WO1992014840A1 - Nucleic acid fragment of the x chromosome region involved in the fragile x syndrome, nucleotide probe and method of diagnosing mental retardation with fragile x - Google Patents
Nucleic acid fragment of the x chromosome region involved in the fragile x syndrome, nucleotide probe and method of diagnosing mental retardation with fragile x Download PDFInfo
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- WO1992014840A1 WO1992014840A1 PCT/FR1992/000145 FR9200145W WO9214840A1 WO 1992014840 A1 WO1992014840 A1 WO 1992014840A1 FR 9200145 W FR9200145 W FR 9200145W WO 9214840 A1 WO9214840 A1 WO 9214840A1
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Definitions
- Mental retardation syndrome with fragile X is an X chromosome-related disease that affects approximately one in every 1,500 boys. Women vectors, about one in seven hundred and fifty, have a 30% risk of having mild or even moderate debility. Mental retardation syndrome with fragile X is the most common cause of inherited mental retardation.
- the diagnosis of the disease is established by a delicate cytogenetic analysis which requires particular culture conditions and the analysis of 50 to 100 mitoses, because the fragile site detectable cytogenetically is only present in 5 to 50% of mitoses.
- the diagnosis of vector women is even more difficult since only 50 to 60% of these women express the fragile site, in a generally low proportion of mitoses.
- the difficulty of the cytogenetic test means that it is not reliably performed except in a limited number of laboratories; moreover, the diagnosis of the disease is often only made after the birth of a second affected child in the same family; however, the early establishment of such a diagnosis is essential for implementing investigations allowing prenatal diagnosis and screening of vector women who transmit the disease to 50% of their male children, in families at risk.
- the high frequency of the disease and the early genetic importance justify systematic neonatal screening for the disease.
- the present invention specifically aims to provide new means of investigation and diagnosis, based on the direct study of DNA, allowing rapid diagnosis of the disease and applicable to a large number of patients.
- hereditary transmission of the disease is unusual compared to other diseases linked to the X chromosome. It has been found that phenotypically normal men transmit the mutation to their daughters, who rarely exhibit the symptoms of the disease, on the other hand. sons and daughters of these women have a high rate of onset of the disease (Sherman et al, Hum. Genêt. (1985)). Several hypotheses have been proposed to explain this phenomenon.
- the invention also provides means for the detection, in non-sick persons, of anomalies and in particular of mutations at the level of the fragile X site.
- the genetic markers proposed in the prior art can only be used to monitor the transmission of the mutation in families where the disease has already been diagnosed by a cytogenetic study, it this is an indirect diagnosis by link study; it is generally necessary to use several markers on either side of the disease locus, and these markers are only informative in varying proportions of families.
- F. Rousseau et al (Am. J. Hum. Genêt. (1991) 48: 108-116) report the detection of seven regions close to the fragile X locus (FRAXA). Two of these regions between DXS369 and DXS296 contain the genetic markers closest to FRAXA known to date, one of these regions contains two probes detecting a polymorphism.
- the inventors cloned as an artificial yeast chromosome (YAC), the approximately 225 kb DNA segment defined between probes St677 and Do33, and located in the Xq27-q28 region of the human X chromosome. Probes from this segment have been prepared for the analysis of mutations, DNA methylations and genes involved in fragile X syndrome.
- YAC yeast chromosome
- CpG sites these are for example sites containing the CpG dinucleotide, such as Hpall, CCGG or Hhal, GCGC, or sites capable of partially covering a CpG dinucleotide such as Avall GG (A or T) CC, in a CGG (A or T) CC or GG (A or T) CGG or even BanI (GG Py Pu CC) sequence; the inventors have demonstrated a surprising correlation between the clinical expression of the disease and the inhibition of the digestibility of CpG sites in the region of approximately 20 kb around the 125 kb position defined on the cloned DNA segment through the inventors, approximately 100 kb from the Do33 probe
- probes are also suitable for the detection of the fragile X locus, without clinical manifestation of the disease, which results from the mutation of the nucleic acid of the X chromosome region described above, by extension of the DNA.
- the lengthening can indeed be more or less significant depending on whether the subject is only a carrier of the anomaly or whether he has clinical signs of mental retardation associated with the fragile X anomaly.
- the probes of the invention advantageously allow the detection of premutations corresponding to an elongation of the nucleic acid from approximately 100 bp (base pairs) to approximately 500 bp and the detection of complete mutations corresponding to an elongation of the nucleic acid at least around 500bp, up to over 3000bp.
