FR2817558A1 - Determining risk of atherosclerosis and related diseases, by detecting specific alleles of the endothelin-converting enzyme-1 gene - Google Patents

Abstract

Ex vivo method for diagnosing subjects at risk of atherosclerosis or reduced life expectancy; or predisposition to atherosclerosis-related disease, by detecting at least one variation either in the ECE-1 (endothelin-converting enzyme-1) gene or close to this gene, in linkage disequilibrium with variants of the gene, is new. Independent claims are also included for the following: (1) an isolated nucleic acid (I) that is a fragment of at least 13 nucleotides, from the ECE-1 gene and containing at least one of the polymorphisms ECE-1A G377A; ECE-1B C25A or C338A, or ECE-1C C102T, or its complements or a sequence antisense to it; (2) allele-specific primers (II) or probes (III) able to detect at least one of the polymorphisms of (1); and (3) a diagnostic kit containing (II) or (III).

Description

<Desc / Clms Page number 1>

 The present invention relates to a method for diagnosing subjects at risk of atherosclerosis and the means used for this method based on the demonstration of polymorphisms of the ECE-1 gene. It also relates to a method for detecting a risk of reduced life expectancy (risk of death earlier than the average for the population) and of predisposition to diseases due to atherosclerosis, such as in particular: coronary disease , myocardial infarction, ischemic stroke, arteriopathy of the lower limbs.

 Atherosclerosis is the main cause of myocardial infarction, stroke and gangrene of the extremities of the limbs.

Atherosclerosis and its complications (thrombosis, restenosis after angioplasty, ...) are very common conditions in industrialized countries.

It is estimated that these conditions are responsible for 50% of total mortality.

 The process of atherosclerosis consists of the formation of fibro-fatty plaques in the wall of the arteries (subendothelial region) in response to an "aggression" by various promoting agents (the most frequent being arterial hypertension and hypercholesterolemia) . These plaques will then increase in volume and will then be able to narrow the caliber of the artery, with under certain conditions a repercussion on the blood flow in the artery.

Finally, the occurrence of a thrombus on the surface of the atheroma plate may completely obstruct the lumen of the artery and lead to necrosis of the downstream organs, by the absence of oxygen supply.

 The etiology of these disorders is multifactorial but includes a genetic component, still poorly identified, whose early knowledge would make it possible to institute appropriate treatments (pharmacogenetic approach).

 Endothelin is one of the most vasoactive biological molecules. Its active form is produced from its precursor (big-endothelin) by the converting enzyme of endothelin-1. Different properties of endothelin (activating and mitogenic effect on smooth muscle cells of the arterial wall, chemotactic effect on circulating leukocytes ...) could raise suspicion of the involvement of the endothelin vasomotor system in the genesis of plaque. atheroma, and thereby the participation of this system in the genetic determinism of the risk of cardiovascular accident. Some studies

<Desc / Clms Page number 2>

ont montré une augmentation de l'expression de l'endothéline et de l'Enzyme de Conversion de l'Endothéline dans le vaisseau athéromateux (Rossi et coil., Circulation. 1999 ; 99 : 1147-1155), ainsi qu'un effet protecteur d'un antagoniste de l'endothéline sur la survenue de lésions d'athérosclérose dans des modèles animaux (Barton et colI., Proc. Natl. Acad. Sci. USA. 1998 ; 95 : 14367-14372).

have shown an increase in the expression of endothelin and of the Endothelin Converting Enzyme in the atheromatous vessel (Rossi et coil., Circulation. 1999; 99: 1147-1155), as well as a protective effect of an endothelin antagonist on the occurrence of atherosclerotic lesions in animal models (Barton et al., Proc. Natl. Acad. Sci. USA. 1998; 95: 14367-14372).

