FR2668162A1 - PCR probes specific for delta-F-508 mucoviscidosis mutated gene - and normal allele, useful for detection of heterozygous mucoviscidosis mutation carriers - Google Patents

Abstract

DNA fragments of formulae (I)-(III), having respectively the Am1, Am2 and Am3 sequences, are new: 5'-CATGCXTTGYTGACGCTTC-3' (I) 5'-CATTAAZGAAZATATCATCTT-3' (II) 5'-CATTWAAGWAAATATCATTGG-3' (III) X = I or T; Y = I or A; Z and W = I or A. Also new is a method (and kit) for detecting carriers of the delta F-508 allele comprising: (1) isolating DNA from cells, fluids or tissue; (2) incubating this DNA with DNA polymerase in a buffer under amplification conditions, using an alpha primer (A) homologous with both the chromosomes carrying the normal and the mutated alleles; and a beta promoter (B) specific for the normal allele and/or a gamma promoter (C) specific for the mutated allele, then (3) detecting the amplified prods. by electrophoresis and staining. USE/ADVANTAGE - The delta F508 mutation is commonly responsible for mucoviscidosis (pancreatic cystic fibrosis) and the method is used to identify carriers of the disease (including heterozygous carriers). This method does not require radioactive reagents nor restriction enzymes, so is simpler than known processes. (I)-(III) are the pref. primers for the amplification

Description

 The present invention relates to a method for detecting the delta-F 508 allele of the mutation responsible for cystic fibrosis. The subject of the invention is also DNA fragments constituting primers allowing the amplification of the part of the human genome carrying this mutation as well as a diagnostic kit containing these primers for the implementation of said method.

 Cystic fibrosis, or cystic fibrosis of the pancreas (CF) is a very serious genetic disease, the treatment of which is still only symptomatic, and very widespread in Caucasian populations. It is a disease with an autosomal recessive hereditary determinism (in the offspring of a couple of asymptomatic carriers, the proportion will be one affected individual for two carriers and one healthy individual).

The incidence of CF in the European population is 1/2500 births (350 out of 750,000 births in France), which gives a gene frequency of 1/50, and a proportion of carriers of 1 in 25 (2 million carriers potential in
France for 50 million inhabitants).

The main mutation responsible for this condition is the delta-F 508 deletion (Kerem et al., 1989 Science, 245, 1073-1080), which is responsible for around 70% of cystic fibrosis cases in Canada. In France, studies show (Lucotte, 1989 Option-Bio, 24, 13-14) that this mutation is responsible for 79.5% of cases (estimate relating to families from the north of the Loire). In Scotland, the reported frequency is 74.4%, while in Spain and
Italy it is only 46.2%.

 The conventional carrier detection technique currently available (Kerem et al., See Supra) involves a relatively complex detection procedure, including: hybridization of the DNA of the subjects with a pair of primers framing the mutation, the amplification of the intermediate sequence by polymerase chain reaction (PCR) and revelation of the mutation by differential hybridization with two complementary oligoprobes, one from the normal chromosome and the other from the mutated chromosome; in addition, these oligoprobes are labeled with 32 p, which requires, for the revelation of the reaction, the use of the autoradiographic method. Thus described, such a procedure, which moreover is not suitable for the sole detection of heterozygous carriers , can only be used in research laboratories with special authorization, and is therefore unsuitable for routine use for analytical purposes.

 Among the recently proposed simplifications of the test, a rapid method has been described (Rommens et al., 1990 Am. J. Hum. Genet., 46, 396-397), which is based solely on the size difference of three pairs of bases (bp) of the amplified fragment, not specific for the delta-F 508 mutation, but common to all deletions of base pair triplets in the region. Even more recently, another simplified method has been proposed (Friedman et al., 1990 Clin. Chem. 36, 695-696), specific because it detects the mutated site, but involves an additional step of digestion with a restriction enzyme.

