FR2764306A1 - METHOD OF INDIRECT GENOTYPIC DIAGNOSIS OF FAMILY HEMIPLEGIC MIGRAINE TYPE 2 - Google Patents

Abstract

The invention concerns an indirect genotypic method for diagnosing type 2 familial hemiplegic migraine in a person, characterised in that it consists in: isolating in said person's DNA the presence of a marker located on the 1a21-q23 chromosome in the locus limited by the D1S2343 and D1S2844 markers, said marker being related with the occurrence of familial hemiplegic migraine in his family.

Description

 The present invention relates in particular to screening for predisposition as well as the diagnosis of familial hemiplegic migraine, hereinafter MHF, and the possibility of highlighting one or more genes involved in this condition.

 Familial hemiplegic migraine is an inherited condition.

 Hemiplegic migraine is a form of migraine with aura in which the aura has a unilateral motor deficit, usually associated with visual and sensitive symptoms, followed by migraine headaches (2, 3). In severe attacks, prolonged deficits, fever, confusion and coma, can occur but usually go away without sequelae (4).

Recent research has shown that the gene responsible for the occurrence of MHF in certain families is located on chromosome 19 (11). This gene,
CACNL1A4, code for an al subunit of a specific calcium channel in the brain (1).

 However, the inventors have demonstrated that MHF is genetically heterogeneous: in particular 9 of the 21 families tested so far were not linked to chromosome 19 (5, 6, 7).

 A linkage analysis conducted on one of these families not linked to chromosome 19 made it possible to locate a second gene responsible for MHF on chromosome 1.

More particularly, the locus of susceptibility, which will be referred to below
MHF2, (as opposed to the locus located on chromosome 19 called MHF1), is located in the lq21-lq23 region within a range of 21 cM (centimorgan) (8) flanked by the two markers D1S2343 and D1S2844.

 The markers mentioned in the present description are known, they are the markers of Genethon and the primers making it possible to demonstrate them are also known. A Lod-score greater than 3 was obtained in this first family with two markers, Dus2635 and Dus2705. Three other markers, D1S2343, DIS2707 and D1S2844 showed lod-scores greater than 2. All of these data made it possible to establish the existence of a second locus for MHF located on chromosome 1.

 Two other families of MEF among the 6 families tested for their binding to chromosome 1 were well linked to chromosome 1. The 4 remaining families were not linked to chromosome 1, thus demonstrating the existence of at least a third locus for MHF since these 4 families were also not linked to chromosome 19.

 Among the 3 families linked to chromosome 1, two of the families had several members in whom the diagnosis of hemiplegic migraine had been very late and difficult because of the nature of the symptoms they presented. The clinical diagnosis in some of these patients had hesitated between hemiplegic migraine and seizure.

 Furthermore, the clinical penetrance (percentage of individuals carrying the sick gene who express the sick phenotype) of the forms of MHF linked to chromosome 1 is incomplete and lower than in the forms linked to chromosome 19. Thus, an affected subject may present as a sporadic case.

 These two observations, the entanglement with epileptic seizures and the incomplete nature of the penetrance, underline the interest of a genotypic diagnostic test of this form of MHF.

 In addition, these forms of MHF linked to chromosome 1 could justify different treatments from other forms of MHF.

 Knowledge of the locus according to the present invention makes it possible to envisage the diagnosis of the disease, in particular within families at risk, and makes it possible to orient the diagnosis during the occurrence of a paroxysmal episode.

This is why, the present invention relates to a method of indirect genotypic diagnosis of familial hemiplegic migraine type 2 in an individual, characterized in that: the presence of a marker is demonstrated in the DNA originating from said individual.
located on chromosome lq21-q23 in the locus limited by the markers D1S2343
and D1S2844, said marker being linked to the occurrence of hemiplegic migraine
family within his family.

This method can be applied in particular in the following two cases:. on the one hand, in front of a table of familial hemiplegic migraine, to highlight
evidence that it is an MHF2, that is to say linked to chromosome 1, and which
could justify special treatments different from those applied in the case
of MHF1 linked to chromosome 19,. on the other hand, to detect in an individual belonging to a family at risk the
risk of suffering from MHF and thus avoid misdiagnosis.

