CA2610057A1 - Method and kits for detecting recessive mutations associated with genetic diseases - Google Patents

Abstract

The present invention relates to a method for determining, way simultaneous presence or absence of multiple genetic mutations in one individual. The mutations contemplated by the present invention are preferably genetic diseases recessive diseases, which include lactic acidosis, also known as variant name Canadian Leigh Syndrome (CFLS), tyrosinemia type 1, neuropathy hereditary sensitivomotrice, with or without agenesis of the corpus callosum (ACCPN) and ataxia spastic from Charlevoix-Saguenay (ARSACS). The present invention relates to also kits for the realization of the proposed method.

Description

METHOD AND KITS FOR DETECTING ASSOCIATED MUTATIONS
WITH RESISTANT GENETIC DISEASES

FIELD OF THE INVENTION

The present invention relates to a method of detecting a plurality of of mutations in at least one targeted DNA region of the human genome, the mutations being associated with genetic recessive diseases. The invention thus allows to identify carriers of mutations associated with genetic recessive diseases.

DESCRIPTION OF THE PRIOR ART

The Saguenay-Lac-St-Jean region (SLSJ) in Canada has a rate of carriers of genetic diseases of a higher recessive nature than in the majority of others Canadian regions. This situation is attributable to certain phenomena historical and demographics, including the founder effect (ie the creation of a new population to from a relatively small number of individuals from a mother population) and one certain homogeneity of the population.

[0003] The most common recessive diseases in this region include acidosis lactic acid, also known as the French Canadian variant of syndrome Leigh (LSFC), tyrosinemia type 1, sensitivomotor neuropathy hereditary, with or without agenesis of the corpus callosum (ACCPN) and the spastic ataxia of Charlevoix-Saguenay (ARSACS).

[0004] Since these genetic diseases are recessive in nature (i.e.
only to be reached, an individual must have two inactive alleles or be homozygous transferred) and that the The majority of individuals carrying these mutations are heterozygous and do not do not develop symptoms associated with these diseases, the identification of individuals with such mutations is difficult. Such identification of carrying individuals is however desirable, see essential, especially when planning births. In effect, children from of procreation between two heterozygous carrier individuals develop, in approximately 25% of cases, recessive genetic disease.
Considering that the level of heterozygous carriers of mutations associated with one of these diseases rises to about 1 in 20 in some populations (including SLSJ), number of descendants with one of these diseases is important.

[0005] In recent years, breakthroughs in engineering human genetics identified different recessive genetic variations associated with at each of these diseases. More specifically, a substitution of cytosine in position 1119 by a thymidine (1119C / T) on the LRPPRC gene (or Leucine-rich PPR motif-containing protein) is associated with lactic acidosis (Mootha VK, et al.
gene causing human cytochrome c oxidase deficiency by integrative genomics. Proc Natl Acad Sci US
A. 2003 Jan 21,100 (2): 605-10). In addition, the mutation IVS12 + 5G / A on the FAH gene (or Fumarylacetoacetate hydrolase) is associated with tyrosinemia type 1 (Grompe Metal., A single mutation of the fumarylacetoacetate hydrolase gene in French Canadians with hereditary tyrosinemia type I. N. Engl. J. Med. 1994 Aug 11; 331 (6): 353-7) while mutation 2436de1G on the gene SLC12A6 (or Solute carrier family 12 potassium chloride transporters member 6) is associated with polyneuropathy (Howard HC, et al., The K-Cl cotransporter KCC3 is mutant in a severe peripheral neuropathy associated with agenesis of the corpus callosum. Nat. Broom. 2002 Nov; 32 (3): 384-92.) Finally, a association was established between the 6594delT and 5254C / T mutations on the SACS gene (or Spastic ataxia of Charlevoix-Saguenay) and the spastic ataxia of Charlevoix-Saguenay (Engert JC, and. al.
ARSACS, a spastic ataxia common in Northeastern Quebec, is caused by mutations in a new gene encoding 11.5-kb ORF. Nat. Broom. 2000, Feb; 24 (2): 120-5).

[0006] The identification of such mutations has allowed the development of some tests of genetic screening. The existing tests rely largely on amplification of fragments of genomic DNA of interest to the individuals screened and by the sequencing said fragments, which tests have been described in the art. Good they arouse some interest, current genetic screening methods present also some disadvantages. For example, screening for the five mutations above identified at an individual requires the taking of nucleic acid samples multiple, which Samples must then be sent to accredited laboratories separate for for analytical purposes.

In the usual way, the samples are subject to five analyzes.
distinct by direct sequencing, ie an analysis for each of the mutations identified in link with recessive genetic diseases described above and the laboratories in which which are performed the analyzes are usually located outside the region of SLSJ. This approach therefore requires sample transport and a large number of manipulations techniques, such as DNA extraction, making this methodology expensive and contributing to increase the time needed to achieve results. In case emergency, as per example when a pregnancy is in progress, additional costs are generated for get the results expeditiously, making the methodologies current not very flexible.
Finally, the volume of tests to be performed is variable and the number of samples to test during each shipment to accredited laboratories is usually small, this which contributes also to maintain high costs associated with such genetic testing.

Since the current screening tests are uneconomical, the programs of Screening is a challenge (eg as part of a program population screening). So, the screening process currently in use in the SLSJ region for the detection of recessive diseases only targets certain groups of individuals and is based on the family cascading blanket. briefly, when individual from a family waterfall comes to the genetic counseling service of his establishment for the detection of a recessive genetic disease, the screening of others Recessive genetic diseases of interest are offered to him. Thus, the individuals belonging not at such family cascades, but nevertheless at risk of being carriers some Genetic mutations have less access to genetic testing.