 According to the present invention, it has been shown, surprisingly, that a simple delta-F 508 mutation detection method can be implemented, requiring the use neither of radioactive molecules, nor of restriction enzymes and allowing specific detection of said mutation.

 The main advantage of such a method compared to the other methods described in the prior art lies not only in its specificity, but also in the fact that it makes it possible, in the same reaction medium, to determine whether the patient is a carrier delta-F 508 mutation and if it is heterozygous.

The first subject of the present invention is DNA fragments having the following sequences:
SEQUENCE Aml: 5 '-CATGCXTTGYTGACGCTTC-3' in which X represents I or T and Y represents I or A
SEQUENCE Am2: 5 '-CATTAAZGAAZATATCATCTT-3' in which Z represents I or A
SEQUENCE Am3: 5 '-CATTWAAGWAAATATCATTGG-3' in which W represents I or A.

The present invention also relates to a method for detecting carriers of the delta-F 508 allele of the mutation responsible for cystic fibrosis, mainly comprising the following steps:
- isolation of DNA from cells, fluids or biological tissues,
- incubation with DNA polymerase under reaction conditions allowing the amplification by chain polymerization (PCR) of a reaction medium comprising: * isolated DNA, * an alpha primer having a sequence homology with the chromosomes carrying the normal allele and the chromosomes carrying the mutated allele, and * a beta primer specifically presenting a sequence homology with the chromosome carrying the normal allele or wild-type allele, and / or a gamma primer exhibiting a sequence homology with the chromosome carrying the mutated allele.

* a buffer system adapted to the reaction conditions.

 - revelation of the amplification products by electrophoresis and coloring, for example with ethidium bromide.

 Preferably, the tissues from which the DNA is isolated are buccal epithelial cells or blood cells.

The method according to the invention has two preferred modes of implementation:
- a detection mode, called monoreaction, in which the three primers alpha, beta and gamma are added simultaneously to the amplification medium. In this case, the electrophoresis is preferably carried out on acrylamide gel,
- A detection mode, called birectional in which the beta and gamma primers are added in two separate reaction media, both containing the alpha primer. In this case, the electrophoresis is preferably carried out on an agarose gel.

Advantageously, the primers consist of DNA fragments of approximately 20 bases, preferably 19 bases, in which case the alpha primer can have the sequence
Am1, the beta primer can have the Am2 sequence and / or the gamma primer can have the Am3 sequence.

Amplification is generally continued for 30 replication cycles and is carried out, for example, by
Taq polymerase.

 The present invention also relates to a diagnostic kit for implementing the method described above, comprising in particular: - a medium allowing the extraction of DNA from the cells, - solutions of the three primers alpha, beta, gamma in the buffer suitable for the polymerization reaction, - an acrylamide or agarose gel, - a staining solution, for example of Ethidium Bromide (BETA) - a sample of marker DNA of molecular weight of suitable size.

 The gel can be in dry form to ensure better conservation.

The invention is illustrated without however being limited by the following examples, in which
FIGS. 1 and 2 represent diagrams of electrophoresis obtained by the method in which the detection is carried out by the two-reaction mode, the primers
Am2 and Am3 being added in two separate reaction media, both containing the primer Aml.

 In Figure 2 the even wells correspond to Ami Am3 the odd wells to Aml Amz.

 FIG. 3 represents an electrophoresis gel in which the detection was carried out by the monoreactional mode, the three primers Am1, Am2 and Am3 being added simultaneously to the reaction medium.

 In Figure 2, H represents heterozygous individuals dela-F 508-N, and N represents normal individuals N / N.

EXAMPLE 1
Dual reaction process.

 The DNA to be tested (at least 500 ng) is placed in each of the two tubes corresponding to each of the reactions: '1) medium for detecting the normal chromosome, 2) medium for detecting the mutated delta F 508 chromosome.

