PROBE FOR DETECTION OF THE FRAGILE X SYNDROME

Abstract: A nucleic acid fragment capable of hybridising selectively with the human X chromosome at the region Xq27.3 - DXS52 is useful in diagnosing fragile X syndrome and other X-linked disorders.

Description: Probe for detection of the fragile X syndrome. The present invention relates to nucleic acid fragments and their use in the diagnosis of fragile X syndrome. The fragile X (Martin-Bell) syndrome (McKusic cat no: 30955) is characterised by mental retardation, and physical anomalies including large ears, oblong face and macro-orchidism in affected males (1,2) . When lymphocytes derived from patients are cultured in thymidine deficient media, 2-50% of the cells express a fragile site at Xq27.3 (4,5). Recent prevalence figures of 0.6-1/1000 among males and 0.2-0.6/1000 among females suggest that the fragile X syndrome is the most common genetic cause of mental retardation after Down's syndrome (6,7). 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 (8,9,10). 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 (11,12,13). Although DNA markers have been described which are linked to the fragile X syndrome, ordering of markers distal to the fragile site has proved difficult because of the higher frequency of recombination between the markers and the fragile site compared to recombination between the markers themselves (refs 14-23) . Some studies support the order FRAXA - DXS52 - F8 - Xqter (24,27) whereas others support FRAXA - F8 - DXS52 - Xqter (26,27). More recently, a cross-over was found which separated DXS134 from F8 and DXS15 (28) . In view of the physical mapping of DXS134 between DXS52 and DXS15 (29) , this data would give the order DXS52 - DXS134 - DXS15 - F8„ The closest proximal marker DXS98 (4D8) has been linked at a recombination fraction between 0.03 and 0.06 (23,30) whereas the distal marker DXS52 (Stl4) is linked at a recombination fraction 0.10 (17). In view of the high frequency of single and double crossover events in this region, these loci are not reliable enough for routine clinical use. One strategy to identify the fragile X locus that "" circumvents the complexities of the inheritence pattern and the lack of closely linked markers is to construct a physical map of Xq27-qter. This should enable marker order to be unequivocally established and for disease loci to be identified. To date, loci have been physically mapped in three unlinked clusters covering approximately 2 megabases of DNA sequence (29, 31-34) . Thus a complete physical map of the region, estimated to be of the order to 12 megabases (32) , requires the isolation of new DNA sequences. We have now surprisingly discovered a new locus designated 1A1 which lies distal to the fragile site but closer than DXS52. It has further been discovered that the 1A1 locus displays restriction fragment length polymorphism (RFLP) and the 1A1 locus should provide a useful flanking marker for the fragile X locus in conjunction with DXS98 on the proximal side. Probes capable of detecting the 1A1 locus will, therefore, be useful in diagnosis of fragile X syndrome and a number of other X-linked disorders, such as X-linked manic depression, X-linked schizophrenia, X-linked bipolar illness, Emery Dreifuss dystrophy and adrenoleukodystrophy (see ref 15) . The locus is different from those previously reported for markers in this region such as F8 and DXS52 and colour blindness (CB) (29, 31-33). Probes for the 1A1 locus may, therefore, have additional diagnostic applications. The present invention therefore provides a nucleic acid fragment capable of hybridising selectively with the human X chromosome at the region Xq27.3 - DXS52. The fragments of the invention may hybridise with the Xq27.3 - DXS52 region under low or high stringency conditions and will be selective in that fragments do not hybridise with other human chromosomal DNA under those conditions. The nucleic acid fragment may be RNA or DNA but the latter is preferred. More preferably the fragment is double stranded DNA. Preferably the nucleic acid fragment will hybridise at the 1A1 locus as hereinafter defined. For use as probes, it will be necessary for the 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, 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 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 a nucleic acid fragment as defined above having a detectable label attached