AU671418B2 - Nucleic acid fragment of the X chromosome region involved inthe fragile X syndrome, nucleotide probe and method of diagnosing mental retardation with fragile X - Google Patents

Abstract

A new nucleic acid fragment (I). of at least 156p or nucleotides, is derived from the Xq27-q28 region of the X chromosome. It is contained within the approx. 225 kb DNA segment defined between probes St677 and Do33. Also new are (1) DNA or RNA fragments (Ia) which hybridise selectively with (I); (2) cDNA (Ib) corresponding to (I); (3) probes (II) which hybridise selectively with any of (I),(Ia) and (Ib); (4) fragments of mRNA able to hybridise to (II); (5) recombinant DNA (Ic) contg. at least one fragment (I),(Ia) or (Ib) plus transcription/translation control elements; (6) cloning and expression vectors contg. (Ic); (7) host cell, transformed with these vectors; (8) proteins (III) produced from (Ic) and (9) antibodies (Ab) raised against (III). More specifically (I) is adjacent to, or contains, CpG sites in the region of 20 kb around the 125 kb position as defined on a genetic map.

Description

OPT DATE 15/09/92 AOJP DATE 29/10/92 APPLN. I D 14136 q2 PCT NUMBER PCT/FR92/l0i45

PCIP

DEMANDE INTERNATIONALE PUBLIEF EN VERTU DU TRAITE DE COOPERATION EN MATIERE DE BREVETS (PCT) (51) Classification internationale des brevets 5 Num~ro de publication internationale WO 92/14840 C12l /,C2N 1/215/7 A (43) Date de publication Internationale: 3 septembre 1992 (03.09.92) (21) Numero de In demande internationale: PGT/FR92/00145 (72) Inventeur: OBERLE, Isabelle (d~c~d6).

(22) Date de dep6t international: 13 f~vrier 1992 (13.02.92) (74) Mandataires: GUTMANN, Ernest etc. ;Ernest Gutmann Yves Piasseraud 67, boulevard Haussmann, F- Donn~es relatives fiIna priorit6: 75008 Paris (FR).

91/01684 13 l'~vrier 1991 (13.02.9 1) FR (81) Etats designes: AT (brevet europ~en), AU, BB, BE (brevet Brevet ou demande principal(e) europ~en), BF (brevet OAP1), BG, BJ (brevet OAP1), (63) Apparent6(e) par continuation BR, CA, CF (brevet OAPI), CG (brevet QAPI), CH us 815,932 (C IP) (brevet europ~en), CI (brevet QAPI), CM (brevet QAPI), D36pos~e le 30 d~cembre 1991 (30,12.9 1) CS, DE (brevet europ~en), DK (brevet europ~en), ES (brevet europ~en), Fl, FR (brevet europ~en), GA (brevet (71) D36posant (US seuletnent): H ElDET, St~phane (h~ritier de OAPI), GB (brevet europ~en), GN (brevet GAPI), GR l'inventeur d~#y~d6) [FR/FR]; 5, rue de la Souffel, F- (brevet europ~en), HU, IT (brevet europ~en), JP, KP, 67450 Lampertheim KR, LK, LU (brevet europ~en), MC (brevet europ~en), MG, ML (brevet OAPI), MN, MR (brevet QAPI), MW, (71) D36posant (pour tolls les Etats dksigti~s saif US): I NSTITUT NL (brevet europ~en), NO, PL, RO, RU, SD, SE (brevet NATIONAL DE LA SANTE ET DE LA RE- europ~en), SN (brevet OAPI), TD (brevet 'OAPI), TG CHERCHE MEDICALE [FR/FR]; 101, ru, de Tolbiac, (brevet GAPI), US.

F-75654 Paris C~dex 13 (FR).

Publice (72) Inventeurs; et Avec rapport de rcheILrch~e internationale.

Inventeurs/Deposants (US seuilenent) MANDEL, Jean- Avant l'expiration du dtlai pr~vu pour la mnodification des Louis [FR/FR]; 9, rue du Barrage, F-67300 Schiltigheim revetldications, sera republieesi de relles modifications sont ROUSSEAU, Franqois [Cix/CA]; 1289 Jean-Des- re~ucs.

quen, Sainte Foy, Quebec GIW 3H5 VINCENT, Anne [FR/FR]; 113, chemin de L. Pomnme, F-69160 Tassin la Demi Lune HEITZ, Dominique [FR/FR]; 46, rue de ]a Garenne, F-67000 Saverne (FR).7 671,1418 (54)Title: NUCLEIC ACID FRAGMENT OF THE X CHROMOSOME REGION INVOLVED IN THE FRAGILE X SYN- DROME, NUCLEOTIDE PROBE AND METHOD OF DIAGNOSING MENTAL RETARDATION WITH FRAGILE X (54)Titre: FRAGMENT D'ACIDE NUCLEIQUE DE LA REGION DU CHROMOSOME X IMPLIQUE DANS LE SYN- DROME X FRAGILE, SONDE NUCLEOTIDIQUE ET PROCEDE POUR LE DIAGNOSTIC DU RETARD MENTAL AVEC X FRAGILE (57) Abstract Nucleic acid fragment containing mutations associated with the fragile X syndrome, or other DNA anomalies resulting from these mutations such as methylations of CpG sites. Nucleotide probe for detecting these mutations and DNA anomalies associated with mental retardation with fragile X and method for diagnosing mental retardation with fragile X.