 By marker linked to the occurrence of MHF within the family of the individual is intended to denote the presence of one or more DNA sequences which are linked with the occurrence of the disorder in the family, some of these markers located in the previous locus are identified more precisely below.

 A marker which can be used in the method according to the present invention can be demonstrated by the binding analysis applied to DNA samples from family members, affected or not. This is a process which is known and which will therefore only be briefly recalled below.

 The first step is to establish the link between the gene responsible for the pathology and one or more markers. This study is conducted on families whose structure lends itself to this type of analysis, that is to say families in which the presence of familial hemiplegic migraine has been clearly established by clinical analyzes as complete as possible.

 Then, a genetic linkage analysis is carried out between the different markers tested, that is to say DNA fragments, and the pathology; preferably, highly polymorphic markers are used for this: microsatellites flanking highly polymorphic CA or GATA repeat units, these markers covering the interval in which the gene involved in the pathology is found, here the locus located between D1S2343 and D1S2844.

The advantage of using microsatellite markers is that they are 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 areas are called microsatellites and are the subject of numerous publications (Weissenbach J. et al., Nature 359, 794-801, 1992; Gyapay et al. 1996, A comprehensive genetic map of the human genome based on 5,264 microsatellites, Nature, vol. 380; Hudson et al., 1992, Genomics 13: 622-629 and
Weber and May, 1989 and Weber J. et al., Am. J. Hum. Gen., 1993, 53, 1079-1095).

 These markers are selected to give the best information for a specific family. The closer these markers are to the gene, the lower the risk of recombination between the gene and these markers and therefore the diagnosis will be all the more reliable.

In the case of familial hemiplegic migraine, the two markers mentioned above are the end markers but other markers could have been used, these are in particular the markers: D1 S2343
D1S2635
D1S2707
D1S2705
D1S2844
These microsatellite markers, as well as the primers which make it possible to highlight them, are known, they have been the subject of numerous publications and in particular appear in the map known as the Généthon Map.

 The alleles are defined by the different sizes of the fragments amplified with the oligonucleotides corresponding to a given microsatellite. The analysis of several members of defined status within a family makes it possible to identify for each marker the allele which is linked to the disease in the family considered.

 Of course, it is possible to highlight other markers in the same locus which could be linked to the occurrence of the disease in the family in question.

 In order to detect the presence of a microsatellite allele, the most commonly used method consists, after having hybridized the primers corresponding to microsatellite markers for example, to carry out an amplification by PCR or by any analogous method, then to separate the product from amplification, in particular by electrophoresis on acrylamide gel, then demonstrated by labeling using polyCA or polyGATA type sequences as a probe for microsatellites.

 A statistical analysis, lod-score method, makes it possible to estimate, from the observed distribution of alleles within the family, whether or not there is a genetic link between the pathology in question and the marker tested.

 The so-called lod-score statistical analysis is a known statistical technique and it is commonly accepted that a lod-score greater than 3 unambiguously establishes the link between the pathology gene and the marker tested.

 If a marker and a gene are very close (we say that they are linked), the portion of chromosome containing them will be transmitted as is 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 is no longer necessarily associated with the presence of the defective gene.

 Of course, when this analysis has been carried out, as is the case in the context of the present invention, it is possible to directly use information thus collected to carry out the screening for a predisposition to familial hemiplegic migraine of the type 2 in a member of this family, it is not necessary to repeat the entire analysis.

 The method according to the invention can be implemented both in adults, in children and in the fetus as a prenatal diagnosis.

 In order to simplify the detection, when different markers are used, 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.

 Of course, it is possible to use other amplification methods than PCR, which are sometimes called PCR-like, or others. Likewise, the disclosure methods are known and will not be described in detail.

 As an indication, a diagnostic protocol is given below on the basis of the polymorphisms which have been described previously. Of course, any method of indirect genotypic diagnosis can be used.

In order to select the most informative markers based on what is known about the family or whether we are trying to establish a diagnosis on the family, we will perform the following operations We obtain DNA from the different members of the family, each of them having
been studied clinically in order to highlight the existence or not of the
illness in the individual in question.