 The gene for the endothelin-1 converting enzyme (ECE-1, an enzyme having a key role in the production of active endothelin) appears to be a candidate in vascular pathology, the authors of the present invention have sought variants ( polymorphisms) in the gene sequence, in order to then carry out association studies. They specifically studied the promoter regions of the gene described above (Valdenaire et al., J.

Biol. Chem. 1995; 270: 29794-29798; Orzechowski et al., J. Mol. Med.

1997 ; 75: 512-521; Funke-Kaiser et al., FEBS Lett. 2000; 466: 310-316).

They thus discovered four binucleotide polymorphisms (called in English "single nucleotide polymorphism" or SNP), located in the three different promoter regions (called A, B, C); the position of these polymorphisms is given relative to their respective sites of initiation of translation (starting ATG codon): - promoter region ECE-1A: polymorphism G-377A (Adenine replacing Guanine); sequence: 5'-TTTTGC (G / A) TGGGAT-3 '(SEQ. ID NO: 1) - promoter region ECE-1 B: polymorphism C-25A; (Adenine replacing Cytosine); sequence: 5'-GGGCAG (C / A) GGGACC-3 '(SEQ. ID NO: 2) - promoter region ECE-1 B: polymorphism C-338A; (Adenine replacing Cytosine); sequence: 5'-GGGCCA (C / A) ATCGAG-3 '(SEQ. ID NO: 3) - promoter region ECE-1 C: polymorphism C-102T; (Thymine replacing Cytosine); sequence: 5'-AGGCAG (C / T) CGAGCC-3 '(SEQ. ID NO: 4)
After genotyping of several hundred control subjects, the allelic frequencies of the rare alleles of these polymorphisms are 30% for the ECE-1 B C-338A polymorphism, 10% for the ECE-1A polymorphisms

<Desc / Clms Page number 3>

 G-377A, ECE-1B C-25A and ECE-1C C-102T. These rare alleles have a strong linkage imbalance between them: absolute between ECE-1A G-377A and ECE-1B C- 25A, complete between ECE-1B C-338A and ECE-1A and G-377A and incomplete between ECE1B C- 338A and ECE-1C C-102T.

 After genetic studies of association between these polymorphisms and vascular pathologies, it has been found that the gene for ECE-1 is a gene for susceptibility to atherosclerosis and that it also influences longevity by increasing the risk of death. premature.

 The inventors have more particularly demonstrated that the carriers of allele A of the C-338A polymorphism are more frequently carriers of atheroma plaques than subjects homozygous CC.

 Thus, these results make it possible to envisage the detection, and in particular the early detection, of subjects at risk of premature atherosclerosis (or more significant at equal age). These results also allow the detection of an increased risk of vascular diseases linked to atherosclerosis, as well as the detection of a risk of premature death.

 The present invention therefore relates to a method for diagnosing subjects at risk of atherosclerosis comprising the identification of at least one variant of the ECE-1 gene or of at least one variant located near the ECE-1 gene in linkage imbalance with one of these gene variants.

 More specifically, the variant highlighted is located in the promoter regions of the ECE-1 gene.

 More particularly, the method is carried out by highlighting at least one variant of the ECE-1 gene or of a neighboring sequence in linkage imbalance with one of these variants, said variant being chosen from polymorphisms following: G-377A; C-25A; C-338A and C-102T.

 It relates more specifically to a method for diagnosing subjects at risk of atherosclerosis by detecting the variant ECE-1B C-338A described above.

 Thus, in the case of the ECE-1B variant C-338A, when the allele A is detected, this subject is diagnosed as predisposed to atherosclerosis.

 It also relates to a method of detecting subjects at risk of reduced life expectancy (risk of death earlier than average

<Desc / Clms Page number 4>

 of the population) or of subjects predisposed to diseases due to atherosclerosis, such as in particular coronary disease, myocardial infarction, ischemic stroke and arteriopathy of the lower limbs, including the detection of at least one variant of the ECE-1, as defined above.

 Thus, the method according to the invention can in particular make it possible to detect subjects at increased risk of clinical complications (notably thombotic) of atherosclerosis and of associated and related cardiovascular diseases.

