WO1994028172A1 - Sondes de detection du syndrome du chromosome x fragile - Google Patents

Sondes de detection du syndrome du chromosome x fragile Download PDF

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Publication number
WO1994028172A1
WO1994028172A1 PCT/GB1994/001175 GB9401175W WO9428172A1 WO 1994028172 A1 WO1994028172 A1 WO 1994028172A1 GB 9401175 W GB9401175 W GB 9401175W WO 9428172 A1 WO9428172 A1 WO 9428172A1
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fragment
fraxe
repeat
region
seq
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PCT/GB1994/001175
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English (en)
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Samantha Jayne Lesley Knight
Kay Elizabeth Davies
Mark Charles Hirst
Angela Veronica Flannery
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Medical Research Council
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Priority to AU68030/94A priority Critical patent/AU6803094A/en
Publication of WO1994028172A1 publication Critical patent/WO1994028172A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to polynucleotides and their use in the diagnosis of susceptibility to mental retardation.
  • Fragile sites appear as non-staining regions, chromatid gaps, or less frequently as breaks in metaphase chromosome spreads.
  • At least twenty six rare fragile sites in the human genome are induced in culture by the use of low folate media. They occur in approximately 5% of the population and are inherited in a Mendelian fashion (Sutherland and Hecht, 1985; Sutherland, 1991) . Common fragile sites are observed at a higher frequency in populations but are only expressed at low levels under specific culture conditions.
  • Fragile sites are highly conserved in evolution and can be found in many species including primates, mouse and horse, suggesting a common structure or function (Sanz et al., 1986; Wurster-Hill et al. 1988; Smeets andretert, 1990). It has been suggested that fragile sites may predispose to chromosome breakage .in vivo and lead to an increased rate of sister chromatid exchange and recombination in their vicinity (Glover and Stein, 1987; Glover and Stein, 1988) . More than fifty per cent of breakpoints that have occurred during chromosome evolution in primates are reported to be at or close to fragile sites (Miro et al., 1987) . The molecular basis of fragile sites is thus of considerable interest.
  • FRAXA fragile site at Xq27.3
  • FRAXE expression was first reported in individuals who are not mentally retarded indicating that the mental retardation may be a chance association due to ascertainment bias (Sutherland and Baker, 1992) .
  • the inventors have located a gene, an associated CpG island and a GCC repeat region which are present in the vicinity of the FRAXE site of the human genome. They have also established that the GCC repeat is usually amplified in individuals suffering from mental retardation and other manifestations related to FRAXE fragility and that the GCC repeat and/or associated CpG island may be hypermethylated in affected individuals.
  • proximal (CA) m repeat region lies between the CpG island and the centromere.
  • distal (CA) n repeat region lies between the CpG island and the telomere on the q arm of the X chromosome in which the FRAXE site is located.
  • the proximal (CA) m repeat region lies at a distance of less than 20kb from the CpG island whilst the distal (CA) n repeat region lies at a distance of less than 5kb from the CpG island.
  • the proximal (CA) m repeat region lies within a 13kb EcoRI fragment of the human X chromosome whilst the distal (CA) n repeat region lies within a 1.5kb Hind III fragment of the human X chromosome.
  • proximal (CA) m repeat region There is allelic variation in the size of the proximal (CA) m repeat region.
  • the inventors have identified nine proximal (CA) m repeat alleles, alleles (A)-(I), whose sizes are given in Table 1 below from which it can be seen that m is 52 or more.
  • allele (F) has been sequenced and found to comprise pure (CA) m repeats.
  • the lengths of the other alleles all differ from that of the sequenced allele by multiples of two base pairs. Therefore, it is believed that the variation in the alleles' sizes arises from variation in the copy number of CA units and that the alleles all comprise pure (CA) m .
  • proximal (CA) m repeat allele F is prevalent in individuals suffering from mental retardation.
  • allelic variation in the size of the distal (CA) n repeat region There is also allelic variation in the size of the distal (CA) n repeat region and the inventors have discovered three distal (CA) n repeat region alleles, alleles (A)-(C), whose sizes are given in Table 2 below, from which it may be deduced that n would be 52 or more if the alleles were pure CA repeats.
  • the (CA) n repeat is interrupted by a different sequence; the reasons for the allelic variation in size are not fully understood. Variation may be due to the size or number of interrupting sequences in the distal (CA) n repeat region or to variation in the copy number of CA units or to any combination of these.
  • the inventors have found that one distal (CA) n allele, allele C, is prevalent in individuals suffering from mental retardation.
  • (CA) n repeat allele C may be suggestive of susceptibility to mental retardation if one or more of the other diagnostic features, such as amplification of the GCC repeat region methylation of the GCC repeat region and methylation of the CpG island is also present.
  • the present invention relates to the diagnosis of susceptibility to mental retardation by detecting the presence or absence of one or more features within the FRAXE region of the human X chromosome.