thereto. The 1A1 locus comprises a region of the X chromosome extending for at least 2Mb between the fragile X site and DXS52 but which does not overlap with the DXS52 locus. Digests of the human X chromosome obtained using the restriction enzymes Mlu I, Not I, Nru I or Sac II produced as described in Example 1 below yield a variety of DNA fragments, which are described in Example 1 below, each of which hybridises with the 1A1 locus. The 1A1 locus is, therefore, defined as that portion of the Xq27-qter region of the human X chromosome where one or more of the aforementioned Mlu I, Not I, Nru I or Sac II fragments hybridise. The aforementioned Mlu I, Not I, Nru I and Sac II fragments are all examples of fragments according to the present invention. Other fragments according to the present invention are nucleic acid fragments which hybridise with the lAl locus as defined above and/or with any one or more of the aforementioned Mlu I, Not I, Nru I, or Sac II fragments. Using known techniques, the fragments of the present invention may be employed to identify further fragments which will hybridise to the human X chromosome between the fragile X site and the lAl locus as defined. Some such fragments will hybridise with one or more of the aforementioned Mlu I, Not I, Nru I or Sac II fragments. Others of the fragments which hybridise between the fragile X site and the 1A1 locus will not hybridise to any of the aforementioned Nlu I, Not I, Nru I, or Sac II 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 lAl locus. Further fragments of the invention hybridise with the human X chromosome between the 1A1 locus and the DXS52 locus; such fragments may or may not hybridise with the aforementioned Mlu I, Not I, Nru I or Sac II fragments. In addition to their use in diagnosis of fragile X disease, fragments according to the present invention which hybridise between the fragile X site and the lAl locus may have further uses in the diagnosis of other X-linked locus may also find uses in diagnosis of other X-linked disorders referred to above. Fragments according to the invention which hybridise between the 1A1 locus and DXS52 disorders . Hybridisation as referred to herein is conducted by admixing the fragments to be hybridised at 42°C in a buffer comprising 3 X SSC, 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°C. SSC is 0.15M sodium chloride, 0.015J1 sodium citrate, pH 7.0 buffer solution. Denhardts solution is 0.02% polyvinyl pyrrolidine, 0.02% Ficoll 400 and 0.02% 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. The terms "hybridisation", "low stringency" and "high stringency" as used herein should be interpreted accordingly. 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 fragments as necessary and selecting fragments capable of hybridisation between Xq27.3 and DXS52, for instance at 1A1. A skilled person would be well aware of the techniques required to conduct the digestion, cloning and screening to produce fragments as hereinbefore defined. As mentioned above, the 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 a nucleic acid fragment or probe as hereinbefore defined under suitable hybridising conditions. Hybridisation of' he fragment or probe may be detected by conventional techiques. 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 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. Fragments 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 a nucleic acid fragment or probe as hereinbefore defined and one or more of the following accessory components: A reagent or reagents for labelling the 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 hybridisations; 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 from a detectable label. The 1A1 locus of the human X chromosome has been found to have a highly conserved sequence indicating that the lAl locus corresponds with one or more functional genes. It is reasonable to predict that the gene or genes at lAl encode one or more proteins and that these proteins will be capable of raising antibodies when used as immunogens. The genes may be obtained and expressed by conventional techniques and proteins express-ed 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 nucleic acid fragments of the present invention as hereinbefore defined, such as a gene corresponding to the 1A1 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 rector; polyclonal and monoclonal antibodies against such