The two pairs of primers correspond, on the one hand to the primer Aml in which X is T and Y is A whose sequence is 5 '-CATGCTTTGATGACGCTTC-3', and on the other hand, that is the primer Am2; in which Z is A whose sequence is 5 '-CATTAAAGAAAATATCATCTT-3', for the detection of the normal chromosome, ie the primer Am3 in which W is A; whose sequence is
5-CATTAAAGAAAATATCATTGG-3 '
for the mutated chromosome.

In each of the two tubes, the reaction volume is 100 μl and comprises, in addition to the amplification buffer (20 mM
Tris-HCl, 1.5 mM MgCl2, 25 mM KCl, 100 Frg / ml of Albumin Serum
Bovine, pH = 8.3), 25 pmol of each primer per couple, 25 HM of each dNTP (dATP, dGTP, dCTP, dTTP) and 2.5 units (u) of Taq polymerase.

 Amplification is carried out as follows: denaturation (950C for 5 minutes), 20 amplification cycles including denaturation (950C, 45 seconds), and an extension (600C, 1 minute); the thirtieth cycle is lengthened (extension to 72 "C, 7 minutes).

 The amplification product is revealed by depositing 25 μl of the reaction product on a 1.5% agarose gel, then stained with ethidium bromide. A migration of 30 minutes at 50 mA is sufficient. Amplification products have an approximate size of - 80 base pairs.

 Figures 1 and 2 give examples of revelations obtained: normal individuals (+) only present a band with the revelation Ami Am2. A weak band, of smaller size (-3 bp) can also be observed with Aml Am3 when the whole of the amplification product is deposited, which makes it possible to judge the efficiency of two reactions. H heterozygous individuals (delta-F 508 / N) carriers present in Ami Am2 and in Aml Am3 two bands of equal intensity, while homozygotes h delta-F 508 / delta-F 508 only present the Aml Am3 band.

 In these Figures ED represents the position of the sample deposits, PA the position of the amplification product and MT denotes the well of the DNA marker of molecular weight.

EXAMPLE 2
Monoreaction process
In order to simplify the presentation and use of the diagnostic kit, a simplified mutation detection system involving only one reaction has been developed. To do this, the three primers are added to the same tube, at concentrations of 50 Pmoles for the primer Aml and 25 Pmoles for each of the primers Am2 and Am3. The reaction volume is always 100 μl, but with 5 mM of MgCl2, 100 μM of each dNTP and 3 U of Taq polymerase. The amplification cycles are the same as in Example 1.

 The development of the amplification product is carried out by depositing 25 μl of the product on an acrylamide gel (migration of at least 10 cm) at 12%, colored with ethydium bromide. The migration is carried out in TEB buffer at 150 V for 1/2 hour.

 Such a 10-well gel, prepared and stored under vacuum, can be part of a ready-to-use kit, together with a sample of molecular weight marker DNA with fragments having sizes corresponding to the region to solve.

Under these conditions (Figure 3) the amplification products are in the form:
of a single band for individuals homozygous normal (well 1) and homozygous delta-F 508 / delta-F 508 (well 2),
two bands for heterozygotes [H] (well 3).

In this figure M represents the well of the molecular weight marker and S and I indicate the respective positions of the upper and lower bands (3 bp apart).

 The technique can be adapted for serial screening. To this end, the DNAs (+) and H were mixed in the proportions 1/1, 2/1, 4/1 and 24/1; in all cases, the lower band appears clearly, which makes it possible to detect a single ADH H in a DNA mixture of 25 individuals (the frequency of the carriers is precisely of the order of 1/25). When a series is positive (the strip of smaller size is present, even in the trace state), it can then be broken down and the individuals tested one by one in order to identify the positives.