It would therefore be desirable to be able to benefit from a technology simple, flexible and economical to determine in a large number of individuals presence of recessive genetic mutations. Such technology could advantageously make it possible to determine, simultaneously and within a even laboratory, the presence or absence of mutations associated with genetic diseases recessive.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention, a method for detect the presence or absence of mutations in the genome of an individual is provided. The method comprises (a) simultaneously amplifying a plurality of acid fragments nucleic the individual to obtain an amplification product, each of said fragments overlapping the position of one of the mutations; (b) hybridize the amplification product with a first game probes including a number of probes corresponding to said plurality of fragments; and (c) detect the presence of specific markers.

According to one aspect of the invention, each of the probes of the first game is suitable to hybridize specifically to one of the amplified fragments when it comprises one of the mutations. Each of the probes is coupled to a specific marker. The detection of a probe-specific marker of the first game indicates the presence of a mutation corresponding in the genome of the individual. The first set of probes includes preferably at least one probe having a sequence corresponding to one of the SEQ. ID.
NO. 12, SEQ. ID. No. 14, SEQ. ID. No. 16, SEQ. ID. NO 18 and SEQ. ID. NO. 20

According to another aspect of the invention, the method also comprises hybridization of the amplification product with a second set of probes, which hybridization has place before detecting the presence of specific markers. The second game of probes comprises a number of probes corresponding to the plurality of fragments and each of probes the second game is adapted to hybridize specifically to one of fragments amplified when it corresponds to a wild genotype. Each of the probes is coupled with specific marker, and the detection of a probe-specific marker of the second game indicates the absence of a corresponding mutation in the individual's genome.
The second probe set preferably includes at least one probe having a sequence corresponding to one of the SEQs. ID. No. 11, SEQ. ID. NO. 13, SEQ. ID. No. 15, SEQ. ID. NO 17 and SEQ. ID.
NO 19.

According to an additional aspect, the nucleic acid fragments are fragments genomic DNA, and preferably gene fragments and so preferential genes are selected from LRPPRC, FAH, SLC12A6 and SACS.

According to another aspect of the present invention, one of the mutations is associated with lactic acidosis and is preferably a 1119C / T substitution on the gene LRPPRC.

[0015] According to another additional aspect, one of the mutations is associated with the tyrosinemia and is preferably an IVS 12 + 5G / A mutation on the FAH gene.

According to a further aspect of the present invention, one of the mutations is associated with hereditary sensitivomotor neuropathy, with or without agenesis callous body and preferably a 2436de1G mutation on the SLC12A6 gene.

According to another additional aspect, one of the mutations is associated ataxia Spastic from Charlevoix-Saguenay and is preferably a 6594delT mutation or a 5254C / T mutation on the SACS gene.

According to an additional aspect of the present invention, the amplification of the fragments is carried out using one of the primer pairs chosen from the SEQ. ID NO. 1 and SEQ. ID. NO. 2; SEQ. ID NO. 3 and SEQ. ID. NO. 4; SEQ. ID NO. 5 and SEQ. ID.
NO. 6;
SEQ. ID NO. 7 and SEQ. ID. NO. 8; and SEQ. ID NO. 9 and SEQ. ID. NO. 10.

100191 Still according to another embodiment of the invention, a kit for simultaneously determine the presence or absence of a plurality of mutations in the genome of an individual is provided. The kit includes a set of primers perrnettant to simultaneously amplify a plurality of nucleic acid fragments of the individual for get an amplification product. The primer set preferably includes least one primer corresponding to one of the sequences selected from SEQ. ID. NO. 1 to SEQ. ID.
NO. 10.

100201 According to an additional aspect, the kit also includes a first game of probes including a number of probes corresponding to the plurality of fragments.
Each of the probes the first game is adapted to hybridize specifically to one of fragments when it involves one of the mutations. Preferably, the first set of probes includes at least one probe having a sequence corresponding to one of the SEQs. ID. NO
12, SEQ.
ID. No. 14, SEQ. ID. No. 16, SEQ. ID. NO 18 and SEQ. ID. NO. 20 According to another additional aspect, the kit also comprises a second game of probes including a number of probes corresponding to the plurality of fragments. Each probes of the second game is adapted to hybridize specifically to one of fragments when it corresponds to a wild genotype. The second set of probes includes preferably at least one probe having a sequence corresponding to one of the SEQs. ID. NO
11, SEQ.
ID. NO. 13, SEQ. ID. No. 15, SEQ. ID. NO 17 and SEQ. ID. NO 19.

According to another embodiment of the invention, a kit for determine simultaneously the presence or absence of a plurality of mutations in a plurality of Nucleic acid fragments of an individual is provided. The kit includes a first game of probes, each of the probes of the first game being adapted to hybridize specifically to one of the nucleic acid fragments when it has one of the mutations. The first game of probes preferably includes at least one probe having a sequence corresponding to one of the SEQs. ID. NO. 12, SEQ. ID. No. 14, SEQ. ID. No. 16, SEQ. ID. NO 18 and SEQ. ID.
NO. 20 100231 According to an additional aspect, each of the probes of the second game is adapted for to hybridize specifically to one of the fragments when it corresponds to a wild genotype.
The second set of probes preferably includes at least one probe having a sequence corresponding to one of the SEQs. ID. No. 11, SEQ. ID. NO. 13, SEQ. ID. No. 15, SEQ.
ID. NO
17 and SEQ. ID. NO 19.