(57) Abr~g2 Fragment d'acide nucl~ique contenant les mutations associ~es au syndrome X fragile, ou d'autres anomalies de l'ADN cons~quentes A ces mutations telles que des m~thylations des sites A CpG. Sonde nucl~otidique pour la detection de ces mutations et anomalies de I'ADN Iikes au retard mental avec X fragile et proc6d6 pour le diagnostic du retard mental avec X fragile.

I

NUCLEIC ACID FRAGMENT OF THE REGION OF THE X CHROMOSOME INVOLVED IN THE FRAGILE X SYNDROME, NUCLEOTIDE PROBE AND PROCESS FOR THE DIAGNOSIS OF MENTAL RETARDATION WITH FRAGILE X.

The syndrome of mental deficiency with fragile X is a disease linked to the X chromosome which affects about one out of one thousand five hundred boys. The women carriers, about one in seven hundred and fifty, have a 30% risk of exhibiting a slight or even moderate debility. The syndrome of mental deficiency with fragile X represents the most frequent cause of hereditary mental deficiency.

The diagnosis of the disease is established by means of a delicate cytogenetic analysis which requires special conditions of culture and the analysis of 50 to 100 mitoses since the fragile site which can be detected cytogenetically is present in only 5 to 50% of the mitoses. The diagnosis of the women carriers is still more difficult since only 50 to 60% of these women express the fragile site in a usually low proportion of mitoses.

The difficulty of the cytogenetic test means that it is performed in a reliable manner in only a limited number of laboratories; furthermore, the diagnosis of the disease is often only made after the birth of a second affected child in the same family; now in fact the early establishment of such a diagnosis is fundamental for the implementation of investigations which make possible the prenatal diagnosis and screening of the women carriers who transmit the desease to 50% of their male children in the families at risk. The high frequency of the disease and the early genetic importance justify a systematic neonatal screening for the disease.

The present invention relates specifically to the provision of novel means of investigation based on the direct study of the DNA and makes possible a rapid diagnosis of the disease which is applicable to a large number of patients.

The syndrome of mental deficiency with fragile X is characterized by the presence of a fragile site in the Xq27.3 region, induced in vitro by folic acid deprivation or by inhibitors of the synthesis of deoxynucleotides. The genetic mapping of the Xq27-q28

'CC

region had made it possible to confirm that the locus of the disease is localized at or adjacent to the fragile site (Oberle et al., Proc.

Natl. Acad. Sci. (1986); Suthers et al., Science (1989); Rousseau et al., J. Hum. Genet. (1991)).

Moreover, it appears that the hereditary transmission of the disease is unusual in comparison with other diseases linked to the X chromosome. It has been observed that phenotypically normal men transmit the mutation to their daughters who rarely exhibit the symptoms of the disease; on the other hand, the disease appears with high frequency in the sons and daughters of these women (Sherman et al., Hum. Genet. (1985)). Several hypotheses have been put forward to explain this phenomenon.

The genetic markers proposed in the prior art can only serve to monitor the transmission of the mutation to the families in which the disease has already been diagnosed by means of a cytogenetic study, it is an indirect diagnosis by means of a linkage study; it is usually necessary to use several markers on both sides of the locus of the disease and such markers are only informative in variable proportions of families.

The international patent application W090/05194 describes a new locus entitled 1AI distant from the fragile X site but the restriction polymorphism of which makes it available as a genotypic marker. The probes capable of detecting the lAl locus make possibla the diagnosis of several diseases linked to the X chromosome and in particular, the syndrome of the fragile X by selective hybridization in the Xq27. DXS52 region of the said X chromosome.

Other probes close to the fragile X locus have also been described, in particular by the inventors. Thus F. Rousseau et al (Am.

J. Hum. Genet. (1991) 48:108-116) report the detection of seven regions close to the fragile X locus (FRAXA). Two of these regions included between DXS369 and DXS296 contain the genetic markers closest to FRAXA known hitherto, one of these regions contains two probes detecting a polymorphism and both of these probes, designated St677 (DX463) and 2-34 (DXS477) detect the polymorphic Taql restriction fragments; another probe also detecting a polymorphism and designated Do33 (DXS465: 42% 3 heterozygocity) is localized in the same region as DXS296, proximal to the point of cleavage of the chromosome corresponding to the locus of the Hunter syndrome (IDS). The use of these probes has made it possible to determine the physical arrangement of the regions just mentioned: Cen-DXS369-DXS476-(DXS453, DXS477)-(DXS296, DXS465)-IDS- DXS304-tel, and their position with respect to the fragile X locus.