- Premutations generally correspond to a subject carrying the anomaly without clinical manifestation, whereas mutations by extension greater than 500 bp are normally associated with the presence of clinical signs.
- mutation will be used to designate the two situations mentioned above and the distinction between mutations associated or not with clinical signs will be made if necessary.
- Probes of interest in the search for a mutation in the fragile X region without clinical expression of mental retardation are, for example the so-called probes adjacent to the CpG site.
- Figure 1 is a schematic representation of the X chromosome region around the 125 kb position located about 100 kb from the Do33 probe in direction of the centromere. On the scale, the location of the probes St677, 2-34, and Do33 is shown. In accordance with the scale, the clones YAC 141 H5 and 209G4 corresponding to the above-mentioned DNA segments have been shown, and the location of restriction sites Eaql, BssII etc., has been represented on the line of clone 141 H5. The indication CpG on the line of clone 141 H5 corresponds to the region whose methylation is abnormal in patients with mental retardation with fragile X.
- This phenomenon of inhibition of digestibility of CpG sites can be detected by the Southern Blot method, using probes originating from the 20 kb region around the 125 kb position defined in FIG. 1, probes close to or containing the sites to CpG.
- the inventors have also demonstrated, by the same method, anomalies in the migration of DNA fragments, not necessarily linked to the phenomenon of methylation, anomalies which are characteristic of subjects carrying a fragile X mutation.
- the germinal mutations responsible for fragile X syndrome and capable of being transmitted to the offspring are located in the region of the X chromosome defined between the probes St677 and Do33, and the methylation of the CpG sites in this region is a consequence not obligatory of these mutations, consequence which is linked to the clinical expression of the disease.
- the invention therefore relates to any nucleic acid fragment originating from the Xq27-q28 region of the X chromosome, adjacent to or containing the CpG sites, located in the region of 10 to 20 kb around the position 125 kb.
- the invention relates to any nucleic acid fragment included in the DNA segment of approximately 225 kb, defined between the probes St677 and Do33.
- nucleic acid fragments comprise at least 15 bp or nucleotides.
- the nucleic acid sequences of the invention are DNA, cDNA or even mRNA sequences.
- nucleic acid sequences of the invention are individualized sequences and therefore they are not in their natural environment.
- the cDNA corresponding to the nucleic acid sequences of the invention can be obtained according to conventional techniques by screening a library of available human cDNA, with a nucleic acid fragment of the invention, the screening making it possible to detect cDNA fragments which selectively hybridize with the nucleic acid fragment of the invention.
- This region of the X chromosome is involved in fragile X syndrome and the probes specific for this region are advantageously used to detect the presence of methylation abnormalities related to the clinical expression of the disease, or to detect the presence of mutations responsible for the fragile X syndrome and its transmission, or to locate, identify and analyze the expression of the gene (s) involved in fragile X syndrome.
- These probes can generally be used to detect DNA abnormalities that are the direct or indirect consequence of mutations associated with fragile X syndrome.
- the invention therefore relates to any nucleotide probe capable of hybridizing selectively with a nucleic acid fragment included in the DNA segment defined between the St677 and Do33 probes and more particularly with a nucleic acid fragment adjacent to or containing the sites at CpG in the region of about 20 kb around the 125 kb position.
- the probes of the invention specifically hybridize under appropriate stringency conditions with the abovementioned nucleic acid fragments but do not hybridize with other regions of the human genome under the same hybridization conditions.
- a support such as a nitrocellulose filter or a nylon membrane
- a pre-hybridization solution having the following composition: 40% formamide, * 0.9 M NaCl; 200 ⁇ g / ml of salmon sperm DNA; 50 mM sodium phosphate pH 6.5; 4% dextran sulfate; 0.08% (weight / volume) of ficoll and of polyvinyl pyrrolidone, hybridization in a solution identical to the pre-hybridization solution, further comprising at least one nucleotide probe according to the invention advantageously labeled, at a temperature at 42 ° C and for 18 hours, 4 successive washes, revealing the hybrid possibly formed by any method suitable for labeling the probe.
- the hybridization conditions defined above constitute preferred conditions, but are in no way limiting and can be
- the probes of the invention are advantageously marked with any marker conventionally used. They can be marked using a radioactive tracer such as 32 P, 35 S, 125 I ,, 14 C and the radioactive marking can be carried out according to any method known to those skilled in the art.
- a radioactive tracer such as 32 P, 35 S, 125 I ,, 14 C and the radioactive marking can be carried out according to any method known to those skilled in the art.
