WO1996033287A1 - Juvenile glaucoma detection process - Google Patents

Juvenile glaucoma detection process Download PDF

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Publication number
WO1996033287A1
WO1996033287A1 PCT/FR1996/000592 FR9600592W WO9633287A1 WO 1996033287 A1 WO1996033287 A1 WO 1996033287A1 FR 9600592 W FR9600592 W FR 9600592W WO 9633287 A1 WO9633287 A1 WO 9633287A1
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marker
sequence
seq
type
description
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PCT/FR1996/000592
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French (fr)
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Henri-Jean Garchon
Jean-François Bach
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
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Publication of WO1996033287A1 publication Critical patent/WO1996033287A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/172Haplotypes

Definitions

  • the present invention relates in particular to family screening for predisposition to juvenile glaucoma as well as the possibility of highlighting the gene responsible for this condition. Juvenile glaucoma, which affects nearly 100,000 people in
  • This juvenile glaucoma comes from the combination of ocular hypertension and damage to the optic nerve.
  • effective treatments exist, notably medical and surgical, which make it possible to stop the disease or to slow down its progression.
  • these treatments are only very effective when glaucoma is detected at an early stage, late screenings lead to heavy surgeries which can leave disabilities.
  • juvenile glaucoma is linked to the presence on chromosome 1 of a particular gene or of a particular structure, the presence of a single copy of which is sufficient for the onset of the disease.
  • the chromosome contains sequences which are very polymorphic, that is to say that they have different compositions according to the individuals although being located in the same place on the chromosome, these zones are called “microsatellites” and make the subject of numerous publications.
  • microsatellites being located in the same place, we can consider highlighting the presence of a particular microsatellite which would be linked to the gene that we are looking for.
  • the maximum correlation rate is obtained for a microsatellite marker which is located very close to the gene in question. Indeed, in this case, these two portions of the chromosome are transmitted as they are to the descendants, on the other hand, if the marker is more distant it can be separated from the gene during transmission ("crossing over") and its presence n is more necessarily associated with the presence of the defective gene.
  • microsatellite marker in any individual has, in principle, no meaning. Indeed, in the absence of particular inheritance, this marker often presents no interest and it should also be understood that even when family history reveals an inherited juvenile glaucoma, the presence of the family marker of another family does not is not necessarily critical in predicting the onset of the disease. It is necessary to redo studies of correlation, even if when the families are very close (same region) and even in the absence of known common ancestors, the marker can in some cases be discriminating (linkage imbalance between the marker and the disease).
  • the present invention relates to a method for screening for a predisposition to juvenile glaucoma in a patient, characterized in that the presence of a microsatellite marker linked with the occurrence of juvenile glaucoma in its family, the microsatellite marker being chosen from the markers afm350yhl, afml22xa3, ngal, afm21, afm248wg5, afm278ye5, afm212xbl0, afml57xe7 and NGA5 of chromosome Iq21q31.
  • the detected microsatellite marker is preferably located in the locus corresponding to the region located between the marker afm248wg5 and afm212xbl0.
  • the techniques used to detect the presence of markers are known; it may be a direct detection using a marker complementary probe, but preferably, the methods using an amplification of the marker sequence will be used, for example by methods of the PCR (Polymerase Chain Reaction) type. using primers, one of which is marked with a fluorochrome.
  • the combinations of primer pairs are tested two by two. Then, if the results allow it, a third pair will be introduced and so on.
  • the presence of this microsatellite marker only indicates a predisposition to the occurrence of juvenile glaucoma and this when family history having been analyzed, a link was made between the presence of these markers and the occurrence of juvenile glaucoma.
  • the present invention also relates to the DNA sequence located between the locus comprising the marker afml22xa3 and afm212xbl0, preferably afml 22xa3 and NGA5, which part is linked to the juvenile glaucoma system. Indeed, thanks to previous developments, it is possible to locate the gene implicated in juvenile glaucoma.
  • FIG. 2 represents a more detailed map of the region D1S210 - L854B9 in which the gene for the susceptibility to juvenile glaucoma is located.
  • the markers thus described made it possible to identify artificial chromosome clones of yeast covering the region. 25 YACs covering a maximum distance of 3 Mbases thus define a first level path.
  • genotyping described will make it possible to identify families where the glaucoma gene located on chromosome 1 is not involved, opening the way to the localization of other genes predisposing to glaucoma in other places in the genome.
  • DNA is extracted from peripheral venous blood after cell lysis, protein digestion, organic partition and finally alcoholic precipitation.
  • the blood (20 ml) is taken by peripheral venipuncture from a tube containing EDTA. It is diluted with a volume of double-distilled water. After 10 minutes, the cells are collected by centrifugation at 1600 g for 10 minutes. This manipulation is repeated.
  • the white cells are lysed in the presence of 20 ml of CLB buffer (10 mM Tris pH 7.6, 5 miM MgCl 2 , 0.32 M sucrose, 1% Triton X-100 (v / v).
  • CLB buffer 10 mM Tris pH 7.6, 5 miM MgCl 2 , 0.32 M sucrose, 1% Triton X-100 (v / v).
  • the nuclei are collected by centrifugation at 1600 g This operation is repeated for 10 minutes.
  • the nuclei are washed once in RSB buffer (10 mM Tris pH 8, 10 mM NaCl, 10 mM EDTA). The pellet is resuspended in 2 ml of RSB buffer to which is added sodium lauryl sulfate (1%) and proteinase K (200 ⁇ g / ml). The mixture is incubated at 55 ° C for at least 3 hours and stirred regularly.
  • the DNA solution thus obtained is then extracted with a volume of phenol balanced with a 50 M Tris pH 8 buffer. This operation is repeated and supplemented by extraction with a volume of chloroform / isoamyl alcohol (24: 1 v / v) .
  • the DNA is precipitated with a volume of isopropanol, rinsed with ethanol (70%), dried and finally resuspended in 1 ml of TE buffer (10 mM Tris pH 8, 0.5 mM EDTA).
  • the DNA concentration is evaluated by measuring the absorbance at 260 mm using the equivalence of 50 ⁇ g / ml of DNA for one unit of absorbance. The DNA concentration is then adjusted to 200 ⁇ g / ml.
  • Genomic DNA (200 ⁇ g / ml) 1 ⁇ l
  • nucleotide triphosphate (each 1.25 M) 4 ⁇ l
  • primers each 10 picomoles / ⁇ l 2 x 1 ⁇ l
  • Taq DNA polymerase TM (5 u / ⁇ l) 0.05 ⁇ l. 10 times concentrated PCR buffer 2.5 ⁇ l
  • composition of the PCR buffer 10 x Tris 0.1 M pH 8.3 (at 20 ° C), KC1 0.5
  • the antibody TaqStartTM (Clontech) is added to the final concentration of 0.056 ⁇ M (i.e. an antibody / enzyme molar ratio of 28: 1).
  • the amplification is carried out in a TechneTM PHC-3 thermal cycler with heated cover. After heating to 94 ° C for
  • Each cycle comprises two one-minute periods each at '55 ° C and 94 ° C successively.
  • a final elongation segment of 2 minutes at 72 ° C ends the amplification.