  • FRAXE region refers to a part of the human X chromosome at Xq28 which contains the following features, listed with the most proximal first; (1) a proximal flanking region; (2) a proximal (CA) m repeat region; (3) a CpG island; (4) a GCC repeat region; (5) a distal (CA) n repeat region; and (6) a distal flanking region.
  • the FRAXE fragile site at which a fragile chromosome actually breaks, is thought to be within the GCC repeat region.
  • the FRAXE region may also include a gene. This is thought to be the case because of the presence of the CpG island, as CpG islands are often associated with genes; also, individuals expressing FRAXE have a defined phenotype, which suggests that expression of a specific gene is disrupted by the chromosome breakage. The exact position of the gene is indeterminate.
  • the FRAXE region also includes intermediate sequences. The exact relationship of regions (l)-(6) within the FRAXE region is not known. Therefore, the exact positions and sizes of the intermediate sequences and the size of the FRAXE region as a whole are uncertain. Nevertheless, the FRAXE region is defined by the presence and the relative positions of regions (l)-(6) as defined above.
  • the present invention provides methods by which the presence of the FRAXE fragile site in an individual may be detected, and also provides nucleic acid fragments and related materials which may be useful in the diagnosis and/or treatment of individuals with fragile X syndrome.
  • the present invention provides, in one aspect, a polynucleotide selected from a Hind III nucleic acid fragment, polynucleotide sections thereof and nucleic acids hybridisable thereto: the Hind III fragment of this aspect of the invention is referred to below as the Hind Ilia fragment, it hybridises to that part of the FRAXE region of the human X chromosome which includes the GCC repeat as illustrated in SEQ.ID.No. 1, and is substantially free of other nucleic acid fragments.
  • the present invention in a further aspect, provides a polynucleotide selected from an EcoRI nucleic acid fragment, polynucleotide sections thereof or nucleic acids hybridisable thereto: this EcoRI fragment is referred to below as EcoRla, it hybridises to the FRAXE site of the human X chromosome and is substantially free of other nucleic acid fragments.
  • the present invention in a further aspect provides a polynucleotide selected from an EcoRI nucleic acid fragment of the human X chromosome, polynucleotide sections thereof or nucleic acids hybridisable thereto: this EcoRI fragment is referred to below as EcoRlb, it hybridises at a site overlapping with and distal to the Hind Ilia fragment defined above and is substantially free of other nucleic acid fragments.
  • the present invention also provides a polynucleotide selected from (i) a 13kb EcoRI nucleic acid fragment of the human X chromosome referred to below as EcoRIc, which hybridises to the proximal (CA) m repeat region and which is substantially free of other nucleic acid fragments, (ii) polynucleotide sections of EcoRIc and (iii) nucleic acids hybridisable thereto.
  • Polynucleotide sections of EcoRIc may hybridise at one or other flank of the proximal (CA) m repeat region and may optionally overlap with at least part of the (CA) m repeat region.
  • Such polynucleotides which do not overlap with the (CA) m repeat region optionally terminate at the boundary of the proximal (CA) m repeat region or terminate one or more nucleotides away from the boundary of the proximal (CA),.. repeat region.
  • the present invention also provides a polynucleotide selected from (i) a 1.5kb Hind III nucleic acid fragment of the human X chromosome, referred to below as Hind Illb, which hybridises to the distal (CA) n repeat region and which is substantially free of other nucleic acid fragments, (ii) polynucleotide sections of Hind Illb and (iii) nucleic acids hybridisable thereto. Polynucleotide sections of Hind Illb may hybridise at one or other flank of the distal (CA) n repeat region and may optionally overlap with at least part of the (CA) n repeat region.
  • Hind Illb 1.5kb Hind III nucleic acid fragment of the human X chromosome
  • Such polynucleotides which do not overlap with the (CA) n repeat region optionally terminate at the boundary of the distal (CA) n repeat region or terminate one or more nucleotides away from the boundary of the distal (CA) n repeat region.
  • the fragments of the invention are the Hind Ilia and Hind Illb fragments and the EcoRla, EcoRlb and EcoRIc fragments defined above and the OxE18 and OxE20 fragments defined below.
  • the fragments of the invention are double stranded DNA.
  • a polynucleotide of the invention other than these fragments generally has at least 100 bases or base pairs, for example at least 200, at least 300, at least 500 or at least 1000 bases or base pairs.
  • a polynucleotide section is a nucleic acid having a sequence contained within a fragment of the invention.
  • a section generally has at least 10 bases or base pairs, for example at least 15 bases or base pairs, at least 20 bases or base pairs, at least 50 bases or base pairs, at least 150 bases or base pairs or at least 300 bases or base pairs.
  • a nucleic acid capable of hybridizing to a fragment of the invention will generally be at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the polynucleotide over a region of at least 20, preferably at least 30, for instance 40, 60 or 100 or more contiguous nucleotides.
  • polynucleotides according to the invention Such fragments, sections and nucleic acids will be referred to below as "polynucleotides according to the invention".