polypetides and proteins and antibody producing cells, such as hybridomas, capable of secreting such antibodies. The invention will be further described with reference to the figures of the accompanying drawings in which: FIG 1. Shows localisation of DNA using somatic cell hybrids. " DNA was digested with Pst I. The blot hybridized to the whole lAl cosmid with cold human DNA competitor in A and to a subfragment 1A1.6 in B. A: 1, total human DNA; 2, human/chinese hamster hybrid containing Xpter-Xqter; 3, Chinese hamster DNA; 4, human/mouse hybrid containing Xpter-Xq26; 5, human/mouse hybrid containing Xq26-qter; 6, human/chinese hmaster hybrid containing Xpter-Xcen. B: 1, Chinese hamster DNA; 2, human male DNA; 3, human female DNA; 4, hybrid B17 containing Xq27-qter. FIG 2. Shows A: lAl RFLP revealed after Pst I digestion of normal individuals, (Qq, alleles of lAl) segregation of the lAl RFLP in a fragile X family. FIG 3. Shows Nru I digestion of human sperm DNA (tracks 1 and 2). Pulse time and length. Hybridization to lAl. The gel was run at 5 C, 100V, 200S, for 60h. FIG 4. Shows Southern blot analysis of Not I digested DNA isolated from EBV lymphoblastoid cell lines of different individuals and hybridized to 1A1 (A and C) and F8 (B and D) . Blots A and B: 1, fragile X male; 2, spastic paraplegia male patient; 3, fragile X carrier female; 4, female with an X;autosome translocation; 5, normal male fragile X carrier. Blots C and D: 1,2, fragile X males; 3,5, normal females; 4, male haemophilia patient; 6, normal male sperm DNA sample. The gel was run at 50V, 20 mins, pulse time for 130h at 5 C. FIG 5. Shows Southern blot analysis of human DNA isolated from EBV lymphoblastoid lines digested with Mlu I (A) and Sac II (B) and hybridised to lAl. A: 1, 5, 6, 7, 9, 10, 11,; 2, great-great grandmother of 1: 3, normal female. B: 1,2, normal males; 3,4,5, fragile X males; 6, normal female; 7, male haemophilia patient; 8, male adrenomyeloneuropathy patient; 9, great-great grandmother of fragile X male in track 10. The gel was run at 50V, 20 mins pulse time, for 130h at 10 C. The invention will now be illustrated by reference to the following Example. EXAMPLE 1 In this Example we describe the genetic and physical mapping of a new locus, 1A1, which lies' distal to the fragile site. We demonstrate that this locus extends the physical map of this region by at least 2 megabases of DNA. In Not I digests, the pulsed filed gel patterns observed for lAl vary between individuals unlike other distal markers. MATERIAL AND METHODS Isolation of cosmids A cosmid library made from human/chinese hamster hybrid DNA containing the human X chromosomes as its only human component was constructed in the LTC vector (35) . The library was screened at low density with nick translated human genomic DNA under standard conditions and washed to 0.1 x SSC at 65°C. Positive clones were miniprepped and a Southern blot of the digested cosmid DNA hybridized against Chinese hamster and human DNA to confirm the presence of human X chromosome sequences. Somatic cell hybrids The human X chromosome only hybrid and the hybrid containing the short arm of the X chromosome have been described previously (36). Somatic cell hybrids containing various regions of the long arm of the human X chromosome were also used. Two reciprocal mouse/human hybrids were derived from the cell line GM3884, 46, X, t (X;16) (q26;q24). One hybrid containing the derivative X as the only human chromosome and the other the derivative 16 as the only human chromosome (37). A third hybrid contained Xq27-qter was derived by irradiation from a microcell hybrid line containing a t(X;19) (Xqter-q24:19ql3.2- pter) as the only human chromosome. This hybrid contains all known Xq28 markers including F8C, DXS52 and DXS115, but none of the markers that map proximal to fragile X such as DXS98 (38; and van Oost et al, unpublished). Southern blotting Southern blots (39) were carried out as previously described (40). Cosmids were labelled by random priming (41) or nick-translation (42), heat denatured and preincubated with heat denatured, sonicated human DNA (1 mg/ml) for 2 hours. The mixture was then diluted 10 fold into the hybridization buffer with the appropriate blot. Filters were washed to 0.1 x SSC at 65°C. Linkage programs The LINKAGE program was used for two-point and multipoint calculations (43,44). Normal families were from the Centre D'Etude du Polymorphisme