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WO1998007884A1 - Procede permettant de diagnostiquer des maladies recessives autosomiques et agents utiles de diagnostic - Google Patents

Procede permettant de diagnostiquer des maladies recessives autosomiques et agents utiles de diagnostic Download PDF

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Publication number
WO1998007884A1
WO1998007884A1 PCT/AU1997/000539 AU9700539W WO9807884A1 WO 1998007884 A1 WO1998007884 A1 WO 1998007884A1 AU 9700539 W AU9700539 W AU 9700539W WO 9807884 A1 WO9807884 A1 WO 9807884A1
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Prior art keywords
mutation
homozygous
individual
gene
disorder
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PCT/AU1997/000539
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English (en)
Inventor
Lawrie William Powell
Elizabeth Catherine Jazwinska
Lara Michelle Cullen
Frances Busfield
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QIMR Berghofer Medical Research Institute
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Queensland Institute of Medical Research QIMR
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Priority claimed from AUPO1849A external-priority patent/AUPO184996A0/en
Priority claimed from AUPO2083A external-priority patent/AUPO208396A0/en
Application filed by Queensland Institute of Medical Research QIMR filed Critical Queensland Institute of Medical Research QIMR
Priority to AU38417/97A priority Critical patent/AU3841797A/en
Publication of WO1998007884A1 publication Critical patent/WO1998007884A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12Q1/6813—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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational 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
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates generally to a method of diagnosing an autosomal recessive disorder or determining the risk of developing such a disorder and to agents useful for same. More particularly, the present invention contemplates a method of diagnosing an autosomal recessive disorder of iron metabolism resultant from or associated with two copies of an abnormal gene (homozygous abnormal). Even more particularly, the present invention contemplates a method of identifying an individual who has developed or who has a propensity to develop hereditary haemochromatosis or an individual who carries one abnormal gene (heterozygote) or an individual who carries no abnormal gene (homozygous normal). The present invention further provides a rapid diagnostic assay for hereditary haemochromatosis providing a high degree of accuracy in diagnosing the disease condition or a propensity to develop or pass on the hereditary disease.
  • a recessive disorder generally results from one or more mutations in a critical gene.
  • the recessive nature of the disorder generally means that the mutation is in a homozygous form for symptoms to develop.
  • heterozygous mutations may result in disease progression where other mutations have occurred as well as other diagnostic difficulties. It is particularly important, therefore, to determine the requirement for homozygosity in diagnosing and treating many of these disease conditions as well as to determine the requirement for diagnosing a heterozygous state (one abnormal gene) or a homozygous normal state (no abnormal gene), particularly within families.
  • HH hereditary haemochromatosis
  • GH genetic haemochromatosis
  • the condition can be treated by a therapeutic regime to remove excess iron provided that therapeutic phlebotomy is instituted early in the course of the disease (6,7).
  • a therapeutic regime to remove excess iron provided that therapeutic phlebotomy is instituted early in the course of the disease (6,7).
  • techniques to identify development of the disease such as following clinical evidence of the disease or to identify individuals with the propensity to develop the disease.
  • the gene subject to mutation in HH development is referred to herein as the "HC" gene and corresponds to GenBank Accession No. U60319 (8).
  • the HC gene has also been referred to as "HLA-H" (8).
  • one difficulty in achieving a suitable diagnostic procedure is the uncertainty as to the nature of the mutation occurring in the HC gene leading to the disease.
  • the inventors genetically and clinically examined HH families.
  • the inventors have identified a method of diagnosing a homozygous mutation in the HC gene which is indicative that an individual with that mutation has or will develop HH as well as a method of detecting a heterozygous mutation (one abnormal gene) or a homozygous normal individual (no abnormal gene) for this disease.
  • one aspect of the present invention contemplates a method of identifying an individual with an autosomal recessive disorder or a predisposition to develop said disorder or to genetically pass on a predisposition to develop such a disorder to an offspring, said method comprising isolating a biological sample from said individual and screening genomic DNA in said biological sample for the presence of a homozygous or heterozygous mutation in a gene, the normal function of which, is required to prevent progression of the disorder.