  • a polynucleotide according to the invention may be produced recombinantly, synthetically, or by any other means available to those of skill in the art.
  • a polynucleotide section or nucleic acid of the invention may be flanked at either or both flanks by sequences unrelated to fragments of the invention.
  • the polynucleotide section or nucleic acid of the invention may be RNA or DNA and may be double or single stranded.
  • nucleic acids and polynucleotide sections of the invention are hybridisable to the fragments of the invention under low stringency conditions.
  • Preferred nucleic acids and polynucleotide sections hybridise under high stringency conditions.
  • low stringency conditions we mean 3X SSC (0.5M sodium chloride pH7.5) at room temperature.
  • high stringency conditions we mean 0.1X SSC (0.01M sodium chloride pH7.5) at 65'C.
  • Preferred polynucleotide sections and nucleic acids hybridise selectively to fragments of the invention.
  • One fragment of the present invention is the Hind Ilia fragment of the human X chromosome which hybridises to that part of the FRAXE region of the human X chromosome containing the (GCC)p repeat region and which generally has from 5 to 6 Kbp in normal individuals, but which in FRAXE family members can show an increase in size of at least lOObp, for example at least 400, at least 1000, at least 10000, or at least 20000 bp.
  • the Hind Ilia fragment may be digested to produce the EcoRla fragment which generally has from 2.7 to 3.5 Kbp in normal individuals but which, in FRAXE family members, can show an increase in size of at least lOObp, for example at least 400, at least 1000, at least 10000, or at least 20000 bp.
  • the Hind Ilia fragment includes the sequence of nucleotides 1 to 2977 of Sequence 1 shown in Table 3 below (SEQ.ID. o. 1).
  • Nucleic acids which hybridise to the Hind Ilia fragment may contain a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of nucleotides 1 to 2977 of Sequence 1 in Table 3 (SEQ.ID. ' No.l) .
  • the sequence of such a polynucleotide of the invention may vary from that of sequence l by deletion of at least one nucleotide, insertion of at least one nucleotide or substitution of at least one nucleotide in the sequence.
  • Nucleic acids which hybridise to the EcoRla fragment may contain a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of nucleotides 1 to 2977 of Sequence 1 in Table 3 (SEQ.ID.No. 1) .
  • the sequence of such a polynucleotide of the invention may vary from that of Sequence 1 by deletion of at least one nucleotide, insertion of at least one nucleotide or substitution of at least one nucleotide in the sequence.
  • Both the Hind Ilia fragment and the EcoRla fragment contain a GCC trinucleotide repeat of at least 3 repeats, for example at least 10 repeats, at least 20 repeats, at least 50 repeats, at least 100 repeats or at least 200 repeats.
  • both the Hind Ilia fragment and the EcoRla fragment usually contain a trinucleotide repeat of at least 15 repeats.
  • the trinucleotide repeat may be methylated and is generally more highly methylated in affected individuals than in unaffected individuals.
  • a preferred polynucleotide section of the invention has the sequence of nucleotides 198 to 450 shown in Sequence 1 of Table 3 (SEQ.ID. o. 1) and .includes the trinucleotide repeat.
  • a further preferred polynucleotide section of the invention has the sequence of nucleotides 1116 to 1974 of Sequence 1 in Table 3 (SEQ.ID.No. 1) and is referred to below as OXE20.
  • a further preferred polynucleotide section of the invention has the sequence of nucleotides 243 to 811 shown in Sequence l of Table 1 (SEQ.ID.No. 1).
  • a yet further preferred polynucleotide section of the invention includes the open reading frame or coding sequence of the gene in the FRAXE region which codes for a protein.
  • the present invention also provides a gene which comprises at least one of the polynucleotide fragments of the invention.
  • the gene comprises at least 3 kb, such as at least 5 kb, for example at least 10 kb, at least 20 kb, at least 30 kb or at least 40 kb.
  • a polynucleotide section or nucleic acid according to the invention may be used as a primer, eg. a PCR primer, or a probe, or the polynucleotide or nucleic acid may be inserted into a vector.
  • PCR and other primers of the invention will be at least 15, preferably at least 20, for example 25, 30 or 40 nucleotides in length. It has been found that a pair of PCR primers designed to anneal at low temperature (ca 65°C) were ineffective at 65°C whereas another pair of PCR primers designed to anneal at high temperature (ca 95 ⁇ C) was effective even at ca 65 ⁇ C, probably due to the influence of the GCC repeat.
  • Probes of the invention will be at least 40 nucleotides in length, preferably at least 50 nucleotides in length, for example 60, 70, 80 or 100 nucleotides in length and may be labelled by conventional means using radioactive or non- radioactive labels, such as an enzyme or a fluorescent moiety or the probes may utilise as labels specific binding agents and their binding partner, for example avidin/biotin or antigen/antibody.