100241 Finally, according to a final aspect of the present invention, the kit comprises also a set of primers to simultaneously amplify the plurality fragments nucleic acids of the individual. Amplification makes it possible to obtain a product amplification. The primer set preferably includes at least one primer corresponding to one of the sequences selected from SEQ. ID. NO. 1 to SEQ. ID. NO. 10.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the present invention should be understood so clearer by referring to the detailed description of the embodiments of the invention conjunction with the accompanying drawings, in which:

FIG. 1 is a graph illustrating the median value of the fluorescence obtained at from different samples using the specific ALSASS probes and ALSASM-2;
FIG. 2 is a graph illustrating the median value of the fluorescence obtained at from different samples using TYRSSS-3 specific probes and TYRSSM-3;
FIG. 3 is a graph illustrating the median value of the fluorescence obtained at from different samples using PNPSSS specific probes and PNPSSM-3;
FIG. 4 is a graph illustrating the median value of the fluorescence obtained at from different samples using the specific probes AS 1 SSS-3 and AS 1 SSM-3;
and FIG. 5 is a graph illustrating the median value of the fluorescence obtained at from different samples using AS2SSS specific probes and AS2SSM-2.
DETAILED DESCRIPTION OF THE INVENTION

The following description and the embodiments described therein, are provided by illustrating one or more example (s) of embodiments of principles and - ô -aspects of the present invention. These examples are provided for a purpose explanation and not limiting the principles of the invention.

In a first embodiment of the present invention, a method of detection of the presence or absence of mutations in the genome of a individual is provided. The method of the present invention is fast, reliable, relatively affordable and limits the experimental manipulations required for the determination of mutations associated with several genetic diseases. In particular, the method of present invention is based on the use of the tools presently available in areas of molecular biology and nanotechnology in order to centralize the analyzes, minimize transportation and consumable costs and limit required time for obtaining the results. So, this method can find a utility in the part of a population screening program where the volume of samples treat by period of time is important and can be planned.

In general, the method of the present invention allows amplification simultaneous multiple nucleic acid fragments of interest and subsequent hybridization of these nucleic acid fragments with probes specific to each of the mutations, the hybridization of the different fragments being carried out simultaneously, from of a single product amplification and under the same hybridization conditions. The method allows also the simultaneous detection of a large number of probes specific to each of the mutations, which probes are each labeled with a specific marker and indicate the presence or absence of mutations associated with genetic diseases recessive, as it will be described in detail below. This approach is multiplexed to signify the application of the method for simultaneous detection, in the same and single test, from to minus two mutations.

Recessive genetic diseases contemplated by the present invention include recessive genetic diseases found more commonly in the region SLSJ, either lactic acidosis or French Canadian variant of Leigh's syndrome (LSFC), the tyrosinemia type 1, hereditary sensitivomotor neuropathy, with or without agenesis corpus callosum (ACCPN) and spastic ataxia of Charlevoix-Saguenay (ARSACS), which diseases are described in more detail in Table 1 below. A
paid person However, the art will appreciate that the method of the present invention may also find a utility in detecting mutations associated with other diseases recessive example, of a common founder effect, such as cystic fibrosis and mucolipidosis type 2. That same person skilled in the art will understand that the invention is not not limit only to detect mutations associated with genetic diseases recessive, but It also extends to the detection of other mutations associated with other diseases any disease associated with at least one mutation in at least one least one region of genomic DNA of an individual.

List of high-incidence recessive diseases in the Saguenay-St.
Lac-St-Jean Diseases OMIM4 GENE OMIM Chromosome Mutation References (Sickness) (Gene) LSFC '220110 LRPPR 607544 2p21 1119C / T Mootha VK et al., (2003) VS

Tyrosinemia 276700 FAH 276700 15q25.1 IVS12 + 5G / A Grump M et al., (1994) (Type 1) ACCPNZ 218000 SLC12 694878 15q13-q15 2436de1G Howard HC et al., (2002) AT

ARSACS3 270550 BAGS 604490 13q12.12 6594de1T Engert JC et al., (2000) LSFC: lactic acidosis or French Canadian variant of Leigh syndrome.
2ACCPN: Hereditary sensitivomotrice neuropathy with or without agenesis of callous body.
3ARSACS: Spastic ataxia of Charlevoix-Saguenay.
40MIM: number in the database Online Mendelian Inheritance in Man.

According to a preferred embodiment, the method of this invention includes the step of simultaneously amplifying a plurality of acid fragments nucleic the individual to obtain an amplification product. In this mode of realization each of amplified fragments overlaps the position of a mutation associated with one of diseases recessive genes. Mutations associated with recessive diseases are preferably selected from mutation 11 19C / T on LRPPRC gene associated with acidosis lactic acid (Leigh syndrome, LSFC), the IVS12 + 5G / A mutation on the FAH gene associated with tyrosinemia of type 1, the 2436de1G mutation on the SLC12A6 gene associated with polyneuropathy, mutation 6594delT on the SACS gene associated with Charlevoix spastic ataxia.
Saguenay and the 5254C / T mutation on the SACS gene, also associated with spastic ataxia of Ch arl evo i x- S aguenay.

The amplification step is preferably carried out by the technique of reaction in polymerase chain or PCR, using a set of primers selected for to permit to amplify simultaneously, in the same test and according to the same conditions amplification, five (5) nucleic acid fragments, each of the fragments amplified overlapping one of the mutations described above. A paid person in art recognize that the amplification may apply to a lower number or superior of nucleic acid fragments, insofar as the primers are adapted to allow such a simultaneous amplification of the fragments.

[0037] The nucleic acid fragments are preferably fragments DNA
genomics, and more preferably gene regions where a polymorphism for a targeted recessive disease exists. Thus, for each recessive disorder targeted, the gene region specific to each of the diseases considered, where the polymorphism for the disease recessive targeted is found, will be the region amplified by PCR. Therefore, each sequences primers used in the PCR reaction corresponds to a region of the genome human containing the gene associated with the recessive disease. The amplification step is so carried out to using primers specific to the targeted regions of the genome.

[0038] Thus, since each of the sequences of the primers used in the PCR reaction corresponds to a region of the gene associated with a recessive disease (see Table 2), during the PCR reaction, the region-specific primers contribute to amplification of this region of the gene, and this, under conditions of amplification favoring production of large amounts of DNA fragments to obtain PCR products. These last can be about 200 nucleotides in length.

