EP0413003A1 - Diagnostic method - Google Patents
Diagnostic methodInfo
- Publication number
- EP0413003A1 EP0413003A1 EP89909007A EP89909007A EP0413003A1 EP 0413003 A1 EP0413003 A1 EP 0413003A1 EP 89909007 A EP89909007 A EP 89909007A EP 89909007 A EP89909007 A EP 89909007A EP 0413003 A1 EP0413003 A1 EP 0413003A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- schizophrenia
- individual
- detect
- kit
- dna sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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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
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
-
- 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
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/172—Haplotypes
Definitions
- This invention relates to a diagnostic method; more particularly, it relates to a method for the diagnosis of individuals suffering from or liable to suffer from schizophrenia.
- Schizophrenia is a common disorder with a lifetime prevalence of 0.85%.
- the illness develops in early adult life and is characterised by psychotic symptoms which include hallucinations and delusions and a constellation of symptoms, such as inappropriate emotional responses, a disorder of thinking, erratic behaviour, as well as social and occupational deterioration. Patients often relapse into a psychotic state when treatments with anti-psychotic drugs are stopped.
- the present inventors have found scientific evidence to show that a single chromosome locus is involved in aetiology of schizophrenia.
- the present inventors have shown that the inheritance of a dominant allele at a single major locus on chromosome 5 is sufficient for the development of schizophrenia.
- schizophrenia is associated with an abnormality on chromosome 5.
- the chromosome 5 schizophrenia locus lies within the cytogenic bands 5gll-5gl3, when defined in terms of cytogenic banding, or, when defined in terms of genetic distances, the schizophrenic locus lies on chromosome 5 at a recombination fraction of between 0.00% and 7% from DNA marker p105-153Ra (D5S39) and 10% from p105-599Ha (D5S76).
- the present invention relates to a method of detection of carrier status for schizophrenia which involves the analysis of a DNA sample using a probe, more particularly one of the four probes (human genomic clones), M4, p105-153Ra, p105-599Ha and PSY5.1.
- probes may be labelled radioactively or non-radioactively by known means.
- the analysis methods to be used for the present purpose detect single base pair mutations, deletions and polymorphisms at or near the chromosome 5 schizophrenia susceptibility locus using (non) radioactive labelling.
- DNA from a human individual may be extracted from blood or other suitable human tissue according to standard protocols (Hodgkinson, S., et al. Nature, 325, 805-806, 1987).
- Amplification of specific genomic regions from patients or their relatives within or linked to in particular the four probes M4, p105-153Ra, p105-599Ha and PSY5.1, as well as at other loci at or closely linked to the chromosome 5 schizophrenia susceptibility locus may be carried out by the use of pairs of oligonucleotide sequences obtained from the probes and the use of a polymerase chain reaction (Saiki, R.K., Science, 239, 487-491, 1988).
- the two oligonucleotide sequences flank the DNA segments to be amplified and are used as primers for Taql polymerase or Klenow extension of the annealed primers.
- Primer oligonucleotide sequences that may be used include:
- Probe M4 which was found by the inventors to be genetically linked to the chromosome 5 schizophrenia locus, was obtained from Dr. A. Retief, University of Whybosch, South Africa.
- the probe PSY5.1 was obtained by screening a human phage library with the clone p105-153Ra and selecting clones that contained at least a fraction of the original sequence. This has enabled further polymorphisms to be detected at this locus.
- Other primers derived from the clones M4 and PSY5.1 may be obtained by known means. These primers hybridise to opposite strands of the target sequence and are oriented so that DNA synthesis by the polymerase proceeds across the region between the two primers. Amplification of intervening and flanking sequences of the oligonucleotide primers may produce sufficient quantities of DNA for the following techniques.
- Restriction fragment length polymorphisms may be identified in the amplified products by using restriction enzymes to cleave the amplified DNA and separating the resultant digested DNA by size on an agarose gel. The relative position and frequency of fragments provide information as to whether disease or normal alleles have been inherited.
- base pair polymorphisms in the amplified products found by DNA sequencing amplification of the sequences at or near the schizophrenia susceptibility gene is carried out as described above with the additional step that direct sequencing techniques are applied.
- a sequencing primer complimentary to the amplified DNA enables the chain termination method to be used (Sanger, F, et al., Proc. Nat. Acad. Sci., USA, 74, 5463-5467, 1977).
- the number and position of base pair polymorphisms or substitutions may be used to indicate carrier status as described by Wong, C, et al, Nature, 330, 384-386, 1987.