  • a preferred probe according to the invention is the
  • OxEl ⁇ section of the Hind Ilia fragment is defined by a Not I Site, which falls within the FRAXE CpG island and a
  • OxE18 is approximately 2kb long.
  • the full sequence of 0xE18 is not known but the sequences of its proximal and distal limits are as follows:
  • the invention provides a preferred pair of primers, one primer having the sequence of nucleotides 239 to 270 (SEQ.ID.No. 19) and one primer having the sequence of nucleotides 555 to 581 (SEQ.ID.No. 20) shown in Table 1 (SEQ.ID.No. 1).
  • the invention also provides another preferred pair of primers, which hybridise at either flank of the proximal (CA) m repeat (SEQ . ID . Nos . 4 , 5 ) .
  • the invention also provides another preferred pair of primers which hybridise at either flank of the distal (CA) n repeat region.
  • One of these primers is represented by SEQ.ID. o. 6.
  • the second is selected from the primers represented by SEQ.ID.Nos. 7 and 8.
  • the invention also provides methods of screening for susceptibility to mental retardation, which methods comprise analysing the FRAXE region of an individual.
  • the invention provides a method of detecting the normal FRAXE GCC repeat array by means of PCR.
  • a preferred example of such a method does not require the use of radio labelled nucleotides. Detection of the normal FRAXE GCC repeat array indicates that an individual's susceptibility to mental retardation is low.
  • the invention also provides a method of diagnosing susceptibility to mental retardation which comprises detecting an amplification in the GCC repeat in the FRAXE region of the human genome.
  • the invention further provides a method of diagnosing susceptibility to mental retardation which comprises detecting methylation of the GCC repeat in the FRAXE region.
  • CpG island also known as an HTF island.
  • CpG island we mean a GC rich region containing a cluster of sites for rare cutting enzymes which are methylation-sensitive.
  • the enzymes usually, but not always or exclusively, include Hpall, BssHIII, Eagl, Notl, Nrul, Narl and SacII.
  • CpG islands are usually associated with genes and are most often found near the 5' end of the gene but may also be found elsewhere in the gene. CpG islands are usually unmethylated in all tissue types. Not all genes contain CpG islands but the presence of an island is suggestive of a gene.
  • the CpG island in the FRAXE region is generally not methylated in individuals unaffected by FRAXE mental retar-dation but is frequently methylated in affected individuals.
  • the invention further provides a method of diagnosing susceptibility to mental retardation which comprises detecting methylation of the CpG island of the FRAXE region.
  • An amplification in the trinucleotide repeat at the FRAXE site may be determined by removing a sample of genomic DNA from the patient, carrying out a polymerase chain reaction (PCR) with primers upstream and downstream of the FRAXE site and determining the amount of nucleic acid produced.
  • PCR polymerase chain reaction
  • PCR generally does not occur to a substantial extent across genomic DNA comprising a GCC repeat of 30 repeats or more.
  • Substantial amounts of nucleic acid are only produced by PCR carried out on a DNA fragment in which there is little or no amplification of the trinucleotide repeat, ie. less than 30 repeats.
  • methylation of either or both of the GCC repeat and the CpG island may be detected by removing a sample of genomic DNA from the patient, fragmenting the DNA with a methylation-sensitive restriction enzyme, carrying out PCR with primers upstream and downstream of the GCC repeat -and/or the CpG island and determining the amount of nucleic acid produced.
  • the restriction enzyme does not cut the DNA at the methylated site and PCR carried out subsequently across the restriction site will produce substantial amounts of nucleic acid.
  • the restriction enzyme cuts the DNA and PCR carried out across the restriction site will not produce substantial amounts of nucleic acid.
  • the genomic DNA from the patient may be fragmented with a methylation-sensitive restriction enzyme and Southern analysis may be carried out on the digested DNA.
  • a combination of Hind III with a suitable restriction enzyme such as Notl, BssHII, Eagl, SacII, Narl or Hpall gives a distinctive pattern in a Southern analysis of the genomic DNA in normal individuals and a different pattern in FRAXE family members. Southern analysis may also be carried out on EcoRI fragments in a similar way.
  • the absence of the Hind Ilia fragment in the Southern analysis indicates that the fragment has been cut and that there is therefore no methylation at the restriction site.
  • the absence of the Hind Ilia fragment is ordinarily accompanied by the detection of the two smaller fragments into which the Hind Ilia fragment has been split.
  • the restriction enzyme used in the method should have a recognition site within the region of interest, for example Notl, BssHII, Eagl, SacII, Narl or Hpall and should also be sensitive to methylation of any nucleotides within that recognition site. For example an enzyme will be selected to recognise a methylation sensitive site within the Hind Ilia fragment.
  • the methods of the invention are carried out using at least one primer of the invention, for example having nucleic acid sequence corresponding to bases 239 to 270 or 561 to 581.
  • the invention also provides a method of diagnosing susceptibility to mental retardation, which method comprises at least one of the methods defined above and which method further comprises detecting the presence or absence of one or more alleles of the proximal (CA) m repeat region and/or detecting the presence or absence of one or more alleles or the distal (CA) n repeat region.