In one embodiment of the invention, the set of primers comprises five pairs of primers, ie the primers comprising the SEQ sequences. ID. NO. 1 and SEQ. ID.
NO. 2; SEQ. ID. NO. 3 and SEQ. ID. NO. 4; SEQ. ID. NO. 5 and SEQ. ID. NO. 6;
SEQ. ID.
NO. 7 and SEQ. ID. NO. 8; and SEQ. ID. NO. 9 and SEQ. ID. NO. 10, which ones sequences are described in more detail in Table 2 below.

~ Q ~ = -, NM d ~ l ~ 00 01 11b Cy v ~ ic ~ n O, -. ~

A .. o 0 0 0 0 ~
wd CD
~ C7 U d HU dd C7 <U
UH d Er U Cdj QQ <~
Q '~ Q <dd C7 H <<~ U
~ ap Qh ÇD d EC7- C'J U ~
h <<u ~ <~
<Cd7 ~ ~ C ~ 7 U d U
dd Udd UHU ~ ~ <
d UUHE ~ -UQ <QUE ~ U

~

w ~ NN ~ N

zz ~ d ~ ~
dddh H aa .-. ~ NN
O F- ~ C7 M Vi Q1 V ~
U ~ O
C ~ i N
ax UU
C7 to w vdi In another preferred embodiment, at least one of the primers used in the PCR reaction was modified prior to the reaction of PCR in order to include a terminal change at the 5 'end. According to this mode of production, the 5 'end of the primer is bound to a terminal molecule, preferably biotin. In this preferred embodiment, biotin is subsequently coupled to the streptavidin in order to allow the possible binding of a chromophore at the 5 'end of each fragments amplified by PCR. The chromophore coupled to streptavidin is preferably the R-phycoerythin (also known as 'Alexa 532' or 'Cy3'), which is excited to a wavelength of 532 nm-13 mW by an aluminum yttrium garnet laser (YAG) of green color. As will be described in detail below, the coupling of a chromophore to amplified fragments by PCR will detect fluorescence of strands acid nucleic acids having a sequence complementary to the hybridization probes used and by the even indirectly evaluate the binding of these nucleic acid strands to probes specific to microspheres.

Although the terminal molecule is preferably biotin, a nobody skilled in the art will understand that the terminal molecule is selected from molecules having particular affinities and known to other molecules and that other terminal modifications can be made to the primers of the reaction of PCR in order to couple, at their 5 'end, a molecule allowing a coupling with other molecules.

It is understood that the amplification step is carried out from a sample nucleic acids of the individual, and preferably from a sample DNA
genomics. Thus, the amplification step may include the steps of obtain a DNA sample of the individual and to prepare the sample in order to make it usable for the PCR reaction. Such a sample can include any type of sample of DNA, including a sample of blood, saliva or tissue. The sample obtained is preferentially treated to isolate the genomic DNA, preferably by cell lysis using from a solution to base of detergents. A person skilled in the art will know other methods isolation and DNA purification, which can also be used for realization of the method of the present invention.

The method of the present invention also includes the step of to hybridize the amplification product of the PCR reaction with a first set of probes. The first probe set includes a number of probes corresponding to the number of fragments amplified, and preferably five (5) probes. Each of the probes of the first game is adapted for to hybridize specifically to one of the amplified fragments when this fragment has a targeted changes. In this embodiment, the probes of the first game are preferably the probes ALSASM-2 (SEQ ID NO: 12), TYRSSM-3 (SEQ ID NO.
14) PNPSSM (SEQ ID NO: 16), ASISSM-3 (SEQ ID NO: 18) and AS2SSM-2 (SEQ ID NO: 18).
20) described in Table 3 below.

In an alternative embodiment, the method of the present invention invention also includes hybridization of the amplified fragments by the reaction of PCR with a second set of probes. Such a hybridization of the second game is preferably done simultaneously with the hybridization of the first set of probes.

The second set of probes preferably includes five (5) probes, ie one number of probes corresponding to the number of amplified fragments. Each of the probes second game is adapted to hybridise specifically to one of said fragments when corresponds to wild genotype, or in other words, when the mutation is absent. In this mode of realization, the probes of the second set are preferably the ALSASS probes (SEQ ID NO.
11), TYRSSS-3 (SEQ ID NO: 13), PNPSSS (SEQ ID NO: 15), AS1SSS-3 (SEQ ID NO.
NO.
17) and AS2SSS (SEQ ID NO: 19) described in Table 3 below.

A person skilled in the art will appreciate that the number, sequence and the combination of probes may vary depending on the number of mutations to detect.
Thus, a single probe having a sequence corresponding to one of the SEQs. ID.
NO. 11 to SEQ. ID. NO. 20 above described could be used in combination with probes having other sequences.

O ~ Q p = - NM ~ V> ~ \ O l- 00 O ~ O
Z

~ M ~ ~ 'O t ~ 00 NM of ~
z MMMMMM ~ d 't7 a0, o 0 0 0 0 0 0 0 0 0 U

O ~
rr W
Q ~ F o 0 0, \ o ~ ao 0 0 00 0, F. U "z NN ~ ~ ~ NNN r r +
~ z ~ -~

w ~
HH
OHFUUUU ~ ~ ~ C7 ÇD7 ÇD7 ~ = C7 UU <
~

'd' zdd ~ ~ d ~ Q UU
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
D Ç Q Q d d d CU CU 7 7 7 Q ~ HH QV ~ d <
~ U Ç7 C7 c ~ <U
ÇD ÇD UU C7 F ~ - ~ d ÇD UU
VUHU C7 d UU
~ ~ d C7 c ~ 7 CU7 H <
d C ~ 7 C ~ 7 ~ ~ ~ i cr1 MMMMN
WA v ~ v ~ v1 v ~ ~ ~ ~ ~ ~ ~
~ zdd ~ cn UD a cn cn Qn V) i! DL ~ ~ f1 ~ G-, - = - + NN
Q <HHZZ <<<<
Q