- CA, CT, (GATA) n and (GACA) n repeat sequence polymorphisms may be found in the amplified products from patients or their relatives and also used to predict the carrier status. In this approach, sequences at or near the schizophrenia locus which possess dinucleotide repeat sequences exhibiting polymorphism are observed. They may be detected by the use of clones derived from M4, p105-153Ra, p105-599Ha and PSY5.1 by the screening of gene libraries. In addition, other plasmid, phage or cosmid clones from chromosome 5q11-q13 containing these specific repeat sequences can be identified and then screened using radioactive repeat probes for the presence of repeat sequences.
- the products may be separated in size on a 6% polyacrylamide gel, the gel may be dried and the alleles detected by autoradiography (Litt, N, and Jeffrey, A, Am. J. Hum. Genet., 44, 397-401, 1989).
- Deletions in genomic DNA from patients and their relatives may be identified from the use of oligonucleotide primers that span either side of a deletion related to carrier status.
- the use of PCR will produce a shorter length product than that of the normal genomic sequence. Detection of this shorter product may be accomplished by agarose gel electophoresis and ethidium bromide fluorescent staining.
- Allele specific oligonucleotide probes may be used to detect base pair polymorphisms or deletions linked to or at the schizophrenia susceptibility gene both in genomic DNA and in PCR amplified products. Allele specific oligonucleotides have single base pair mismatches between normal and affected alleles.
- the dot blot system of hybridization, autoradiography and allele detection may be employed as described by Engelhe et al, Proc. Nat. Acad. Sci., USA, 544, 1988.
- RNA probes produced by suitable cloning vectors may be used in Southern hybridization experiments and denaturing gel electrophoresis may be used to detect polymorphisms at or closely linked to the schizophrenia susceptibility locus.
- Denaturing gel electrophoresis may be used to identify base pair or other polymorphisms or mutations at or closely linked to the schizophrenia susceptibility locus (Myers, R.M., et al. Nature, 313, 495-498, 1985).
- DNA from the patient and other affected and unaffected relatives is extracted from blood, for example, digested, made single stranded and reannealed to probe RNA or DNA which has also been made single stranded.
- the resultant heteroduplexes may be separated electrophoretically in a formamide denaturing gradient gel and the relative position of heteroduplexes versus homoduplexes provides information as to which alleles an individual has inherited.
- the ligase mediated gene detection system may also be used to identify carriers.
- two oligonucleotide probes are hybridised to the denatured target DNA sampled from patients or their relatives so that the 3' end of one oligonucleotide is immediately adjacent to the 5' end of the other.
- the DNA ligase may then join the two juxtaposed oligonucleotides by the formation of a phosphodiester bond, provided that the nucleotides at the junction are correctly base paired with the target strand.
- the ligation event thus positively identifies the sequences complementary to the two nucleotides.
- a heterozygous sample is therefore scored as positive for both alleles.
- oligonucleotides may be demonstrated by labelling one of the oligonucleotides with biotin and the other with 32 P, for example. After ligation, the biotinylated oligonucleotides are bound to streptavidin immobilised on a solid support. Radioactive oligonucleotides that have been ligated remain on the support after washing and may be autoradiographed (Landegren, U., et al, Science, 241, 1077-1080, 1988).
- Polymorphisms or mutations at or near the chromosome 5 schizophrenia susceptibitlity locus may be identified by use of the "arms" method of identification or the 'COP' method (competitive oligonucleotide primers) (Gibbs, et al, (1989) Nucleic acids research, 17; and Newton, C.R. et al , (1989), Nucleic acids research, 17, 2503).
- an allele specific oligonucleotide with one or more base pair mismatches compared to the wild type, together with a second primer without a mismatch are used to amplify the intervening DNA.
- no amplified products will be detectable at autoradiography or with non-radioactive labelling or on agarose gel electrophoresis.
- RNAse protection system may also be used to detect mutations and polymorphisms related to schizophrenia.
- mismatches between probe RNA and genomic sequence are detected according to the method of Myers, R. (1985b) Science, 230, 1242.
- Various techniques of such DNA analysis to provide the desired indication are known and some have been exemplified. The presently-preferred approach will be illustrated in more detail below.
- the present invention provides a method for the analysis of a genomic DNA sample taken from a human individual to detect a genetic abnormality predisposing or causing the said individual or a related individual to suffer from schizophrenia, which method comprises analysing the said genomic DNA sample involving the use of one or more DNA probes which are human genomic clones to detect the presence of a polymorphism or a mutation indicative of an allele linked to or associated with the disease allele for schizophrenia.
- the present invention relates to such a method which comprises digesting the said genomic DNA sample with a suitable restriction enzyme, probing the restriction fragments with one or more DNA probes which hybridise to chromosome 5q11-5q13 region and investigating the length of the restriction fragments hybridising to the probe or probes to detect the presence of a restriction fragment polymorph indicative of an allele linked to or associated with the disease allele for schizophrenia.