- the probes can be labeled in 3 ′ by addition of one or more deoxynucleotides or ribonucleotides or of a dideoxynucleotide, marked in alpha by 32 P, in the presence of the Terminal Deoxynucleotidyl Transferase, or in 5 ′ by transfer of a group radioactive phosphate of a deoxynucleotide or free dideoxynucleotide labeled in the gamma position, in the presence of T4 DNA ligase. Probes can still be labeled using DNA polymerase by "Nick Translation "or” Random Priming "or” Polymerase Chain Reaction ".
- the method of detection of hybridization will depend on the radioactive marker used and may be based on autoradiography, liquid scintillation, gamma counting or any other technique making it possible to detect the emission of the radiation emitted by the radioactive marker.
- non-radioactive labeling can also be used, by combining with the probes groups having immunological properties such as an antigen, a specific affinity for certain reagents such as a ligand, physical properties such as fluorescence or luminescence, properties allowing the completion of the reactions enzymes like an enzyme or an enzyme substrate.
- Non-radioactive labeling can also be done directly by chemical modification of DNA, such as photobiotinylation or sulfonation.
- the probes of the invention are used for the in vitro detection, mutations responsible for fragile X syndrome, as well as anomalies in methylation of CpG sites or other DNA anomalies resulting from the presence of these mutations and associated to the syndrome.
- the mutation detection aims to follow the family transmission and allows in particular a diagnosis of the non affected people likely to transmit the disease, such as women vectors or normal men transmitters of the mutation.
- methylation abnormalities allows a diagnosis of the disease, expressed clinically or likely to be expressed in the context of an antenatal or birth diagnosis.
- the methods for detecting mutations responsible for fragile X syndrome, as well as abnormal methylation of CpG sites, or other DNA abnormalities associated with the syndrome rely on the use of at least one probe according to the invention and at least one suitable restriction enzyme so that the fragment detected by the probe is different depending on whether or not it contains the methylation mutation or anomaly or any other anomaly DNA.
- Such a process can be carried out according to the so-called "Southern Blot” method (Sa brook, Fritsh and Maniatis; Molecular cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press), comprising the following steps: treatment of genomic DNA extracted for example from leukocytes or chorionic villi or from any other source of genomic DNA, by at least one specific restriction enzyme, under conditions allowing the obtaining of restriction fragments resulting from the cleavage of DNA at its level restriction sites recognized by said enzyme; separation of the fragments by electrophoresis; bringing into contact a nucleic probe according to the invention capable of hybridizing specifically with the preceding fragments under conditions allowing the formation of hybridization complexes; detection of hybridization complexes; measurement of the size of the restriction fragments engaged in the hybridization complexes.
- the search for methylation anomalies for the in vitro diagnosis of fragile X syndrome in patients patients suffering from mental retardation is advantageously carried out according to the preceding method using at least one restriction enzyme with a site sensitive to methylation, with at least one probe according to the invention advantageously labeled.
- the above-mentioned method can be carried out with a single methylation-sensitive restriction site enzyme such as BssHII, Eaql, Avall, BanI; or again with two enzymes, one with a restriction site sensitive to methylation and the other with a restriction site not sensitive to methylation.
- the probes of the invention used for hybridization with the fragments obtained are advantageously adjacent to or contain the site for the enzyme with a site sensitive to methylation.
- the Southern Blot method is also used to detect mutations associated with fragile X syndrome, or mutations existing in vector vectors, without clinical signs. These mutations are, for example, DNA sequence deletions or insertions, or other DNA abnormalities such as conformational or replicative abnormalities, which are consequential to these mutations.
- the detection of these mutations for the in vitro diagnosis of fragile X syndrome or more generally of the mutation of the fragile X site, in patients with or without mental retardation is carried out according to the Southern Blot method by subjecting the genomic DNA to a digestion with any suitable restriction enzyme not necessarily sensitive to methylation such as EcoRI or HindIII.
- the restriction fragments are demonstrated by hybridization with a probe of the invention advantageously labeled and the abnormal electrophoretic migration of said fragments is correlated either with the clinical expression of the disease, or with the presence of the mutation even in the very absence of clinical expression of the disease.
- This abnormal migration of the fragments corresponds to the presence of fragments elongated by the insertional mutation of a nucleotide sequence.
- the invention also relates to the kits for implementing the above method.
- nucleic acid fragments of the invention those of 20 to 30 nucleotides can be advantageously used as primers in a method of genetic amplification of a nucleic acid fragment of the 20 kb region around the position 125.
