The present invention relates to nucleic acid fragments and their use in the diagnosis of fragile X syndrome.

The fragile X (Martin-Bell) syndrome (McKusick cat no: 30955) is characterised by mental retardation, and physical anomalies including large ears, oblong face and macro- orchidism in affected males. When lymphocytes derived from patients are cultured in thymidine deficient media, 2 to 50% of the cells express a fragile site at Xq27.3. Recent prevalence figures of 0.6 to 1/1000 among males and 0.2 to

0.6/1000 among females suggest that the fragile X syndrome is the most common genetic cause of mental retardation after Down's syndrome. However, reliable carrier detection and prenatal diagnosis for this disorder are not yet available because of the variability in the expression of the fragile site in lymphocytes and amniocytes. Genetic counselling is also made difficult because of the inheritance of the mutation through apparently phenotypically normal males and the variable expression in heterozygous females. Although DNA markers have been described which are linked to the fragile X syndrome, none of them are very informative. DXS396 lies proximal to the mutation at a recombination of 0.05 (Oostra et al. Science. 1989) and DXS304 lies at a similar genetic distance distal from the fragile site (Dahl

et al. , Am. J. Med. Genet.. 45; 304-309, 1989). The locus DXS269 shows no recombination with the fragile site (Suthers et al. , Am. J. Hum. Genet.. in press 1989) but is not very informative and shows no differences in fragile X patients compared to normal individuals in conventional Southern blots or by pulsed field gel electrophoresis. These loci are being used clinically in conjunction with cytogenetic evaluation of the expression of the fragile site.

We have now surprisingly discovered a new locus designated M54 which lies in the region of the fragile site closer than DXS304. The locus is different from those previously reported for markers in this region such as DXS304, DXS296 and DXS396. In situ hybridisation of radio-labelled M54 to a human male karyotype reveals grains in photographic emulsion at Xq27 and 12 autosomal sites, 9 of which are fragile sites and one of which is a telomere.

The present invention therefore provides an oligonucleotide or nucleic acid fragment comprising at least 17 contiguous nucleotide bases in a sequence:

(a) according to Figure 1

(b) complementary to a sequence (a) or (c) hybridisable with a sequence (a) or (b) .

Oligonucleotides and fragments according to the invention may be single or double stranded, RNA or DNA. When double

stranded they contain at least 17 contiguous nucleotide base pairs. Preferred oligonucleotides and fragments of the invention are at least 20 bases or base pairs in length, more preferably at least 50 bases or base pairs in length and most preferably about 110 bases or base pairs in length although yet longer oligonucleotides and fragments are also within the scope of the invention. All oligonucleotides and fragments must contain at least 17 contiguous bases or base pairs having a sequence (a) , (b) or (c) above; oligonucleotides and fragments longer than 17 bases or base pairs preferably contain a sequence according to Figure 1, complementary thereto or hybridisable therewith of at least 20, more preferably at least 50 and most preferably about 110 bases or base pairs in length. Particularly preferred oligonucleotides and fragments of the invention contain the entire sequence of Figure 1 or are perfectly complementary thereto.

Oligonucleotides and fragments which contain a sequence hybridisable to a sequence of at least 17 contiguous nucleotide bases or base pairs of Figure 1, or a complementary sequence will hybridise at least under low stringency conditions defined for the present invention as 3 x SSC at 65°C. More preferably such oligonucleotides and fragments will hybridise under high stringency conditions, i.e, 0.1 x SSC at 65°C oligonucleotides and fragments hybridising under low stringency must be at least 70%

homologous with the corresponding portion of the sequence of Figure 1 (or the complementary sequence) . To hybridise at high stringency it is necessary for there to be 90 or 95% ho ology. Differences may arise by way of substitution, deletion or insertion.

The oligonucleotides fragments of the invention are capable of hybridising selectively with the human X chromosome in the region Xq27.3.

The oligonucleotides and fragments of the invention hybridise with the Xq27.3 region under low or high stringency conditions. Under low stringency conditions hybridisation with chromosomal DNA blots will occur between the oligonucleotides or fragments of the invention and a large class of sites of related sequence in various parts of the human genome. Such hybridisation has uses in identifying fragile sites other than the fragile X site and mobile regions with the human genome. Under increasingly high stringency conditions the cross-hybridisation is progressively reduced and at the highest stringency (0.1 x SSC at 65°C) only a few sites are identified.

Hybridisation at high stringency reveals a number of loci related to M54 adjacent the fragile X site with Xq27.3. Particular oligonucleotides and fragments of the invention therefore include several copies of the sequence of Figure 1, or complementary sequences or sequences hybridisable thereto.