  • NUCLEOTIDE SEQUENCE MARKER (size in bp) of PRIMERS (upstream then downstream) afm350y l JOE- 5 '-TCTTCCCACCACTGCC
  • NGA5 (L854B9) CTGAAACTGAGATAGGAGTGC GAAATGGGAGTTGAGTTACCC
  • amplification products (2 ⁇ l) of the same individual are combined, coprecipitated with ethanol (2.5 volumes, 2 hours at -20 ° C) and allowed to migrate on the same track of acrylamide gel (6% ) - urea (8M), after resuspension in a loading buffer (formamide 2 ⁇ l, 0.5 ⁇ l Blue ABITM, 0.5 ⁇ l GeneScan 2500 Rox, 1 ⁇ l H 2 O) and after thermal denaturation.
  • the electrophoresis is carried out in an ABI 373 automatic sequencer, at a power of 30 Watts for eight hours, the laser beam being placed at a height of 24 cm.
  • Raw data is analyzed with GeneScan Analysis software
  • the likelihood of the presence of the disease gene is estimated using the LIN AGE software.

Abstract

The invention concerns a process for detecting a predisposition to juvenile glaucoma in a person, the process being characterized in that microsatellite markers associated with the occurrence of juvenile glaucoma in the person's family are characterized in a biological sample taken from said person. These markers are afm350yhl, afm122xa3, ngal, afm21, afm248wg5, afm278ye5, afm212xb10, afm157xe7 and NGA5 of chromosome 1q21q31.

Description

DEPISTAGE DU GLAUCOME TUNENILETUNENILE GLAUCOMA SCREENING
La présente invention concerne notamment le dépistage familial de la prédisposition au glaucome juvénile ainsi que la possibilité de mettre en évidence le gène responsable de cette affection. Le glaucome juvénile qui touche près de 100 000 personnes enThe present invention relates in particular to family screening for predisposition to juvenile glaucoma as well as the possibility of highlighting the gene responsible for this condition. Juvenile glaucoma, which affects nearly 100,000 people in
France y constitue une des principales causes de cécité.France is one of the main causes of blindness there.
Comme son nom l'indique, Les premiers symptômes se manifestent souvent avant 40 ans par une diminution du champ visuel. Cette affection indolore et d'évolution très progressive est souvent méconnue par le patient et n'est mise en évidence qu'à un stade avancé, souvent irréversible où elle évoluera vers la cécité.As the name suggests, The first symptoms often appear before 40 years of age by a decrease in the visual field. This painless and very progressive condition is often overlooked by the patient and is only revealed at an advanced, often irreversible, stage where it will progress to blindness.
Ce glaucome juvénile provient de la combinaison d'une hypertension oculaire et d'une lésion du nerf optique. Actuellement des traitements efficaces existent, notamment médicaux et chirurgicaux, qui permettent de stopper la maladie ou d'en ralentir l'évolution. Toutefois, ces traitements ne sont très efficaces que lorque le glaucome est dépisté à un stade précoce, les dépistages tardifs conduisent à des chirurgies lourdes qui peuvent laisser des handicaps.This juvenile glaucoma comes from the combination of ocular hypertension and damage to the optic nerve. Currently effective treatments exist, notably medical and surgical, which make it possible to stop the disease or to slow down its progression. However, these treatments are only very effective when glaucoma is detected at an early stage, late screenings lead to heavy surgeries which can leave disabilities.
Depuis quelques années, on a pu mettre en évidence le rôle majeur des facteurs génétiques dans la survenue du glaucome junvénile. Il est maintenant certain qu'il s'agit dans la plupart des cas d'une maladie transmise sur le mode mendélien simple dominant; le gène en cause étant situé sur le bras long du chromosome 1 .In recent years, we have been able to highlight the major role of genetic factors in the occurrence of glaucoma. It is now certain that in most cases it is a disease transmitted in the dominant simple Mendelian mode; the gene in question being located on the long arm of chromosome 1.
C'est-à-dire que le glaucome juvénile est lié à la présence sur le chromosome 1 d'un gène particulier ou d'une structure particulière dont la présence d'une seule copie suffit pour la survenue de la maladie.In other words, juvenile glaucoma is linked to the presence on chromosome 1 of a particular gene or of a particular structure, the presence of a single copy of which is sufficient for the onset of the disease.
Il est donc nécessaire, pour le dépistage précoce de la maladie, de déterminer si ce gène est ou non présent chez le patient. Actuellement, on connaît la région où se situe le gène que l'on nomme "locus de susceptibilité" mais pas sa position exacte ni sa structure. Un "locus de susceptibilité" peut comporter de l'ordre de 10 millions de bases, ce qui rend évidemment la localisation très compliquée d'autant que l'on ignore tout sur la structure du gène. Il n'est donc pas possible de mettre en évidence directement la présence du gène en cause, c'est pourquoi on doit avoir recours à un diagnostic mettant en oeuvre la technique des "microsatellites". Le chromosome comporte des séquences qui sont très polymorphes, c'est-à-dire qu'elles ont des compositions différentes selon les individus bien qu'étant situées au même endroit sur le chromosome, ces zones sont dénommées "microsatellites" et font l'objet de nombreuses publications.It is therefore necessary, for early detection of the disease, to determine whether or not this gene is present in the patient. Currently, we know the region where the gene called the "susceptibility locus" is located, but not its exact position or its structure. A "susceptibility locus" can contain around 10 million bases, which obviously makes localization very complicated, especially since everything about the structure of the gene is unknown. It is therefore not possible to directly demonstrate the presence of the gene in question, this is why we must have recourse to a diagnosis using the technique of "microsatellites". The chromosome contains sequences which are very polymorphic, that is to say that they have different compositions according to the individuals although being located in the same place on the chromosome, these zones are called "microsatellites" and make the subject of numerous publications.
Dans le cas d'une affection de type familial et de caractère dominant, il est possible d'étudier la corrélation de la survenu--, du Q-laucoine juvénile avec un allèle d'un marqueur microsatellite déterminé. Pour un taux de corrélation élevé, on pourra considérer que la présence de l'allèle du microsatellite est un indice de la présence du gène morbide.In the case of a family type disorder and of a dominant character, it is possible to study the correlation of the occurrence-- of juvenile Q-laucoine with an allele of a determined microsatellite marker. For a high correlation rate, it may be considered that the presence of the microsatellite allele is an index of the presence of the morbid gene.
Ces microsatellites étant situés au même endroit, on peut envisager de mettre en évidence la présence d'un microsatellite particulier qui serait lié au gène que l'on recherche.These microsatellites being located in the same place, we can consider highlighting the presence of a particular microsatellite which would be linked to the gene that we are looking for.
Il est donc nécessaire d'étudier la famille du patient et de disposer de son "histoire", c'est-à-dire d'avoir pour chaque individu de la famille étudiée à la fois la présence de la maladie et la présence d'un marqueur spécifique, en fait allèle d'un microsatellite, qui soit nécessairement ou très probablement associé à la maladie.It is therefore necessary to study the family of the patient and to have his "history", that is to say to have for each individual of the family studied both the presence of the disease and the presence of a specific marker, in fact allele of a microsatellite, which is necessarily or very probably associated with the disease.