  • One particularly preferred method of screening for susceptibility to mental retardation comprises analysing an individual's FRAXE region in the following manner. Firstly, the individual's X chromosome is analysed for the normal FRAXE GCC repeat array by means of PCR. If the individual has an amplified GCC repeat array, very little or no PCR products are generated. The individual's FRAXE region is then analysed for the presence of the proximal (CA) m and/or distal (CA) n repeat alleles that are prevalent in individuals suffering from mental retardation (proximal (CA) m repeat allele F and distal (CA) n repeat allele C) . Then a Southern analysis is performed wherein Hind III blots of genomic DNA are probed with one or more probes as defined above in order to confirm that an expansion of the FRAXE GCC repeat exists.
  • a further embodiment of the invention provides vectors for the replication or expression of a polynucleotide according to the invention.
  • the vectors may be, for example, plasmid, virus, phage vectors, cosmids or yeast artificial chromosome, provided with an origin of replication, and expression vectors will contain a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the expression vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • the expression vectors may be used in vitro, for example for the production of RNA corresponding to DNA, or used to transfect or transform a host cell.
  • a further embodiment of the invention provides host cells transformed or transfected with the vectors for the replication and expression of a polynucleotide according to the invention, including the DNA SEQ.ID.No. 1 or the open reading frame thereof.
  • the cells will be chosen to be compatible with the vector and may for example be bacterial, yeast, insect or mammalian.
  • a polynucleotide according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA.
  • Antisense RNA may also be produced by synthetic means. Such antisense RNA may be used in a method of controlling the levels of the protein encoded by the FRAXE site in a cell.
  • the invention further provides a protein encoded by the gene in the FRAXE region in substantially purified form, homologues thereof, and fragments of the sequence and its homologues.
  • a further embodiment of the invention provides a polynucleotide, such as DNA or RNA, for example mRNA, coding for the protein or a fragment thereof.
  • the protein in substantially purified form will generally comprise the protein in a preparation in which more than 90%, eg. 95%, 98% or 99% of the protein in the preparation is that of the encoded protein.
  • An encoded protein or peptide homologue of the encoded protein will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the encoded protein over a region of at least 5, preferably at least 10, for instance 20, 40, 60 or 100 or more contiguous amino. acids.
  • Such protein or peptide homologues will be referred to below as a peptide according to the invention.
  • peptide fragments of the encoded protein or its homologues will be at least 10, preferably at least 15, for example 20, 25, 30, 40, 50 or 60 amino acid residues in length, and are also encompassed by the term "a peptide according to the invention" as used herein.
  • the invention also provides monoclonal or polyclonal antibodies to a peptide according to the invention.
  • the invention further provides a process for the production of monoclonal or polyclonal antibodies to the encoded protein or to a peptide of the invention.
  • the antibodies used in the system of the invention may be monoclonal or polyclonal.
  • the term "antibody”, unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies.
  • the antibodies and fragments thereof may be humanised antibodies, eg. as described in EP-A- 239400.
  • the antibodies may be produced by conventional hybridoma techniques or, in the case of modified antibodies or fragments, by recombinant DNA technology, eg. by the expression in a suitable host vector of a DNA construct encoding the modified antibody or fragment operably linked to a promoter.
  • suitable host cells include bacterial (eg. E.coli ⁇ , yeast, insect and mammalian.
  • the present invention also provides pharmaceutical vector compositions containing as active ingredient a polynucleotide of the invention, a peptide of the invention, or an antibody or fragment thereof to the peptide, and a diluent.
  • compositions according to the invention may be administered to humans by any route appropriate to the condition to be treated, suitable routes including oral, rectal, nasal, topical (including buccal and sublingual) , vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) . It will be appreciated that the preferred route may vary with, for example, the condition, of the recipient.
  • a suitable, effective dose will be in the range 1 ⁇ g to lg per kilogram body weight of recipient per day, preferably in the range 10 ⁇ g to 100 mg per kilogram body weight per day and most preferably in the range 100 ⁇ g to 10 mg per kilogram body weight per day for example 200 ⁇ g, 400 ⁇ g 600 ⁇ g or 1 mg.
  • the dose may, if desired, be presented as two, three, four or more sub-doses administered at appropriate intervals throughout the day.
  • sub-doses may be administered in unit dosage forms, for example, containing 10 to 1000 mg, preferably 20 ⁇ g to 500 mg and most preferably 100 ⁇ g to 400 mg of active ingredient per unit dosage form. While it is possible for the compounds to be administered alone it is preferable to present them as pharmaceutical formulations.
  • the formulations of the present invention comprise at least one active ingredient, as above defined, together with one or more acceptable carriers thereof and optionally other therapeutic ingredients.
  • the carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipients thereof.