F ~ ~ n ç: ~ â ~
HU + ~ ~ U
~
v > M
N ~ ~
<
NOT
W
ÇD ~~~
aw cn cn cn According to one aspect of the present invention, each of the probes is coupled with specific marker to simultaneously determine the presence of each of probes in the sample. Thus, the hybridization step of the PCR products may be preceded by the binding of each of the probes to specific markers, such microspheres. Preferably, a modification at the 5 'end is made to each of probes. This modification is preferably of the "5'Uni-link amino" type so that to allow probes to bind to hydroxyl groups on the surface of microspheres specific.
More particularly, each of the microspheres can be defined usually as an excipient in the form of a small sphere whose diameter is between a few microns and a millimeter, solid and solid, having on its surface a substance adhesive, preferably polystyrene. Active ingredients, preferably at number of two, are dissolved or dispersed in the microspheres, which are usually added according to a predefined quantity, according to the marking technique of the microsphere that will ensue. Such microspheres are available in many suppliers and are selected from 100 available microspheres. The choice of microspheres coupled at each probes is usually determined randomly since these are very similar and therefore generally interchangeable. In addition, it should be noted that a person skilled in the art will know that the adhesive substance on the surface of the microsphere can be of a another type of molecule, such as for example a ligand, an antibody or a protein.

According to a preferred embodiment of the invention, the active ingredients which are dissolved or dispersed in each of the microspheres are chromophores. The chromophores are usually colored molecules, which can be part of the family of nitrates, monazoids, diazo compounds, triphenylmethanes, quinoneimines, xanthenes and anthraquinones. The list of these families of chromophores is not comprehensive. By example, the active ingredients, preferably red and orange chromophores are mixed in the microsphere and excited with a 635nM-10mW diode laser, Red color, during the detection stage by the cytofluorometric device, as it will be described in detail below. The color that will be observed during the detection and quantification microspheres is due to a resonance phenomenon between a double bond chemical and the excitation light of the selected chromophore and is specific to each of the probes used.
In a preferred embodiment, the probes ALSAS, ALSASM-2, TYRSSS-3, TYRSSM-3, PNPSSS, PNPSSM, ASISSS-3, ASISSM-3, AS2SSS and AS2SSM-2 are coupled to the microspheres marketed by LUMINEX under the numbers 033, 035, 036, 037, 038, 042, 043, 044 and 045, respectively. So once that they will be linked microspheres, the specific probes will have the task of binding, by hybridization, PCR products obtained during the amplification step and to allow the determination of the presence or absence of mutations on the fragments.

The use of a chromophore for binding to the microsphere is not not the only way to mark these microspheres. Yes, if a detection device allow it, a another type of active ingredient may be used. For example, a person versed in art recognize that an active principle of a radioactive, thermal or other nature, for example, can be used to detect a PCR / probe product. A person paid in art will also understand that other types of changes on the probes used can achieve the same goal.

The techniques relating to the hybridization of probes are generally known in the art and do not require exhaustive description. The stage of hybridization of PCR products to probes coupled to microspheres can then be completed in order to produce complexes comprising PCR products linked to coupled probes Has microspheres. After hybridization, these complexes are recovered by centrifugation and suspended in a solution containing a combined agent. For the purposes of accuracy so he is understood that the hybridization step includes a purification step aimed at eliminate the probes not having hybridized with a complementary fragment of amplified nucleic acid and that during of detection, only probes having hybridized with such fragments Additional will be detected. One skilled in the art will understand that the term hybridization specific or any similar term indicates hybridization usually specific according to the experimental conditions and that such a hybridization can However involve a number of non-specific hybridizations defining a some noise from background during the experiment.

The method of the present invention finally comprises a step of detection of presence of markers specific to each of the probes of the first set and, the where appropriate, markers specific to each of the probes of the second game. It will be understood that the detection of a probe specific marker of the first set indicates the presence of corresponding mutation in the genome of that individual, whereas the detection a marker specific to a probe of the second game indicates the absence of mutation corresponding.

According to a preferred embodiment of the invention, the detection of complex products from PCR / probe is performed in a device allowing simultaneous detection by laser of a complex linked to a combined agent. This device is preferably a cytofluorometer. So, the detection stage makes it possible to identify, preferably by measurement of the fluorescence chromophores, the simultaneous presence of the different microspheres and PCR products (i.e., the PCR amplification product comprising a molecule of biotin, from streptavidin and a chromophore). Thus, the simultaneous detection of a microsphere related to a specific probe (which probe can be hybridized or not with a fragment DNA complement) and the complementary DNA fragment is indicative of hybridization of the DNA strand concerned to the probe. precisely.

[0054] A person in the field will understand that the detection step of the present method involves a step of quantifying the product complexes of PCR / probes, which can be calculated, for example, by a median value of the fluorescence chromophores. In the case where the invention is carried out using a brand cytofluorometer LUMINEX, the simultaneous realization, in a single reaction, of 100 tests on different samples, will be allowed, making it an ideal template for the needs of population screening of a limited number of genes or gene regions.
A person skilled in the art will understand that another type of apparatus can achieve the same goal and that the type of device used will depend on the nature of the terminal modification at the end 5 ' performed on the primers and on the first element of the combined agent.

