FR2742758A1 - Probes for diagnosing Alzheimer's disease - Google Patents

Abstract

A pair of probes for detecting a mutation in the locus of chromosome 14 associated with a presenile form of Alzheimer's disease is claimed, where each of the probes hybridises with one of the two human chromosomal DNA segments cloned in the CEPH yeast artificial chromosome (YAC) library under the accession numbers YAC 934A3 (identifiable by genetic marker D14S251) and YAC 854F5 (identifiable by genetic marker D14S76). Also claimed is a DNA segment of the locus of chromosome 14 associated with a presenile form of Alzheimer's disease, where the segment hybridises with at least one of the above probes.

Description

 The invention relates to the identification of a human chromosomal segment associated with one of the early forms of Alzheimer's disease, and to a greater number of genetic markers carried by it, as well as their use as DNA probes for diagnosis. of said form of the disease.

Alzheimer's disease is the most common form of dementia. According to the age of onset of the first symptoms, a "late" form ("senile dementia", beginning after age 60) and an "early" form ("presenile dementia", before age 60) can be distinguished. It is a multifactorial disease, showing in 30-50% of cases inherited inherited dominantly (McKusick, 1992). The formation of amyloid plaques in the brain is one of the hallmarks of the disease. It contains a precipitated accumulation of several proteins, the major species being the A4 peptide (St. George-Hyslop et al., 1990, McKusick, 1992;
Hardy and Higgins, 1992).

 Alzheimer's disease is heterogeneous: it can be caused by various genetic anomalies, on different chromosomes - the late form is the most common. It seems that the ApoE4 allele carried by chromosome 19 is an important predisposing factor for this form of the disease (Corder et al., 1993).

- the early form represents about 20% of the family cases. This form is divided into
a major form, associated with a locus on chromosome 14, at 14q24.3 (St. George-Hyslop et al., 1992, Van Broeckhoven et al., 1992, Schellenberg et al 1992, Mullan et al. )
a minor form, associated with certain mutations of the API gene (precursor of amyloid A4 protein), located on chromosome 21 (St. George-Hyslop et al., 1987).

Finally, some families have been described for which the disease is not linked to either chromosome 14 or chromosome 21 ("Germans of the Volga" for example, St
George-Hyslop et al., 1992)
Due to the late onset of the disease, many cases occur in families that are too small (eg, deceased parents) to allow genetic linkage analysis.

 As the biochemical mechanisms affected by these different genetic forms of the disease are different, it will be important, in the attempts to develop therapies, to have simple diagnostic methods to distinguish, unambiguously, a particular form of the disease. Alzheimer's disease, in other forms or even in neighboring pathologies.

 The present invention relates to the early form of Alzheimer's disease associated with chromosome 14.

 Until very recently (St. George-Hyslop et al.

1992; Mullan et al. 1992; Van Broeckhoven et al. 1992;
Schellenberg et al. 1992) the causative locus was circumscribed in a fairly large region of the chromosome, localized at 14q (estimated between 15 and 18 cM), bounded by markers D14S57 and D14S59 and centered on the marker
D14S43 (these markers are defined in the report of the
Chromosome Coordinating Meeting 1992). The physical size of this region was estimated at 8-10 Mb. (cM = centi-Morgan,
Mb = megabases)
The Applicant has endeavored, first of all, to establish the physical mapping of the region D14S57-D14S43
D14S59 by constituting an "contig" (or continuous assembly) of overlapping YAC clones (YAC: Yeast Artificial
Chromosome, Burke et al., 1987).

 The YAC clones come from the human genomic library built at the CEPH (Center for the Study of Human Polymorphism - Paris) which is described by Cohen et al. (1993).

 The Applicant has observed that the 18 cM range identified above is characterized by the absence of genetic recombination events, in the families of patients which led them to search in this chromosomal segment, the existence of a genetic accident explaining this phenomenon and which could be the cause of the pathology.

In the 14q region limited by markers
D14S57 and D14S59 and centered on D14S43, the Applicant has identified a chromosomal segment of interest, which is limited by the following segments, cloned in YACs, oriented from upstream to downstream in the centromere - + telomere direction
939A6.934A3 --- 905C2 --- 854F5.746B4 (the numbers correspond to a clone representative of YAC, other clones corresponding to the same chromosomal segment are available in the same bank).

 YAC clones 939A6 and 934A3 overlap on a segment that contains the D14S251 marker. YAC clones 854F5 and 746B4 overlap on a segment that contains the D14S76 marker.