  • the formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual) , vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and .then, if necessary, shaping the product.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose) , lubricant, inert diluent, preservative, disintegrate (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) , surface-active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxpropylmethylcellulose in varying proportions to provide desired release profile.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacterios.tatis and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Injection solutions and suspensions may be prepared extemporaneously from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.
  • formulations of this invention may include other agents conventional in the art 5 having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • YACs pulsed field gel electrophoresis and yeast artificial chromosomes
  • the map shows the location of a CpG island 600kb distal to FRAXA (Bell et al,
  • Fluorescence in situ hybridisation (FISH) analysis indicates that FRAXE maps distal to FRAXA and proximal to DXS296 close to this CpG island (Sutherland and Baker, 1992; Flynn et al, 1993) .
  • Cosmid and phage clones from the contig were analyzed by FISH to metaphase chromosomes from patients expressing FRAXE. Of particular interest are the analyses with cosmid F9/8 and phage 2A3.3.
  • Panel (a) in Figure 2 shows the hybridisation of 2A3.3 to chromosomes from a normal individual and demonstrates positive hybridisation signals specific to Xq27-q28.
  • F9/8 maps consistently distal to FRAXE
  • 2A3.3 maps distal, proximal on the fragile site
  • FRAXE CpG island is shown in two FRAXE families ( Figure 3 and Figure 4).
  • Normal individuals have a 5.2kb Hind III fragment.
  • family 1 Figure 3
  • the phenotypically normal carrier females also have an increased fragment size but one which is smaller than that seen in the affected males and is not associated with fragility in those tested.
  • the size of the altered fragment increases when passed from the great- grandmother (individual 2) to her affected sons (individuals 4 and 10) and then decreases again when passed to their carrier daughters (individuals 6, 11 and 14).
  • This is the same family as described previously (Nakahori et al., 1991; Dennis et al., 1992). Whilst this study was carried out on a cell line established from lymphocytes, the mosaicism is still likely to be representative of his peripheral blood.
  • GCC GlobalCalculation Center
  • SEQ.ID.No. 10 the longest observed allele (GCC) 25 (SEQ.ID.No. 10) showed normal Mendelian inheritance and was stable in 6 meioses (data not shown) . No PCR products were observed in FRAXE positive males suggesting that these individuals have amplifications which are too large to amplify in this assay. Only a single allele in the normal range was observed in carrier females.
  • This method allows the detection of the normal array without the use of radiolabelled tracer.
  • PCR amplification across the FRAXE GCC trinucleotide array was carried out using primers 598 (SEQ.ID.No. 11) and 603 (SEQ.ID.No. 12) under the following conditions. PCR reactions were cycled through 35 cycles of 98°C, 15 seconds and 70°C, 10 minutes with each lO ⁇ l reaction contained lOOng genomic DNA, 20mM Tris-HCl (pH8.8), lOmM KCl, 1.5mM MgCl 2 , lO ⁇ m (NH 2 S0 4 , 0.1% Triton, lOO ⁇ gm/ml BSA, 0.5 ⁇ m each oligonucleotide, 200 ⁇ m dATP, 200 ⁇ m dTTP, 200 ⁇ M dGTP, 200 ⁇ M dCTP, 5% denaturing polyacrylamide gels. Products on agarose gels were visualised with .ethidium bromide and products resolved on denaturing polyacrylamide gels were visualised by silver
  • primers 598 and 603 are as follows: 598 5' GCG AGG AAG CGG CGG CAG TGG CAC TGG G 3'
  • the proximal (CA) m repeat regions were analysed by mean of PCR.
  • primer pair G8228 and G80682 give a product of 116bp corresponding to allele F of the polymorphism.
  • PCR reactions were carried out in a lO ⁇ l volume containing lOOng genomic DNA, SpMoles of each primer, 50nM Tris-HCl (pH ⁇ .O), 10mM KC1, 1.5mM MgCl 2 , 0.5 ⁇ M each oligonucleotide, 25 ⁇ M dATP, 200 ⁇ M dTTP, 200 ⁇ M dGTP, 200 ⁇ M dCTP, 2.5 ⁇ Ci alpha 35 S dATP and 0.5 units of 'Amplitaq' (Perkin Elmer Cetus) . PCR reactions are cycled through 1 cycle of 95 C for 3 minutes, 35 cycles of 95°C, l minute, 64 C 1 minute, 72 C 1 minute and a final extension of 72°C, 4 minutes.
  • Amplification products were then resolved on a 6% denaturing polyacrylamide gel for 2 hrs at 65W. Dried gels were exposed to X-ray film overnight or until the (CA)n alleles were clearly visible.
  • the proximal (CA) m repeat lies within a 13kb EcoRI fragment and is less than 20kb from the FRAXE CpG island. In a sample of 100 normal unrelated X chromosomes of Caucasian origin, the allele frequencies of this repeat and the allele sizes are as given in Table 1. Table 1
  • Allele I (104bp) 28%
  • the distal CA repeat (DXS1691) lies within a 1.5kb Hind III fragment and is less than 5kb from the FRAXE CpG island.