 The present invention also relates to the application, as desired, of a treatment with agents, in particular pharmacological agents, acting on the various components of the endothelin vasomotor system (for example endothelin antagonists or inhibitors of the enzyme converting endothelin). Indeed, one can consider for example a therapeutic action different from the various classes of prophylactic or therapeutic anti-atheromatous treatments in subjects carrying a risk allele of the ECE-1 gene. The detection of risk alleles of the ECE-1 gene makes it possible to envisage the establishment of a suitable preventive treatment in carriers.

 The demonstration of the alleles of one of the variants mentioned above, such as the variant ECE-1B C-338A, can be done according to different methods well known to those skilled in the art.

 This demonstration generally comprises two main stages, that is the extraction of a sample from the genomic DNA and the detection of the variant.

 Advantageously, the extracted DNA is amplified by any method known per se, such as the PCR (polymerase chain reaction) method, before analysis of the allelic variation.

 There are a large number of analytical methods that can be used to detect an allelic variation of polymorphisms as previously described. Detection can be performed by an automated method or by a direct visual method.

<Desc / Clms Page number 5>

 In general, this detection requires a mutation detection technique, possibly an amplification reaction and optionally a signal generation system.

 Thus, this detection of variants can be carried out by implementing one or more methods of different types, such as in particular: - sequencing methods, such as direct DNA sequencing, sequencing by hybridization (with the use of probes of oligonucleotides), - hybridization-based methods, such as allele specific hybridization (ASO), the 5'-exonuclease technique (Taqman), hybridization on DNA chips (microarrays or oligo-chips), - ligation techniques, such as the oligonucleotide ligation technique (OLA), - incorporation techniques, such as mini sequencing, - physicochemical techniques, such as high pressure liquid chromatography under denaturing conditions (DHPLC), mass spectrometry, - techniques with restriction enzymes, such as PCR generating restriction sites or the RFLP (Restriction Fragment Length Polymorphism) technique.

 This identification of a variant can more particularly comprise the following stages: extraction of a sample from the genomic DNA of a subject; - amplification of DNA; - optionally transfer of the amplified DNA to a variant detection support; - variant detection.

 The extraction of a sample of genomic DNA is done from an individual's tissue or fluid. This tissue or fluid more particularly corresponds to blood, plasma or lymph fluid. This extraction can therefore be done by blood sample.

 The present invention also relates to the means used in particular for this diagnostic method based on polymorphisms of the ECE-1 gene.

<Desc / Clms Page number 6>

 The present invention thus relates to an isolated nucleic acid (DNA or RNA) corresponding to a fragment of the gene for ECE-1 at least 13 nucleotides in length, in which at least one of the polymorphisms mentioned above is present, or a complementary strand of this nucleic acid or an antisense sequence of this nucleic acid.

 The fragment has at least 13 bases, preferably at least 17 and better still at least 30. Preferably, it has less than 200 nucleotides and even more preferably less than 100 bases.

 The fragment according to the invention can come from a natural component or be synthesized by techniques well known to those skilled in the art.

Preferably, it is at least 30% by pure weight, more preferably at least 60% pure, even more preferably 90% by pure weight.

 By nucleic acid in which at least one of the polymorphisms mentioned above is understood according to the invention is meant a nucleotide fragment having a fragment of the nucleotide sequence of said gene in which one of the two allelic variants of at least one of said polymorphisms is present.

 According to one aspect of the invention, the use of this nucleic acid comprising at least one of the polymorphisms mentioned above is proposed in a readable computer medium as databases, in particular for analyzing and / or identifying the homologs, haplotypes, as a screening tool to detect the presence of one of these polymophisms.

 According to another aspect of the present invention, the use of this nucleic acid comprising at least one of the polymorphisms mentioned above is proposed to identify compounds capable of modifying the expression of the ECE-1 gene. Modification of gene expression includes inhibition or increase of gene expression.