-19-According to a preferred embodiment of the invention, the quantification of the complexes product PCR / probe makes it possible to establish a ratio of the fluorescence between the complexes formed with the PCR product with a mutation (or mutated complex) and complexes trained with the PCR product with the wild-type genotype (or wild-type complex . Of preferably, the values of these ratios make it possible in their turn to establish a diagnostic clinical presentation of the presence of the recessive mutation in a specimen and so to identify carriers of this mutation. For example, a complex report value mutated / complex wild population varying between 0.4 and 0.6 tends to indicate that the individual diagnosed is homozygous for wild alleles (ie it does not carry the mutation concerned, then a ratio value approaching 1.0 (eg, ranging from 0.9 to 1.0) indicates the presence of the mutation on one of the alleles of the diagnosed individual. Finally, a value of the report mutated complex / wild complex approaching 2 tends to indicate a genotype homozygote mutated in the individual. One skilled in the art will appreciate that the value of these reports are arbitrary and can be modified without departing from the spirit of the invention. This same person skilled in the art will appreciate that establishing this report value may take into account certain factors, including especially the sound of background and values of positive and negative controls.

[0056] Thus, according to one embodiment, each of the wells contains the reagents necessary to screen at least one of the mutations concerned. Each plate includes samples of 40 people in duplicate, positive controls represented by at least one carrier of each of the targeted mutations, a wild homozygote known in addition a Negative control consisting of a PCR reaction in the absence of genomic DNA.
The invention extends to the method performed in plates of different sizes and does not is not limited to its execution on a 96-well plate.

In another preferred embodiment, a kit for determining simultaneously the presence or absence of a plurality of mutations in the genome of a individual is provided. The kit preferably includes a set of primers allowing to simultaneously amplify a plurality of nucleic acid fragments of the individual. Such

-20-previously described, the nucleic acid fragments concerned are preferably fragments of genomic DDN of the individual. Thus, the kit allows obtaining several distinct amplified fragments by performing a single reaction of PCR, in a same reaction tube. The set of primers to which reference is made can include, by for example, one or more primers corresponding to the SEQs. ID. NO. 1 to SEQ. ID.
NO. 10.
One skilled in the art will appreciate that the nature, number and combination of primers is adapted according to the mutations to be identified.

100581 The kit may also include a set of probes capable of to hybridize to PCR products. The probe set includes a number of probes corresponding to to numbers amplified fragments and each of the probes is adapted to bind to way relatively specific to amplified fragments when they contain one of the mutations interest. Thus, the probe set may include one or more probes having a sequence corresponding to one of the SEQs. ID. NO. 12, SEQ. ID. No. 14, SEQ. ID. No. 16, SEQ.
ID. NO
18 and SEQ. ID. NO. 20 Similarly, the kit may comprise a second set of probes.
Unlike the first set of probes, the second set includes probes adapted for hybridize to amplified fragments when they have the genotype wild or, in other words, when the fragments are free of mutations. The second game waves preferably includes one or more probes having a corresponding sequence to one of SEQ. ID. No. 11, SEQ. ID. NO. 13, SEQ. ID. No. 15, SEQ. ID. NO 17 and SEQ. ID. NO
19.
[0060] Alternatively, a kit could include only one or the other first and second set of probes described above. Such a kit could find a utility, by for example, when the nucleic acid fragments are amplified using other primers than those described above. Also, a person skilled in the art will appreciate that establishing a complex mutated / wild complex relationship may not be desired in all situations. So, it can be useful, in some circumstances, to use only one only sets of probes. This same person skilled in the art will understand that the kit

-21 -can therefore include one or more of the (1) sets of primers, (2) first set of probe and (3) second probe set or some just some of their components (eg 2 Having described the general principles of the invention, a method for determine the presence or absence of mutations in the genome of an individual is going now to be described using an example.

METHOD OF SCREENING OR DETECTING CARRIERS OF DISEASES RESPONSIVE TO
THE HELP OF PROBES COUPLED TO MICROSPHERES

Materials and methods:

Extraction of genomic DNA (gDNA) According to a preferred embodiment of the invention, the extraction of the DNA
genomics (GDNA) was performed according to a modification of the standard method from of paper FTA (Whatman (I)) The method used is that described by Caggana and his contributors in 1998 (Caggana M. Conroy JM, Pass KA Rapid efficient method for multiplex amplification from filterpaper. Human mutation 11: [M. Conroy] 404-409 (1998)).
Several methods of genomic DNA extraction are available. The samples of Blood collected on FTA paper makes it possible to obtain from a 2 mm circle an amount Acceptable DNA array. The circles or punches are in the presence of methanol and left to free air all night to which 25 L of sterile water are added.
The samples are heated at 95 ° C for 10 minutes, then centrifuged and the supernatant containing the DNA
is transferred to a sterile tube. A fraction of this sample is used in the reaction PCR, performed in multiplex.

Preparation of the mastermix

-22-According to a preferred embodiment of the invention, the preparation of the mastermix for the PCR reaction for the amplification of the genome regions targeted by this invention details as follows:
at. Thaw reagents (buffer, dNTPs, primers, gDNA) and store at 4 C;
b. Dilute and combine the primers to obtain a final concentration of 10 M for each of the primers (see Table 2). Primers are designed to amplify the mutated region of the genome where a polymorphism for each of the targeted recessive diseases is already identified;
vs. Calculate the required volume according to the number of reactions to be performed in using a value of 25 L per PCR reaction;
d. Mix the reagents in the following order:

Reagents volume conc. final Buffer 10X 10 L 1 X
mM dNTPs 1 L 250 M
10 M Primers 3 L 0.3 M
H20 * 75 L
Taq polymerase 1 μL 5 units 20 Final volume 90 L
e. Put 18 L of mastermix per well f. Add 2 L of genomic DNA
boy Wut. Seal the plate and deposit it in the PCR device when the temperature 25 reaches 94 C

Programming of the PCR device.

