOCATION: replace (21, "t")
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: allele(A) NAME / KEY: allele
(B) EMPLACEMENT:replace(64, "c")(B) LOCATION: replace (64, "c")
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: allele(A) NAME / KEY: allele
(B) EMPLACEMENT:replace(68, "a")(B) LOCATION: replace (68, "a")
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: allele(A) NAME / KEY: allele
(B) EMPLACEMENT:replace(78, "t")(B) LOCATION: replace (78, "t")
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 9: GCACGGCTGT CCAAGGAGCT GCAGGCGGCG CAGGCCCGGC TGGGCGCGGA CATGGAGGAC GTGTGCGGCC GCCTGGTGCA GTACCGCGGC (2) INFORMATIONS POUR LA SEQ ID NO: 10:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: GCACGGCTGT CCAAGGAGCT GCAGGCGGCG CAGGCCCGGC TGGGCGCGGA CATGGAGGAC GTGTGCGGCC GCCTGGTGCA GTACCGCGGC (2) INFORMATION FOR SEQ ID NO: 10:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 30 acides aminés(A) LENGTH: 30 amino acids
(B) TYPE: acide aminé(B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide
(iii) HYPOTHETIQUE: NON(ii) TYPE OF MOLECULE: peptide (iii) Hypothesis: No
(vi) ORIGINE:(vi) ORIGIN:
(A) ORGANISME: Homo sapiens(A) ORGANIZATION: Homo sapiens
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: Modified-site(A) NAME / KEY: Modified-site
(B) EMPLACEMENT:22(B) LOCATION: 22
(D) AUTRES INFORMATIONS:/product= "arg"(D) OTHER INFORMATION: / product = "arg"
(ix) CARACTERISTIQUE:(ix) CHARACTERISTIC:
(A) NOM/CLE: Modified-site(A) NAME / KEY: Modified-site
(B) EMPLACEMENT:23(B) LOCATION: 23
(D) AUTRES INFORMATIONS:/product= "asp"(D) OTHER INFORMATION: / product = "asp"
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 10:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:
Ala Arg Leu Ser Lys Glu Leu Gin Ala Ala Gin Ala Arg Leu Gly Ala 1 5 10 15Ala Arg Leu Ser Lys Glu Leu Gin Ala Ala Gin Ala Arg Leu Gly Ala 1 5 10 15
Asp Met Glu Asp Val Cys Gly Arg Leu Val Gin Tyr Arg GlyAsp Met Glu Asp Val Cys Gly Arg Leu Val Gin Tyr Arg Gly
20 25 30
20 25 30
Claims
1. Procédé de détection, dans une préparation d'ADN, d'une mutation intervenant dans la maladie d'Alzheimer caractérisé en ce que l'on détermine la présence, dans le gène de l 'apolipoprotéine E, d'une des séquences nucléotidiques SEQ ID N*l, SEQ ID N*3 ou SEQ ID N*5 suivantes:1. A method for detecting, in a DNA preparation, a mutation intervening in Alzheimer's disease characterized in that the presence in the apolipoprotein E gene of one of the nucleotide sequences is determined. SEQ ID N * 1, SEQ ID N * 3 or SEQ ID N * 5:
SEQ ID N'1: TGC GGC CGC SEQ ID N*3: TGC GAC CGCSEQ ID N'1: TGC GGC CGC SEQ ID NO 3: GAC TGC CGC
SEQ ID N'5: CGC GAC CGCSEQ ID NO: CGC GAC CGC
2. Procédé selon la revendication 1 caractérisé en ce que la présence de la séquence nucléotidique est déterminée par séquençage de l'ADN. 2. Method according to claim 1 characterized in that the presence of the nucleotide sequence is determined by sequencing of the DNA.
3. Procédé selon la revendication 1 caractérisé en ce que la présence de la séquence nucléotidique est déterminée par amplification spécifique de l'ADN du gène de l 'apolipoprotéine E ou d'une partie de ce gène. 3. Method according to claim 1 characterized in that the presence of the nucleotide sequence is determined by specific amplification of the DNA of the apolipoprotein E gene or a part of this gene.
4. Procédé selon la revendication 3 caractérisé en ce que l'amplification est effectuée par la méthode d'amplification en chaîne par polymerase (PCR).4. Method according to claim 3 characterized in that the amplification is performed by the polymerase chain reaction method (PCR).