- the amplification may be intended to reveal the presence of mutations in this region, in particular by lengthening of the DNA, or / and determined for the detection of a fragment containing the CpG sites.
- This amplification method consists in specifically fixing by hybridization on the genomic DNA extracted for example from leukocytes, two oligonucleotides judiciously chosen from those containing the nucleic acid fragments of 20 to 30 nucleotides of the invention which are adjacent to the fragment to amplify, then implement an enzymatic extension process using DNA polymerase, followed by a denaturation process, and repeat the hybridization-extension-denaturation cycle known as the PCR "Polymerase cycle Chain Reaction ", a sufficient number of times to increase the quantity of the fragment to be amplified in an exponential proportion compared to the number of cycles carried out.
- the amplification method described can be applied to the detection of DNA abnormalities which are the consequence of the presence of the above mutations.
- the in vitro diagnosis of fragile X syndrome, in particular in patients with mental retardation, or in vector vectors is advantageously carried out by genetic amplification carried out in parallel on the DNA digested with at least one restriction enzyme chosen from the enzymes not sensitive to methylation or on the contrary sensitive to methylation, and on undigested DNA. Analysis for diagnostic purposes is done by comparing the amplification product of undigested DNA to that of DNA digested with the chosen restriction enzyme (s).
- This analysis of the amplification products can be carried out by visualization of the fluorescence induced by ethidium bromide, after electrophoresis on agarose or polyacrylamide gel, or by the detection of radioactive or non-radioactive labeling, such as by a system. biotin-avidin, priming oligonucleotides or nucleotides incorporated into the amplified fragment.
- the number of amplification cycles is limited so as to maintain a linearity of the amplification and therefore a quantitative estimate of the digestibility of the sites of the enzyme or enzymes used.
- internal markers are used, in particular other fragments of the co-amplified genome also containing a site of the chosen enzyme, as a witness for the action of the restriction enzyme, and as a witness for the efficiency of amplification, a fragment not containing such a site.
- the quantitative measurement of the characteristic fragments of the mutations, in particular resulting from the elongation, as well as the quantitative measurement of the methylation of the genomic DNA at the site studied can be done by adapting the method. amplification described above, in particular by using in addition to the oligonucleotides according to the invention, so-called “linker” oligonucleotides and DNA Ligase, a method called “Ligation mediated PCR” described by Steigerwald et al (Nucleic ACids Res. (1980 ) 18: 1435-1439).
- Deletions or any other rearrangement constituting mutations liable to give rise to fragile X syndrome, in particular by lengthening of nucleic acid can also be detected by the so-called multiplex DNA amplification method, using pairs of oligonucleotides which can be mixed in a single reaction to amplify several fragments of the region around the 125 kb position and to detect the disappearance or modification of some of these fragments.
- the detection of point mutations is advantageously carried out by amplification of the DNA segments, followed by an analysis by SSCP (single strand conformation polymorphism) or DGGE (denaturing gradient gel electrophoresis) methods, or even by detection of heteroduplex.
- kits for implementing the above method include: oligonucleotides labeled or not labeled for the amplification of nucleic acid fragments exhibiting an anomaly resulting from a mutation, in particular by lengthening of the DNA and / or of nucleic acid fragments containing a site sensitive to methylation according to the invention; oligonucleotides labeled or not for the amplification of the control nucleic acid segments; so-called "linker”oligonucleotides; - buffers, nucleotides and enzymes suitable for amplification.
- a group of probes which are particularly advantageous for carrying out the invention includes probes, the detailed description of which is given in the experimental part.
- These probes include the fragments 9B12.3 (now designated StB12.3), 9B12.5 (StB12.5), 9B12.4, 9B12.2 and 9B12.1 located in relation to the DNA located between the probes Do33 and St677 , represented in FIG. 2.
- Other probes are the stA22 (Avall-Avall) fragments of approximately 1500bp, stX21 of approximately 3800bp and stX21E (centromeric of the Eaql site).
- the 9B12.3 probe can be used to detect anomalies at the fragile X site of nucleic acid fragments, and more particularly mutations by lengthening of the DNA, after a step of digestion with the enzyme of EcoRI restriction.
- FIG. 7 A diagram of the results obtained on restriction fragments resulting from digestion with EcoRI, detected by the probe 9B12.3, showing a normal or mutated fragment is given in FIG. 7.
- probes 9B12.3 (stB12.3) and 9B12.5 (stB12.5) is shown in Figure 8, with its complementary sequence.