The programming of the PCR apparatus used for the amplification of fragments of nucleic acids is summarized below:

-23-step temperature time 1. 94 C 10 min 2. 94 C 30 sec 3. 54 C 60 sec 4. 72 C 60 sec 5. Repeat 35 times steps 2 to 4 90 min 6. 72 C 10 min 7. store 4 C

Hybridization According to a preferred embodiment of the invention, the hybridization of the fragments amplified to probes that have been previously coupled to a specific microsphere selected from 100 different microspheres are performed. These probes correspond to sequences conserved or mutated regions where polymorphisms are found responsible for one of the 4 recessive diseases under study (see Table 3). The Luminex device has a laser whose function is to detect chromophores and therefore identify with precision one of the 100 spheres available.
at. Re-suspend the microspheres by vortex for 20 seconds and by sonication for 20 seconds.
b. Dilute the microspheres to a concentration of 50 microspheres per TMAC solution at 1.5X (3M TMAC, 50 mM Tris-HCl, pH8.0, 4 mM EDTA, pH8.0, 0.1% sarkosyl);
vs. Put 10 L of TE buffer per well.
d. Add 25 μL of the diluted microsphere solution.
e. Add 2.5 L of the PCR reaction and mix using a pipettor multiwell f. Seal the plate.
boy Wut. Incubate at 95 ° C for 5 min and at 52 ° C +/- 5 ° C for 15 min.
h. Centrifuge the plate at 3000 xg for 5 min at 22 C.

-24-i. Remove the supernatant by inversion.

Marking a protein, biotin, to a chromophore, phycoerythrin.

Once the hybridization has been completed, the complexes comprising the microspheres coupled to probes with PCR products, are recovered by centrifugation and re-suspended in a solution containing a protein, preferably biotin, bound to a chromophore, of preferably phycoerythine. This stage of the invention is detailed as follows:

at. Add 75 μL of a 1X TMAC solution containing streptavidin phycoerythrin at 4 g per mL.
b. Re-suspend the pellets using a multi-well pipette.
vs. Incubate for 10 minutes at room temperature in the dark.
Analysis at. Prepare the run file of the LUMINEX device using, for example, example a volume of 50 L analyzed for 45 seconds at the temperature to detect a minimum of 100 microspheres for each of the probes used.
b. Remove the plate in the appliance.
vs. Start the analysis.

The cytofluorometric apparatus of LUMINEX brand has a laser which has for function to detect chromophores and therefore to identify accurately one of 100 microspheres available. The sampling tray specific to the device LUMINEX has been retained because it allows simultaneous realization in a single reaction 100 tests out of 96 different samples, making it an ideal caliber for screening needs. Of Moreover, the costs of acquiring the device and that of the reactions are lower than those already used. The experimental conditions are unique, fast and specific to a cost significantly lower than what currently exists. This device allows measure the presence

-25-two fluorescent elements simultaneously. Thus, the presence of the principles dissolved assets in the microsphere on the one hand, and the chromophore constituting the second agent element combined, will report the presence of the targeted DNA regions, corresponding to the DNA sequence of the first and second subregions.

Report (Optional but preferable step for the analysis of the results) at. Transfer and save the results to an Excel file or other.
b. Use the median intensity fluorescence values to generate a histogram.
vs. Automated interpretation of the result;
at. Validation of the result by accredited person.

The detection device therefore produces the raw data that must be then analyze. Data transfer and recording can be done in a file of type Excel ™. Such a transfer is illustrated in FIGs. 1 to FIG. 5, which figures demonstrate the results of the method carried out as part of the determination of the genotype of a large number of individuals for 1119C / T mutations on the LRPPRC gene, IVS12 + 5G / A
on the gene FAH, 2436de1G on the gene SLC12A6, 6594delT on the gene SACS and 5254C / T and on the SACS gene, respectively.

The compilation of the generated data allows the establishment of a report mutated complex / wild complex, as previously described. In this example, a mutated complex ratio value / wild complex varying between 0.4 and 0.6 tends to indicate that the diagnosed individual is homozygous for the wild alleles (i.e.
that he is not carrier of the mutation concerned), while a value of the report approaching of 1.0 (ex.
ranging from 0.9 to 1.0) indicates the presence of the mutation on one of the alleles of the individual diagnostic. The interpretation of complex mutated / wild complex relationships allows to establish the clinical diagnosis.

-26-[0070] Thus, a median intensity value of higher fluorescence or equal to 100 is considered as a positive and indicative value that the origin of the specimen of DNA taken carries the mutation of a targeted recessive disease.

[0071] The invention also makes it possible to detect or detect at least one mutation in at least one targeted DNA region of the human genome, said mutation being associated to one recessive disease, characterized in that the method is exercised in order to to detect or to detect at least two mutations simultaneously, that is to say, in the same and unique test.
Thus, the invention makes it possible to develop a carrier detection test of diseases recessive, which is also fast and effective at a significant cost less than what exists currently on the market.

Although a preferred embodiment of the present invention has been described in detail above and illustrated in the drawings accompanying the application, it must be understood that the invention is not limited to this particular embodiment and that many changes and modifications can be made without departing from the scope or spirit of the present invention.

Claims (36)

1. A method for determining the presence or absence of mutations in the genome of an individual, said method comprising:

(a) simultaneously amplifying a plurality of nucleic acid fragments said individual to obtain an amplification product, each of said fragments overlapping the position of one of said mutations;