 By using polymerase chain reaction (PCR) DNA probes on each of these YACs and labeled with different fluorochromes, the different chromosomal segments could be located relative to one another on chromosome 14, by the technique. in situ hybridization (FISH) and the observation and photography of chromosomes labeled with fluorescence microscopy. In normal individuals, they are placed in the order indicated above.

 On the other hand, in one patient, and with probes corresponding to several clones of each type, one of the two chromosomes 14 has a displacement of the signal corresponding to the 934A3 type marker which approaches the telomere and the signal corresponding to the 854F5 marker. away from the telomer, the signals corresponding to markers 936A6, 905C2 and 946B4 remaining in the same place as on the chromosomes of the controls. All these observations correspond to a microinversion of the segment 934A3 - 854F5, around the pivot represented by the marker 905C2.

 This small genetic rearrangement is not detectable in classical cytogenetics, the karyotypes made on the mitotic preparations of the patients having no abnormal character. In particular, no reshuffling is visible at chromosome 14.

 The Applicant has thus demonstrated the existence of a genetic rearrangement involving a small segment of the 14q locus associated with an early form of Alzheimer's disease. The Applicant has taken advantage of this surprising phenomenon to make this chromosomal segment a genetic marker of the disease. For this purpose, she designed DNA probes and the method of using them to reveal the genetic anomaly on metaphase chromosome preparations of patients, this method allows a simple and discriminatory diagnosis of a form of Alzheimer's disease.

According to a first aspect, the invention therefore relates to a pair of DNA probes for detecting genetic rearrangement in the chromosome 14 locus associated with an early form of Alzheimer's disease. The DNA of these probes hybridizes to selected human chromosomal DNA segments, cloned and available in the YACs library.
CEPH (cited above). The first probe hybridizes to the YAC carrying the access nO 934A3, which can be identified by the genetic marker D14S251 which is close to its upstream end (remote from the telomere). The second probe hybridizes to
YAC bearing access No. 854F5, which can be identified by the genetic marker D14S76 which is close to its downstream end (towards the telomere). These markers D14S .. are listed in the international nomenclature of the human genome taken up by Gyapay et al. 1994.

It is obvious that the probes thus defined also hybridize with all the segments carried by
YACs of the same group, that is, whose location on the chromosome largely overlaps the YAC chosen as the type.

They also hybridize to shorter segments within the chosen segment; such shorter segments may also be cloned into another type of vector, for example in a cosmid. The preparation of the same probes can be carried out from such vectors, to simplify the manipulation (the resulting probe will always hybridize to the YAC segment which defines the probe according to the invention).

 The pair of probes according to the invention is made visible under the microscope by a distinct labeling of each probe, chosen to allow the simultaneous detection of both probes and their relative locations on the chromosome, after in situ hybridization. The labeling is preferably carried out using 2 fluorochromes with distinct spectra.

Another type of marking using chemical markers or radioactive isotopes could also be applied.

The invention also relates to the use of the pair of DNA probes for the diagnosis of the form of Alzheimer's disease linked to chromosome 14. The implementation of the diagnostic test comprises the following steps: the preparation on slides of chromosome microscope
metaphases of lymphoblastoid cells taken from
the patient and put in culture, - the putting in contact, on this preparation of chromo
somes, of the pair of probes marked under the conditions
for hybridization of DNAs, - the detection of probes hybridized under the conditions
(with a fluorescence microscope equipped with
filters adapted to selected fluorochromes) and
relative location of the 2 probes on a number
significant number of chromosome pairs 14.

 This test is performed by comparison with control chromosome preparations; in addition, one of the two chromosomes of the patient also serves as a reference since the genetic accident concerns only one of the two chromosomes.

 Variations of this test can be envisaged.

 Thus, in the pair of probes, one of the two can be replaced by a probe which hybridises to the segment considered pivotal in the microinversion movement and which is represented by the YAC 905C2. In this case we will observe, on one of the chromosomes of the patient, a displacement of the other marker around this pivot.

 This embodiment of the test can be implemented systematically in addition to the test described above, to confirm the result.

In another aspect, the invention also relates to the DNA segment of the chromosome 14 locus, as identified, and which hybridizes to at least one of the probes according to the invention. Said DNA segment has a genetic rearrangement identifiable by a microinversion of genetic markers, one or the other of the bounds is probably located within a gene associated with the pathology or in regulatory regions controlling the expression of this gene. This segment of DNA associated with the pathology is close to at least one of the two markers
D14S251 and D14S76. In addition, it comprises at least one of two regions common to overlapping YAC clones 939A6 and 934A3 or 854F5 and 746B4, or a portion of said common regions.

