GB2380194A

GB2380194A - Mitochondrial mutation associated with schizophrenia

Info

Publication number: GB2380194A
Application number: GB0123577A
Authority: GB
Inventors: Roger Michael Marchbanks
Original assignee: Individual
Current assignee: Individual
Priority date: 2001-10-01
Filing date: 2001-10-01
Publication date: 2003-04-02
Anticipated expiration: 2021-10-01
Also published as: GB2380194B; GB0123577D0

Abstract

This invention relates to methods for detecting and quantitating a heteroplasmic mutation at position 12027 of mitochondrial DNA in various human tissues. The mutation is a <I>t</I> T <I>c</I> substitution that changes the protein structure of NADH-Ubiquinone reductase causing oxidative stress by increased superoxide production and reduced electron transfer. The mutant form predominates in 24% of the mtDNA in lymphocytes from normals and in 47% of a sample of 180 schizophrenics. Several other illnesses such as cancer, cardiovascular disorder and age related neurological, ophthalmological and dermatological age related degeneration are also associated with oxidative stress. The invention thus affords methods of predicting the likelihood of schizophrenia and other ailments before the onset of clinical symptoms. Also disclosed are methods of alleviating the illnesses caused by the mutation by replacing mtDNA or preventing its expression and methods of using quercetin and its glycosides to reduce superoxide emission without compromising the energy transfer capabilities of the reductase.

Description

Title: A MITOCHONDRIAL MUTATION ASSOCIATED WITH SCHIZOPHRENIA AND OXIDATIVE STRESS

Technical field This invention describes methods for detecting and quantitating the ocurrence of a heteroplasmic mutation in human mitochondrial DNA that is associated with schizophrenia. The mutation causes changes in the protein structure of NADH-Ubiquinone reductase and in doing so causes dysfunctional mitochondrial energy metabolism and increased superoxide production. The invention thus affords methods of predicting the likelihood of schizophrenia and other ailments associated with oxidative stress before the onset of clinical symptoms. Also disclosed are molecular and pharmacological methods by which the effects of the mutation may be nullified or avoided thus enabling rational means for treatment of the consequent illnesses.

Background It has been observed that brains of patients afflicted with schizophrenia carry RNA transcripts of mitochondrial DNA (mtDNA) altered in both amount and nucleic acid sequence (Mulcrone et al Schizophrenia Res 1995; 14: 203-213). Mitochondria carry the enzymes of the respiratory chain that produce adenosine triphosphate which is essential for all cellular processes particularlythose of nerve cells. In addition mitochondria produce the precursors of many important neurotransmitters such as acetylcholine, glutamate and the inhibitory neurotransmitter gamma-aminobutyric acid so that synaptic transmission is dependent mitochondrial function. Dysfunctional mitochondria also produce oxidative stress because the

enzyme NADH-Ubiquinone reductase produces superoxide (Turrens & Boveris Biochem 1980 ; 191: 421-427) which is readily converted to the highly destructive free radical OH in the tissues.

Mitochondrial DNA is transmitted from the mother and demonstrates the phenomena of heteroplasmy in that different tissues contain slightly different types of mitochondria within their cells recognisable by differences in the mtDNA sequence and its variants between different cell types. Furthermore it is probable that the same cell type or even the same mitochondrion may contain one or more mtDNA variants (intracellular heteroplasmy). Mitochondrial DNA is particularly prone to somatic mutation in which clonal expansion of a single mutational event causes substantial populations of variant DNA in particular cell types that give the variant or the cell selective advantage.

Schizophrenia is a mental illness characterised by delusional experiences and thought disorder that results in a serious and often unresolvable erosion of the mind and personality of the afflicted. The lifetime risk of both schizophrenia and depression is approximately 1 % of the population and the many cases where the symptomatology is intermediate have suggested (Crow Br J Psychiatry 1986; 149: 419-429) that they are both manifestations at different ends of the clinical spectrum of the same underlying illness. The afflictions are socially and economically debilitating both for affected individuals, their families and society at large. Some drugs such as phenothiazine derivatives, other neuroleptics, tricyclics and monoamine oxidase inhibitors are available to attenuate the more florid aspects of these illnesses but in general drug therapies have undesirable side effects and do not restore normal mental function to the patient.

Increased liability to both schizophrenia and depression occurs in families. The

expectation of the illnesses is increased approximately ten fold in the children or siblings of affected individuals and for identical twins the expectation is raised a further five fold to about 50%. A family history of both illnesses together is commonly found. The familial distribution suggests that genetic factors are involved but since identical twins are not always similarly afflicted there must also be environmental or social triggering events. It is generally thought that the underlying susceptibility is genetic in origin but that the precipitating factors are to be found in the life experience. Genetic studies of schizophrenia and depression have not so far identified how many genes are involved or what the mode of genetic transmission is. Maternal transmission of schizophrenia has been shown to predominate (Wolyniec et al J Psychiat Res 1991; 1: 17-27) and it is clear that Mendelian rules do not apply.

Tissues such as lymphocytes from schizophrenic patients have been shown to produce increased amounts of superoxide (Melamed et al Psychiat Res 1998; 77: 29-34) though this effect will be obscured by the effect of neuroleptic drug administration in reducing NADHubiquinone reductase activity and superoxide production (Whatley et al Molecular Psychiatry 1998; 3: 227-237). Superoxide (02-) is injurious in itself and is readily converted in tissues in the presence of Fe to the highly destructive free radical OR. These compounds jointly damage proteins and DNA and form the condition known as oxidative stress. associated with various illnesses.