  • Another aspect of the present invention contemplates a method of identifying an individual with one copy (heterozygous), two copies (homozygous) or zero copies (homozygous normal) of a mutation in a gene controlling or associated with an autosomal recessive disorder, said method comprising isolating a biological sample from said individual and screening genomic DNA is said sample for the presence of a mutation in said gene.
  • the present invention is directed to a method of identifying an individual with HH or a predisposition to develop HH or to genetically pass on HH to an offspring, said method comprising isolating a biological sample and amplifying a region of genomic DNA in said biological sample encompassing all or part of the DNA between D6S265 and D6S276, and detecting a mutation in at least one homozygous or heterozygous mutation in a nucleotide within said region.
  • the present invention is directed to a method of identifying an individual with HH or a predisposition to develop HH, or a potential carrier (heterozygotes) of HH or a homozygous normal individual, said method comprising isolating a biological sample from said individual screening genomic DNA in said biological sample for the presence of a homozygous mutation or its equivalent in HC at amino acid position 282 of HC or heterozygous (one mutation) or homozygous normal (zero mutation).
  • Another aspect of the present invention provides a method of identifying the risk of an offspring of two parents of developing HH, said method comprising detecting the presence of a homozygous or heterozygous mutation or its equivalent in genomic DNA from said two parents at amino acid position 282 of HC wherein if both parents are homozygous for said mutation then the offspring will develop HH or when the parents are both heterozygous for the mutation, the offspring has approximately a 25% chance of developing HH.
  • HC is considered in accordance with the present invention to control the development of HH. It has now been determined that a homozygous mutation at amino acid 282 (Cys) in association with the development of HH.
  • the mutation is preferably a substitution mutation from Cys to Tyr and is referred to herein as a "Cys282Tyr" mutation.
  • Cys282Tyr A mutation and in particular a homozygous Cys282Tyr mutation in HC results in development of HH.
  • the HC gene is located between the D6S265 which is near HLA-A and the more telomeric marker D6S276.
  • the marker order (and ancestral alleles in parentheses) is considered to be D6S265 (1), HLA-A (3), HLA-F (2), D6S258 (4), D6S306 (3), D6S105 (8), D6S464 (6) and D6S1260 (4) [see Table 1 and ref 13].
  • the HC gene is GenBank Accession No. U60319 (8).
  • the Cys282Tyr mutation may be analysed in any of a number of ways including PCPv analysis and antibody binding patterns.
  • the HC region is subjected to PCR using the method and primers of Feder et al. (8) or primers homologous to gene sequences encoding HLA class I-like molecules and then detecting the Cys282Tyr missense mutation by, for example, first nucleotide change analysis [FNC] (see International Patent Application Publication No. WO 90/09455) or restriction enzyme cleavage.
  • FNC relies on an allele specific extension of a mutation detecting an oligonucleotide abutting the mutant base.
  • the oligonucleotide is generally labelled with a reporter molecule capable of giving an identifiable signal such as a radioisotope, chemiluminesce molecule or a fluorescent molecule.
  • a particularly useful reporter molecule involves biotinylation.
  • Another useful detection system involves antibodies directed to the mutant allele or to the Cys282Tyr mutation itself.
  • the presence of the Cys282Tyr mutation or its equivalent or closely linked marker may be accomplished immunologically in a number of ways such as by Western blotting and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653. These, of course, include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
  • Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-mutation target complex, a second antibody specific to the mutation, labelled with a reporter molecule capable of producing a detectable signal, is then added and incubated, allowing time sufficient for the formation of another complex of antibody-mutation target-labelled antibody.
  • any unreacted material is washed away, and the presence of the mutation is determined by observation of a signal produced by the reporter molecule.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.
  • Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody.
  • a first antibody having specificity for the Cys282Tyr mutation or mutant allele or antigenic parts thereof or linked marker/mutation is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.
  • an aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes) and under suitable conditions (e.g. 25 °C) to allow binding of any subunit present in the antibody.
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.
  • a second labelled antibody specific to the first antibody is exposed to the target- first antibody complex to form a target- first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen- bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta- galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • a particularly preferred method is a rapid diagnostic assay using sequence specific oligonucleotides to determine the presence or absence of a single nucleotide substitution, addition and or deletion.