- the probes of the invention also allow access to the region in which the gene or genes involved in the clinical expression of fragile X syndrome are located. Indeed, the identification of CpG sites such as BssHII, EagI, SacII in the region close to the 125 kb position, indicates the presence of adjacent genes, the expression of which is controlled by this region (CpG islet).
- the probes corresponding to these genes are useful for the detection of the corresponding messenger RNAs, or for the construction of vectors for expression of the corresponding proteins for therapeutic application or for the production of specific antibodies which may also be used in diagnosis .
- a fragment according to the invention is characterized in that it selectively hybridizes with an mRNA present in human cells, for example leukocytes and in that it corresponds to a gene whose 5 'end is contained or adjacent to the CpG island in position 125 kb on the genetic map shown in Figure 2A.
- Another particular fragment of the invention is characterized in that it is included in the DNA defined between the Do33 probe and the 125 kb position at approximately 100 kb of the Do33 probe of the Xq27-q28 region of the X chromosome, and that it selectively hybridizes with an mRNA present in human cells, for example leukocytes.
- the invention relates to any recombinant DNA containing a nucleic acid fragment of the invention associated with any sequence allowing transcription and / or translation in the host cell into which said recombinant DNA is capable of being introduced.
- the invention therefore also relates to the cloning and expression vectors containing one of the nucleic acid fragments of the invention.
- the introduction of the recombinant DNA is advantageously carried out using a vector, in particular of the plasmid type, able to replicate in the host cell and to allow the transcription of the corresponding RNA and the expression of the proteins. encoded by the nucleic acid fragment contained in the recombinant DNA.
- the host cells mentioned above can be prokaryotic cells, in particular E. coli cells, or, more advantageously, eukaryotic cells, which make it possible to obtain glycosylated and mature proteins (yeasts, CHO cells, or insect cells infected with bacculovirus). These proteins can advantageously be used for the preparation of specific antibodies useful for the diagnosis of fragile X syndrome and in therapeutic applications.
- DNA segments corresponding to probes St677 and Do33 can be obtained from human DNA by genetic amplification using the following oligonucleotides:
- the amplification conditions in a standard medium are as follows:
- the probes of the invention were obtained by subcloning the nucleic acid fragments of the invention originating from the digestion of DNA segments contained in the region of approximately 225 kb defined between the probes St677 and Do33, and located in the Xq27-q28 region of the human X chromosome, cloned as an artificial yeast chromosome (YAC) and represented in FIG. 1.
- YAC yeast chromosome
- subcloning aims to obtain specific probes for fragments located in the region of 20 kb around the position 125 kb and corresponding therefore specifically to the nucleic acid fragments adjacent to or comprising the CpG sites.
- a probe is obtained by subcloning of fragments prepared by double digestion Eaql, B ll of the clone 141H5.
- the probe obtained 9B12 corresponds to an Eagl-Bgll fragment of approximately 7 kb containing the Eaql site at the position 125 kb on the map of FIG. 1.
- FIG. 2 is a diagrammatic representation of the map of the probe 9B12 and of its subfragments 9B12.1, 9B12.2, 9B12.3, 9B12.4 and 9B12.5, oriented inversely to FIG. 1. This figure has the following 7 lines:
- line (1) is a scale in kb on which is mentioned in 0 the position 125 kb defined on the map of the clone segment of FIG. 1 between the probes St677 and Do33 at approximately 100 to 125 kb from the probe Do33.
- line (2) is a representation in agreement with the scale of the probe 9B12 and its subfragments 9B12.1, 9B12.2, 9B12.3, 9B12.4 and 9B12.5.
- line (3) represents the location, in agreement with the scale, of the BanI sites (GGPyPuCC) and in particular the polymorphic BanI site noted RFLP and the BanI site sensitive to methylation noted Me.
- line (5) represents the location, in accordance with the scale of line (1), of the BglII sites (AGATCT).
- - line (6) represents the location, in line with the scale of line (1), of the EcoRI sites (GAATCC).
- - line (7) represents the location, in accordance with the scale of line (1), of the HindIII sites (AAGCT).
- the BanI, XmnI and EcoRI site maps correspond to altered fragments in carriers of the fragile X mutation.
- Genomic DNA extracted from leukocytes from a patient with mental retardation is cut by the enzymes Avall, BanI or XmnI.
- the DNA fragments obtained are analyzed by electrophoresis on 0.9% agarose gel in TAE buffer, then transferred to a nylon membrane.