The analysis of the fragile X locus with the previouslymentioned probes and, more particularly, with the probe Do33 (DXS465) by means of pulsed field electrophoresis has led the inventors to observe that the patients with fragile X and mental deficiency differ from normal subjects or from subjects who are bearers of the fragile X mutation but who show no clinical expression of the disease by a partial or total inhibition of the digestion of restriction sites close to the probe Do33, and in particular sites for the enzymes BssHII, SacII, EagI.

The inventors have cloned in a yeast artificial chromosome (YAC) the DNA segment of about 225 kb defined between the probes St677 and Do33, and situated in the Xq27-q28 region of the human X chromosome.

Probes derived from this segment were prepared for the analysis of mutations, of methylations of the DNA and of the genes implicated in the fragile X syndrome.

Starting from the DNA of leucocytes, these probes have made possible in particular the study of the digestibility of the restriction sites BssHII, EagI, SacII and in a general manner of restriction sites, the digestion of which by the corresponding enzyme is inhibited when the site contains a methylated cytosine; these sites are designated CpG sites, for example they are sites containing the dinucleotide CpG such as HpaII, CCGG or HhaI, GCGC, or sites likely to partially conceal a CpG dinucleotide such as Avail GG(A or T)CC, in a sequence CGG (A or T)CC or GG(A or T)CGG or also BanI (GG Py Pu CC); the inventors have detected a surprising correlation between the clinical expression of the disease and the inhibition of the digestibility of the CpG sites in the region of about 20 kb surrounding the position 125 kb defined on the DNA segment cloned by the inventors, at about 100 kb from the Do33 probe in the direction of the centromere.

ri 4 Figure 1 is a schematic representation of the region of the X chromosome around the position 125 kb located at about 100 kb from the probe Do33 in the direction of the centromere. On the scale, the locations of the probes St677, 2-34 and Do33 are shown. In alignment with the scale the YAC clones 141 H5 and 209G4 are shown, corresponding to the above-mentioned DNA segments, and the locations of the restriction sites EagI, BssII have been shown on the line representing the 141 F5 clone. The mention CpG on the line representing the clone 141 H5 corresponds to the region in which the methylation is abnormal in the patients suffering from mental deficiency associated with fragile X.

This phenomenon of inhibition of digestibility of the CpG sites can be detected by the Southern Blot method, by using probes derived from the 20 kb region surrounding the position 125 kb defined in figure 1, probes close to or containing the CpG sites. The inventors have also detected by the same method anomalies in the migration of the DNA fragments, not necessarily linked to the phenomenon of methylation, anomalies which are characteristic of the subjects bearing i a fragile X mutation. These anomalies have been detected in particular in the DNA digested by the enzymes EcoRI or HindIII after hybridization with a probe specific of the above-mentioned region.

It appears that the germinal mutations responsible for the fragile X syndrome and which are likely to be transmitted to the progeny are situated 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 not inevitable consequence of these mutations, a consequence which is linked to the clinical expression of the disease.

Consequently, the invention relates to any nucleic acid fragment derived from the Xq27-q28 region of the X chromosome, adjacent to or containing the CpG sites situated in the region of 10 to 20 kb surrounding the 125 kb position.

In a general manner, the invention relates to any nucleic acid fragment included in the DNA segment defined between the probes St677 and Do33.

This region of the X chromosome is implicated in the fragile i I- X syndrome and the probes specific for this region are advantageously used to detect the presence of methylation anomalies linked to the clinical expression of the disease, or to detect the presence of mutations responsible for the fragile X syndrome and its transmission, or also to localize, identify and analyse the expression of the gene(s) implicated in the fragile X syndrome. The probes can usually be used to detect the anomalies in the DNA which are the direct or indirect consequence of the mutations associated with the fragile X syndrome.

Hence, the invention relates to any nucleotide probe capable of hybridizing selectively with a nucleic acid fragment included in the segment of DNA defined between the probes St677 and Do33 and more particularly with a nucleic acid fragment adjacent to or containing the CpG sites in the region of about 20 kb surrounding the 125 kb position.

The probes of the invention hybridize specifically under appropriate conditions of stringency with the above-mentioned nucleic acid fragments but do not hybridize with other regions of the human genome under the same conditions of hybridization.