  • genomic DNA around the HC gene region is amplified, such as in a microtitre tray well.
  • the amplified DNA is then transferred to another solid support such as a membrane, gel or other suitable matrix and subjected to hybridization under specific stringency conditions using one of at least two oligonucleotides where one spans the normal, ie.
  • a homozygous mutation at amino acid position 282 or its functional equivalent will result in substantially no hybridisation of the normal probe but high hybridisation with the mutant probe.
  • a homozygous normal nucleotide sequence will permit hybridisation of the normal probe but not the mutant 5 probe.
  • a heterozygous mutation will result in hybridisation by both probes. Hybridisation is readily detected by any number of procedures such as, but not limited to, the use of radioactive isotopes, chemiluminescence and/or immunologically-based detection systems.
  • said method comprising isolating a biological sample from said individual, amplifying genomic DNA in said biological sample in a region of DNA between D6S265 and D6S276 and transferring multiple aliquots of said amplified DNA to a solid support and contacting one of said aliquots with an oligonucleotide encompassing a non-mutated HC region and contacting another of said
  • the mutated oligonucleotide is designed with the mismatch in the middle of the molecule and the hybridisation washing and annealing conditions are designed that hybridisation requires a perfect match.
  • This aspect of the present invention extends to mismatches of more than one oligonucleotide such as from two to five nucleotide mismatches.
  • the methods according to the present invention are particularly efficacious in detecting individuals homozygous normal or abnormal as well as heterozygous individuals.
  • greater than about 80% of homozygous abnormal individuals may be detected by the methods of the present invention, preferably, greater than about 85%, even more preferably, greater then about
  • individuals with HH or having a predisposition to develop HH are homozygous for Cys282Tyr in HC or its equivalent.
  • Individuals heterozygous for Cys282Tyr do not develop the disease but can be detected by the methods described herein which is important in diagnosis, differential diagnosis and prognostications.
  • kits for detecting individuals having HH or a propensity to develop HH or heterozygotes or homozygous normal subjects comprising in compartmental form a first compartment adapted to contain an agent for detecting a Cys282Tyr mutation in HC or in a mutation in a linked gene and a second compartment adapted to contain reagents useful for facilitating the detection by the agent in the first compartment.
  • Further compartments may also be included, for example, to receive a biological sample.
  • the agent may be an oligonucleotide or antibody or other suitable detecting molecule.
  • Further components of the kit may include a solid support such as filter paper or a gel matrix to receive amplified portions of individual's genome. A component or an agent of the kit may, therefore, require amplifying reagents.
  • Another aspect of the present invention is directed for use of an agent such as an oligonucleotide or antibody in the manufacture of a reagent for detecting a homozygous or heterozygous Cys282Tyr mutation in HC.
  • an agent such as an oligonucleotide or antibody in the manufacture of a reagent for detecting a homozygous or heterozygous Cys282Tyr mutation in HC.
  • the present invention may, in one particular embodiment, be useful for diagnosing a subset of people of European descent such as those from the geographical regions of Australia, Australasia, New Zealand, Asia and the Pacific rim. Even more particularly, people of European descent in Australia and surrounding regions.
  • Reference herein to a biological sample include whole blood such as from a pin-prick, buccal swabs or washings, hair follicles, skin scrapings or other convenient sources of DNA.
  • the present invention may be used for the screening of individuals, families and populations.
  • Figure 1 is a photographic representation of Sna Bl digests of DNA from a family segregating for HH.
  • Figure 2 is a photographic representation showing the results of sequence specific oligonucleotide (SSO) detection of homozygous normal, homozygous abnormal and heterozygous individuals, in respect of the C282Y mutation.
  • SSO sequence specific oligonucleotide
  • HH families Twenty six genetically and clinically well characterised HH families were examined. Probands were also analysed in an additional 38 families together with a further five subjects who were clearly homozygous normal at the HH locus. This gave a total of 1 12 HH patients, 87 HH heterozygotes and 25 homozygous normal controls.
  • the Cys282Tyr mutation was analysed in ⁇ C in the subjects of Example 1 using two different methods; each sample was analysed by both methods and in all cases the results using the two methods were completely concordant.
  • PCR of the region containing the missense mutation was carried out using the previously described PCR primers (8). PCR conditions were as follows: 94°C for 10 min, then 35 cycles of 94°C 1 min, 62°C 1 min, 72°C 1 min, followed by a final step of 72°C for 10 min.
  • the two methods to detect the mutation were first nucleotide change (FNC) and restriction enzyme cleavage.