Si la famille est suffisamment nombreuse, on pourra déterminer quels sont les marqueurs qui sont statistiquement le plus souvent présent lorsque la maladie est détectée .If the family is large enough, we can determine which markers are statistically most often present when the disease is detected.
Le taux maximum de corrélation est obtenu pour un marqueur microsatellite qui est situé très près du gène en cause. En effet, dans ce cas, ces deux portions du chromosome sont transmises telles quelles à la descendance, par contre, si le marqueur est plus éloigné il peut se trouver séparé du gène lors de la transmission ( "crossing over") et sa présence n'est plus nécessairement associée à la présence du gène défectueux.The maximum correlation rate is obtained for a microsatellite marker which is located very close to the gene in question. Indeed, in this case, these two portions of the chromosome are transmitted as they are to the descendants, on the other hand, if the marker is more distant it can be separated from the gene during transmission ("crossing over") and its presence n is more necessarily associated with the presence of the defective gene.
Il est donc nécessaire de comprendre que la mise en évidence d'un marqueur microsatellite chez un individu quelconque n'a pas, en principe de signification. En effet, en l'absence d'hérédité particulière, ce marqueur ne présente souvent aucun intérêt et il faut également comprendre que même lorsque l'histoire familiale fait apparaître un glaucome juvénile héréditaire, la présence du marqueur familial d'une autre famille n'est pas nécessairement déterminante pour prédire l'apparition de la maladie. Il est nécessaire de refaire des études de corrélation, même si lorsque les familles sont très proches (même région) et même en l'absence d'ancêtres communs connus, le marqueur peut dans certains cas être discriminant (déséquilibre de liaison entre le marqueur et la maladie). C'est pourquoi la présente invention concerne un procédé de dépistage d'une prédisposition au glaucome juvénile chez un patient, caractérisé en ce que l'on détecte, dans un prélèvement biologique chez ledit patient, la présence d'un marqueur microsatellite lié avec la survenue du glaucome juvénile dans sa famille, le marqueur microsatellite étant choisi parmi les marqueurs afm350yhl, afml22xa3, ngal, afm21, afm248wg5, afm278ye5, afm212xbl0, afml57xe7 et NGA5 du chromosome Iq21q31.It is therefore necessary to understand that the detection of a microsatellite marker in any individual has, in principle, no meaning. Indeed, in the absence of particular inheritance, this marker often presents no interest and it should also be understood that even when family history reveals an inherited juvenile glaucoma, the presence of the family marker of another family does not is not necessarily critical in predicting the onset of the disease. It is necessary to redo studies of correlation, even if when the families are very close (same region) and even in the absence of known common ancestors, the marker can in some cases be discriminating (linkage imbalance between the marker and the disease). This is why the present invention relates to a method for screening for a predisposition to juvenile glaucoma in a patient, characterized in that the presence of a microsatellite marker linked with the occurrence of juvenile glaucoma in its family, the microsatellite marker being chosen from the markers afm350yhl, afml22xa3, ngal, afm21, afm248wg5, afm278ye5, afm212xbl0, afml57xe7 and NGA5 of chromosome Iq21q31.
De façon plus précise, le marqueur microsatellite détecté est de préférence situé dans le locus correspondant à la région située entre le marqueur afm248wg5 et afm212xbl0. Les techniques mises en oeuvre pour détecter la présence des marqueurs sont connues; il peut s'agir d'une détection en direct grâce à une sonde complémentaire du marqueur, mais on utilisera, de préférence, les méthodes mettant en oeuvre une amplification de la séquence marqueur par exemple par des méthodes de type PCR (Polymerase Chain Reaction) à l'aide d'amorces dont l'une est marquée par un fluorochrome.More precisely, the detected microsatellite marker is preferably located in the locus corresponding to the region located between the marker afm248wg5 and afm212xbl0. The techniques used to detect the presence of markers are known; it may be a direct detection using a marker complementary probe, but preferably, the methods using an amplification of the marker sequence will be used, for example by methods of the PCR (Polymerase Chain Reaction) type. using primers, one of which is marked with a fluorochrome.
Afin de simplifier la détection des différents marqueurs en cause, il est possible d'utiliser la PCR "multiplex". Le principe est de réaliser plusieurs réactions de PCR simultanément à partir d'un même échantillon d'ADN génomique. L'intérêt est d'économiser les réactifs ainsi que l'ADN à typer, et de réduire le risque d'erreur en diminuant le nombre de manipulations.In order to simplify the detection of the various markers involved, it is possible to use "multiplex" PCR. The principle is to carry out several PCR reactions simultaneously from the same genomic DNA sample. The interest is to save the reagents as well as the DNA to be typed, and to reduce the risk of error by reducing the number of manipulations.
Dans ce but, les combinaisons de paires d'amorces sont testées deux à deux. Puis, si les résultats le permettent, une troisième paire sera introduite et ainsi de suite. Comme cela a été indiqué précédemment, la présence de ce marqueur microsatellite n'indique qu'une prédisposition à la survenue du glaucome juvénile et ceci lorsque l'histoire familiale ayant été analysée, il a été fait un lien entre la présence de ces marqueurs et la survenue du glaucome juvénile. La présente invention concerne également la séquence d'ADN située entre le locus comportant le marqueur afml22xa3 et afm212xbl0, de préférence afml 22xa3 et NGA5, laquelle partie est liée au système du glaucome juvénile. En effet, grâce aux développements précédents, il est possible de localiser le gène mis en cause dans le glaucome juvénile.For this purpose, the combinations of primer pairs are tested two by two. Then, if the results allow it, a third pair will be introduced and so on. As previously indicated, the presence of this microsatellite marker only indicates a predisposition to the occurrence of juvenile glaucoma and this when family history having been analyzed, a link was made between the presence of these markers and the occurrence of juvenile glaucoma. The present invention also relates to the DNA sequence located between the locus comprising the marker afml22xa3 and afm212xbl0, preferably afml 22xa3 and NGA5, which part is linked to the juvenile glaucoma system. Indeed, thanks to previous developments, it is possible to locate the gene implicated in juvenile glaucoma.
La caractérisation des hapLotypes recomb.n-.ms» sv sein de grandes familles, grâce aux marqueurs microsatellites décrits ci-dessus, permet d'ordonner les marqueurs les uns par rapport aux autres. Chez les sujets glaucomateux, ces haplotypes recombinants permettent de mieux cerner la région chromosomique contenant le gène morbide. Une région de trois centiMorgans a été ainsi définie (figure 1 ). La figure 2 représente une carte plus détaillée de la région D1S210 - L854B9 dans laquelle est situé le gène de susceptibilité au glaucome juvénile. Les marqueurs ainsi décrits ont permis d'identifier des clones de chromosome artificiels de levure couvrant la région. 25 YAC couvrant une distance maximale de 3 Mbases définissent ainsi un chemin de premier niveau.The characterization of the hapLotypes recomb.n-.ms »sv within large families, thanks to the microsatellite markers described above, makes it possible to order the markers with respect to each other. In glaucomatous subjects, these recombinant haplotypes allow a better understanding of the chromosomal region containing the morbid gene. A region of three centiMorgans was thus defined (Figure 1). FIG. 2 represents a more detailed map of the region D1S210 - L854B9 in which the gene for the susceptibility to juvenile glaucoma is located. The markers thus described made it possible to identify artificial chromosome clones of yeast covering the region. 25 YACs covering a maximum distance of 3 Mbases thus define a first level path.