  • the allele frequencies of this repeat for primer pair F0322 and FOlO are as given in Table 2
  • primer pair FOlO and F332 give a product of 102bp corresponding to allele C of the polymorphism.
  • PCR reactions were carried out as described above for the proximal (CA) m repeat.
  • YACs were grown in YPD broth (1% yeast extract, 2% peptone, 10% glucose, pH5.8). Yeast DNA was prepared and BssHII, SacII and Not I digested DNA was subjected to restriction mapping with pYAC4 vector arm probes as described previously (Hirst et al., 1991a) .
  • Yeast DNA was prepared in low melting point agarose using standard protocols and partially digested with Mbol (NEB) for 5-15 mins at 37°C. Sticky ends were partially filled with Klenow polymerase and dGTP/dATP and the DNA precipitated after digestion with GELASE (Cambio) . These fragments were ligated to lambda-Gemll-XhoI half site arms (Promega) , packaged (Gigapack II XL, Stratagene) and plated at high density using LE392 host cells. Duplicate filter lifts were probed with Cot-1 human DNA and pYAC4. Phage DNA was prepared by liquid lysate method (Sambrook et al.
  • YAC derived phage clones were radiolabelled (Feinberg and Vogelstein, 1983) and used to screen cosmid library grids after pre-annealing with 200ngm of sonicated human DNA to remove repetitive elements.
  • Hybridisation against filters from the ICRF Reference Library (Lehrach et al., 1990) and the 5X-L cosmid library (Holland et al., 1993) was carried out at 65°C in 0.5M sodium phosphate pH 7.4 , 7% SDS, 5% dextran sulphate overnight.
  • Cosmid H,7R (ICRFcl04G0875) , D7/13 (5X-L:269/D7) and F9/8 (5X-L:424/F9) were identified and DNA prepared by the alkaline lysis method (Sambrook et al., 1989).
  • Human Cot-1 competitor DNA (BRL) was used at 500ng/ ⁇ l for F9/8 and lOOng/ ⁇ l for 2A3.3.
  • Hybridisation mix (lO ⁇ l) containing probe and" competitor DNA was denatured and preannealed for 15 mins at 37°C, then placed on slides overnight at 42°C. Slides were then washed to 0.1 X SSC at 65°C without formamide.
  • Biotinylated probes were detected by alternate layers of fluorescein conjugated avidin (DCS, Vector) and biotinylated anti-avidin antibody (Vector) both diluted to 5 ⁇ g/ml in 4 X SSC with 5% non-fat dried milk.
  • DNA extracted from blood samples or EBV transformed cell lines by standard techniques was digested with restriction endonucleases Hind III, Hpall, Notl, Eagl, SacII or BssHII (Boehringer) , electrophoresed on 1% agarose gels, and after denaturation and neutralisation was transferred to Hybond N membranes (Amersham) in 10X SSC. Hybridisation conditions were as described for cosmid library screening.
  • Oligonucleotides 598 (5'- GCG AGG AAG CGG CGG CAG TGG CAC TGG G -3') (SEQ.ID.No. 11) and 603 (5'- CCT GTG AGT GTG TAA GTG TGT GAT GCT GCC G -3') (SEQ.ID.No. 12) were derived from sequences flanking the GCC repeat were used as primers to amplify genomic DNA from normal individuals and from patients.
  • Amplification was carried out with 200ngm genomic DNA in a reaction containing 50mM KCL, lOmM Tris.HCl pH8.4, 1.5mM MgCl 2 , 200 ⁇ dATP, 200 ⁇ dTTP, 200 ⁇ M dCTP, 50 ⁇ M dGTP, 150 ⁇ M 7-deaza-dGTP, 1.5 ⁇ Ci ⁇ -[ 32 P]-dCTP (Amer ⁇ ham) , 10% DMSO, 0.5 units Amplitaq (Perkin Elmer) and 0.5 ⁇ M of oligonucleotides 598 and 603 in a lO ⁇ l reaction volume.
  • each reaction was overlayed with mineral oil and cycled through 95°C, 10 mins; 65°C, 1 min; 72°C, 2 mins followed by 29 cycles of 95°C, 1 min 30 sees; 65°C, 1 min; 72°C, 2 mins.
  • 0.5 ⁇ M of each primer for the marker M125 (Bell et al, 1991) were included in the reaction (M125F 5'-CGCTGACCAAACCAAAATGAAGAG (SEQ.ID.No. 17), 125R 5'-ACCTTGAGGGGTAGTTCTATCTAA (SEQ.ID.No. 18)) .