- the membrane on which the DNA fragments are fixed is subjected to a pre-hybridization treatment at 42 "C for 2 hours with a solution having the following composition: 40% formamide; 0.9 M NaCl; 200 ⁇ g / ml d '' Salmon sperm DNA; 50 mM sodium phosphate pH 6.5; 4% dextran sulfate; 0.8% (weight / volume) of ficoll and polyvinyl pyrrolidone; then the pre-hybridization solution is replaced by a hybridization solution identical to the pre-hybridization solution, and further comprising the probe 9B12.3 labeled with dCTP alpha ⁇ P and of specific activity 2 10 B dpm / ⁇ g; after incubation for 18 hours at 42 "C and successive washings with a 0.5 SSC solution at 65 ° C., autoradiography is carried out at -80 ° C.
- FIG. 3 is a schematic representation of a autoradiography image on 0.9% agarose gel, of DNA of leukocytes or chorionic villi of normal men (1 and 5), of a man with X fragile (3), of a vector woman (4) and of a normal woman, digested by BanI and revealed by the probe 9B12.3.
- FIG. 4 is a schematic representation of a autoradiography image on 0.9% agarose gel, of chorionic villus DNA from a normal man (2), from a man with fragile X (3) , of a fetus with fragile X, digested with BanI and revealed by the probe 9B12.3.
- the 9B12.3 probe detects in the genomic DNA of a normal man, after digestion with the enzyme BanI, either a single fragment of 2.9 kb (allele 1) or two fragments of 2.3 and 0.6 kb (allele 2).
- BanI enzyme-catalyzed kinase
- the existence of two alleles is due to the presence of a polymorphic BanI site indicated RFLP in FIG. 2.
- the 2.9 kb fragment is replaced more or less completely by a 3.5 kb fragment.
- the 0.6 kb fragment is replaced more or less completely by a 1.2 kb fragment.
- the anomaly was detected not only on DNA from leukocytes, but also on DNA from chorionic villi of fetuses carrying a fragile X mutation. It can therefore be used in postnatal or antenatal diagnosis in males.
- fragments of 3.5 kb and 1.2 kb can be detected in normal women (methylation of the BanI site on the inactive X chromosome). However, their intensity appears stronger in women vectors with a clinical or cytogenetic expression of fragile X syndrome.
- FIG. 5 is a schematic representation of a autoradiography image on 0.9% agarose gel, of DNA of leukocytes of normal men (1 and 10), of men with mental retardation linked to fragile X (3, 4, 6, 9), vector women (5, 7, 8) and a normal woman (2), digested with XmnI and revealed by the probe 9B12.3
- the 9B12.3 probe detects a 9 kb fragment in men and women as well as in carriers of a fragile X mutation not expressing the disease clinically or cytogenetically.
- the 9 kb fragment is replaced in whole or in part by fragments of apparent size greater than 10 kb. The appearance of such fragments is also characteristic of female vectors with clinical and / or cytogenetic expression.
- FIG. 6 is a schematic representation of an autoradiography image on 0.9% agarose gel, of DNA of leukocytes of normal women (9, 12, 13) and of vector vectors (1 to 8, 10 and 11) of the fragile X mutation not all expressing clinical or cytogenetic signs, digested with EcoRI and revealed by the probe 9B12.3
- the 9B12.3 probe detects an EcoRI fragment of approximately 6 kb in a normal man or a normal woman.
- an additional larger fragment, of variable size according to the individuals (6 to 7 kb) is present. This anomaly appears in vector women without clinical and / or cytogenetic expression, and can therefore be used for the diagnosis of vector women.
- the normal fragment is replaced by a fragment or series of fragments of larger apparent size. Very similar results are obtained with the enzyme HindIII.
- FIG. 9 is another representation of the detection of mutations at the level of the fragile X region, by digestion of the DNA with EcoRI using the probe stB12.3.
- Figure 9 Detection of fragile X mutations in genomic DNA digestion products by EcoRI.
- the leukocyte DNA was fully digested with EcoRI.
- the arrows indicate the size of the normal fragment (5.2 kb) and certain mutated fragments.
- On the left ⁇ refers to the increase in size of the mutated fragment.
- the symbols describing the subjects and their relationships within the family are presented above. The squares and circles represent men and women respectively. Open symbols that are not cut in half indicate subjects who are apparently normal and have not been tested cytogenetically. For symbols cut in half, the right part represents the sequence of the fragile X site and the left part the degree of mental retardation.
- the symbols contained in a triangle correspond to samples obtained from a fetus.
- the dotted symbols show subjects for which EcoRI digestion has been carried out and which do not provide information to clarify the family relationship of the other subjects.