Il est alors possible d'isoler le gène par : - recherche simultanée de nouveaux marqueurs polymorphes et de nouveaux haplotypes recombinants qui permettront de rétrécir encore la région contenant le gène, recherche directe de gènes par plusieurs méthodes : . sélection de transcrits par hybridation . piègeage d'exonsIt is then possible to isolate the gene by: - simultaneous search for new polymorphic markers and new recombinant haplotypes which will make it possible to further shrink the region containing the gene, direct search for genes by several methods:. selection of transcripts by hybridization. trapping of exons
. séquences phylogénétiquement conservées. phylogenetically conserved sequences
. îlots CpG. CpG islands
. expansion de triplets. expansion of triplets
Enfin, le génotypage décrit permettra d'identifier des familles où le gène du glaucome localisé sur le chromosome 1 n'est pas impliqué, ouvrant la voie à la localisation d'autres gènes prédisposant au glaucome en d'autres endroits du génome.Finally, the genotyping described will make it possible to identify families where the glaucoma gene located on chromosome 1 is not involved, opening the way to the localization of other genes predisposing to glaucoma in other places in the genome.
D'autres caractéristiques et modalités de mise en oeuvre du procédé pourront être déduits de la lecture de l'exemple qui suit. ExempleOther characteristics and methods of implementing the method can be deduced from reading the example which follows. Example
Méthode de typaee de l'ADN génomique humain avecTyping method for human genomic DNA with
9 marqueurs microsatellites de la région Iq21q31.9 microsatellite markers of the Iq21q31 region.
A) L'ADN est extrait du sang veineux périphérique après lyse cellulaire, digestion protéique, partition organique et finalement précipitation alcoolique.A) DNA is extracted from peripheral venous blood after cell lysis, protein digestion, organic partition and finally alcoholic precipitation.
Le sang (20 ml) est prélevé par ponction veineuse périphérique sur un tube contenant de l'EDTA. II est dilué avec un volume d'eau bidistillée. Après 10 minutes, les cellules sont collectées par centrifugation à 1600 g pendant 10 minutes. Cette manipulation est répétée.The blood (20 ml) is taken by peripheral venipuncture from a tube containing EDTA. It is diluted with a volume of double-distilled water. After 10 minutes, the cells are collected by centrifugation at 1600 g for 10 minutes. This manipulation is repeated.
Les cellules blanches sont lysées en présence de 20 ml de tampon CLB (Tris 10 mM pH 7.6, 5 miM MgCl2, sucrose 0.32 M, Triton X-100 1% (v/v). Les noyaux sont collectés par centrifugation à 1600 g pendant 10 minutes. Cette manipulation est répétée.The white cells are lysed in the presence of 20 ml of CLB buffer (10 mM Tris pH 7.6, 5 miM MgCl 2 , 0.32 M sucrose, 1% Triton X-100 (v / v). The nuclei are collected by centrifugation at 1600 g This operation is repeated for 10 minutes.
Les noyaux sont lavés une fois dans le tampon RSB (Tris 10 mM pH 8, NaCl 10 mM, EDTA 10 mM). Le culot est resuspendu dans 2 ml de tampon RSB auquel est ajouté du lauryl sulfate de sodium ( 1 %)et la protéinase K (200 μ g/ml). Le mélange est incubé à 55° C pendant au moins 3 heures et régulièrement agité.The nuclei are washed once in RSB buffer (10 mM Tris pH 8, 10 mM NaCl, 10 mM EDTA). The pellet is resuspended in 2 ml of RSB buffer to which is added sodium lauryl sulfate (1%) and proteinase K (200 μ g / ml). The mixture is incubated at 55 ° C for at least 3 hours and stirred regularly.
La solution d'ADN ainsi obtenue est ensuite extraite avec un volume de phénol équilibré avec un tampon 50 M Tris pH 8. Cette opération est répétée et complétée par une extration avec un volume de chloroforme/alcool isoamylique (24:1 v/v).The DNA solution thus obtained is then extracted with a volume of phenol balanced with a 50 M Tris pH 8 buffer. This operation is repeated and supplemented by extraction with a volume of chloroform / isoamyl alcohol (24: 1 v / v) .
L'ADN est précipité avec un volume d'isopropanol, rincé à l'éthanol (70 %), séché et enfin resuspendu dans 1 ml de tampon TE (Tris 10 mM pH 8, EDTA 0.5 mM). La concentration d'ADN est évaluée par mesure de l'absorbance à 260 mm en utilisant l'équivalence de 50 μg/ml d'ADN pour une unité d'absorbance. La concentration d'ADN est alors ajustée à 200 μg/ml. B ) Amplification de l'ADN génomique pour les marqueurs microsatellites fluorescents. Neuf microsatellites sont utilisés pour déterminer les haplotypes associés à la région Iq21q31 sur le bras long du chromosome 1. Leur liste avec la description des amorces (séquence et addition d'un fluorochrome) est donnée dans le tableau 1.The DNA is precipitated with a volume of isopropanol, rinsed with ethanol (70%), dried and finally resuspended in 1 ml of TE buffer (10 mM Tris pH 8, 0.5 mM EDTA). The DNA concentration is evaluated by measuring the absorbance at 260 mm using the equivalence of 50 μg / ml of DNA for one unit of absorbance. The DNA concentration is then adjusted to 200 μg / ml. B) Amplification of genomic DNA for fluorescent microsatellite markers. Nine microsatellites are used to determine the haplotypes associated with the Iq21q31 region on the long arm of chromosome 1. Their list with description of primers (sequence and addition of a fluorochrome) is given in table 1.
Les conditions d'amplification communes à ces marqueurs sont les suivantes : Mélange réactionnel :The amplification conditions common to these markers are as follows: Reaction mixture:
. ADN génomique (200 μg/ml) 1 μl. Genomic DNA (200 μg / ml) 1 μl
. nucléotides triphosphate (chacun 1,25 M) 4 μl. nucleotide triphosphate (each 1.25 M) 4 μl
. amorces (chacune 10 picomoles/μl) 2 x 1 μl. primers (each 10 picomoles / μl) 2 x 1 μl
. Taq DNA polymérase TM (5 u/μl) 0,05 μl . tampon de PCR 10 fois concentré 2 .5 μl. Taq DNA polymerase TM (5 u / μl) 0.05 μl. 10 times concentrated PCR buffer 2.5 μl
. H2O, qsp 25 μl. H 2 O, qs 25 μl
Composition du tampon de PCR 10 x : Tris 0.1 M pH 8.3 (à 20° C), KC1 0.5Composition of the PCR buffer 10 x: Tris 0.1 M pH 8.3 (at 20 ° C), KC1 0.5
M, Mg Cb 15 mM, gélatine (SigmaTM G2500) 1 mg/ml.M, Mg Cb 15 mM, gelatin (SigmaTM G2500) 1 mg / ml.