Humain (C.E.P.H.) in Paris. Probes We are grateful to the following people for making probes available: Dr. J-L Mandel for Stl4 (DXS52)(17) and 55E (DXS105) (45), Dr. J. Gitschier for F8 (46), Professor Pearson for 767 (DXS115) (45) and Dr. R. Nussbaum for 4D8 (47). P lsed field gel electrophoresis This was carried out using a hexagonal array of electrodes as previously described (33,48). Enzyme digestions of DNA in agarose blocks were performed according to manufacturer's instructions and detailed in Kenwrick et al (49). Results Isolation and localisation of 1A1 A library constructed from a somatic cell hybrid cell line containing the X chromosome as its only human component was screened at low density with total human DNA in order to select those cosmids containing human DNA sequences. DNA sequences mapping around the fragile site were localised using a somatic cell hybrid panel containing two hybrids derived from a cell line with an X;16 translocation. One hybrid contained the derivative X chromosome and the other contained the derivative chromosome 16. The labelled cosmids were hybridized to the blots in the presence of total human competitor DNA (see methods) although a signal was not seen for every cosmid tested possibly due to the high level of repetitive sequences. One sequence, 1A1, which hybridized well under these conditions, gave a signal indicating its localization to Xq26-qter (see figure 1A). The pattern seen in tracks 2 and 5 is not absolutely identical leading us to investigate the possibility of a Pst I RFLP (see below). In order to establish whether the sequence lay distal or proximal to the fragile site, a single copy fragment of the cosmid 1A1 was hybridized to DNA from a hybrid containing Xq27-Xqter, B17 (see methods). The result In. figure IB shows that this fragment gives a clear positive signal with this hybrid DNA indicating localisation of 1A1 distal to the locus DXS98 which gives a negative signal with B17 DNA. Genetic mapping of 1A1 Single copy PstI fragments from the 1A1 cosmid were taken and hybridized against DNA from normal individuals digested with various different restriction enzymes. One particular fragment, designated 1A1.1, revealed a Pst I restriction fragment length polymorphism (RFLP) in the white Caucasian population studied. The alleles (a band at 5.2 kb or two bands at 2.9 and 2.3 kb) are shown in figure 2. Thirty-five per cent of the females tested were heterozygous at the 1A1 locus. Normal DNA from the C.E.P.H. panel, which we had previous l y typed with other markers in the Xq26-qter region, were typed for the 1A1 polymorphism. The two point lod scores are presented in table 1. Multipoint analysis suggests that, of the most likely orders, the order Xcen - DXS105 - 1A1 - DXS52 - F8C - DXS115 - Xqter is twice as likely as DXS105 - DXS115 - F8C - DX52 - 1 A 1 - Table 1 Recombination fraction (Q) Locus 1 Locus 2 Zmax θ ax 0.001 0.01 0.05 0.1 0.15 0.2 0.3' 0.4 -1.79 -0.22 0.45 0.74 0.73 0.31 -0.91 -0.42 -0.19 -0.07 0.019 0.014 3.95 3.76 3.41 2.99 1.94 0.75 4.60 4.55 4.20 3.72 2.45 0.94 2.67 . 2.80 2.66 2.38 1.56 0.60 J.35 3.588 3.378 2.287 0.887 Xqter. No significant data were obtained for DXS98 as the RFLP was only informative in one family. 1A1 was also studied in fragile X families. The two point lod scores are given in table 1. The data in this and other families are consistent with the localisation of 1A1 distal to the fragile site but proximal to DXS52 (location score xxxx). Pulsed field gel electrophoresis studies Pulsed field gel electrophoresis studies were carried out as described in methods such that bands in the molecular weight range 800 - 2000kb could be resolved. An example of the results obtained in given in figure 3. 1A1 identifies a 1.2 megabase (Mb) band in Nru I digests of human sperm DNA (figure 3). This 1A1 fragment is different from those previously reported for markers in this region such as F8 and DXS52 and color blindness (CB) (29, 31-33). An unusual finding with 1A1 is the variability in the band sizes observed after Not I digestion of different individuals (figure 4A). This is not a gel artefact as the same blot hybridised to F8 gives the same molecular weight band in all individuals (figure 4B). The pattern is also reproducible from gel to gel. Samples of sperm and lymphocyte DNA from the same individual give the same band pattern (data not shown). In view of the proximity of 1A1 to the fragile