 This can in particular be achieved by measuring the level of expression of a reporter gene (for example ss-galactosidase) under the control of the promoter in host cells transfected in the presence and in the absence of the test compound.

 The present invention also relates to nucleotide primers for detecting the polymorphisms mentioned above.

<Desc / Clms Page number 7>

 Thus, the present invention also relates to a nucleotide primer specific for an allele capable of detecting at least one of the polymorphisms mentioned above.

 A nucleotide primer is used, generally in conjunction with a common primer, in an amplification reaction, such as PCR, which makes it possible to distinguish alleles by selective amplification of an allele at a particular position in the sequence.

 The specific allele primer according to the invention preferably has 17 to 50 nucleotides, more preferably 17 to 30 nucleotides.

 The specific allele primer preferably corresponds exactly to the allele to be detected, but their derivatives may also be suitable, derivatives in which 6 to 8 of the nucleotides at the 3 ′ end correspond to the allele to be detected and in which up to to 10 (8.6, 4.2 or 1) of the remaining nucleotides may vary without significantly affecting the properties of the primer.

 The primers can be prepared according to the methods of synthesis known to those skilled in the art. Examples of methods can be found in standard books, for example 'Protocols for oligonucleotides and analogues; Synthesis and properties, "Methods in Molecular Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN: 089603-247-7; 1993; 1 Edition.

 The PCR reaction techniques are described in numerous publications, mention may in particular be made of patents EP 0200362, EP 0201184, US 4683195, US 4800159 and US 4683202.

 The primers according to the invention can be marked, prior to their use. For this, several techniques are within the reach of those skilled in the art such as, for example, fluorescent, radioactive, chemiluminescent or enzymatic labeling.

 The variant can therefore be detected by reference to the loss or gain of sites, possibly created, recognized by restriction enzymes.

 Thus, according to a particular variant of the invention, the demonstration of variants of the ECE-1 gene can be carried out in the following manner: - a) amplification of the DNA by PCR in the presence of primers as notably described above ;

<Desc / Clms Page number 8>

 b) subjecting the amplified DNA according to a) to the action of at least one restriction enzyme allowing the differentiation of the alleles; - c) separation of the fragments obtained in b); - d) identification of the allele by comparison of the fragments separated in c) with controls having undergone at least steps b) and c) and presenting the various genotypings of the polymorphisms previously described.

 The separation of the DNA fragments can in particular be carried out by gel electrophoresis, preferably on polyacrylamide gel.

 More particularly, the appropriate enzyme is used in step c), knowing that: the allele A of the ECE-1A polymorphism G-377A creates a Nia Ili restriction site, the allele A of the ECE polymorphism 1B C-25A removes a restriction site MspA1 1, - the T allele of the ECE-1 polymorphism C C-102T creates a restriction site Taq 1.

NO : 5) - amorce spécifique de l'allèle A : 5'-GTATCAGGAAGGTGCCCTCTATT-3' (SEQ. ID NO : 6) - amorce spécifique de i'a) iè) e C : 5'-GTATCAGGAAGGTGCCCTCGATG-3' (SEQ. ID NO : 7), Chaque échantillon d'ADN doit donc subir, dans ce cas (sans création et suppression de sites de restriction), deux amplifications pour déterminer d'abord la présence ou l'absence de l'allèle A, puis la présence ou l'absence de l'allèle C. Chaque amplification est une réaction de PCR duplex , avec amplification conjointe d'un fragment contrôle de plus grande taille (témoin positif du succès de la réaction de PCR). In addition, more particularly, for the ECE-1B C-338A polymorphism which neither creates nor removes restriction sites, its genotyping was carried out by an amplification technique by PCR (Polymerase Chain Reaction) specific for allele, in using the following oligonucleotide primers: - common primer: 5'-CCTCTGGAAATCTGCTGGGTAAG-3 '(SEQ. ID