 The identification of the DNA segment according to the invention opens therapeutic perspectives for the form of the Alzheimer's disease associated therewith.

 The following examples illustrate a mode of characterization and embodiment of the invention, without limiting its scope.

EXAMPLE I. Preparation of samples and probes for
in situ hybridization (FTSH).

 The following preparations are performed for the location of the chromosomal fragment identified as a marker of Alzheimer's disease on chromosome 14 by the FISH technique.

 T 1 Preparation of metaphase chromosomes.

 Lymphoblastoid lines derived from blood samples from patients identified or whose diagnosis is to be specified are used.

 High resolution metaphase chromosome preparations are obtained following the experimental protocol described by Chérif et al. (1990).

 The synchronization is carried out by adding, in the exponential growth phase, methotrexate (10wu, for 17h), then 5-bromodeoxyuridine (0.1mu, for 6h). 1 mg / ml of colcemide is added 15 minutes before stopping the culture. Plating on slides of chromosomes is carried out in hypotonic solution (75mM KC1). After spreading and fixing with a solution of methanol / glacial acetic acid 3/1 (vol / vol), the slides are stored at -200C and protected from light, until use.

 T.2. DNA Preparation of YAC Clones.

Cloning of large DNA fragments into artificial yeast or YAC chromosomes has been described by
Burke et al. (1987) and the method was applied to construct libraries of human chromosomal material (Cohen et al 1993). The library built at CEPH (Center for the Study of Human Polymorphism - Paris) contains 33,000 clones with inserts with an average length of 0.9 megabases and has been tested with more than 2,000 genetic markers distributed over 90% of the genome. This material is available to the scientific community for the detailed establishment of the physical map of human chromosomes. The access references to this bank are given in Cohen et al. 1993 already mentioned.

 The YAC clones used hereinafter come from this bank.

The DNA of the YAC clones of interest is prepared from inclusion blocks assayed at 4x109 yeast cells / ml, in 0.5% low-melting agarose (SeaPlaques GTG agarose,
FMC BioProducts, USA). Five blocks (approximately 400 ml) are equilibrated (3x30 min at 40 ° C.) with 5 volumes of Agarase buffer (10 mM pH 10.6 Bis Tris-HCl, 5 mM EDTA). The blocks are then melted 5mn at 650C, then tempered at 400C. After addition of 4 units of β-Agarase (New England Biolabs, USA), the whole is incubated for 4 hours at 400 ° C., then the total DNA is extracted with phenol / chloroform and precipitated with ethanol.

 I.3 Preparation of the probes by PCR.

 The total DNA of the YAC clones is used to prepare hybridization probes, by PCR (polymerase chain reaction), or directly by random priming (the random initiation of the enzymatic polymerization reaction, by means of a mixture of hexanucleotides), or by Alu-PCR followed by random priming.

In the latter case, primers SR1 (5'-CCACTGCACTCCAGCCTGGG-3 ', Romana et al., 1993) and BK33 (5'-CTGGGATTACAGGCGTGAGC-3', Sinke et al., 1992) are used simultaneously, according to a protocol described by
Romana et al. (1993).

 The use of these primers which correspond to sequences close to the ends of the repetitive regions Alu makes it possible to avoid the marking of the Alu regions and the background noise of them, on reading the FISH test.

 The PCR products are precipitated with ethanol, redissolved in 10mM: 1mM TE (Tris-HCl: EDTA), quantified on 1.3% agarose gel before being used for random priming.

 For experiments performed in single staining, the labeling of the probes is performed using dUTP-11biotin. For double staining experiments the labeling of the probes is performed using dUTP-11biotin for one probe and digoxigenin for the other probe.

I.4. In situ hybridization (FTSH).

 The technique of non-radioactive labeling of chromosomes in metaphase, by in situ hybridization with probes labeled with two different fluorescent dyes, makes it possible to establish high resolution physical maps, to locate a chosen marker with respect to the R bands (Camargo and Cervenka 1980 and 1982, ISCN - 1991) or a novel trait with respect to a reference marker whose position on the physical map is already known.

Recently this technique is commonly applied to map human genome segments cloned into cosmid libraries or YACs.

 The FISH technique is carried out according to the protocol described by Chérif et al. (1990). Hybridization is carried out in a 1OCL1 volume containing 250ng of probe (or each probe for double staining experiments), 120l of Cotl DNA (Gibco BRL) and 10mg of sonicated salmon sperm DNA (SSS DNA) .