Such a hybridization comprises the following steps: the pre-hybridization treatment of a support, such as a nitrocellulose filter or a nylon membrane on which is bound the DNA to be tested and capable of hybridizing with the above-mentioned probes, at F temperature of 42 0 C and for 2 hours with a pre-hybridization solution of the following composition: 40% formamide; 0.9 M NaCl; 200 pg/ml of salmon sperm DNA; 50 m of sodium phosphate, pH 6.5; 4% of dextran sulfate; 0.08% (weight/volume) of Ficoll and polyvinyl pyrrolidone, the hybridization in a solution identical with the pre-hybridization solution containing in addition at 1 ast one, advantageously labelled nucleotide probe in conformity with the invention at a temperature of 42 0 C and for 18 hours, -4 successive washings, the revelation of the hybrid possibly formed by any method appropriate to the label borne by the probe.

The hybridization conditions defined above constitute preferred conditions, but are in no way limiting and may be modified without I r affecting the pairing properties of the probes of the invention and the above-mentioned nucleotide sequences.

The probes of the invention are advantageously labelled by any label classically used. They may be labelled with the aid of a radioactive tracer such as 3 2 p, 3 5 S, 125, 3 H, 14C and the radioactive labelling may be performed by any method known to the person skilled in the art.

The probes may be labelled at the 3' end by the addition of one or more deoxynucleotides or ribonucleotides or by a dideoxynucleotide label]ed in the alpha position by means of 32 P, in the presence of the terminal deoxynucleotidyl transferase, or at the end by transfer of a radioactive phosphate group of a free deoxynucleotide or dideoxynucleotide labelled in the gamma position in the presence of the T4 DNA Jigase. The probes may also be labelled by using a DNA polymerase by means of "nick translation" or "random priming" or "polymerase chain reaction".

The method of detection of the hybridization will depend on the radioactive label used and may be based on autoradiography, liquid scintillation, gamma counting or any other technique making possible the detection of the radiation emitted by the radioactive label.

Non-radioactive labelling may also be used by combining with the probes groups exhibiting imnunological properties such as an antigen, a specific affinity for certain reagents such as a ligand, physical properties such as fluorescence or luminescence, properties making possible the completion of enzymatic reactions such as an enzyme or an enzyme substrate. The non-radioactive labelling may also be performed directly by chemical modification of the DNA, such as photobiotinylation or sulfonation.

The probes of the invention are used for the in vitro detection of the mutations responsible for the fragile X syndrome as well as methylation anomalies of the CpG sites or other anomalies in the DNA consistent with the presence of these mutations and linked to the syndrome.

The aim of the detection of a mutation is to monitor the I 1 familial transmission and make possible, in particular, a diagnosis of unaffected persons capable of transmitting the disease, such as women carriers or normal men who transmit the mutation.

The detection of the methylation anomalies makes possible a diagnosis of the disease, expressed clinically or capable of being so in the context of an antenatal diagnosis or a diagnosis at birth.

In a general manner, the procedures for the detection of the mutations responsible for the fragile X syndrome as well as anomalies of methylation of the CpG sites, or other anomalies of the DNA associated with the syndrome are based on the use of at least one probe conforming to the invention and of at least one suitable restriction enzyme in a manner such that the fragment detected by the probe is different depending on whether it contains or not the mutation or methylation anomaly or any other anomaly of the DNA.

Such a procedure may be performed according to the method called "Southern Blot" (Sambrook, Fritsh and Maniatis; Molecular cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press), comprising the following steps: treatment of the genomic DNA extracted, for example, from leucocytes or chorial villosities or from any other source of genomic DNA, by at least one specific restriction enzyme under conditions leading to the production of restriction fragments as a consequence of the cleavage of the DNA at the restriction sites recognized by the said enzyme; separation of the fragments by means of electrophoresis; the placing in contact of a nucleic acid probe according to the invention which is capable of hybridizing specifically with the fragments thus produced under conditions leading to the formation of hybridization complexes; detection of the hybridization complexes; measurement of the size of the restriction fragments forming part of the hybridization complexes.

The investigation of methylation anomalies for the in vitro diagnosis of the fragile X syndrome in patients suffering from mental deficiency is advantageously performed according to the previous 8 procedure making use of at least one restriction enzyme at site sensitive to methylation with at least one, advantageously labelled probe conforming to the invention.

The above-mentioned procedure may be implemented with a single enzyme specific for a restriction site sensitive to methylation such as BssHII, EagI, Avail, BanI; or also with two enzymes, one specific for a restriction site sensitive to methylation and the other specific for a restriction site insensitive to methylation. The probes of the invention used for the hybridization with the fragments obtained are advantageously adjacent to or contain the site for the enzyme specific for a site sensitive to methylation.

*The method of Southern Blot is also used to detect the mutations associated with the fragile X syndrome, such as deletions or insertions of a DNA sequence, or other anomalies of the DNA such as anomalies of conformation or replication, which are a consequence of these mutations.

