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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
  • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• 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 (1, 2).
• 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
• amniocytes (8, 9, 10). Genetic counseling 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).
• 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).
• 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,
• RFLP restriction fragment length polymorphism
• 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.
• fragment will hybridise at the 1A1 locus as hereinafter defined.
• probes 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
• 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 restr iction 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
• 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.
• 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.
• 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 1A1 locus.
• 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.
• 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, 0.015M 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.
• 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
• nucleic acid fragments of the present invention particularly when bearing a
• 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 the 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
• 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
• the diagnostic process is conducted to screen for carriers of fragile X syndrome and/or as a pre-natal screening to identify
• 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
• 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 cond itions ; 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 1A1 locus corresponds with one or more functional genes. It is reasonable to predict that the gene or genes at 1A1 encode one or more proteins and that these proteins will be capable of raising antibodies when used as
• 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,
• the present invention provides cloning and expression vectors
• 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
• polyclonal and monoclonal antibodies against such polypetides and proteins and antibody producing cells, such as hybridomas, capable of secreting such antibodies are included in the expression rector; polyclonal and monoclonal antibodies against such polypetides and proteins and antibody producing cells, such as hybridomas, capable of secreting such antibodies.
• 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.
• 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: 1A1 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
• 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. Shows Southern blot analysis of human DNA
• 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.
• 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
• 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-q24:19q13.2- 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
• 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.
• 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 al le les ( 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.
• 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).
• 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).
• 1A1 detects any of three different bands at approximately 0.85Mb, 1.2Mb and 1.4Mb. Two bands are
• 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).
• 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.
• 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
• 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.
• 1A1 gives large fragments (a megabase or more) with several enzymes with CpG in their recognition sites. 1A1 therefore lies 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.

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