NO: 5) - specific primer for allele A: 5'-GTATCAGGAAGGTGCCCTCTATT-3 '(SEQ. ID NO: 6) - specific primer for i'a) iè) e C: 5'-GTATCAGGAAGGTGCCCTCGATG-3' (SEQ ID NO: 7), Each DNA sample must therefore undergo, in this case (without creation and removal of restriction sites), two amplifications to first determine the presence or absence of the allele A, then the presence or absence of the C allele. Each amplification is a duplex PCR reaction, with joint amplification of a larger control fragment (positive control for the success of the PCR reaction).

 This genotyping technique used for the ECE-1B C-338A polymorphism being longer and more expensive than a usual PCR reaction

<Desc / Clms Page number 9>

 (because double amplification under very strict conditions), it is preferable to use a PCR-RFLP reaction, combining an amplification of the fragment of interest with oligonucleotide primers, one of which is modified so as to create a cleavage site for an enzyme of restriction, such as in particular the enzyme Tsp509 l, in the presence of the allele in question, such as allele A, followed by digestion with the restriction enzyme.

The primers used for said amplification are: - sense primer: 5'-TAGGGTTATAGGAGAGGGCTCAGG-3 '(SEQ. ID NO: 8) - antisense primer: 5'-AAGTATCAGGAAGGTGCCCTCAAT-3' (SEQ. ID NO: 9)
According to another aspect of the invention, there is provided an oligonucleotide probe specific for an allele of one of the polymorphisms described above capable of detecting a polymorphism as previously described.

 The probe according to the invention preferably has 17 to 50 nucleotides, more preferably from 17 to 35 nucleotides, even more preferably from 17 to 30.

 In general, these probes comprise base sequences entirely complementary to the position of the variant in the gene. However, it is possible to introduce one or more mismatches, as long as the discriminatory power of the probe is not substantially affected.

 The probes according to the invention can be marked, prior to their use. For this, several techniques are within the reach of those skilled in the art such as, for example, fluorescent, radioactive, chemiluminescent or enzymatic labeling.

 The present invention also relates to a kit for diagnosing subjects at risk of premature atherosclerosis (or greater at equal age), subjects at increased risk of vascular diseases linked to atherosclerosis, or subjects at increased risk of death premature, which comprises a probe or a primer as previously described.

 The kit can further comprise at least one compound for amplifying DNA. This compound is advantageously a polymerase enzyme which can be used in a PCR reaction. This compound can for example be Taq polymerase.

<Desc / Clms Page number 10>

 The kit may also include a reagent for performing gel electrophoresis, preferably on polyacrylamide gel.

 Concrete but nonlimiting examples of the invention will now be presented.

EXAMPLES:
The ECE-1B C-338A polymorphism was genotyped in subjects participating in the GENIC multicenter study (Elbaz et al., Blood 2000; 95-586-591), i.e. 477 control subjects, 452 of whom had undergone an examination of the carotid arteries by Doppler ultrasound, and 457 patients with cerebral infarction of various causes.

30 secondes, 72 C pendant 30 secondes). Le génotypage a ensuite été simplifié par l'utilisation d'une technique de PCR-RFLP. La polymerase AmpliTaq Gold (Perkin-Elmer) a été utilisée avec un cycle de 10 minutes de dénaturation à 95 C suivi de 35 cycles d'amplification (95OC pendant 30 secondes, 56 C

pendant 30 secondes, 72 C pendant 30 secondes), et d'une extension finale de 10 minutes à 72OC. Cette amplification a été suivie d'une digestion par l'enzyme de restriction Tsp5091 pendant 2 heures à 65OC. Pour les deux méthodes de génotypage, les fragments d'ADN obtenus ont été ensuite soumis à une électrophorèse sur gel d'Agarose (2 % dans le premier cas, 3 % dans le deuxième), avec révélation par coloration au bromure d'éthidium. Genotyping was first performed by an allele-specific PCR amplification technique, using AmpliTaq Gold polymerase (PerkinElmer), with a 10-minute cycle of denaturation at 95 C followed by 35 cycles of amplification ( 95OC for 30 seconds, 58 C for