. Nuclear DNA can, for example, be isolated from leukocytes in the blood
peripheral, lymphoblastoid cells, amniotic fluid cells, put
in culture or otherwise, treated appropriately.

. Then we amplify the polymorphic alleles of microsatellites by the method
PCR using a thermal cycler,. then the size of the amplification products is analyzed by gel electrophoresis,
membrane fixation and hybridization with appropriate probes as was
previously mentioned.

It is also possible to perform an analysis by RFLP, that is to say analysis of
Polymorphic restriction fragments on an agarose gel after Southern blotting.

. The information obtained from the autoradiographies is analyzed by the
statistical program.

 The confirmation of the link or the absence of link between the analyzed fragments and the MHF were the subject of a statistical analysis for which a link score higher than 3 establishes in an unambiguous way the link between the gene responsible for pathology and the marker tested. A positive lod-score but less than 3 can be obtained when linked to the MHF2 locus, due to the small size of the family explored. A calculation of the probability that the family is related can then be performed using the HOMOG method (12).

 This then makes it possible to determine the most informative microsatellites for the family in question.

The diagnostic protocol for an individual at risk of a disease implements the information previously obtained: the most informative markers for the family in question are selected, the DNA of the individual is separated by appropriate methods,. the sequences located in the locus of onset of the disease are amplified, such as
this has been described previously,
the amplification products are analyzed,. we then use the polymorphic markers and the results found previously
so as to be able to calculate the risk of occurrence of the pathology in the individual in
cause.

 The present invention also relates to the DNA sequence and the genes involved in MHF2. The localization of the MHF2 gene in fact makes it possible, through a functional cloning strategy, to identify this gene.

It is possible to isolate the gene, in particular by. simultaneous search for new polymorphic markers and new
recombinant haplotypes that will further shrink the region containing the
uncomfortable; direct search for genes by several methods
selection of transcripts by hybridization
trapping exons
phylogenetically conserved sequences CpG islets
expansion of triplets
This is why the present invention also relates to the DNA sequence located on the locus located between the markers DIS2343 and D1S2844 and linked to the onset of familial hemiplegic migraine as well as the gene involved in the occurrence of hemiplegic migraine type 2. and located on a DNA according to the invention.

 Other characteristics and methods of implementing the method can be deduced from reading the example which follows.

 Figure 1 shows schematically the pedigree of the 3 MHF families linked to chromosome I q21 -q23. Affected individuals are represented by black symbols, unaffected individuals are represented by empty symbols, when the status of the individual is unknown, the symbol is hatched, individuals with no information are crossed out.

 The position of the various markers in the region has also been shown.

The alleles for the markers are represented for each individual, the blackened parts indicate regions which co-aggregate with the disease.

EXAMPLE
GenotvPaze method of human genomisue DNA with microsatellite markers of the la21-lu23 region
The P family is a large Caucasian family of 3 generations from the north of France. Families C, D and V are from southern France, families H and N from central France and family W from northern Spain. A total of 102 subjects including 20 spouses were examined as part of this study. In family P, 12 members suffer from characteristic hemiplegic migraine attacks (2). In addition to the typical attacks, 5 of them suffered severe episodes in which hemiplegia was associated with fever, confusion and coma. In the other 6 pedigrees, 35 patients were identified fulfilling the international criteria
Headache Society. The 47 subjects suffering from lvlHF had a normal neurological examination outside the attacks. These 47 members have been classified as affected. 25 subjects over the age of 15 have no history of hemiplegic migraine, no migraine with non-hemiplegic aura or other episode not determined such as aseptic meningitis or episodic confusion. These 25 were classified as healthy. 9 subjects suffering from migraine with sensitive and visual aura, or having children in whom there was a diagnostic doubt, were classified with unknown status. Blood was collected from all consenting members and DNA was prepared as described. Due to the presence of 1 or 2 asymptomatic subjects who carry the mutated gene in each family, the penetrance for the linkage analysis was set at 80%.