- the sample on line 22 had a weak cloud at 7.5 kb in the EcoRI digestion product. This cloud is more visible as a band at 3 kb in the BqlII digestion product from the same sample, shown on the right; it is located just above the normal BqlII fragment which is 12 kb in length.
- the genomic DNA extracted from leukocytes of a patient suffering from mental retardation is subjected to a digesiton by an enzyme with a site sensitive to methylation such as Avall, BanI or XmnI and the amplification is carried out in parallel on the genomic DNA digested with the methylation sensitive restriction enzyme, and on undigested DNA.
- an enzyme with a site sensitive to methylation such as Avall, BanI or XmnI
- Two oligonucleotides of 20 to 30 nucleotides adjacent to the genomic DNA fragment to be amplified, containing the site for the enzyme sensitive to methylation, are hybridized with the DNA subjected to digestion and the DNA not subjected to digestion. Then an enzymatic extension process using DNA polyerase, followed by a denaturation process is implemented; the hybridization-extension-denaturation cycle is repeated a number of times sufficient to increase the quantity of the fragment containing the CpG sites in an exponential proportion relative to the number of cycles carried out.
- the number of amplification cycles is limited so as to maintain a linearity of the amplification and therefore a quantitative estimate of the digestibility of the site of the enzyme sensitive to methylation.
- internal markers are used, in particular other fragments of the co-amplified genome containing a site of the methylation-sensitive enzyme, not methylated in humans, or of fragments not containing such a site.
- the analysis of the amplification products is carried out by visualization of the fluorescence induced by ethidium bromide, after electrophoresis on agarose or polyacrylamide gel. If the methylation-sensitive site is methylated, the amplified fragment is present in both reactions, if the methylation-sensitive site is unmethylated, the amplified fragment is present only in the reaction with undigested DNA.
- the 2.8 kb fragment was absent in men with a fragile X mutation, while women with the mutation had both normal fragments (2.8 and 5.2 kb) and the mutated fragments. Premutations were detected as EcoRI-Eaql fragments of 2.9 to approximately 3.3 kb in men (line 3) and were detected on the active X chromosome of women (lines 8, 24, 26 and the like) whereas EcoRI fragments of 5.3 to 5.7 kb corresponded to a premutation on the inactive X chromosome. The fragments corresponding to complete mutations were not digested by Eaql and therefore appear as simple or heterogeneous bands above 5.7 kb (lines 10, 12, 15 and the like).
- FIG. 10 represents the detection of the fragile X mutations and of the methylation scheme for the DNA digested with EcoRI and Eaql: the DNA of leukocytes was simultaneously digested completely with the enzymes EcoRI and Eaql. The sizes are indicated on the left for some of the mutated fragments and for the normal fragments on the active and inactive X chromosomes (2.8 and 5.2 kb respectively). ⁇ corresponds to the increase in size of the mutated fragments. Hybridization patterns are identified on the right. The "/" trait denotes a deceased family member. For the other symbols, see Figure 9. The results for lines 6 and 16 are poor. Hybridization of these samples with a control probe showed that these lines contained as much DNA as the others and did not exhibit any migration artifacts.
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Application Number | Priority Date | Filing Date | Title |
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DE69225797T DE69225797T2 (en) | 1991-02-13 | 1992-02-13 | NUCLEIC ACID FRAGMENT OF THE X-CHROMOSOME AREA PARTICIPATING IN THE SENSITIVE X-SYNDROME, NUCLEOTIDIC PROBE AND METHOD FOR THE DIAGNOSIS OF MENTAL RESIDUAL WITH SENSITIVE X |
AU14136/92A AU671418B2 (en) | 1991-02-13 | 1992-02-13 | Nucleic acid fragment of the X chromosome region involved inthe fragile X syndrome, nucleotide probe and method of diagnosing mental retardation with fragile X |
CA002104110A CA2104110C (en) | 1991-02-13 | 1992-02-13 | Nucleic acid fragment from the chromosome x region involved in the fragile x syndrome, nucleotide probe and method for the diagnosis of mental retardation with fragile x |