Two of these sites are located either side of the fragile X site itself and thus define a region containing the fragile X site. Fragments comprising the whole of the region between these two sites and optionally also containing flanking DNA from one or both of the proximal and distal regions are particularly useful as probes for the fragile X site and because such fragments can be used to identify coding sequences disrupted by the fragile X site.

The most preferred oligonucleotides and fragments of the invention are double stranded DNA fragments containing the full sequence of Figure 1 and capable of hybridising under high stringency conditions at a M54 locus.

Other oligonucleotides and fragments of the invention comprise the sequence (I) :

5 3'

GGGGGTGX (I)

wherein X is A or C (using the internationally recognised 1- letter codes for nucleotide bases) or the sequence (II) :

5 ^« 3' (II) YCACCCCC

wherein Y is T or G.

Sequence (I) appears twice in the M54 locus sequence of Figure 1 and is believed to be unique.

Oligonucleotides and fragments containing this sequence are therefore useful as probes for the M54 locus. Preferred such oligonucleotides and fragments contain two copies of the sequence (I) , more preferably spaced apart by about 50 nucleotide residues, most preferably spaced apart by the 50 residues in the sequence of Figure 1. Sequence (II) is complementary to sequence (I) and oligonucleotides and fragments containing this sequence, or preferred such oligonucleotides and fragments as described for sequence (I) are also useful as probes.

For use as probes, it will be necessary for the oligonucleotides and fragments of the invention to be detectable following hybridisation with human DNA and this may be achieved by any known labelling technique. Typically the probe will be labelled by incorporation of radio- isotopes such as ³² P, for instance by nick translation, which may be detected by auto-radiography. Other labelling techniques include provision of directly detectable labels such as fluorescent labels and indirectly detectable labels such as biotin or enzyme labels. A skilled person will be aware of the techniques required for introduction of these and other known labels and for the detection of such labels. The invention therefore also provides a probe comprising an oligonucleotide or nucleic acid fragment as defined above having a detectable label attached thereto.

The M54 locus comprises a region of the X chromosome

extending around the fragile X site and including the sequence of Figure 1. It does not overlap with any previously reported locus. Digests of the human X chromosome obtained using the restriction enzymes Rsa I, Pvu II, ECoRI or Hind III produced by conventional techniques yield a variety of DNA fragments, each of which hybridises with the M54 locus. The M54 locus is, therefore, defined as that portion of the Xq27-qter region of the human X chromosome where one or more of the aforementioned Rsa I, Pvu II, EcoRI or Hind III fragments hybridise. The present invention further provides oligonucleotide and nucleic acid fragments and probes which hybridise with the M54 locus as defined above and/or with any one or more of the aforementioned Rsa I, Pvu II, EcoRI and Hind III fragments. The aforementioned Rsa I, Pvu II, EcoRI or Hind III fragments are all examples of fragments according to the present invention. Using known techniques, these fragments and probes may be employed to identify further fragments which will hybridise to the human X chromosome between the fragile X site and the M54 locus as defined.

Oligonucleotides and fragments and probes which hybridise to the human X chromosome between the fragile X site and the M54 locus as defined form a further aspect of the invention. Some such oligonucleotides and fragments will hybridise with one or more of the aforementioned Rsa I, Pvu II, EcoRI

and Hind III fragments. Others of the oligonucleotides and fragments which hybridise between the fragile X site and the M54 locus will not hybridise to any of the aforementioned fragments but will nevertheless be useful in probing human X chromosomes for diagnosis of fragile X disease because they are capable of identifying regions nearer the fragile X site than the M54 locus.

In addition to their use in diagnosis of fragile X disease oligonucleotides and fragments according to the present invention which hybridise at the M54 locus or between the fragile X site and the M54 locus may also find uses in diagnosis of other X-linked disorders. In addition the use of oligonucleotides and fragments according to the invention to detect other fragile sites and mobile elements elsewhere in the human genome may enable diagnosis and therapy of various other diseases such as cancers and other syndromes which arise from rearrangements involving human chromosome 2.

,.*Tt has been found that probes for the M54 locus incorporating the sequence given in Fig.l or a part thereof hybridise to human X-chromosomal DNA in a region near to or including the fragile X site which has a very high new mutation rate. For instance in a family blot of four siblings, three showed classical Mendelian inheritance of parental alleles and one showed the presence of a new allele. The exact location of the mutation may be within the Fig.l sequence or within flanking proximal or distal DNA regions.

Oligonucleotides and fragments containing a mutant of the Fig.l sequence and oligonucleotides or fragments of the invention containing to Fig.l sequence or a part thereof and mutant flanking sequences are therefore also useful in diagnosis or therapy of X-linked disorders in accordance with the invention.