(b) hybridizing the amplification product with a first set of probes including a number of probes corresponding to said plurality of fragments, each said probes of the first set being adapted to hybridise specifically to one of said amplified fragments when it comprises one of said mutations, each of said probes being coupled to a specific marker; and (c) detecting the presence of said specific markers, detecting a said probe-specific markers of said first game indicating the presence of a corresponding mutation in the genome of said individual. 2. The method of claim 1, further comprising hybridizing said amplification product with a second set of probes before detecting the presence of said specific markers, said second set of probes comprising a number of probes corresponding to said plurality of fragments, each of said probes of second game being adapted to hybridise specifically to one of said amplified fragments when it corresponds to a wild-type genotype, each of said probes being coupled to a marker specific, wherein the detection of one of said probe specific markers of said second game indicates the absence of a corresponding mutation in the genome of said individual. 3. The method of claim 1 wherein said acid moieties nucleic acids are fragments of genomic DNA. 4. The method of claim 3 wherein said DNA fragments genomics are fragments of genes. 5. The method of claim 4, wherein said genes are chosen among LRPPRC, FAH, SLC12A6 and SACS. The method of claim 5, wherein one of said mutations is associated with lactic acidosis. 7. The method of claim 6 wherein said mutation is a 1119C / T substitution on the LRPPRC gene. The method of claim 7 wherein the amplification of one of said fragments is made using a pair of primers corresponding to SEQ. ID
NO. 1 and SEQ.
ID. NO. 2. 9. The method of claim 1 wherein one of said probes of said first game has a sequence corresponding to SEQ. ID. NO. 12. The method of claim 2 wherein one of said probes of said second game has a sequence corresponding to SEQ. ID. NO. 11. 11. The method of claim 4 wherein one of said mutations is associated with tyrosinemia. 12. The method of claim 11 wherein said mutation is a mutation IVS12 + 5G / A on the FAH gene. 13. The method of claim 12 wherein the amplification of a said fragments is made using a pair of primers corresponding to SEQ. ID
NO. 3 and SEQ.
ID. NO. 4. 14. The method of claim 1 wherein one of said probes of said first game has a sequence corresponding to SEQ. ID. NO. 14. 15. The method of claim 2 wherein one of said probes of said second game has a sequence corresponding to SEQ. ID. NO. 13. 16. The method of claim 4 wherein one of said mutations is associated with hereditary sensorimotor neuropathy. 17. The method of claim 16 wherein said neuropathy hereditary sensitivomotrice is hereditary sensitivomotor neuropathy with agenesis of corpus callosum or hereditary sensitivomotrice neuropathy without agenesis of callous body. 18. The method of claim 16 wherein said mutation is a mutation 2436delG on the SLC12A6 gene. 19. The method of claim 18 wherein the amplification of a said fragments is made using a pair of primers corresponding to SEQ. ID
NO. 5 and SEQ.
ID. NO. 6. 20. The method of claim 1 wherein one of said probes of said first game has a sequence corresponding to SEQ. ID. NO. 16. 21. The method of claim 2 wherein one of said probes of said second game has a sequence corresponding to SEQ. ID. NO. 15. 22. The method of claim 4 wherein one of said mutations is associated with spastic ataxia of Charlevoix-Saguenay. 23. The method of claim 22 wherein said mutation is a mutation 6594delT on the SACS gene. 24. The method of claim 23 wherein the amplification of a said fragments is made using a pair of primers corresponding to SEQ. ID
NO. 7 and SEQ.
ID. NO. 8. 25. The method of claim 1 wherein one of said probes of said first game has a sequence corresponding to SEQ. ID. NO. 18. 26. The method of claim 2 wherein one of said probes of said second game has a sequence corresponding to SEQ. ID. NO. 17. 27. The method of claim 22 wherein said mutation is a mutation 5254C / T on the SACS gene. 28. The method of claim 27 wherein the amplification of a said fragments is made using a pair of primers corresponding to SEQ. ID
NO. 9 and SEQ.
ID. NO. 10. 29. The method of claim 1 wherein one of said probes of said first game has a sequence corresponding to SEQ. ID. NO. 20. 30. The method of claim 2 wherein one of said probes of said second game has a sequence corresponding to SEQ. ID. NO. 19. 31. A kit to simultaneously determine the presence or absence of a plurality of mutations in the genome of an individual, the kit comprising a primer set for simultaneously amplifying a plurality of acid fragments nucleic acid an individual to obtain an amplification product, said set of primers including at least one primer corresponding to one of the sequences selected from SEQ. ID. NO. 1 to SEQ.
ID. NO.
10. 32. The kit of claim 31, further comprising a first set of probes including a number of probes corresponding to said plurality of fragments, each of said probes of the first game being adapted to hybridize specifically to a of said fragment when it includes one of said mutations, said first set of probes including at least one probe having a sequence corresponding to one of the SEQs. ID. NO. 12, SEQ. ID. NO

SEQ. ID. No. 16, SEQ. ID. NO 18 and SEQ. ID. NO. 20 33. The kit of claim 32 further comprising a second set of probes including a number of probes corresponding to said plurality of fragments, each of said second game probes being adapted to hybridise specifically to one of said fragment when it corresponds to a wild genotype, said second set of probes including at least one probe having a sequence corresponding to one of the SEQs. ID. No. 11, SEQ. ID. NO

SEQ. ID. No. 15, SEQ. ID. NO 17 and SEQ. ID. NO 19. 34. A kit to simultaneously determine the presence or absence of a plurality of mutations in a plurality of nucleic acid fragments of a individual, the kit comprising a first set of probes, each of said probes of said first game being adapted to hybridise specifically to one of said acid moieties nucleic when it includes one of said mutations, said first set of probes including at least one probe having a sequence corresponding to one of the SEQs. ID. NO. 12, SEQ. ID. NO

SEQ. ID. No. 16, SEQ. ID. NO 18 and SEQ. ID. NO. 20 35. The kit of claim 34 further comprising a second set of probes, each of said probes of the second set being adapted to hybridize specifically to a said fragment when it corresponds to a wild genotype, said second game waves including at least one probe having a sequence corresponding to one of the SEQs.
ID. No. 11, SEQ. ID. NO. 13, SEQ. ID. No. 15, SEQ. ID. NO 17 and SEQ. ID. NO 19. 36. The kit of claim 34 further comprising a set of primers allowing simultaneously amplifying said plurality of nucleic acid fragments said individual to obtain an amplification product, said set of primers including at least a primer corresponding to one of the sequences selected from SEQ. ID. NO. 1 to SEQ. ID.
NO. 10.