site in the genetic analyses, we also investigated the 1A1 Not I pattern in fragile X positive males and females (track 1, 3 and 5, figure 4A and tracks 1 and 2, figure 4C). The variation is observed in unrelated fragile X positive males as well as normal individuals. After Not I digestion, 1A1 detects any of three different bands at approximately 0.85Mb, 1.2Mb and 1.4Mb. Two bands are sometimes seen in both male and females suggesting that the pattern is generated by variable methylation of the restriction enzyme sites rather than by a fragment length polymorphism. Mlu I digests of human DNA hybridised to 1A1 also show a variable pattern that appears in both fragile X negative and positive individuals (figure 5A). In this experiment we also tested the growth state of the cells to determine whether this altered the gel pattern. As can be seen from tracks 5 and 6 of figure 5A where the cells were harvested in early and late log phase respectively, the band pattern is identical. Sac II digests of individuals also show several bands when hybridized to 1A1. However, in this instance, the band patterns observed are similar for all samples (figure 5B). (Minor apparent variations are attributable to loading artefacts). None of the fragments detected by 1A1 overlap with any observed for loci previously mapped in this region. For example the Not I fragments for 1A1, DXS52 and F8C are 1.2Mb, 1.0Mb and 1.8Mb respectively. Thus 1A1 is a physically distinct locus from DXS52. None of the PFGE bands overlap with bands observed for the closest proximal marker, DXS98 (Bell and Davies, unpublished observations). Discussion We have localised a DNA sequence, 1A1, to the region Xq27- qter using somatic cell hybrids containing various portions of the telomeric region of the long arm of the human X chromosome. Thus 1A1 should be a useful new marker for segregation analysis of the fragile X syndrome. Genetic analysis of 1A1 in the C.E.P.H. families indicate close linkage between 1A1 and both DXS52 and F8C, the closest distal markers to the fragile site at Xq27.3. The multipoint analysis of the data in these normal families favours the order where 1A1 lies proximal to DXS52 and F8C. Close linkage is not observed across the fragile site region in these families between 1A1 and the markers in Xq26/q27 such as DXS105 (55E). In the fragile X families, close linkage between 1A1 and DXS52 is again observed. Multipoint analysis of the segregation of markers supports the order suggested from the normal families with 1A1 lying proximal to DXS52. In view of the high frequency of the 1A1 RFLP in the population, 1A1 should be a useful flanking marker for the fragile X locus in conjunction with DXS98 on the proximal side. This locus should also be useful in the study of other syndromes which have been found to be closely linked to DXS52 such as X- linked bipolar illness, Emery-Dreifuss dystrophy and adrenoleukodystrophy (see ref 15). Physical mapping studies using PFGE demonstrate that 1A1 defines a new region of Xq27 non-overlapping with previously reported loci (29,31-34). A Not I map derived from the largest fragments detected by the probes used is summarised below: FRAXA - 1A1- DXS52, DXS33, DXS15 - G6PD, F8, DXS115 - Xqter Mb 1.2 1.0 0.30, 1.2 The largest Not I fragment seen by the color blindness probe is less than 300 kb (32,50) but this locus has not been ordered relative to the other loci given above. Thus 1A1 significantly extends the physical map. No overlapping fragments are seen between 1A1 and DXS52 in spite of the fact that bands greater than 1Mb are observed for the enzymes Not I, Nru I, Mlu I and Sac.II. The largest Mlu I band identified by 1A1 extends over 2Mb which does not overlap with the closest adjacent locus DXS52 which detects a partial digest band at approximately 550 kb in Mlu I digests of human DNA (31). DXS52 hybridised to the same f lter as in Figure 5A does not hybridise to any high molecular weight band. PFGE studies with 1A1 consistently give multiple band patterns indicating a variable degree of methylation between individuals in this region of Xq27/28. The band patterns are reproducible for each individual DNA sample. It was particularly striking that several bands were obtained in blots after digestion of the DNA with Sac-II. In our experience, this enzyme has always given almost complete digestion around all the other loci tested from this region consistent with the observation that many Sac II sites are associated with the unmethylated CpG dinucleotide in HTF islands (51). 