Hybridisation as referred to herein is conducted by admixing the oligonucleotides or fragments to be hybridised at 65°C in a buffer comprising SSC (at a specified concentration), 0.1% sodium dodecyl sulphate (SDS), 10% dextran sulphate and 1 x Denhardts solution followed by washing in high or low stringency buffer at 65 ^β C. 1 x SSC is 0.15M sodium chloride, 0.015. sodium citrate, pH 7.0 buffer solution. Denhardts solution is 0.02% polyvinyl pyrrolidine, 0.02% Ficoll 400 and 0.2% bovine serum albumin (BSA) . Low stringency buffer comprises 3 x SSC and 0.1% SDS whereas high stringency buffer comprises 0.1 x SSC and 0.1% SDS.

.•Oligonucleotides and fragments according to the invention may be obtained by digestion of human chromosome X or, preferably of the Xq27-qter region of the X chromosome, cloning the oligonucleotides and fragments as necessary and selecting oligonucleotides and fragments capable of hybridisation around Xq27.3, for instance at M54. A skilled person would be well aware of the techniques required to conduct the digestion, cloning and screening to produce oligonucleotides and fragments as hereinbefore defined.

As mentioned above, the oligonucleotide and nucleic acid fragments of the present invention, particularly when bearing a detectable label, may be used for the diagnosis of fragile X syndrome. The invention therefore further provides a process for diagnosing fragile X syndrome comprising contacting DNA from an individual potentially at risk of contracting fragile X syndrome with an oligonucleotide or nucleic acid fragment or probe as hereinbefore defined under suitable hybridising conditions. Hybridisation of the fragment or probe may be detected by conventional techniques. The diagnostic process of the invention preferably further comprises similar screening of DNA samples from close and distant relatives of the individual potentially at risk, particularly grandparents, parents, aunts and uncles, siblings and/or children of the individual. The DNA samples used may be freshly obtained or the process may be applied to archival material such as that obtained shortly after birth as'part of the Guthrie test. Such testing of relatives, coupled with case histories indicating the emergence of fragile X syndrome is used to trace the susceptible DNA through the family tree based on patterns of hybridisation to individual copies of M54 or related sequences in the vicinity of the fragile X site and thus determine whether the individual potentially at risk has, or has not inherited a susceptible X chromosome. In a particular aspect of the invention, the diagnostic process is conducted to screen for

carriers of fragile X syndrome and/or as a pre-natal screening to identify at-risk foetuses.

Oligonucleotides and fragments and probes according to the present invention may also be used in similar manner to diagnose other X-linked disorders as mentioned above.

The invention further provides a kit for conducting a diagnostic test for fragile X syndrome comprising an oligonucleotide or nucleic acid fragment or probe as hereinbefore defined and one or more of the following accessory components: A reagent or reagents for labelling the oligonucleotide or nucleic acid fragment; one or more restriction enzymes for the digestion of the patient's DNA; a buffer or buffers for conducting the digestion and/or loading the digestion DNA onto a support or substrate for hybridisation; a support or substrate such as a hybridisation filter on which to conduct hybridisation; a buffer or buffers for washing the hybridisation filter; a buffer or buffers for conducting hybridisation under low or high stringency conditions; a reagent or reagents for detecting a probe as hereinbefore defined; and control reagents such as fragile X positive and/or negative samples of human DNA and/or samples of non-human DNA, as well as standards for comparison of the signal form a detectable label.

The M54 locus of the human X chromosome has been found to have a highly conserved sequence (it also appears in mouse, pig, cow, baboon and chicken and yeast chromosomes)

indicating that the M54 locus may be functionally important and be adjacent to or part of a gene sequence. It is reasonable to predict that the gene or genes at or adjacent the M54 locus encode one or more proteins and that these proteins will be capable of raising antibodies when used as i munogens. The genes may be obtained and expressed by conventional techniques and proteins expressed by the genes may be used to raise antibodies by well known methods. The proteins and antibodies against the proteins may, themselves, have uses in the diagnosis of fragile X syndrome.

Accordingly, in further aspects the present invention provides: cloning and expression vectors containing one or more oligonucleotide or nucleic acid fragment of the present invention as hereinbefore defined, such as a gene corresponding to the M54 locus or cDNA corresponding to such a gene, the expression vector further comprising initiation and termination signals, regulatory and promoter sequences in correct position, orientation and reading frame as appropriate; host cells transformed with said cloning or expression vectors; polypeptides or proteins expressed by culturing such transformed host cells containing an expression vector; polyclonal and monoclonal antibodies against such polypeptides and proteins and antibody producing cells, such as hybridomas, capable of secreting such antibodies.