  • 4 ⁇ l of each reaction was mixed with formamide loading buffer, heated for 5 minutes at 95°C and the products seperated by electrophoresis through a 5% denaturing polyacrylamide gel
  • MOLECULE TYPE DNA (genomic)
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Abstract

Polynucléotide sélectionné à partir de fragments d'acide nucléiq ue (i) à (vii), sections de polynucléotides desdits fragments et acides nucléiques pouvant s'hybrider auxdits fragments; chacun desdits fragments d'acide nucléique (i) à (vii) peut s'hybrider à au moins une partie de la région FRAXE du chromosome humain, ladite région FRAXE comprenant une répétition proximale (CA)m où m est un entier positif, une répétition distale (CA)n où n est un entier positif, et un ilôt CpG intermédiaire et une répétition (GCC)p, où p est au moins 3 et où lesdits fragments d'acide nucléique (i) à (vii) sont définis comme suit: (i) un fragment Hind III, Hind IIIa, s'hybridant à la partie de la région FRAXE du chromosome X comprenant la répétition de GCC comme l'illustre SEG. ID. No. 1; (ii) un fragment EcoRI, EcoRIa, s'hybridant à la partie de la région FRAXE du chromosome humain comprenant le répétition de GCC, comme l'illustre SEQ. ID. No. 1; (iii) un fragment EcoRI, EcoRIb, s'hybridant à la partie de la région FRAXE du chromosome X humain distale au fragment Hind IIIa, et chevauchant celui-ci, défini à (i) ci-dessus; (iv) un fragment EcoRI, EcoRIc, s'hybridant à la partie de la région FRAXE du chromosome X humain comprenant la répétition proximale (CA)m; (v) un fragment Hind III, Hind IIIb, s'hybridant à la partie de la région FRAXE du chromosome X comprenant la répétition distale (CA)n; (vi) un fragment OxE 20, comprenant les bases 1116 à 1974 de SEQ. ID. No. 1; (vii) un fragment OxE 18 délimité par un site EcoRI au niveau de sa limite proximale et par un site Not1 au niveau de sa limite distale et qui s'hybride à un site à l'intérieur de la région FRAXE du chromosome X humain chevauchant l'ilôt CpG et proximale à celui-ci; lesdits fragments d'acide nucléique (i) - (vii) étant sensiblement exempts d'autres fragments d'acide nucléique.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998056950A1 (fr) * 1997-06-11 1998-12-17 Fondation Jean Dausset-Ceph Sequences d'adn riches en triplet repete utiles dans le diagnostic de maladies a repetition trinucleotidique
US5876949A (en) * 1995-05-31 1999-03-02 The Trustees Of The University Of Pennsylvania Antibodies specific for fragile X related proteins and method of using the same
DE19935303A1 (de) * 1999-07-28 2001-02-08 Aventis Pharma Gmbh Oligonukleotide zur Inhibierung der Expression von humanem eg5
DE10019058A1 (de) * 2000-04-06 2001-12-20 Epigenomics Ag Detektion von Variationen des DNA-Methylierungsprofils

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992012262A1 (fr) * 1991-01-04 1992-07-23 Washington University Sequences d'adn associees au syndrome de chromosome x fragile isole
WO1992020825A1 (fr) * 1991-05-24 1992-11-26 Baylor College Of Medicine Diagnostic du syndrome du chromosome x fragile

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992012262A1 (fr) * 1991-01-04 1992-07-23 Washington University Sequences d'adn associees au syndrome de chromosome x fragile isole
WO1992020825A1 (fr) * 1991-05-24 1992-11-26 Baylor College Of Medicine Diagnostic du syndrome du chromosome x fragile

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Title
HIRST ET AL., HUMAN MOLECULAR GENETICS, vol. 2, no. 2, February 1993 (1993-02-01), OXFORD, UK, pages 197 - 200 *
KNIGHT ET AL.: "Trinucleotide repeat amplification and hypermethylathion of a CpG islands in FRAXE mental retardation", CELL, vol. 74, 16 July 1993 (1993-07-16), CAMBRIDGE, NA US, pages 127 - 134 *
WANG ET AL.: "cytogenetic versus DNA diagnosis in routine referrals for fragile X syndrome", LANCET THE, vol. 342, 23 October 1993 (1993-10-23), LONDON GB, pages 1025 - 1026 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5876949A (en) * 1995-05-31 1999-03-02 The Trustees Of The University Of Pennsylvania Antibodies specific for fragile X related proteins and method of using the same
WO1998056950A1 (fr) * 1997-06-11 1998-12-17 Fondation Jean Dausset-Ceph Sequences d'adn riches en triplet repete utiles dans le diagnostic de maladies a repetition trinucleotidique
FR2764611A1 (fr) * 1997-06-11 1998-12-18 Fondation Jean Dausset Ceph Sequences d'adn riches en triplet repete utiles dans le diagnostic de maladies a repetition trinucleotidique
DE19935303A1 (de) * 1999-07-28 2001-02-08 Aventis Pharma Gmbh Oligonukleotide zur Inhibierung der Expression von humanem eg5
DE10019058A1 (de) * 2000-04-06 2001-12-20 Epigenomics Ag Detektion von Variationen des DNA-Methylierungsprofils

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