  • FNC is a mutation screening method (See International Patent Application Publication No.
  • WO 90/09455 that relies on allele specific extension of a mutation detection oligonucleotide abutting the mutant base.
  • a biotinylated FNC oligonucleotide (5'-biotin-GGAAGAGCAGAGATATACGT-3' [SEQ ID NO:l]) was annealed to denatured PCR product encompassing the Cys282Tyr mutation, then bound to strepavidin coated microtitre plates and extended with either fluorescein labelled dideoxy ATP (mutant) or dideoxy GTP (normal). Incorporation was detected using anti-fluorescein alkaline phosphatase conjugate antibody followed by colorimetric detection of substrate turnover at 410nm.
  • HLA-H Cys282Tyr mutation in 100% of well characterised HH patients with widely variant haplotypes indicates that HC is the primary HH locus and that the Cys282Tyr mutation is likely to be of ancient origin and its high prevalence in European populations may be attributed to a selective advantage conferred by it against iron deficiency (16).
  • the absence of genetic heterogeneity within this locus indicates that the gene is not highly mutable.
  • Homology searches indicate that HC is clearly a member of the Ig superfamily of receptors and the finding that a single amino acid substitution can explain all HH indicates that this substitution is likely to have a profound effect on receptor function.
  • HC is the primary HH locus, although there are likely to be other 6p- linked modifying genes which would explain both the HLA-linked haplotype variation in expression of the disorder and the large region of linkage disequilibrium present in all populations and spanning at least 4.5Mb distal of D6S265.
  • SSO SEQUENCE SPECIFIC OLIGONUCLEOTIDE
  • Genomic DNA is isolated from a biological sample such as blood from a pin-prick or buccal wash and DNA in the HC region amplified. Approximately 1cm 2 grids are drawn on Hybond N + and 1.5 ⁇ l (approximately 50ng) of PCR product spotted on each square. Membranes are soaked for approximately 10 minutes in 0.4 NaOH, rinsed briefly in 2xSSPE for neutralisation. The membranes are then baked at approximately 120°C for 30 minutes.
  • the reagent mixture is incubated to 37°C for 20 minutes and 1 ⁇ l of 200 mM EDTA and 1 ⁇ l of glycogen added and then placed on ice to stop the reaction. Approximately 2 ⁇ l of this labelled probe is used in 10 ml of standard prior hybridisation solution (lpmol/ml) to facilitate hybridisation.
  • Membranes are prehybridised for approximately 30 minutes before use in 30 ml of standard prehybridisation solution at 68°C. The membrane is included with known samples with each hybridisation for control purposes.
  • Labelled probe (4 ⁇ l) is added in 20 ml standard prehybridisation solution to the filters and allowed to hybridise for at least two hours at the optimum temperature. The probe can then be recovered and stored at -20°C for other uses.
  • the optimum temperatures are as follows:
  • the membranes are washed for 5 minutes at room temperature in 50 ml of 2xSSC/0.1% w/v SDS.
  • the membranes are washed for 40 minutes in 50 ml of TMAC wash solution at the following temperatures:
  • the membranes can then be stored in buffer 1 (see below) at 4°C ready for detection.
  • Chemiluminescence based detection systems are particularly appropriate for the present invention.
  • the membranes are rinsed for 2 minutes in 50 ml of buffer 1 with 0.3% v/v Tween-20.
  • the membranes are incubated for 10 minutes at room temperature in 40 ml of 1% v/v washing solution.
  • Antibodies diluted 1 in 1000 in 1% blocking reagent (ie. 48 ⁇ l added to the 40 ml 1% blocking solution) and membranes incubated at 30 minutes at room temperature.
  • the antibody solution can be recovered and stored at 4° for 24 hours for re-use purposes.
  • the membranes are washed twice for 5 minutes at room temperature in 50 ml buffer 1.
  • the presence of 0.3% v/v Tween-20 The membranes were rinsed for 25 minutes in 50 ml buffer 3.
  • CSPD is diluted 1 in 200 in buffer 3 (80 ⁇ l in 16 ml) and membrane incubator for 20 minutes at 37°C. Diluted CSPD solution can be recovered and stored in the dark at 4°C and re-used 1 to 2 times. The membranes are wrapped under damp conditions in glad wrap and exposed to X-ray film for between 30 minutes and 3 hours at 37°C.
  • Blocking reagent 50 ml of 10% Blocking reagent stock
  • Blocking reagent is dissolved in Buffer 1 to a final concentration of 10% w/v with shaking and heating. Autoclaved and stored at 4°C or -20°C.
  • Buffer 3 500ml 1 litre lOOmM Tris 6.057g 12.114g lOOmM NaCl 2.922g 5.844g 50mM MgCl 2 5.0825g 10.165g pH adjusted to 9.5 with HCl. Filter sterilise. (Dissolve powder in -lOOmls water, adjust pH and filter sterilise then dilute to 500ml with sterile water).
  • Blocking reagent stock 10 ml of Blocking reagent stock + 90 ml of Buffer 1 (Final cone, is 1% blocking reagent).
  • TMAC wash solution 500ml 3M TMAC 300ml of 5M TMAC
  • the membrane is soaked for 10 mins in 0.4M NaOH, rinsed briefly in 2xSSPE and baked 120°C for 30 mines.
  • the mutant oligonucleotide hybridises to the heterozygous DNA sample and the homozygous mutant DNA sample but not the homozygous normal DNA sample.
  • the normal (non-mutated) oligonucleotide hybridises to the wildtype and heterozygous DNA sample but not the homozygous mutant.
  • MOLECULE TYPE DNA (oligonucleotide)