Pour l'amplification du marqueur ngal , l'anticorps TaqStartTM (Clontech) est ajouté à la concentration finale de 0.056 μM (soit un rapport molaire anticorps/enzyme de 28:1).For the amplification of the ngal marker, the antibody TaqStartTM (Clontech) is added to the final concentration of 0.056 μM (i.e. an antibody / enzyme molar ratio of 28: 1).
L'amplification est réalisée dans un thermocycleur TechneTM PHC-3 avec couvercle chauffant. Après un chauffage à 94° C pendantThe amplification is carried out in a TechneTM PHC-3 thermal cycler with heated cover. After heating to 94 ° C for
5 minutes, 30 cycles sont effectués. Chaque cycle comprend 2 segments d'une minute chacun à ' 55° C et 94° C successivement. Un segment final d'élongation de 2 minutes à 72° C termine l'amplification. 5 minutes, 30 cycles are performed. Each cycle comprises two one-minute periods each at '55 ° C and 94 ° C successively. A final elongation segment of 2 minutes at 72 ° C ends the amplification.
Tableau. Liste des marqueurs microsatellites utilisés pour haplotyper la région du gène du glaucome juvénile sur le chromosome Iq21q31.Board. List of microsatellite markers used to haplotype the region of the juvenile glaucoma gene on chromosome Iq21q31.
MARQUEUR SÉQUENCE NUCLÉOTIDIQUE (taille en pb) des AMORCES ( amont puis aval ) afm350y l JOE- 5 ' -TCTTCCCACCACTGCCNUCLEOTIDE SEQUENCE MARKER (size in bp) of PRIMERS (upstream then downstream) afm350y l JOE- 5 '-TCTTCCCACCACTGCC
(189 bp) 5 ' -TGTATTCCTACTGCCCA(189 bp) 5 '-TGTATTCCTACTGCCCA
af l22xa3 FITC-5 ' -CCTCAGTTCATTCCCATAAaf l22xa3 FITC-5 '-CCTCAGTTCATTCCCATAA
(D1S210 ) ( 121 pb) 5 ' -AGCTGAATCTCACCCAATAACTA(D1S210) (121 bp) 5 '-AGCTGAATCTCACCCAATAACTA
ngal 5'-JOE CCAACTGAGAATTCTATATTTAACCngal 5'-JOE CCAACTGAGAATTCTATATTTAACC
(200 bp) 5'-TCTGGTAGGGCAGATCTGCTAGAA(200 bp) 5'-TCTGGTAGGGCAGATCTGCTAGAA
afm21 JOE -5 ' -CCTTCCTTTCTAAGGCTGafm21 JOE -5 '-CCTTCCTTTCTAAGGCTG
( 121 bpN) 5 ' -TCTTATCAGTCAGGCA(121 bpN) 5 '-TCTTATCAGTCAGGCA
afm248wg5 FITC-5 ' -TAATGGGTTCAGTGGACCTTafm248wg5 FITC-5 '-TAATGGGTTCAGTGGACCTT
(D1S452 ) (223 pb) 5 ' -TGCAGTTCCATATTCCAGGT(D1S452) (223 bp) 5 '-TGCAGTTCCATATTCCAGGT
afm278yθ5 JOE-5 ' -TGAGCCGAGATTGAGCCafm278yθ5 JOE-5 '-TGAGCCGAGATTGAGCC
( 240 bp) 5 ' -CCAGGTCAGAGATGTTGG(240 bp) 5 '-CCAGGTCAGAGATGTTGG
a£m212xbl 0 5'-TAJMRA-TCTACCACTTGAATTCCTGTa £ m212xbl 0 5'-TAJMRA-TCTACCACTTGAATTCCTGT
(D1S242 ) (219 bp) 5 -ACCACTCCAGTTTGAGCAAC(D1S242) (219 bp) 5 -ACCACTCCAGTTTGAGCAAC
afml 57xθ7 5 ' -FAM-TGTAAAAGCAAACTGTAGACGATafml 57xθ7 5 '-FAM-TGTAAAAGCAAACTGTAGACGAT
( D1S218 ) (274 pb) 5 ' -TTTATGTTATCACCAAGGCTTCT(D1S218) (274 bp) 5 '-TTTATGTTATCACCAAGGCTTCT
NGA5 (L854B9) CTGAAACTGAGATAGGAGTGC GAAATGGGAGTTGAGTTACCC Les produits d'amplification (2 μl) d'un même individu sont rassemblés, coprécipités à l'éthanol (2.5 volumes, 2 heures à -20° C) et mis à migrer sur une même piste de gel d'acrylamide (6 %) - urée (8M), après resuspension dans un tampon de charge (formamide 2 μl, 0.5 μl Bleu ABITM, 0.5 μl GeneScan 2500 Rox, 1 μl H2O) et après dénaturation thermique.NGA5 (L854B9) CTGAAACTGAGATAGGAGTGC GAAATGGGAGTTGAGTTACCC The amplification products (2 μl) of the same individual are combined, coprecipitated with ethanol (2.5 volumes, 2 hours at -20 ° C) and allowed to migrate on the same track of acrylamide gel (6% ) - urea (8M), after resuspension in a loading buffer (formamide 2 μl, 0.5 μl Blue ABITM, 0.5 μl GeneScan 2500 Rox, 1 μl H 2 O) and after thermal denaturation.
L'électrophorèse est réalisée dans un séquenceur automatique ABI 373, sous une puissance de 30 Watts pendant huit heures, le faisceau laser étant placé à une hauteur de 24 cm. Les données brutes sont analysées avec le logiciel GeneScan AnalysisThe electrophoresis is carried out in an ABI 373 automatic sequencer, at a power of 30 Watts for eight hours, the laser beam being placed at a height of 24 cm. Raw data is analyzed with GeneScan Analysis software
(ABI). Les pics de fluorescence sont identifiés avec le logiciel Genotyper (ABI), permettant ainsi d'assigner les allèles des marqueurs microsatellites. Interprétation des résultats. Ces résultats de typage sont interprétés en fonction du contexte :(ABI). The fluorescence peaks are identified with the Genotyper software (ABI), thus making it possible to assign the alleles of the microsatellite markers. Results interpretation. These typing results are interpreted according to the context:
- dans une famille où un haplotype de susceptibilité pour le glaucome associé cette région du chromosome 1 a déjà été caractérisé, la comparaison des allèles de l'échantillon testé avec ceux de l'haplotype familial permet de façon simple de déterminer la présence ou l'absence du gène de susceptibilité. En l'état actuel des connaissances, la présence de ce dernier confère un risque de développer la maladie qui est estimé à 80 %. En son absence, le risque rejoint celui de la population générale ( 1 à 2 % selon les études épidémiologiques).- in a family where a haplotype of susceptibility for glaucoma associated with this region of chromosome 1 has already been characterized, the comparison of the alleles of the test sample with those of the family haplotype makes it possible to determine the presence or the absence of the susceptibility gene. In the current state of knowledge, the presence of the latter confers a risk of developing the disease which is estimated at 80%. In its absence, the risk meets that of the general population (1 to 2% according to epidemiological studies).