 After in situ hybridization, immersion in Earle solution at 870C for 2 to 4 minutes reveals an R band system (Cherif et al., 1990, R bands according to the ISCN international nomenclature, 1991).

 The slides are analyzed by fluorescence microscopy and photographed as described by Chérif et al. Specific signals appear as symmetrical spots on the 2 chromatids of at least one of the homologous chromosomes. The technique being based on the use of 2 fluorochromes with distinct spectra allows the identification of bands and specific signals (or 2 different specific signals) on the same preparation, by simple filter change.

example It highlights a micro-reshuffle
generic associated with an early form of Alzheimer's disease.

 Chromosomal preparations were made from cultures of lymphoblastoid lineages of patients for whom the diagnosis of early familial Alzheimer's disease was established.

 II.1. Patients.

Patient # 1 comes from a French family for which the mutation could be demonstrated associated with chromosome 14, by a genetic linkage study using markers D14S43, D14S71 and D14S77
Patients n02 (V-249, line AG 07877) and n03 <VII-14; line AG 07638B) are from independent families, of Italian origin (FAD4) and Canadian (FAD1) respectively (ADF: Familial Alzheimer's Disease), listed for a similar form of the disease. For these two families, the mutation could also be genetically associated with a locus carried by chromosome 14 (St.

George-Hyslop et al., 1992). The lymphoblastoid lineages of patients 2 and 3 (as well as the description of the families
FAD4 and FAD1) were obtained from the Coriell Institute for Medical Research (NIGMS Human Genetic Mutant Cell
Repository, 401 Haddon Avenue, Camden NJ 08103, USA).

Il.2. Genetic map of useful markers of locus 14q24.3
The work of different teams (cited on p.2) identified a locus associated with an early form of Alzheimer's disease on chromosome 14, in position 14q24.3, centered around marker D14S43, and delimited by markers. D14S57 and D14S59.

This region has been analyzed in detail - The physical mapping of the region D14S57 - D14S43
D14S59 was specified by the constitution of a "contig" of overlapping YAC clones. The YAC clones come from the genomic library built at the CEPH.

The genetic markers D14S ... are described in the references cited above and in "The 1993-94
Genethon human genetic linkage map ", Gyapay et al (1994).

- The elements of this map that have been used in FISH experiments are summarized in the following diagram

<tb> CEN <SEP> - // - <SEP> - 1 / - <SEP> AB <SEP> - // - <SEP> 90su2 <SEP> - // - <SEP > CD <SEP> - // - <SEP> - / 1-- <SEP> TEL
<tb><SEP> T <SEP> T
<tb><SEP> D14S57 <SEP> T <SEP> D14S43 <SEP> t <SEP> D14S59
<tb><SEP> D14S251 <SEP> D14S76
<tb> in which CEN represents the centromere
TEL represents the telomere
A, B, 905C2, C, D represent groups of
YACs
tD14S .. represents the position of the markers
genetic.

 The following YAC clones belong to group A: 939A6, 938E10, 707F8.

 The following YAC clones belong to group B: 934A3, 676E4, 895B8, 924C7, 962D8.

 The YACs of Group A and Group B overlap and share a segment containing the AFM203zb2 microsatellite marker (D14S251).

 Group C is represented by clone YAC 854F5 and group D by clone YAC 746B4. These overlap and share a segment containing the microsatellite markers AFM214zg3 (D14S76) and AFM283va9 (D14S273) which are confused on the genetic map (distance O cM).

 - The region D14S57 - D14S43 - D14SS9 was more particularly analyzed on chromosomes 14 of the 3 patients described above.

 In FISH experiments performed in single staining, the distance of the hybridization signals was evaluated for the two chromosomes of the patient nO 1 relative to the telomer and compared to the signals obtained on reference mitotic preparations, from normal individuals. .

 The results show that the signal obtained with the probe derived from clone 934A3 is slightly closer to telomere for one of the chromosomes of patient 1, while it remains at the position indicated by the reference mitotic preparation for the other chromosome. Clone 939A6, on the other hand, shows no variation.

 In the same way, the 854F5 clone has a telomere remoteness on one of the chromosomes of the patient 1, relative to its reference position, while the clone 746B4 does not vary from its reference position.

 Double staining FISH experiments were performed using clone 905C2 as the pivot point. The results highlight a switchover of the hybridization signals for all clones of group B in telomeric position relative to the pivot point, on one of chromosomes 14 of patient 1 (compared to the reference situation observed on mitotic preparations from normal individuals), whereas the signals relating to the clones of group A show no displacement.