The detection of these mutations for the in vitro diagnosis of the fragile X syndrome in patients suffering from mental deficiency or not is performed according to the method of Southern Blot by subjecting the genomic DNA to digestion by means of any appropriate restriction enzyme not necessarily sensitive to methylation such as EcoRI or HindIII, The restriction fragments are detected by hybridization with an advantageously labelled probe of the invention and the abnormal electrophoretic migration of the said fragments is 3 25 correlated either with the clinical expression of the disease or with the presence of the mutation even in the absence of the clinical expression of the disease.

The invention also relates to kits for the implementation of the aforementioned procedure.

Among the nucleic acid fragments of the invention, those with 20 to 30 nucleotides may be advantageously used as primers in a genetic amplification procedure of a nucleic acid fragment of the kb region surrounding the 125 position and, more particularly, of a fragment containing the CpG sites. This amplification procedure consists of binding specifically by hybridization on the genomic DNA 9 extracted, for example, from leucocytes two oligonucleotides carefully chosen from those containing the nucleic acid fragments of 20 to nucleotides of the invention which are adjacent to the fragment to be amplified, then of implementing an enzymatic extension process with the aid of DNA polymerase, followed by a denaturation process, and of repeating the hybridization-extension-denaturation cycle known by the name of the PCR ("Polymerase Chain Reaction") cycle a number of times suficient to increase the amount of the fragment to be amplified in a proportion which is exponential with respect to the number of cycles performed.

The amplification method described may be applied to the detection of anomalies of the DNA which is the consequence of the presence of the above-mentioned mutations.

The in vitro diagnosis of the fragile X syndrome, in particular in patients suffering from mental deficiency, is advantageously performed by means of a genetic amplification carried out in parallel on the DNA digested by the restriction enzyme sensitive to methylation, and on the non-digested DNA. The analysis for diagnostic' purposes is made by comparing the amplification product of the nondigested DNA with that of the DNA digested by the selected restriction enzyme. This analysis of the amplification products may be performed by visualization of the fluorescence induced by ethidium bromide after electrophoresis on agarose or polyacrylamide gel, or by detection of the radioactive or non-radioactive label, such as by means of a biotinavidin system, of the oligonucleotide primers or nucleotides incorporated into the amplified fragment. The number of amplification cycles is limited so as to preserve a linearity of the amplification and hence a quantitative estimation of the digestibility of the site of the enzyme sensitive to methylation. Advantageously, internal labels are used, in particular other fragments of the genome co-amplified and also containing a site for the enzyme sensitive to methylation, not methylated in man as a control of the action of the restriction enzyme, and as control of the efficiency of amplification a fragment not containing such a site. The quantitative measurement of the methylation of the genomic DNA at the site under study may be done by adaptation of the amplification method previously described, in particular by using in addition to the oligonucleotides of the invention, oligonucleotides called "linkers" and DNA ligase, a method designated as "ligation mediated PCR" described by Steigerwald et al.

(Nucleic Acids Res. (1989) 18:1435-1439).

The invention also relates to kits for the implementation of the aforementioned procedure. Such kits contain: labelled or unlabelled oligonucleotides for the amplification of nucleic acid fragments containing a site sensitive to methylation according to the invention; labelled or unlabelled oligonucleotides for the amplification of the control nucleic acid segments; oligonucleotides called "linkers"; buffers, nucleotides and enzymes required for the amplification.

The deletions or any other rearrangement capable of generating mutations responsible for the fragile X synd:romemay also be detected by the DNA amplification method. The method is basod on the use of oligonucleotide couples which may be mixed in a single reaction according to the amplification method called multiplex which makes it possible to amplify several fragments of the region surrounding the 125 kb position and to detect the disappearance or modification of some of these fragments. The detection of a point mutation is advantageously carried out by amplification of the DNA segments, followed by an analysis by the methods of SSCP (single strand conformation polymorphism) or DGGE (denaturing gradient gel electrophoresis), or also by the detection of heteroduplexes.

The probes of the invention correspond to the region in which are located the gene(s) implicated in the clinical expression o£ the fragile X syndrome, in fact, 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 pf the corresponding messenger RNAs, or for the construction of expression vectors for the corresponding proteins with a view to their therapeutic application or for the 4& ,11 production of specific antibodies which are also capable of being used in diagnosis.

The person skilled in the art has at his disposal means which make it possible to identify, indeed even to select, those of the nucleic acid fragments corresponding to the genes present in the region between the probes St677 and Do33 on either side of the 125 kb position, these genes are capable of being directly inactivated by mutation or indirectly as a result of the methylation of th& CpG sites present around the 125 kb position.

Therefore, the invention relates to any recombinant DNA containing a nucleic acid fragment of the invention combined with any sequence which makes possible the transcription and/or the translation in the host cell in which the said recombinant DNA is likely to be introduced. Consequently, the invention 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 performed with the aid of a vector, in particular of the plasmid type, capable of being replicated in a cell host and of giving rise there to the transcription of the corresponding RNA and to the expression of the proteins encoded by the nucleic acid fragment contained in the recombinant DNA.