- dans une famille où un haplotype de susceptibilité pour le gène du glaucome associé à cette région du chromosome 1 n'a pas été déjà caractérisé, la vraisemblance de la présence du gène de la maladie est estimée à l'aide du logiciel LIN AGE.- in a family where a susceptibility haplotype for the glaucoma gene associated with this region of chromosome 1 has not already been characterized, the likelihood of the presence of the disease gene is estimated using the LIN AGE software.
Les résultats observés sur différentes familles ont démontré l'excellent caractère prédictif des marqueurs mis en oeuvre. LISTE DE SEQUENCESThe results observed on different families have demonstrated the excellent predictive nature of the markers used. LIST OF SEQUENCES
(1) INFORMATIONS GENERALES:(1) GENERAL INFORMATION:
(i) DEPOSANT:(i) DEPOSITOR:
(A) NOM: INSTITUT NATIONAL DE LA RECHERCHE MEDICALE(A) NAME: NATIONAL INSTITUTE OF MEDICAL RESEARCH
CINSERM)CINSERM)
(B) RUE: 101 RUE DE TOLBIAC(B) STREET: 101 RUE DE TOLBIAC
(C) VILLE: PARIS (E) PAYS: FRANCE(C) CITY: PARIS (E) COUNTRY: FRANCE
(F) CODE POSTAL: 75013(F) POSTAL CODE: 75013
(ii) TITRE DE L' INVENTION: DEPISTAGE DU GLAUCOME JUVENILE (iii) NOMBRE DE SEQUENCES: 18(ii) TITLE OF THE INVENTION: SCREENING FOR JUVENILE GLAUCOMA (iii) NUMBER OF SEQUENCES: 18
(iv) FORME DECHIFFRABLE PAR ORDINATEUR:(iv) COMPUTER-DETACHABLE FORM:
(A) TYPE DE SUPPORT: Floppy disk(A) TYPE OF SUPPORT: Floppy disk
(B) ORDINATEUR: MACINTOSH APPLE (C) SYSTEME D' EXPLOITATION: MAC-OS SYSTEME 7(B) COMPUTER: MACINTOSH APPLE (C) OPERATING SYSTEM: MAC-OS SYSTEM 7
(D) LOGICIEL: WORD PERFECT VERSION 2.0(D) SOFTWARE: WORD PERFECT VERSION 2.0
(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: 16 paires de bases(A) LENGTH: 16 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple (D) CONFIGURATION: linéaire(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique(ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AMONT" af 350yhl(A) DESCRIPTION: / desc = "UPSTREAM NUCLEOTIDE PRIMER" af 350yhl
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 1:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
TCTTCCCACC ACTGCC 16 (2) INFORMATIONS POUR LA SEQ ID NO: 2:TCTTCCCACC ACTGCC 16 (2) INFORMATION FOR SEQ ID NO: 2:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 17 paires de bases(A) LENGTH: 17 base pairs
(B) TYPE: nucléotide (C) NOMBRE DE BRINS: simple(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique(ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AVAL" afm350yhl (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 2:(A) DESCRIPTION: / desc = "DOWNLOAD NUCLEOTIDE PRIMER" afm350yhl (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
TGTATTCCTA CTGCCCA 17TGTATTCCTA CTGCCCA 17
(2) INFORMATIONS POUR LA SEQ ID NO: 3:(2) INFORMATION FOR SEQ ID NO: 3:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR: 19 paires de bases (B) TYPE: nucléotide(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique (A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE(ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "NUCLEOTIDE PRIMER
AMONT" afml22xa3UPSTREAM "afml22xa3
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 3: CCTCAGTTCA TTCCCATAA 19(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CCTCAGTTCA TTCCCATAA 19
(2) INFORMATIONS POUR LA SEQ ID NO: 4:(2) INFORMATION FOR SEQ ID NO: 4:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 23 paires de bases(A) LENGTH: 23 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire (ii) TYPE DE MOLECULE: Autre acide nucléique(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AVAL" afml22xa3(A) DESCRIPTION: / desc = "DOWNSTREAM NUCLEOTIDE PRIMER" afml22xa3
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 4:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
AGCTGAATCT CACCCAATAA CTA 23AGCTGAATCT CACCCAATAA CTA 23
(2) INFORMATIONS POUR LA SEQ ID NO: 5: (i) CARACTERISTIQUES DE LA SEQUENCE:(2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 25 paires de bases(A) LENGTH: 25 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique(ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AMONT" ngal (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 5:(A) DESCRIPTION: / desc = "UPSTREAM NUCLEOTIDE PRIMER" ngal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
CCAACTGAGA ATTCTATATT TAACC 25 (2) INFORMATIONS POUR LA SEQ ID NO: 6:CCAACTGAGA ATTCTATATT TAACC 25 (2) INFORMATION FOR SEQ ID NO: 6:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR: 24 paires de bases(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire (ii) TYPE DE MOLECULE: Autre acide nucléique(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AVAL" ngal(A) DESCRIPTION: / desc = "DOWNLOAD NUCLEOTIDE PRIMER" ngal
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 6:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
TCTGGTAGGG CAGATCTGCT AGAA 24TCTGGTAGGG CAGATCTGCT AGAA 24
(2) INFORMATIONS .POUR LA SEQ ID NO: 7: (i) CARACTERISTIQUES DE LA SEQUENCE:(2) INFORMATION. FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 18 paires de bases(A) LENGTH: 18 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique(ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AMONT" afm21 (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 7:(A) DESCRIPTION: / desc = "UPSTREAM NUCLEOTIDE PRIMER" afm21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:
CCTTCCTTTC TAAGGCTG 18CCTTCCTTTC TAAGGCTG 18
(2) INFORMATIONS POUR LA SEQ ID NO: 8:(2) INFORMATION FOR SEQ ID NO: 8:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 16 paires de bases(A) LENGTH: 16 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple (D) CONFIGURATION: linéaire(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique(ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AVAL" afm21(A) DESCRIPTION: / desc = "DOWNLOAD NUCLEOTIDE PRIMER" afm21
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 8: TCTTATCAGT CAGGCA 16 (2) INFORMATIONS POUR LA SEQ ID NO: 9:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: TCTTATCAGT CAGGCA 16 (2) INFORMATION FOR SEQ ID NO: 9:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 20 paires de bases (B) TYPE: nucléotide(A) LENGTH: 20 base pairs (B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique (A) DESCRIPTION: /desc = "AMORCE" NtrCtbUllUlLiUb'(ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = " PRIMER " NtrCtbUllUlLiUb '
AMONT" afm248wg5UPSTREAM "afm248wg5
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 9: TAATGGGTTC AGTGGACCTT 20(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: TAATGGGTTC AGTGGACCTT 20
(2) INFORMATIONS POUR LA SEQ ID NO: 10:(2) INFORMATION FOR SEQ ID NO: 10:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR: 20 paires de bases(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire (ii) TYPE DE MOLECULE: Autre acide nucléique(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AVAL" afm248wg5(A) DESCRIPTION: / desc = "DOWNSTREAM NUCLEOTIDE PRIMER" afm248wg5