30 seconds, 72 C for 30 seconds). Genotyping was then simplified by the use of a PCR-RFLP technique. AmpliTaq Gold polymerase (Perkin-Elmer) was used with a 10 minute cycle of denaturation at 95 C followed by 35 cycles of amplification (95OC for 30 seconds, 56 C

for 30 seconds, 72 C for 30 seconds), and a final extension of 10 minutes at 72OC. This amplification was followed by digestion with the restriction enzyme Tsp5091 for 2 hours at 65 ° C. For the two genotyping methods, the DNA fragments obtained were then subjected to agarose gel electrophoresis (2% in the first case, 3% in the second), with revelation by staining with ethidium bromide.

 This genotyping of the GENIC study controls made it possible to obtain the following results.

de sujets AA varie selon la classe d'âge : 11, 1% chez les moins de 60 ans, 6, 7% chez les plus de 60 ans. First result: The homozygous allele A carriers (AA subjects, representing 8% of the total workforce of this population of ages between 20 and 89) had an average age of 60.2 years, significantly different from that of AC and CC subjects combined, i.e. 66.4 years (p = 0.007). Furthermore, the proportion

of AA subjects varies by age group: 11.1% in those under 60, 6.7% in those over 60.

<Desc / Clms Page number 11>

OR=2, 28 [1, 44-3, 59]). Après ajustement sur l'âge, 1"'Odds Ratio"des porteurs d'au moins un allèle A (AC + AA) est de 1,90 [1, 22-2, 95] (p=0,004). L'"Odds Ratio" (désigné OR) est un facteur d'approximation du risque relatif de développer des plaques d'athérome. Second result: carriers of allele A are more frequently carriers of atheroma plaques than subjects homozygous CC. This is directly visible by comparing the frequency of plaques between AC and CC subjects, whose ages are not statistically different: 53.1% of AC subjects carry carotid atheroma plaques, against 37.8% of CC (p = 0, 0004;

OR = 2.28 [1, 44-3, 59]). After adjusting for age, the "Odds Ratio" of carriers of at least one A allele (AC + AA) is 1.90 [1.22-2.95] (p = 0.004). The "Odds Ratio" (designated OR) is an approximation factor for the relative risk of developing atherosclerotic plaques.

 Taking into account the second component of the GENIC study, namely 457 subjects who had an ischemic stroke, gives a concordant result: the case-control analysis of the group of patients with cerebral infarction linked to atherosclerosis shows an excess of allele A carriers in the group of patients compared to controls matched on age and sex (OR = 1.54 [0, 80-2, 90], after adjustment for cardiovascular history ).

Claims (19)

 CLAIMS 1. Ex vivo method of diagnosing subjects at risk of atherosclerosis, of detecting subjects at risk of shortening of life expectancy or of subjects predisposed to diseases due to atherosclerosis, which comprises the demonstration of at least one variant in the ECE-1 gene or at least one variant located near the ECE-1 gene in linkage disequilibrium with variants of the gene.  2. Method according to claim 1, characterized in that the variant is located in the promoter regions of the ECE-1 gene.  3. Method according to one of the preceding claims 1 or 2, characterized in that the variant is chosen from one of the following polymorphisms: ECE-1A G-377A; ECE-1B C-25A; ECE-1B C-338A and ECE-1C C-102T.  4. Method according to the preceding claim, characterized in that the variant is the ECE-1 B C-338A polymorphism.  5. Method according to any one of the preceding claims, characterized in that it comprises the following steps: - extraction of a sample from the genomic DNA of a subject, - optionally amplification of the DNA, - optionally transfer DNA amplified on a variant detection support, - detection of the variant.  6. Method according to claim 5, characterized in that the DNA is amplified by the PCR reaction <Desc / Clms Page number 13>