The host cells mentioned above may be prokaryotic cells, in particular E.coli cells or, more advantageously, eukaryotic cells, which makes it possible to produce mature and glycosylated proteins (yeasts, CHO cells or insect cells infected by the baculovirus). These proteins can advantageously be used for the preparation of specific antibodies useful for the diagnosis of the fragile X syndrome and in therapeutic applications.

In addition to the preceding characteristics, the invention includes other characteristics which will become apparent in the course of the description which follows and which refers to examples made use of i.l the present invention and which are illustrated by the figure, it being understood that these examples in no way constitute any kind of limitation to the scope of the invention.

I 12 I Preparation of the probes St677 and Do33 DNA segments corresponding to the probes St677 and Do33 may be obtained starting from human DNA by means of genetic amplification by using the following oligonucleotides: in the case of ST677 5'ACTCCTTCACAAATCTTCCG3' and 5'ATCATGCAAGACAGTTCTCTGC3' in the case of Do33 5'GTGAAGCAATTTGAGTTGGTGGGAG3' and 5'CCCAAGTTTAGCTTCCGTAGAAGCC3' The amplification conditions in a standard medium are the following: hybridization 1 minute at 60 0

C,

elongation 8 minutes at 72 0

C,

denaturation 1 minute at 94°C II Preparation of the probes of the invention The probes of the invention were obtained by subcloning the nucleic acid fragments of the invention derived from digestion of DNA segments contained in the region of about 225 kb defined between the probes St677 and Do33, and localized in the Xq27-q28 region of the human X chromosome, cloned in a yeast artificial chromosome (YAC) and shown in figure 1.

In particular, the subcloning is designed to produce specific probes of fragments situated in the 20 kb region surrounding the 125 kb position and hence corresponding specifically to the nucleic acid fragments adjacent to or containing the CpG sites. As an example, such a probe is obtained by subcloning fragments prepared by double digestion using EagI and BglI digestion of the 141H5 clone. The 9B12.3 probe obtained corresponds to a EagI-BglI fragment of about 7 kb containing the EagI site at the 125 kb position on the map of figure 1.

Figure 2 is a schematic representation of the map of the probe 9B12 and of its sub-fragments 9B12.1, 9B12.2, 9B12.3, 9B12.4 and 9B12.5, in the opposite orientation to that of figure 1. This figure contains the following 7 lines: line is a scale in kb on which is shown at 0 the 125 kb position defined on the map of the segment cloned in figure 1 between the probes St677 and Do33 at about 100 to 125 kb from the probe Do33.

line shows, in alignment with the scale, the probe 9B12 and its sub-fragments 9B12.1, 9B12.2, 9B12.3, 9B12.4 and 9B12.5.

line shows, in alignment with the scale, the localization of the BanI sites (GGPyPuCC) and in particular the polymorphic BanI site indicated by RFLP and the BanI site sensitive to methylation and indicated by Me.

line shows, in alignment with the scale, the localization of the XmnI sites (GAANNNNTTC).

line shows, in alignment with the scale of line the localization of the BglII sites (AGATCT).

line shows, in alignment with the scale of line the localization of the EcoRI sites (GAATCC).

line shows, in alignment with the scale of line the localization of the HindIII sites (AAGCT).

The maps of the BanI, XmnI and EcoRI sites correspond to the modified fragments in the carriers of a fragile X mutation.

III Diagnosis of fragile X mental deficiency by means of detection of methylation anomalies according to the method of Southern Blot The genomic DNA extracted from leucocytes of a patient suffering from mental deficiency is cut by the enzymes AvaII, BanI

I

14 or XmnI. The DNA fragments obtained are analysed by electrophoresis on a 0.9% agarose gel in TAE buffer, then transferred to a nylon membrane.

The membrane on which the DNA fragments are bound is subjected to a pre-hybridization treatment at 42 0 C for 2 hours with a solution of the following composition: 40% formamide; 0.9 M NaCI; 200 pg/ml of salmon sperm DNA; 50 mM of sodium phosphate pH 6.5; 4% of dextran sulfate; 0.8% (weight/volume) of Ficoll and polyvinyl pyrrolyidone; then the pre-hybridization solution is replaced by a hybridization solution identical with the pre-hybridization solution and containing in addition the probe 9B12.3 labelled in the alpha position of dCTP with3 P of specific activity 2.10 dpm/pg; after incubation for 18 hours at 42°C and successive washings with a 0.5 SSC solution at 65 0

C,

autoradiography is performed at -80 0

C.

All or part of the Avail, BanI or XmnI fragments detected in the normal subjects is replaced in the subjects expressing the fragile X syndrome by larger fragments. In particular with the enzyme XmnI, the appearance of several bands quite close to each other is very characteristic of the subjects exhibiting the phenotypic expression of the syndrome.