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 10: TGCAGTTCCA TATTCCAGGT 20(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: TGCAGTTCCA TATTCCAGGT 20
(2) INFORMATIONS POUR LA SEQ ID NO: 11: (i) CARACTERISTIQUES DE LA SEQUENCE:(2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 17 paires de bases(A) LENGTH: 17 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique(ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AMONT" afm278ye5 (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 11:(A) DESCRIPTION: / desc = "UPSTREAM NUCLEOTIDE PRIMER" afm278ye5 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:
TGAGCCGAGA TTGAGCC 17 (2) INFORMATIONS POUR LA SEQ ID NO: 12:TGAGCCGAGA TTGAGCC 17 (2) INFORMATION FOR SEQ ID NO: 12:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 18 paires de bases (B) TYPE: nucléotide(A) LENGTH: 18 base pairs (B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique (A) DESCRΓ TTON: /desc = "AMORCT iwaEσrTDrQUEr(ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRΓ TTON: / desc = "AMORCT iwaEσrTDrQUEr
AVAL" afm278ye5DOWNSTREAM "afm278ye5
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 12: CCAGGTCAGA GATGTTGG 18(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: CCAGGTCAGA GATGTTGG 18
(2) INFORMATIONS POUR LA SEQ ID NO: 13:(2) INFORMATION FOR SEQ ID NO: 13:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR: 20 paires de bases(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire (ii) TYPE DE MOLECULE: Autre acide nucléique(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AMONT" afm212xbl0(A) DESCRIPTION: / desc = "UPSTREAM NUCLEOTIDE PRIMER" afm212xbl0
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 13: TCTACCACTT GAATTCCTGT 20(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: TCTACCACTT GAATTCCTGT 20
(2) INFORMATIONS POUR LA SEQ ID NO: 14: (i) CARACTERISTIQUES DE LA SEQUENCE:(2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 20 paires de bases(A) LENGTH: 20 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique(ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AVAL" afm212xbl0 (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 14:(A) DESCRIPTION: / desc = "PRIMER NUCLEOTIDE DOWNSTREAM" afm212xbl0 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14:
ACCACTCCAG TTTGAGCAAC 20 (2) INFORMATIONS POUR LA SEQ ID NO: 15:ACCACTCCAG TTTGAGCAAC 20 (2) INFORMATION FOR SEQ ID NO: 15:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 23 paires de bases (B) TYPE: nucléotide(A) LENGTH: 23 base pairs (B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique (A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE(ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "NUCLEOTIDE PRIMER
AMONT" afml57xe7UPSTREAM "afml57xe7
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 15: TGTAAAAGCA AACTGTAGAC GAT 23(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: TGTAAAAGCA AACTGTAGAC GAT 23
(2) INFORMATIONS POUR LA SEQ ID NO: 16:(2) INFORMATION FOR SEQ ID NO: 16:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR: 23 paires de bases(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire (ii) TYPE DE MOLECULE: Autre acide nucléique(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AVAL" afml57xe7(A) DESCRIPTION: / desc = "DOWNSTREAM NUCLEOTIDE PRIMER" afml57xe7
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 16: TTTATGTTAT CACCAAGGCT TCT 23(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: TTTATGTTAT CACCAAGGCT TCT 23
(2) INFORMATIONS POUR LA SEQ ID NO: 17: (i) CARACTERISTIQUES DE LA SEQUENCE:(2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 21 paires de bases(A) LENGTH: 21 base pairs
(B) TYPE: nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique(ii) TYPE OF MOLECULE: Other nucleic acid
(A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE AMONT" NGA5 (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 17:(A) DESCRIPTION: / desc = "UPSTREAM NUCLEOTIDE PRIMER" NGA5 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17:
CTGAAACTGA GATAGGAGTG C 21 (2) INFORMATIONS POUR LA SEQ ID NO: 18:CTGAAACTGA GATAGGAGTG C 21 (2) INFORMATION FOR SEQ ID NO: 18:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR: 21 paires de bases (B) TYPE: nucléotide(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: Autre acide nucléique (A) DESCRIPTION: /desc = "AMORCE NUCLEOTIDIQUE(ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "NUCLEOTIDE PRIMER
AVAL" NGA5DOWNSTREAM "NGA5
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 18: GAAATGGGAG TTGAGTTACC C 21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: GAAATGGGAG TTGAGTTACC C 21

Claims

REVENDICATIONS
1. Procédé de dépistage d'une prédisposition au glaucome juvénile chez un individu, caractérisé en ce qu'on caractérise dans un prélèvement biologique chez ledit individu, des marqueurs microsatellites liés avec la survenue du glaucome juvénile dans sa famille, les marqueurs microsatellites étant afm350yhl, afml2 xa3- "gα 1 qft"7l af ^ft^g'V afm278ye5, afm212xbl0, afml57xe7 et NGA5 du chromosome Iq21q31.1. Method for screening for a predisposition to juvenile glaucoma in an individual, characterized in that a microsatellite markers linked to the occurrence of juvenile glaucoma in his family are characterized in a biological sample, the microsatellite markers being afm350yhl , afml2 xa3- "g α 1 qft " 7 l af ^ ft ^ g'V afm278ye5, afm212xbl0, afml57xe7 and NGA5 of chromosome Iq21q31.
2. Procédé selon la revendication 1 , caractérisé en ce que le marqueur est situé sur le locus correspondant à la région située entre le marqueur afm248wg5 et afm212 xblO.2. Method according to claim 1, characterized in that the marker is located on the locus corresponding to the region located between the marker afm248wg5 and afm212 xblO.
3. Procédé selon l'une des revendications 1 et 2, caractérisé en ce que la présence dudit marqueur est détectée par amplification de la zone du marqueur. 3. Method according to one of claims 1 and 2, characterized in that the presence of said marker is detected by amplification of the marker area.
4. Procédé selon la revendication 3 , caractérisé en ce que l'amplification est effectuée par PCR.4. Method according to claim 3, characterized in that the amplification is carried out by PCR.
5. Procédé selon la revendication 4, caractérisé en ce qu'on utilise une amplification PCR multiplex.5. Method according to claim 4, characterized in that a multiplex PCR amplification is used.
6. Séquence ADN située entre le locus correspondant au marqueur afml22xa3 et afm 212xbl0 et liée à la survenue du glaucome juvénile.6. DNA sequence located between the locus corresponding to the marker afml22xa3 and afm 212xbl0 and linked to the occurrence of juvenile glaucoma.
7. Séquence ADN selon la revendication 6, caractérisée en ce qu'elle est située entre le marqueur afml22xa3 et NGA5. 7. DNA sequence according to claim 6, characterized in that it is located between the marker afml22xa3 and NGA5.