JP4506537A JPH06507310A (en) | 1991-02-13 | 1992-02-13 | Nucleic acid fragment of the X chromosome region involved in fragile X syndrome, diagnostic method for mental retardation associated with fragile X syndrome, and nucleotide probe |
EP92907135A EP0580621B1 (en) | 1991-02-13 | 1992-02-13 | Nucleic acid fragment of the x chromosome region involved in the fragile x syndrome, nucleotide probe and method of diagnosing mental retardation with fragile x |
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FR91/01684 | 1991-02-13 | ||
FR9101684A FR2672618B1 (en) | 1991-02-13 | 1991-02-13 | NUCLEIC ACID FRAGMENT OF THE X CHROMOSOME REGION INVOLVED IN FRAGILE X SYNDROME, NUCLEOTIDE PROBE AND METHOD FOR DIAGNOSING MENTAL DELAY WITH X FRAGILE. |
US81593291A | 1991-12-30 | 1991-12-30 |
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Cited By (9)
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US5741645A (en) * | 1993-06-29 | 1998-04-21 | Regents Of The University Of Minnesota | Gene sequence for spinocerebellar ataxia type 1 and method for diagnosis |
US5834183A (en) * | 1993-06-29 | 1998-11-10 | Regents Of The University Of Minnesota | Gene sequence for spinocerebellar ataxia type 1 and method for diagnosis |
US5876949A (en) * | 1995-05-31 | 1999-03-02 | The Trustees Of The University Of Pennsylvania | Antibodies specific for fragile X related proteins and method of using the same |
US6197500B1 (en) | 1991-01-04 | 2001-03-06 | Adelaide Medical Centre For Women And Children | DNA sequences related to fragile X syndrome |
US8409805B2 (en) | 2009-02-13 | 2013-04-02 | Asuragen, Inc. | Method of amplification of GC-rich DNA templates |
US8679757B2 (en) | 2009-03-24 | 2014-03-25 | Asuragen, Inc. | PCR methods for characterizing the 5′ untranslated region of the FMR1 and FMR2 genes |
US9371560B2 (en) | 2012-07-20 | 2016-06-21 | Asuragen, Inc. | Comprehensive FMR1 genotyping |
CN110923305A (en) * | 2019-11-25 | 2020-03-27 | 广州市达瑞生物技术股份有限公司 | DNA molecular weight standard suitable for fragile X syndrome southern blot hybridization detection |
US11497816B2 (en) * | 2015-10-06 | 2022-11-15 | The Children's Hospital Of Philadelphia | Compositions and methods for treating fragile X syndrome and related syndromes |
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WO1986005512A1 (en) * | 1985-03-13 | 1986-09-25 | Centre National De La Recherche Scientifique (Cnrs | Dna probe intended to the anti-natal diagnosis of certain chromosomal anomalies |
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Cited By (12)
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US6197500B1 (en) | 1991-01-04 | 2001-03-06 | Adelaide Medical Centre For Women And Children | DNA sequences related to fragile X syndrome |
US6242576B1 (en) | 1991-01-04 | 2001-06-05 | Women's And Children's Hospital | Monoclonal and polyclonal antibodies relating to fragile X syndrome |
US5741645A (en) * | 1993-06-29 | 1998-04-21 | Regents Of The University Of Minnesota | Gene sequence for spinocerebellar ataxia type 1 and method for diagnosis |
US5834183A (en) * | 1993-06-29 | 1998-11-10 | Regents Of The University Of Minnesota | Gene sequence for spinocerebellar ataxia type 1 and method for diagnosis |
US5876949A (en) * | 1995-05-31 | 1999-03-02 | The Trustees Of The University Of Pennsylvania | Antibodies specific for fragile X related proteins and method of using the same |
US8409805B2 (en) | 2009-02-13 | 2013-04-02 | Asuragen, Inc. | Method of amplification of GC-rich DNA templates |
US8679757B2 (en) | 2009-03-24 | 2014-03-25 | Asuragen, Inc. | PCR methods for characterizing the 5′ untranslated region of the FMR1 and FMR2 genes |
US9783848B2 (en) | 2009-03-24 | 2017-10-10 | Asuragen, Inc. | PCR methods for characterizing the 5′ untranslated region of the FMR1 and FMR2 genes |
US9371560B2 (en) | 2012-07-20 | 2016-06-21 | Asuragen, Inc. | Comprehensive FMR1 genotyping |
US11497816B2 (en) * | 2015-10-06 | 2022-11-15 | The Children's Hospital Of Philadelphia | Compositions and methods for treating fragile X syndrome and related syndromes |
CN110923305A (en) * | 2019-11-25 | 2020-03-27 | 广州市达瑞生物技术股份有限公司 | DNA molecular weight standard suitable for fragile X syndrome southern blot hybridization detection |
CN110923305B (en) * | 2019-11-25 | 2023-12-29 | 广州市达瑞生物技术股份有限公司 | DNA molecular weight standard suitable for southern blot hybridization detection of fragile X syndrome |
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