 The result of this analysis is shown in Figure 1 attached according to the usual representations.

The analysis of the markers appearing in this figure was carried out as follows:
DNA is extracted from the blood of different individuals by known methods.

 5 markers were chosen from the Genethon link map. The DNA was amplified by the PCR method using the PHC3 Techne device. The reactions are carried out in a final volume of 50 μl containing 200 ng of genomic DNA, 125 mM 4dNTP mixture, I × PCR buffer Cetus Taq polymerase, I U BRL Taq polymerase, 1 mM of each primer. The samples are treated for 30 temperature cycles (1 cycle, 94 "C for 5 min; 28 cycles including a denaturation step at 92" C for 1 min and a fixing step at 55 "C for 1 min and an extension to 720C for 1 min, the last cycle allows an extension to 72 "C for 10 min). After addition of 75 μl of loading buffer, the samples are denatured for 5 minutes at 94 ° C. and then placed on a 6% acrylamide gel. After transfer to nylon membranes, these are fixed in 0.4 M of sodium hydroxide and hybridized with a probe labeled with P32 (CA) 12 for 14 hours The amplified products are then visualized using an X-ray sensitive film after 4 to 6 hours of exposure.

 The bipoint link analysis is performed using the Linkage program (9).

 The parameters of the linkage analysis were as follows: autosomal dominant mode of transmission, penetrance estimated at 80%, frequency of the mutated gene fixed at 0.0001, recombination frequencies assumed to be identical in both sexes.

The allelic frequency for each locus is obtained from the Genome
Data Base.

Analysis of results
30 members of the P family have been genotyped with 260 microsatellite markers covering all chromosomes.

 A significant binding score of 3.64 was obtained at e = 0.05 for DIS2705. 4 additional markers near DIS2705 were then tested in the P family (Table 1). The marker DIS2635 showed a significant bond with a Zmax of 3.33 at 0 = 0.05. To confirm that a FMH gene was found on the long arm of chromosome 1, a linkage analysis was performed in 6 other families. 2 showed positive binding scores with different markers (Table 1). With D1S2635 the maximum combined binding score for the 3 families showing positive lod-scores was 4.21. This maximum binding score was identified with a recombination fraction of 0.07 indicating that some individuals were recombinant.

 4 families, C, N, H and W, show negative binding scores with all the markers tested in the interval D1 S2343-D 1 S2844. This information demonstrates the existence of at least a third locus for MHF.

 The region where the MHF2 gene has been located contains two candidate genes for this condition, GIRK3 (15, 16, 17) which codes for a potassium channel, and CACNL1A6 (13, 14) which codes for a calcium channel.

 The invention also relates to the use of the hGIRK3 and CACNL1A6 genes for the detection of familial hemiplegic migraine.