1A1 gives large fragments (a megabase or more) with severa l enzymes with CpG in their recognition sites. 1A1 therefore l ies in a relatively less CpG-rich region compared to F8C, G6PD and DXS52. The biological significance of this observation with regard to genomic structure remains to be determined. Recently GC-rich regions have been shown to be correlated with lightly staining G-bands containing Alu sequences (52) and it may be that 1A1 lies within the darkly staining band Xq27. On the other hand, 1A1 might define a region of different function within Xq28. Further physical mapping will be required to resolve this question. The size variations seen with Mlu I and Not I are of the order of several hundred kb and are not consistent with a fragment length polymorphism. Individuals giving the smaller band in the Mlu I digest do not always give the smallest band in the Not I digest and vice versa. In some Not I digested male and female DNA samples faint bands, in addition to the main hybridizing band, could be visualized. The pattern is therefore likely to be due to differential methylation of CpG sites in this region of Xq27/28. Laird (53) recently proposed an imprinting theory to account for the segregation pattern observed in the fragile X syndrome and suggested that methylation might be one way in which this imprinting could be achieved. The experiments described here were undertaken in EBV lines which may not have maintained the active X chromosome methylation pattern. 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Claims: CLAIMS 1. A nucleic acid fragment capable of hybridising selectively with the human X chromosome at the region Xq27.3 - DXS52. 2. A fragment according to claim 1 capable of hybridising under high stringency conditions. 3. A double stranded DNA fragment according to claim 1 or claim 2. 4. A fragment according to any one of claims 1 to 3 which hybridises at the 1A1 locus. 5. A nucleic acid probe comprising a fragment according to any one of claims 1 to 4 and a detectable label attached thereto. 6. A probe according to claim 5 wherein the label is a radiolabel, a fluorescent label or an enzyme label. 7. A process for diagnosing fragile X syndrome or other X-linked disorder comprising contacting DNA from an individual potentially at risk of contracting fragile X syndrome or other X-linked disorder with a nucleic acid fragment or probe according to any one claims 1 to 5 under suitable hybridising conditions. 8. A process according to claim 7 for diagnosing fragile X syndrome. 9. A process according to claim 7 for diagnosing X-linked manic depression, X-linked schizophrenia, X-linked bipolar illness, Emery Dreifuss dystrophy or adrenoleukodystrophy. 10. A kit for conducting a diagnostic test for fragile X syndrome or other X-linked disorder comprising a nucleic acid fragment or probe according to any one of claims 1 to 6 and one or more of the following accessory components: A reagent or reagents for labelling the 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 on which to conduct hybridisations; 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; one or more control reagents selected from fragile X positive and negative samples of human DNA and samples of non-human DNA; and one or more standards for comparison of the signal from a detectable label. 11. A cloning or expression vector containing one or more nucleic acid fragments according to any one of claims 1 to 4, the expression vector further comprising initiation and termination signals, regulatory and promoter sequences in correct position, orientation and reading frame as appropriate. 12. A host cell transformed with a cloning or expression vector according to claim 11. 13. A polypeptide or protein obtained by culturing host cell transformed with an expression vector according to claim 11. 14. A polyclonal or monoclonal antibody against a polypeptide or protein according to claim 13. 15. An antibody producing cell capable of secreting an antibody according to claim 14.

Inventors: BELL MARTIN, DAVIES KAY ELIZABETH, PATTERSON MARK, BELL, MARTIN, DAVIES, KAY, ELIZABETH, PATTERSON, MARK
Applicants: MEDICAL RES COUNCIL, MEDICAL RESEARCH COUNCIL
Application year: 1989
Main classification: C12Q1
IPC codes: A61K49, A61K51, C07K14, C07K16, C12N5, C12N15, C12P21, C12Q1, C12R1, G01N33