 Likewise, the hybridization signal of clone 854F5 (group C) switches centromerically with respect to the pivot point on one of the chromosomes of patient 1, whereas the signal corresponding to clone 746B4 (group D) is invariant.

 Similar experiments carried out on the lines from patients 2 and 3 demonstrate a similar chromosomal micro-rearrangement phenomenon (switchover of signals for clones 934A3 (group B) and 854F5 (group C).

 In these patients, no chromosomal rearrangement was visible by conventional karyotype analysis.

 The results of in situ hybridization experiments (FISH) clearly indicate the existence of a microinversion of a segment of one of chromosomes 14 of the studied patients.

 The proximal and distal limits of this rearrangement could be localized, for the patient nO 1, inside or in the immediate vicinity of the DNA segment shared by the clones YAC 939A6 and 934A3 (groups A and B respectively) for the proximal terminal; within or in the immediate vicinity of the segment common to clones 854F5 and 746B4 on the distal side.

 It is likely that either of the boundaries of the inversion is located within the gene associated with the pathology or in regulatory regions, controlling its expression.

Example III. Diagnostic applications.

The characterization of the microinversion event of a segment of chromosome 14, described in Example II, can be applied to the development of a diagnostic test specific to the familial form of Alzheimer's disease linked to the chromosome 14, according to the following protocol
The YAC clones of reference 934A3 (B), 854F5 (C) and 905C2 (pivot) are used to prepare fluorescent probes for FISH test, as described in Example I, probes B and C or probes B or C and pivot being labeled with two different fluorochromes.

 Lymphoblastoid cells are removed from the patients to be diagnosed, they are cultured and synchronized to produce the metaphase chromosome preparations, as described in Example I.

Approximately 20 chromosomal preparations per patient will be analyzed.

 - Hybridization of the probes on the chromosome preparations and their analysis by fluorescence microscopy will show a reversal of the fluorescent signals on one of the two chromosomes 14, the other presenting the same image as a control chromosome preparation, in case of a positive diagnosis . In healthy subjects or those suffering from another form of Alzheimer's disease, the 2 fluorescent signals will be located in the same place, as in control subjects.

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Claims (11)

 1. A pair of DNA probes for the detection of genetic rearrangement in the locus of chromosome 14 associated with one of the early forms of Alzheimer's disease, characterized in that each of said probes hybridizes to one of of the two segments of human chromosomal DNA cloned and available in the CEPH (Center of Human Polymorphism) YACs (1Least Airtificial hromosomes) library under access numbers YAC 934A3 and YAC 854F5, the YAC 934A3 that can be identified by the genetic marker D14S251 and YAC 854F5 can be identified by the genetic marker D14S76.  2. Pair of probes according to claim 1, characterized in that the two probes carry a distinct marking chosen to allow simultaneous detection of both probes and their relative locations on the chromosome, after in situ hybridization.  3. Pair of probes according to claim 1 or 2, characterized in that the labeling is carried out using 2 fluorochromes with distinct spectra.  4.- Use of the pair of DNA probes according to any one of claims 1 to 3, for the diagnosis of the form of Alzheimer's disease associated with chromosome 14, characterized in that it comprises - the preparation on microscope slides of chromosomes  metaphases of lymphoblastoid cells of the patient; - bringing into contact, on this preparation of  chromosomes, the pair of probes according to  according to one of the preceding claims,  hybridization of the DNAs; the detection of the hybridized probes and the localization  relative of the 2 probes on a significant number of  pairs of chromosomes 14.  5.- Use according to claim 4, characterized in that it comprises - bringing into contact, on the chromosome preparation, a probe selected from one of the two probes according to claims 1 to 3 and a another probe that hybridises to YAC 905C2 from the same bank.  6. A DNA segment of the chromosome 14 locus associated with one of the early forms of Alzheimer's disease, characterized in that it hybridises to at least one of the probes according to claim 1.  7. A DNA segment according to claim 6, characterized in that it has an identifiable genetic rearrangement by a microinversion of genetic markers.  8. A DNA segment according to claim 6 or 7, characterized in that one of its ends is close to the marker D14S251.  9. A DNA segment according to claim 6 or 7, characterized in that one of its ends is close to the D14S76 marker.  10. DNA segment according to any one of claims 6 to 8, characterized in that it comprises the common region overlapping YAC clones 939A6 and 934A3 of the same bank, or a portion of said region.  11. DNA segment according to any one of claims 6, 7 and 9, characterized in that it comprises the common region overlapping YAC clones 854F5 and 746B4 of the same bank, or a portion of said region.