 7. Method according to claim 5 or '6, characterized in that the detection of variants can be carried out by using one or more methods of different types chosen from: - sequencing methods, such as direct DNA sequencing, sequencing by hybridization (with the use of oligonucleotide probes), - methods based on hybridization, such as allele specific hybridization (ASO), the 5'-exonuclease technique (Taqman), hybridization on DNA chips (microarrays or oligo-chips), - ligation techniques, such as the oligonucleotide ligation technique (OLA), - incorporation techniques, such as mini sequencing, - physicochemical techniques, such as chromatography high pressure liquid under denaturing conditions (DHPLC), mass spectrometry, - techniques with restriction enzymes, such as PCR generating restriction sites or the RFLP (Restriction Fragment Len) technique gth Polymorphism).  8. Method according to one of claims 5 to 7, in which the steps implemented are the following: - a) amplification of the DNA by PCR in the presence of primers; b) subjecting the amplified DNA according to a) to the action of at least one restriction enzyme allowing the differentiation of the alleles; - c) separation of the fragments obtained in b); d) identification of the allele by comparison of the fragments separated in c) with controls having undergone at least steps b) and c) and having the different genotypings of the variants defined according to one of claims 1 to 4.  9. Method according to one of claims 5 to 8 in which two allele specific PCR reactions are used to genotype the <Desc / Clms Page number 14>  ECE-1 B polymorphism C-338A, with the following allele specific antisense oligonucleotides: allele A specific primer: 5'- GTATCAGGAAGGTGCCCTCTATT-3 '(SEQ. ID NO: 6) allele C specific primer: 5'- GTATCAGGAAGGTGCCCTCGATG-3 '(SEQ. ID NO: 7).  10. Method according to one of claims 5 to 8, in which an amplification by the PCR technique is used with at least one oligonucleotide primer modified so as to create a cleavage site for a restriction enzyme, the amplification being followed by 'a cleavage by the restriction enzyme, in particular for genotyping the ECE-1 B C-338A polymorphism.  11. Method according to claim 10, in which the amplification is carried out with the following primers: - sense primer: 5'-TAGGGTTATAGGAGAGGGCTCAGG-3 '(SEQ. ID NO: 8)

 - antisense primer: 5'-AAGTATCAGGAAGGTGCCCTCAAT-3 '(SEQ. ID NO: 9). 12. Method according to claim 11, in which the cleavage is carried out by the restriction enzyme Tap509 1.  13. Isolated nucleic acid corresponding to a fragment of the gene for ECE-1 at least 13 nucleotides in length, in which at least one of the polymorphisms mentioned in claim 3 is present, or a strand complementary to this nucleic acid or an antisense sequence of this nucleic acid. <Desc / Clms Page number 15>  14. Nucleotide primer specific for an allele capable of detecting the ECE-1B C-338A polymorphism, having one of the following sequences: primer specific for allele A: 5'- GTATCAGGAAGGTGCCCTCTATT-3 '(SEQ. ID NO: 6) specific primer for allele C: 5'- GTATCAGGAAGGTGCCCTCGATG-3 '(SEQ. ID NO: 7).  15. Diagnostic kit for implementing the method according to one of claims 1 to 12, which comprises a probe or an oligonucleotide primer specific for an allele capable of detecting at least one of the polymorphisms mentioned in claim 3.  16. Kit according to the preceding claim, characterized in that it further comprises at least one compound for amplifying DNA.  17. Kit according to claim 15 or 16, characterized in that it further comprises a reagent for carrying out electrophoresis on gel, preferably on polyacrylamide gel.  18. Use of a nucleic acid according to claim 13 to identify compounds capable of modifying the expression of the ECE-1 gene.  19. Use of a nucleic acid according to claim 13 as a screening tool for detecting the presence of one of the polymorphisms mentioned in claim 3.