EXAMPLE 3
Simplified method for obtaining cellular DNA from epithelial cells of the oral mucosa
In general, people do not wish (fear of blood tests, AIDS ...) to donate blood. A simplified method for obtaining cellular DNA from the epithelial cells of the oral mucosa has therefore been developed according to the following protocol:
The subjects rinse their mouths for 10 seconds with 15ml of a sampling solution which may be contained in the box described above. The mouth cells are centrifuged at 500g for 10 minutes on an ordinary bench-top centrifuge. The pellet is resuspended in 1 ml of SDS (Sodium Dodecyl Sulfate) at 1% and incubated for 1 hour at 550C with 0.1 mg / ml of proteinase K. The cell debris is centrifuged for 30 seconds, and each take of 50 μl of the supernatant can constitute the substrate of a PCR amplification reaction.

EXAMPLE 4
Validation of the test on CF families and in a population
The method of Example 1 was tested by studying a collection of 60 CF families (carrier parents and index case reached). In all cases, the carrier parents were detected by the test when they were heterozygotes. The test was also applied to the cases of 19 families where the CF index case had died. From the detection of delta-F 508 carriers, it was possible to predict the state of carriers for relatives (uncles, aunts, nephews, nieces, allies, etc.).

 This test was also applied to the detection of heterozygotes on a population of 138 independent DNAs independent of the two sexes. In this population, the number of heterogyzotes detected is 4, a proportion that is not significantly different from the theoretical one of 1/25 to 1/30.

Claims (15)

 1. DNA fragment having the following Aml sequence: 5 '-CATGCXTTGYTGACGCTTC-3' in which X represents I or T, and Y represents Y or A.  2. DNA fragment having the following Am2 sequence:  5'-CATTAAZGAAZATATCATCTT-3 'in which Z represents I or A.  3. DNA fragment having the following Am3 sequence:  5'-CATTWAAGWAAATATCATTGG-3 in which W represents I or A.  4. Method for detecting carriers of the delta allele F-508 of the mutation responsible for cystic fibrosis mainly comprising the following stages:  - isolation of DNA from cells, fluids, or biological tissues,  - incubation with DNA polymerase under reaction conditions allowing amplification by DNA polymerase; of a reaction medium comprising: * the isolated DNA, * an alpha primer having a sequence homology with the chromosome carrying the normal allele and the chromosome carrying the mutated allele, and * a beta primer specifically presenting homology with the chromosome carrying the normal allele and / or a gamma primer presenting specifically homology with the chromosome carrying the mutated allele, and * a buffer system adapted to the reaction conditions,  - revelation of the amplification products by electrophoresis and coloring.  5. Method according to claim 4 characterized in that the three primers alpha, beta and gamma are added simultaneously to the amplification reaction medium.  6. Method according to claim 5, characterized in that the electrophoresis is carried out on acrylamide gel.  7. Method according to claim 4 characterized in that the primers beta and gamma are added in two separate reaction media, both containing the alpha primer.  8. Method according to claim 7, characterized in that the electrophoresis is carried out on an agarose gel.  9. Method according to one of claims 4 to 8, characterized in that the primers consist of DNA fragments of about 20 bases and preferably 19 bases.  10. Method according to one of claims 4 to 9, characterized in that the alpha primer has the sequence of the DNA fragment according to claim 1.  11. Method according to one of claims 4 to 9, characterized in that the beta primer has the sequence of the DNA fragment according to claim 2.  12. Method according to one of claims 4 to 9, characterized in that the gamma primer has the sequence of the DNA fragment according to claim 3.  13. Method according to one of claims 4 to 12, characterized in that the DNA is isolated from buccal epithelial cells or from blood cells.  14. Method according to one of claims 4 to 14, characterized in that the amplification is carried out for approximately 30 replication cycles.  15. Diagnostic kit for implementing the method according to one of claims 4 to 14, characterized in that it comprises in particular:  - Preparations of the alpha, beta and gamma primers preferably in a solution containing a buffer system suitable for the reaction of the polymerization and a DNA polymerase.  - a molecular weight marker DNA preparation.  - a coloring solution and  - an acrylamide or agarose gel;