5. Séquence nucléotidique caractérisée en ce qu'elle comprend l'une des séquences SEQ ID N*3, ou SEQ ID N'5.5. Nucleotide sequence characterized in that it comprises one of the sequences SEQ ID N * 3, or SEQ ID N'5.
6. Oligosondes pour réaction d'amplification en chaîne par polymerase ( PCR) présentant la séquence selon la revendication 5.6. Oligosondes for polymerase chain reaction (PCR) having the sequence of claim 5.
7. Procédé selon la revendication 4, caractérisé en ce que la réaction est mise en oeuvre à l'aide d'amorces oligonucléotidiques dans le gène choisies de part et d'autre de la région à amplifier.7. Method according to claim 4, characterized in that the reaction is carried out using oligonucleotide primers in the gene chosen on either side of the region to be amplified.
8. Procédé selon la revendication 7 caractérisé en ce que l'une des amorces est celle selon la revendication 6.8. Process according to claim 7, characterized in that one of the primers is that according to claim 6.
9. Produit issu de l'amplification par un procédé selon l'une des revendications 3, 4, 7 et 8 caractérisé en ce qu'il présente une taille comprise entre environ 100 et 400 paires de bases.9. Product resulting from amplification by a Process according to one of Claims 3, 4, 7 and 8, characterized in that it has a size of between approximately 100 and 400 base pairs.
10. Procédé de détection dans une préparation d'ARN d'une mutation intervenant dans la maladie d'Alzheimer caractérisé en ce que l'on détermine la présence, dans l'ARN correspondant au gène de 1 'apolipoprotéine E, d'une des séquences SEQ ID N*5, SEQ ID N*7 ou SEQ ID N*8 suivantes: SEQ ID N' 7 UGC GGC CGC10. A method for detection in an RNA preparation of a mutation intervening in Alzheimer's disease, characterized in that the presence, in the RNA corresponding to the apolipoprotein E gene, of one of the sequences SEQ ID N * 5, SEQ ID N * 7 or SEQ ID N * 8 following: SEQ ID No. 7 UGC GGC CGC
SEQ ID N' 8 UGC GAC CGCSEQ ID NO: 8 UGC GAC CGC
SEQ ID N" 5 CGC GAC CGCSEQ ID NO: 5 CGC GAC CGC
11. Séquence nucléotidique caractérisée en ce qu'elle comprend l'une des séquences SEQ ID N*7 ou SEQ ID N°8. 11. Nucleotide sequence characterized in that it comprises one of the sequences SEQ ID N * 7 or SEQ ID No. 8.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR95/01884 | 1995-02-17 | ||
FR9501884A FR2730745B1 (en) | 1995-02-17 | 1995-02-17 | METHOD FOR DETECTING MUTATION IN ALZHEIMER'S DISEASE AND NUCLEOTIDE SEQUENCES FOR IMPLEMENTING IT |
Publications (2)
Publication Number | Publication Date |
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WO1996025517A1 WO1996025517A1 (en) | 1996-08-22 |
WO1996025517A9 true WO1996025517A9 (en) | 1996-11-28 |
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PCT/FR1996/000263 WO1996025517A1 (en) | 1995-02-17 | 1996-02-19 | Method for detecting a mutation involved in alzheimer's disease and nucleotide sequences for implementing same |
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FR (1) | FR2730745B1 (en) |
WO (1) | WO1996025517A1 (en) |
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US6156503A (en) * | 1997-03-03 | 2000-12-05 | The Regents Of The University Of California | Diagnosing asthma patients predisposed to adverse β-agonist reactions |
DE19855469C2 (en) * | 1998-12-01 | 2001-01-11 | Michael Esrich | Method for determining the apolipoprotein E genotype in a human sample |
CN108588207A (en) * | 2018-04-02 | 2018-09-28 | 南昌艾迪康医学检验实验室有限公司 | Detect the other primer of APOE genotype and method |
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DE625212T1 (en) * | 1992-10-13 | 2000-11-02 | Duke University, Durham | METHOD FOR DISCOVERING DISEASE OF ALZHEIMER. |
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1995
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1996
- 1996-02-19 WO PCT/FR1996/000263 patent/WO1996025517A1/en active Application Filing
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