- a suitable restriction enzyme for digesting the said genomic DNA sample with a suitable restriction enzyme, probing the restriction fragments with one or more DNA probes which hybridise to chromosome 5q11-5q13 region and investigating the length of the restriction fragments hybridising to the probe or probes to detect the presence of a restriction fragment polymorph indicative of an allele linked to or associated with the disease allele for schizophrenia.
- Specific clones which may be used as probes in such methodology include M4, p105-599Ha, p105-153Ra or PSY5.1.
- the genomic DNA sample may be digested with Taql, Xbal
- Southern blots of the genomic DNA sample which may be extracted from a blood sample or a buccal smear, and which have been digested by the restriction enzyme, are hybridised to the probe.
- the above method may be practiced using a kit. Such a kit will always include the necessary probe(s), but the other necessary reagent(s) will vary depending upon the analysis approach to be adopted.
- the present invention also provides a kit for use in the analysis of a genomic DNA sample taken from a human individual to detect a genetic abnormality predisposing or causing the said individual or related individual to suffer from schizophrenia by analysing the said DNA sample involving the use of one or more DNA probes which are human genomic clones to detect the presence of a polymorph or mutation indicative of an allele linked to or associated with the disease allele for schizophrenia, the said kit comprising at least one DNA probe which is a human genomic clone and at least one other reagent suitable for use in the method.
- the presently-preferred kit functions by hybridising the said DNA sample, following treatment with a restriction enzyme, to one or more DNA probes and investigating the length of the restriction fragments hybridising to the probe or probes to detect the presence of a restriction fragment polymorph indicative of an allele linked to or associated with the disease allele for schizophrenia, the said kit comprising at least one labelled DNA probe which hybridises to chromosome 5q11-5q13 region and at least one other reagent suitable for use in the method, in particular a restriction enzyme, such as Taql, Xbal or Mspl.
- the DNA probes are generally those mentioned above, which may be radioactively labelled or non-radioactively labelled.
- the precise means by which the method according to the invention is applied to an individual will depend on the probe which is available and, in particular, how close the site of hybridization of the probe is to the site of the schizophrenia mutation. In the case of a probe which hybridises to a site linked to the site of the schizophrenia mutation, it will not be possible to diagnose with certainty the presence of the schizophrenia mutation in an individual. As well as applying the method of the invention to the individual, it will also be necessary to obtain similar data from parents and/or other relatives which will enable a prediction to be made with a reasonable degree of certainty as to whether the individual carries the schizophrenia mutation. A similar method can also be applied in genetic counseling.
- Examples of gene probes which can be used in the manner described above are clones p105-599 Ha and p105-153 Ra. These clones are random genomic DNA clones mapping to the 5q11-5q13 region of human chromosomes and were obtained from Dr. John Wasmuth, University of California at Irvine, USA. Use of these clones to probe Southern blots of genomic DNA digested with Taq I and Msp I, respectively, can be used to detect individuals likely to carry the schizophrenia mutation by means of restriction fragment length polymorphism. Further details of the use of these probes in a particular study are given below. Other gene probes mapping to the same region of the human chromosomes can be used in a similar way.
- Gene probes associated with schizophrenia can be found by creating gene libraries that originate from the area of chromosome referred to herein which has been shown to be involved in schizophrenia (5q11-5q13). These libraries will be screened to select clones (probes) that show zero recombination fraction with schizophrenia and are therefore very close in terms of genetic distance. This will manifest itself as an association between schizophrenia and restriction fragment length polymorphisms that are found with the chromosome 5 region specific clones.
- probes will enable the method according to the invention to be used to detect the schizophrenia mutant in individuals without any need for data to be obtained from relatives.
- the present invention also provides a kit for use in the method described above.
- a DNA sample must be obtained from the patient.
- This DNA sample may be extracted from a blood sample or buccal smear and is digested with a suitable restriction enzyme.
- the resultant fragments will be separated by size, for example, using gel electrophoresis and transferred to a nylon filter. The fragments may then be examined using the kit.
- the kit according to the invention comprises DNA corresponding to the nucleic acid sequence identified as being polymorphic and found to be in association with schizophrenia.
- This DNA sequence will be labelled in a suitable manner, for example using a radioactive or biotin label, and hybridised with the genomic DNA from the patient. The size of fragments present will indicate which alleles of the schizophrenia linkage marker system are present. The results may be interpreted as a probability that the chromosome 5 schizophrenia mutation is present or absent.
- the present invention is based on work using the probes p105-599 Ha and p105-153 Ra referred to above and a summary of this work is as follows.