TABLE i
WFH binding score and chromosome 1 markers
(80% penetrance)
Family Locus 0.00 0.01 0.05 0.10 0.20 0.30 0.40 Zmax at 8
D1S2343 D -3.19 -0.81 -0.18 0.02 0.12 0.09 0.03 0.12 0.20
P -0.98 1.67 2.72 2.86 2.47 1.72 0.80 2.87 0.09
V -2.63 -0.46 0.13 0.30 0.32 0.22 0.09 0.34 0.16
Total -6.81 0.40 2.67 3.18 2.92 2.04 0.92 3.22 0.12 DIS2635 D -2.89 0.51 0.09 0.27 0.33 0.24 0.11 0.33 0.17
P -1.26 2.98 3.33 3.18 2, '9 1.60 0.69 3.33 0.05
V 0.84 0.82 0.75 0.67 0.49 0.32 0.16 0.84 0.00
Total -3.31 3.29 4.18 4.12 3.31 2.16 0.95 4.21 0.07
D1S2707 D 0.60 0.59 0.53 0.46 0.32 0.17 0.05 0.60 0.00
P -6.06 1.26 2.34 2.51 2.19 1.52 0.66 2.51 0.10
V 1.40 1.37 1.25 1.09 0.77 0.46 0.19 1.39 0.00
Total -4.06 3.22 4.11 4.07 3.28 2.15 0.90 4.15 0.07 Dol 52705 D -4.01 -1.60 -0.88 -0.57 -0 .26 -0.10 -0.02 -
P -1.63 3.28 3.64 3.49 2.81 1.90 0.85 3.64 0.05
V 1.34 1.31 1.19 1.05 0.76 0.46 0.19 1.34 0.00
Total -4.31 2.99 3.95 3.98 3.32 2.26 1.02 4.02 0.08 D1S2844 D -6.55 -2.21 -0.88 -0.38 0.02 0.14 0.12 0.02 0.20
P -3.08 1 1.00 2.13 2.36 2.12 1.50 0.69 2.36 0.11
V 0.78 0.77 0.68 0.58 0.37 0.18 0.05 0.76 0.00
Total -8.87 -0.46 1.90 2.53 2.49 1.81 0.85 2.64 0.14 D 1 S2635 C -3.79 -3.23 -1.93 -1.28 -0.61 -0.25 -0.06
M -8.16 -5.23 -2.80 -1.17 0.67 -0.22 -0.03
N -6.35 -3.73 -1.99 -1.19 -0.49 -0.18 -0.03
W -4.43 -2.66 -1.81 -1.34 -0.78 -0.43 -0.17
D1S2707 C -4.41 -1.88 -1.11 -0.73 -0.34 -0.14 -0.03
H -12.04 -5.96 -3.27 -2.09 -0.99 -0.45 -0.15
N -3.06 -1.40 -0.73 -0.46 -0.21 -0.08 -0.02
W -4.09 -1.80 -1.07 -0.75 -0.44 -0.28 -0.14
D1S2705 C -3.63 -1.12 -0.45 -0.19 -0.01 0.02 0.01
M -8.44 -4.49 -2.38 -1.47 -0.62 -0.23 -0.05
N -2.79 -2.50 -1.42 -0.88 -0.38 -0.15 -0.03
W -4.13 -3.34 -2.01 -1.40 -0.78 -0.43 -0.18 D1S2844 C -4.51 -2.39 -1.28 -0.76 0.28 0.01 0.01
H -11.87 -6.82 -3.96 -2.52 -1.18 -0.52 -0.16
N -3.10 -2.50 -1.28 -0.75 -0.30 -0.10 -0.02
W -4.13 -3.35 -2.01 -1.40 -0.79 -0.43 -0.18
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Claims (10)

 his family.  said marker being linked to the occurrence of familial hemiplegic migraine within  chromosome 1q21-q23 in the locus limited by the markers DIS2343 and D1S2844,  we highlight in the DNA of said individual the presence of a marker located on the  Method for indirect genotypic diagnosis of familial hemiplegic migraine type 2 in an individual, characterized in that  2) Method according to claim 1, characterized in that the marker is chosen from microsatellite markers of the defined locus.  3) Method according to one of claims I and 2, characterized in that the marker is highlighted after amplification of the locus of said marker.  4) Method according to claim 3, characterized in that the amplification is carried out by PCR.  5) Method according to one of claims I to 4, characterized in that the microsatellite markers are chosen from  From 52343  D1S2635  From 52707  D1S2705 Dol 52844  6) Method according to one of claims I to 5, characterized in that the biological sample is a blood sample.  7) Method according to one of claims I to 6, characterized in that the DNA is taken from an individual, the microsatellite markers linked to the occurrence of familial hemiplegic migraine within his family are amplified, highlights the presence or absence of microsatellite alleles linked to the onset of MHF in his family to assess the risks of onset of pathology.  8) DNA sequence located on the locus between the markers DIS2343 and Du 52844 and linked to the occurrence of familial hemiplegic migraine type 2.  9) Gene involved in the occurrence of hemiplegic migraine type 2 and located on a DNA according to claim 8.  10) Gene involved in the occurrence of type 2 hemiplegic migraine according to claim 9, characterized in that it is chosen from the hGIRK3 and CACNLI A6 genes.  il) Use of the hGIRK3 gene or of the gene coding for the calcium channel CACNLIA6 according to claim 9 for the demonstration of familial hemiplegic migraine.