This distinction between anomalies linked to the clinical and/or cytogenetic expression and anomalies present in persons who are carriers of the fragile X mutation without clinical and/or cytogenetic expression is explained in the following examples in which the said anomalies are detected by the method of Southern Blot.

1) Anomalies linked to clinical and/or cytogenetic expression a) Digestion by the BanI enzyme (GG Py Pu CC site) Figure 3 is a schematic representation of an autoradiograph on a 0.9% agarose gel of DNA from leucocytes or chorial villosities of normal men (1 and a man with fragile X a woman carrier and a normal woman, digested by BanI and revealed by the probe 9B12.3.

Figure 4 is a schematic representation of an autoradiograph on a 0.9% agarose gel of DNA from chorial villosities of a normal man a man with fragile X a foetus with fragile X, digested by BanI and revealed by the probe 9B12.3.

The probe 9B12.3 detects in the genomic DNA of a normal man, after digestion by the enzyme BanI, either a single fragment of 2.9 kb (allele 1) or two fragments of 2.3 and 0.6 kb (allele The existence of two alleles is due to the presence of a polymorphic BanI site indicated by RFLP on figure 2. In a male patient suffering from mental deficiency with fragile X and carrier of the allele 1, the 2.9 kb fragment is replaced more or less completely by a fragment of kb. In a patient carrier of the allele 2, the fragment of 0.6 kb is replaced more or less completely by a fragment of 1.2 kb. These specific differences in the case of mental deficiency with fragile X in the male are due to the methylation of a BanI site, indicated by Me on figure 2. This site is included in the following sequence: TCGGTGCCGA, compatible with inhibition as a result of methylation of cytosine.

The anomaly was detected not only in leucocyte DNA, but also in DNA of chorial villosities of foetuses who are carriers of a fragile X mutation. It thus can be used in post-natal or ante-natal diagnosis in males.

The.fragments of 3.5 kb and 1.2 kb can, on the other hand, be detected in normal women (methylation of the BanI site on the inactive X chromosome). However, their intensity appears more strongly in women carriers with clinical or cytogenetic expression of the fragile X syndrome.

b Digestion with the other enzyme XmnI (GAANNNNTTC site) Figure 5 is a schematic representation of an autoradiograph i on a 0.9% agarose gel of leucocyte DNA of normal men (1 and 10), men with mental deficiency linked to fragile X 4, 6, 9) women carriers 7, 8) and a normal woman digested by XmnI and revealed by the 9B12.3 probe.

The probe 9B12.3 detects a fragment of 9 kb in men and women _1 16 as well as in carriers of the fragile X mutation who show no clinical or cytogenetic expression of the disease. In men suffering from mental deficiency with fragile X, the 9 kb fragment is replaced wholly or in part by fragments of apparent size greater than 10 kb. The appearance of such fragments is also characteristic of women carriers with clinical and/or cytogenetic expression.

2) Anomalies present in persons carrying a fragile X mutation without clinical and/or cytogenetic expression Figure 6 is a schematic representation of an autoradiograph on a 0.9% agarose gel of leucocyte DNA of normal women 12, 13) and women carriers (1 to 8, 10 and 11) of the fragile X mutation not expressing all of the clinical or cytogenetic signs, digested by EcoRI and revealed by the probe 9B12.3 The probe 9B12.3 detects a EcoRI fragment of about 6 kb in a normal man or a normal woman. On the other hand, in a woman carrier of a fragile X mutation, an additional larger fragment of variable size depending on the individuals (6 to 7 kb) is present. This anomaly appears in women carriers without clinical and/or cytogenetic expression, and may thus be used for the diagnosis of women carriers.

In affected men, the normal fragment is replaced by a fragment or a series of fragments of larger apparent size. Very similar results are obtained with the enzyme HindIII.

IV Diagnosis of mental deficiency with fragile X by means of genetic amplification of the fragment containing a methylation anomaly.

The genomic DNA extracted from leucocytes of a patient suffering from mental deficiency is subjected to digestion by an enzyme specific for a site sensitive to methylation such as AvaII, BanI or XmnI and the amplification is carried out in parallel on the genomic DNA digested by the restriction enzyme sensitive to methylation and on undigested DNA.

17 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 with the undigested DNA. Then an enzymatic extension process with the aid of DNA polymerase, followed by a denaturation process is carried out; the hybridization-extension-denaturation cycle is repeated a sufficient number of times in order to increase the amount of the fragment containing the CpG sites in an exponential proportion with respect to the number of cycles performed.

The number of amplification cycles is limited so as to preserve a linearity of amplification and hence a quantitative estimation of the digestibility of the restriction enzyme site sensitive to methylation. Advantageously, internal markers are used, in particular other fragments of the genome containing an enzyme site sensitive to methylation, not methylated in man, or fragments not containing such a site are co-amplified.