PCT/FR1996/000592 1995-04-18 1996-04-18 Juvenile glaucoma detection process WO1996033287A1 (en)

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FR9504590A FR2733251B1 (en) 1995-04-18 1995-04-18 Screening for juvenile glaucoma
FR95/04590 1995-04-18

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998024932A1 (en) * 1996-12-05 1998-06-11 Clark Abbot F Methods for diagnosing glaucoma and discovering anti-glaucoma drugs
WO1998036098A1 (en) * 1997-02-13 1998-08-20 The University Of Connecticut Diagnosis and treatment of glaucoma
US5925748A (en) * 1994-04-28 1999-07-20 The University Of Iowa Research Foundation DNA diagnostics for glaucoma
US6127128A (en) * 1999-05-07 2000-10-03 University Of Connecticut Diagnosis of primary congenital glaucoma
US6171788B1 (en) 1997-01-28 2001-01-09 The Regents Of The University Of California Methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
EP1088900A1 (en) * 1999-09-10 2001-04-04 Epidauros Biotechnologie AG Polymorphisms in the human CYP3A4, CYP3A7 and hPXR genes and their use in diagnostic and therapeutic applications
US6271026B1 (en) 1997-03-21 2001-08-07 The University Of Iowa Research Foundation Glaucoma compositions
US6403307B1 (en) 1997-03-21 2002-06-11 University Of Iowa Research Foundation Glaucoma therapeutics and diagnostics
US6475724B1 (en) 1997-01-28 2002-11-05 The Regents Of The University Of California Nucleic acids, kits, and methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
US6956103B2 (en) 1994-04-28 2005-10-18 The University Of Iowa Research Foundation Glaucoma therapeutics and diagnostics
US7138511B1 (en) 1997-01-28 2006-11-21 The Regents Of The University Of California Nucleic acids, kits and methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
US7220546B2 (en) 1996-12-05 2007-05-22 Alcon Manufacturing, Ltd. Methods for diagnosing glaucoma and discovering anti-glaucoma drugs

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
GRAFF ET AL.: "Confirmation of linkage to 1q21-31 in a Danish autosomal dominant juvenile-onset glaucoma family and evidence of genetic heterogeneity", HUMAN GENETICS, vol. 96, September 1995 (1995-09-01), pages 285 - 289, XP002010905 *
GYAPAY ET AL.: "The 1993-1994 Genethon human genetic linkage map", NATURE GENET. 7:246-339(1994), XP000560918 *
MEYER ET AL.: "Liaison du glaucome juvenile au chromosome 1q dans deux familles francaises.", C R ACAD SCI III, (1994 JUN) 317 (6) 565-70, XP000564959 *
MORISSETTE ET AL.: "A common gene for juvenile and adult-onset primary open angle glaucomas confined on chromosome 1q", AMERICAN JOURNAL OF HUMAN GENETICS, vol. 56, June 1995 (1995-06-01), CHICAGO, US, pages 1431 - 1442, XP002010904 *
RAYMOND V ET AL: "A single gene for juvenile and middle-age onset open-angle glaucomas confined within a small interval on chromosome 1q.", 44TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF HUMAN GENETICS, MONTREAL, QUEBEC, CANADA, OCTOBER 18-22, 1994. AMERICAN JOURNAL OF HUMAN GENETICS 55 (3 SUPPL.). 1994. A201. ISSN: 0002-9297, XP002010898 *
RAYMOND V ET AL: "A SPECIFIC HAPLOTYPE ON CHROMOSOME 1Q21-Q31 DEFINES THE LOCUS OF JUVENILE OPEN ANGLE GLAUCOMA IN A LARGE FRENCH-CANADIAN PEDIGREE", JOURNAL OF CELLULAR BIOCHEMISTRY, 1 January 1994 (1994-01-01), pages 200/201, XP000560687 *
RICHARDS ET AL.: "Mapping of a gene for autosomal dominant juvenile-onset open-angle glaucoma to chromosome Iq.", AM J HUM GENET, (1994 JAN) 54 (1) 62-70., XP002010901 *
SEGHATOLESLAMI M R ET AL: "Fine mapping of juvenile primary open angle glaucoma (POAG) on 1q21 -q31 and exclusion of adult-POAG from the respective region.", 44TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF HUMAN GENETICS, MONTREAL, QUEBEC, CANADA, OCTOBER 18-22, 1994. AMERICAN JOURNAL OF HUMAN GENETICS 55 (3 SUPPL.). 1994. A203. ISSN: 0002-9297, XP002010897 *
SHEFFIELD ET AL.: "Genetic linkage of familial open angle glaucoma to chromosome 1q21-q31", NAT GENET, (1993 MAY) 4 (1) 47-50., XP002010902 *
WEISSENBACH ET AL.: "A second generation linkage map of the human genome", NATURE, vol. 359, LONDON GB, pages 794 - 801, XP002010900 *
WIGGS ET AL.: "Genetic linkage of autosomal dominant juvenile glaucoma to 1q21-q31 in three affected pedigrees.", GENOMICS, (1994 MAY 15) 21 (2) 299-303., XP002010899 *
WIGGS J ET AL: "Further Evidence for a Locus for Autosomal Dominant Juvenile Glaucoma on Chromosome 1q and Evidence for Genetic Heterogeneity.", 44TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF HUMAN GENETICS, MONTREAL, QUEBEC, CANADA, OCTOBER 18-22, 1994. AMERICAN JOURNAL OF HUMAN GENETICS 55 (3 SUPPL.). 1994. A206. ISSN: 0002-9297, XP002010903 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5925748A (en) * 1994-04-28 1999-07-20 The University Of Iowa Research Foundation DNA diagnostics for glaucoma
US6956103B2 (en) 1994-04-28 2005-10-18 The University Of Iowa Research Foundation Glaucoma therapeutics and diagnostics
US7220546B2 (en) 1996-12-05 2007-05-22 Alcon Manufacturing, Ltd. Methods for diagnosing glaucoma and discovering anti-glaucoma drugs
WO1998024932A1 (en) * 1996-12-05 1998-06-11 Clark Abbot F Methods for diagnosing glaucoma and discovering anti-glaucoma drugs
AU728438B2 (en) * 1996-12-05 2001-01-11 Abbot F. Clark Methods for diagnosing glaucoma and discovering anti-glaucoma drugs
US6475724B1 (en) 1997-01-28 2002-11-05 The Regents Of The University Of California Nucleic acids, kits, and methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
US7138511B1 (en) 1997-01-28 2006-11-21 The Regents Of The University Of California Nucleic acids, kits and methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
US6171788B1 (en) 1997-01-28 2001-01-09 The Regents Of The University Of California Methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
US6046009A (en) * 1997-02-13 2000-04-04 The University Of Connecticut Diagnosis and treatment of glaucoma
US5962230A (en) * 1997-02-13 1999-10-05 The University Of Connecticut Diagnosis and treatment of glaucoma
WO1998036098A1 (en) * 1997-02-13 1998-08-20 The University Of Connecticut Diagnosis and treatment of glaucoma
US6271026B1 (en) 1997-03-21 2001-08-07 The University Of Iowa Research Foundation Glaucoma compositions
US6403307B1 (en) 1997-03-21 2002-06-11 University Of Iowa Research Foundation Glaucoma therapeutics and diagnostics
US6207394B1 (en) 1999-05-07 2001-03-27 University Of Connecticut Diagnosis of primary congenital glaucoma
US6127128A (en) * 1999-05-07 2000-10-03 University Of Connecticut Diagnosis of primary congenital glaucoma
EP1088900A1 (en) * 1999-09-10 2001-04-04 Epidauros Biotechnologie AG Polymorphisms in the human CYP3A4, CYP3A7 and hPXR genes and their use in diagnostic and therapeutic applications

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