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Abstract

L'invention concerne un procédé permettant de diagnostiquer une maladie récessive autosomique ou de déterminer le risque de développer cette maladie, ainsi que des agents utiles pour la mise en application de ce procédé. Elle concerne, plus particulièrement, un procédé de diagnostic d'une maladie récessive autosomique du métabolisme du fer provenant de deux copies, ou associée à ces deux copies, d'un gène anormal (homozygote anormal). Elle concerne, encore plus particulièrement, un procédé d'identification d'un individu ayant développé ou susceptible de développer une hémochromatose héréditaire ou d'un individu porteur d'un gène anormal (hétérozygote) ou d'un individu porteur d'aucun gène anormal (homozygote normal). Cette invention concerne, de plus, une méthode rapide permettant de diagnostiquer l'hématochromatose héréditaire et présentant un niveau élevé de précision du diagnostic de la maladie ou de la tendance à développer ou à transmettre cette maladie héréditaire.
PCT/AU1997/000539 1996-08-23 1997-08-22 Procede permettant de diagnostiquer des maladies recessives autosomiques et agents utiles de diagnostic Ceased WO1998007884A1 (fr)

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AUPO1849 1996-08-23
AUPO1849A AUPO184996A0 (en) 1996-08-23 1996-08-23 A method of diagnosis and agents useful for same
AUPO2083 1996-09-03
AUPO2083A AUPO208396A0 (en) 1996-09-03 1996-09-03 A method of diagnosis and agents useful for same - II

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997016568A1 (fr) * 1995-10-31 1997-05-09 Bowie Lemuel J DETECTION DES MUTATIONS DE LA THALASSEMIE α HUMAINE ET LEUR UTILISATION COMME MOYENS DE PREDICTION DES TROUBLES ASSOCIES AU SANG

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997016568A1 (fr) * 1995-10-31 1997-05-09 Bowie Lemuel J DETECTION DES MUTATIONS DE LA THALASSEMIE α HUMAINE ET LEUR UTILISATION COMME MOYENS DE PREDICTION DES TROUBLES ASSOCIES AU SANG

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM., Vol. 272, No. 22, 30 May 1997, FEDER J.N. et al., "The Hemochromatosis Founder Mutation in HLA-H Disrupts Beta2-Microglobulin Interaction & Cell Surface Expression", pages 14025-14028. *
NATURE GENETICS, Vol. 13, No. 4, 1 August 1996, FEDER J.N. et al., "A Novel MHC Class 1-Like Gene is Mutated in Patients with Hereditary Haemochromatosis", pages 399-408. *

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