The complementary strand nucleotide base sequence of Fig.

1, contains two open reading frames and the sequence encodes polypeptides of amino acid residue sequences shown in Fig. 3. Such polypeptides are useful in generating antibodies which can be used in research and may also be used diagnostically or therapeutically.

The present invention therefore provides a polypeptide or protein comprising at least 5 contiguous amino acid residues in a sequence according to Fig. 3.

Preferably the polypeptide or protein comprises at least 10 contiguous amino acid residues in a sequence according to Fig. 3 more preferably at least 20, for example at least 30 and most preferably all of the amino acid residues shown in Fig. 3.

Polypeptides of the invention longer than 5 amino acid residues may include portions homologous to further portions of the sequence of Fig. 3, differing from that sequence by substitutions, deletions and/or insertions provided that they contain a contiguous sequence of 5 or more amino acid residues identical to the sequence of Fig. 3. The invention further provides antibodies, whether polyclonal, monoclonal or produced by recombinant DNA techniques, against proteins or polypeptides as defined above.

The proteins, polypeptides and antibodies may be produced and, where desired labelled or immobilised by conventional techniques.

The invention will be further described with reference to the figures of the accompanying drawings in which:

FIG 1. shows the sequence of the M54 locus (one strand only) and, boxed, sequences (I) appearing therein.

FIG 2. shows the in situ hybridisation pattern produced according to Example 1 below.

FIG 3. shows the amino acid residue sequences encoded by the various reading frames in the nucleic acid strand of Fig. 1.

In Fig. 3. the 3 translational phases are indicated.

—^ The codon abbreviations in lower case letters are for the "sense" strand; *→ the codon abbreviations in upper case letters are for the "antisense" strand. There is a "stop" (*.* ^' * in the amino acid sequence corresponds to a "stop" codon) in all reading frames except the two underlined.

The invention will now be illustrated by the following Examples, which are not intended to limit the scope of claims in any way.

EXAMPLE 1

³² P labelling was introduced into a double stranded DNA incorporating the sequence of Fig. 1 using conventional nick

translation technique:

Chromosome preparations

Male human lymphocytes were stimulated with phytohe agglutinin, and after culture 72h at 37°C, bro o deoxyuridine was added to block the cell cycle, as described by Zabel et al. (1983). The cells were washed with medium 16h later and then incubated in thymidine-rich medium for a further 6.5h.

In Situ hybridisation Slides were treated with RNase (100 ug/ml in 2 x SSC) at 37'C for lh, rinsed in 2 x SSC, and dehydrated through an alcohol series. They were denatured at 65 ^β C for 4 min in 70% formamide and 0.1 mM EDTA diluted with 2 x SSC (pH 7), rinsed in 2 x SSC, and dehydrated. Labelled probe was lyophilized, resuspended at 0.3 μg/ml in hybridisation buffer containing 5Q% formamide, 5 x Dehardt's solution, 5 x SSPE (0.9M NaCl/50mM NaH ₂ P0 ₄ /5 mM EDTA), pH 7.2, 10% dextran sulfate, an 200 ug/ml salmon solution was placed on each slide, covered with a coverεlip, and incubated overnight at 43°C. Subsequently, the slides were rinsed in 5 x SSC to remove the coverslips and washed in three changes of 2 x SSC at room temperature over a period of 2h. The slides were then washed in 1 x SSC at 65°C for lh, followed by 0.2 x SSC and 0.1 x SSC for 30 min (each at room temperature) and finally

dehydrated.

The slides were then dipped in Ilford L4 emulsion, exposed for 14 days at 4°C, developed in Kodak D19 for 7 min at 20°C, stained with Hoechst 33258 (10 μg/ l in 2 x SSC) for 30 min, exposed to UV light in 2 X SSC for lh, and then stained with 10% Giemsa for 15 min. The resulting silver grain distribution pattern is shown in Fig. 2. Only grains actually touching the chromosomes were scored. Clusters of grains were noted, but scored as a single hybridisation event on the ideograms illustrating grain distribution. This procedure enabled the ready identification of G-banded chromosomes and the distribution of silver grains on them. Fig. 2 shows the pattern of grains in an autoradiograph of the spread. Grain distribution in 53 cells following jLn situ hybridisation of M54 to replication-banded normal male chromosomes. Closed triangles _i denote sites of hybridisation which are equal to or greater than that scored Xq2 ^' 7, the location of the FRAXA locus. Open triangles A denote where these sites of hybridisation corresponds to the location of a fragile site.