The analysis of the amplification products is carried out by visualization of the fluorescence induced by ethidium bromide, after electrophoresis on an agarose or polyacrylamide gel. If the site sensitive to methylation is methylated, the amplified fragment is present in the two reactions, if the site sensitive to methylation is not methylated, the amplified fragment is present only in the reaction with the undigested DNA.

Claims (13)

1. Nucleic acid derived from the Xq27-q28 region of the X chromosome, characterized in that it is included in the DNA segment of about 225 kb defined between the probes St677 and Do33.

2. Nucleic acid fragment according to Claim i, characterized in that it is adjacent to or contains the CpG sites situated in the 20 kb region surrounding the 125 kb position.

3. Nucleotide probe to detect in vitro mutations responsible for the fragile X syndrome, or other anomalies consistent with the presence of the said mutations or to localize the gene(s) implicated in the fragile X syndrome, characterized in that it is capable of hybridizing selectively with a nucleic acid fragment according to one of the Claims 1 or 2.

4. Nucleotide probe according to Claim 3, characterized in that it is labelled radioactively or by, in particular, an enzymatic, ligand, luminescent or fluorescent label.

Procedure for the in vitro detection of the mutations associated with the fragile X syndrome or other anomalies of the DNA consistent with the presence of these mutations, characterized in that it comprises the following steps: the treatment of the genomic DNA with at least one restriction enzyme specific for a site sensitive or not to methylation; 3 the separation of the fragments by electrophoresis and their hybridization with at least one probe according to Claim 4; the visualization of the hybrids formed and the comparison with a control. .7

6. Procedure for the detection of anomalies of the DNA according to Claim 5 for the in vitro diagnosis of the fragile X syndrome, characterized in that it comprises the following steps: the treatment of the genomic DNA by at least one restriction enzyme specific for a site sensitive to methylation; the separation of the fragments by electrophoresis and their hybridization with at least one probe specific for a nucleic acid fragment adjacent to or containing the CpG sites situated in the 19 kb region surrounding the 125 kb position; the visualization of the hybrids formed and comparison with a control.

7. Procedure for the in vitro diagnosis of the fragile X syndrome according to Claim 6, characterized in that the digestion of the genomic DNA is carried out by a restriction enzyme specific for a site sensitive to methylation and by a restriction enzyme specific for a site not sensitive to methylation.

8. Genetic amplification procedure of a nucleic acid fragment containing mutations or other anomalies consistent with these mutations, associated wi.th the fragile X syndrome, characterized in that it consists of binding by hybridization to the genomic DNA two labelled oligonucleotides flanking the said fragment, and judiciously selected from among the nucleic acid fragments of 20 to 30 nucleotides according to Claim 1 or 2, then of implementing an enzymatic extension process with the aid of DNA polymerase, followed by a denaturation process, and of repeating the hybridization-extension-denaturation cycle a number of times sufficient to increase the amount of the fragment in an exponential proportion with respect to the number of cycles performed.

9. Procedure for the in vitro diagnosis of the fragile X syndrome, characterized in that a genetic amplification procedure according to Claim 8 is performed in parallel on the one hand on the DNA subjected to digestion by an enzyme specific for a site sensitive to methylation and, on the other, to the DNA not digested by the said enzyme, then in that the fragments obtained are compared by visualization with ethidium bromide or detection as a consequence of the primer oligonucleotides being labelled.

Procedure for detection of a mutation associated with the fragile X syndrome, characterized in that a genetic amplification according to Claim 8 of one or several nucleic acid fragments of the 20 kb region surrounding the 125 kb position is carried out and in that the disappearance or modification of some of these fragments is detected.

11. Recombinant'DNA containing at least one nucleic acid fragment according to any one of the Claims 1 and 2, associated with a sequence which makes possible its trarcription and/or translation in a host cell in which the said recombinant DNA is likely to be introduced.

12. Cloning and expression vector characterized in that it contains a recombinant DNA according to Claim 11.

13. Cell host characterizeJ in that it is transformed by a recombinant actor according to Claim 12. ABSTRACT NUCLEIC ACID FRAGMENT FROM THE REGION OF THE X CHROMOSOME IMPLICATED IN THE FRAGILE X SYNDROME, A NUCLEOTIDE PROBE AND PROCEDURE FOR THE DIAGNOSIS OF MENTAl DEFICIENCY WITH FRAGILE X. Nucleic acid fragment containing the mutations associated with the fragile X syndrome, or other anomalies of the DNA consistent wi;h these mutations such as methylations of the CpG sites. A nucleotide probe for the detection of these mutations and anomalies of the DNA linked to mental deficiency with fragile X and a procedure for the diagnosis of mental retardation with fragile X. 1