- DSM-III and RDC schizotypal features
- DSM-III paranoid or schizoid personality disorders
- Blood was taken for blood group and serum marker determinations, for DNA extraction and for the establishment of lymphoblastoid cell lines. standard methods for DNA extraction and restriction mapping were employed (Hodgkinson et al, loc cit). Paternity was confirmed using a combination of blood group markers and minisatellite DNA finger prints (Gurling et al, A Genetic Perspective for Schizophrenia and Related Disorder, Eds. Smeraldi and Kidd, Edi-Hermes, Milan, (in press 1988) and Jeffreys gt al. Nature, 314, 67-73, (1985)).
- the random genomic DNA clones (p105-599 Ha and P105-153 Ra) mapping to the 5q11-5q13 region were used to probe Southern blots to detect restriction fragment length polymorphisms segregating in the effective kindreds.
- the lod score method was used to calculate the probability of linkage between marker and disease alleles at specific recombination fractions (genetic distances) as opposed to non-linkage (50% recombination). These were calculated by the LINKAGE programs.
- a quantitative expression for the likelihood of linkage between loci is given as the lod score which is the log to the base 10 of the relative probability of observing the family at a recombinant fraction of less than 0.5 (i.e. the marker is linked to the disease locus) to the probability of the recombinant fraction being 0.5 (i.e. no linkage).
- Lod scores that are higher than 3 represent the likelihood that linkage has occurred between the marker and disease alleles as opposed to non-linkage at a value of about 50:1 taking prior probability into account.
- a lod score of 3 or higher is considered good evidence for linkage.
- Figures 1 and 2 demonstrate families showing strong evidence of linkage.
- the mean age of onset of schizophrenia was twenty-eight years (SD +- 9.3) and there were three individuals who developed schizophrenia after the age of thirty. Because almost all of the unaffected individuals were past the age of risk, it was not possible to carry out an analysis with penetrance set according to the age of the unaffected individuals.
- a more detailed analysis of penetrance (Table 4) showed that the maximum lod scores did not vary very much for penetrance values for schizophrenia of between 95% and 65%.
- Figure 2 shows the pedigree diagram of F20 which was informative for both markers and shows linkage when the markers are combined into haplotypes.
- the chromosome 5 schizophrenia susceptibility gene may be followed as it is transmitted from parents to children within specific families with the use of a test kit for the schizophrenia locus.
- This consists of the DNA probes p105-153Ra and PSY5.1 at a recombination fraction (genetic distance) of between 0.00 and 0.07 (0.00% and 0.07%) from the schizophrenia locus.
- recombination probe p105-599Ha is linked to schizophrenia on the centromeric side of p105-153Ra.
- the test consists of the hybridization of the probes, after they have been made radioactive with 32 P to genomic DNA extracted from patients and their family members and digested with the relevant enzymes (Taql, Xbal, Mspl).
- MODEL PF Robust, "penetrance free” model. Linkage information between marker and disease obtained only from schizophrenic and spectrum cases, unaffected cases scored as unknown at the schizophrenia locus.
- MODEL DOMS Assuming dominant inheritance and complete penetrance schizophrenia cases scored as affected, schizophrenia spectrum cases excluded. Unaffected obligate carriers scored as unknown at the disease locus.
- MODEL INCSS Penetrance for schizophrenia genotype under a dominant model with incomplete penetrance varied from 95% to 50%. Individuals with schizophrenia and schizophrenia spectrum disorders given positive affection status.
- MODEL DOMSS Assuming dominant inheritance and complete penetrance. Schizophrenia and spectrum cases scored as affected. Unaffected obligate carriers scored as unknown at the disease locus.
Abstract
On a mis au point une technique d'analyse d'un échantillon d'ADN génomique prélevé sur un sujet humain afin de détecter une anomalie génétique prédisposant ledit individu ou un individu apparenté à souffrir de schizophrénie. Le procédé consiste en l'analyse dudit échantillon d'ADN génonique par l'utilisation d'une ou de plusieurs sondes d'ADN qui sont des clones génomiques humains afin de détecter la présence d'un polymorphe ou mutation indicatrice d'un allèle lié ou associé à l'allèle de la maladie. On a également mis au point un kit de mise en pratique de ladite technique.A technique has been developed for analyzing a genomic DNA sample taken from a human subject in order to detect a genetic abnormality predisposing said individual or a related individual to suffer from schizophrenia. The method consists in analyzing said gene DNA sample by the use of one or more DNA probes which are human genomic clones in order to detect the presence of a polymorph or mutation indicative of a linked allele or associated with the disease allele. We have also developed a kit to practice this technique.
Description
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GB888818076A GB8818076D0 (en) | 1988-07-29 | 1988-07-29 | Diagnostic method |
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WO1990001561A1 (en) | 1990-02-22 |
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