Oxidative stress has been implicated not only in the causation of mental and neurological illnesses such as schizophrenia, endogenous depression, Parkinson's, Alzheimer's and other dementias as well as cardiovascular disease, cancer, auto-immune disease, reduced resistance to infection. (Matés et al Clin Biochem 1999; 32: 595-603), age related degeneration such as osteoporosis (Varanasi et al Osteoporosis Int. 1998 ; 10: 143-149), skin

wrinkling and hyperkeratinisation (Keller & Fenske J Am Acad Dermatol. 1998; 39: 64-86) and glaucoma (Tamm et al Investigative Ophthalmol. 1996 ; 37: 2402-2413.

Disclosure of the invention.

The present invention discloses the methods by which the existence of a mutation in mitochondrial DNA from the brain of schizophrenics and in lymphocytes from both schizophrenics and normals may be determined (Table 1). The mutation is a substitution at position 12027 of the"Cambridge"mitochondrial DNA sequence reported by Anderson et al Nature 1981 ; 290: 457-465. HGMP accession number: J01415. The substitution at position 12027 is oc and results in the change of an amino acid from isoleucine to threonine at position 423 of the amino-acid sequence of the ND4 subunit of NADH-Ubiquinone reductase. The mutation causes an increased production of superoxide and decreased transfer of electrons from NADH to ubiquinone (Table 3) with a consequent reduction in ATP synthesis.

The utility of this invention is that it provides methods for predicting the likelihood of schizophrenia and the other ailments associated with oxidative stress before the onset of clinical symptoms. This will enable appropriate medical, social and economic supportive arrangements to be made for patients before the full clinical manifestation of the illness. This invention also provides enabling information for the development of rational methods for the alleviation of schizophrenia and the other illnesses due to oxidative stress. The strategies for alleviation include intervention at the genetic coding level, at the gene expression and control stage, manipulation of the protein products of gene expression and finally pharmacological and dietary interventions rationally designed to alleviate the enzymic deficiency or nullify toxic products.

TABLE 1 Frequency of mutation1 at MtDNA position 12027 in various human tissues

Tissue source Number Normal: Mixed: Mutant: Frequency of of samples 12027 t 12027 c variant forms c/ (c+t) 0. 5-1 Lymphocytes from normals 184 76"* 96 12'". 24"' schizophrenics 181 32'"111 38'". 47'" Brain tissue from normals 9 2 7 0. 11* schizophrenics 15 3 10 2. 53* Lymphocytes from 12 multiple affected families normal schizophrenic mothers 0 12 2 8 2. 50 fathers 12 0 2 8 2. 25 sibs 10 26 10 14 12.36

NOTES (1) The mutant form 12027 c and heteroplasmic mixtures are determined as the fraction c of the total (c + t) at position 12027. For convenience values of cl (c+t) 0-. 25 r . HM"/ ! y7 ? M/" ;. 2-. 7 < M"/MK" ;. 7-7. 0 as "mutant". The term "variant forms" as in column &num;6 is used to designate samples having the proponion cI (c+ t) O. 5 1. 0 at position 12027. (j p < . 05, r' < .

Description The methods and utility of the present invention will now be described in general terms with examples that indicate but do not delimit its application. Throughout this application references to the prior art as revealed in the scientific journals cited in the text are hereby incorporated in order to describe more fully the methods utilised.

Extraction and quantitation of mtDNA Whole cell DNA including mtDNA can be extracted from most tissues by the standard methods disclosed in the prior art (Sambrook et al 1989 Molecular cloning. A Laboratory Manual 2nd Edition, Cold Spring Harbour, New York.) reagents for which are now commercially available. This invention does not require the specific separation of mitochondrial DNA because the methods of mutation detection are sufficiently specific to detect the mitochondrial mutation in the presence of large quantities of nuclear DNA. In the preferred embodiment DNA is extracted from the platelets and lymphocytes in a whole blood sample of 10ml from the patient by a procedure involving lysis, SDS/proteinase K digestion, phenol/chloroform amyl alcohol extraction followed by ethanol precipitation. With modification to ensure efficient initial maceration the above procedure can be used on tissues available from cheek swabs, skin punch, biopsy or postmortem from patients. After extraction the amount of DNA is measured spectrophotometrically or fluormetrically, diluted to a stock concentration of 1 Ong/JLl and stored at-70"C.

Amplification of specific sequences of mtDNA For the determination of the presence or plification of spec fic seg. uences of mtDNA absence of the mutation the mtDNA fragment surrounding the mutation must be specifically

amplified. This can be done by repeated application of the thermostable polymerase mediated primer extension reaction well known in the prior art as the polymerase chain reaction (PCR).

The primers are oligonucleotide sequences that specifically recognise mtDNA sequences that encompass the position of the mitochondrial mutation at a suitable distance for detection of the amplified fragment. Table 2 discloses suitable oligonucleotide sequences and conditions of hybridisation for amplification of fragments encompassing the mutation at position 12027 of mtDNA and which do not amplify any DNA from other parts of the genome, such as nuclear pseudomitochondrial DNA. The suggestions shown in Table 2 are illustrative and do not in anyway delimit the invention since other oligonucleotides may be suitable within the general spirit of this invention. The PCR is carried out with 21LI of the stock DNA (20 ng).

31 extension cycles under conditions shown in Table 2 are sufficient for most purposes disclosed by this invention.

Detection of the presence or absence of the mutation The determination of the existence or otherwise of the previously described mutations in DNA samples from patients and their correlation with the risk of illness is central to application of this invention. The prior art has disclosed many methods of detecting mutations, those which have been found useful in the development of this disclosure are describled below but the invention is in no way delimited to these and may be embodied in others not described below or yet to be developed.

Restriction enzyme digestion. The mutation at 12027 destroys a digestion site for the commercially available restriction enzyme Msel. In fragments amplified with primer ID 1 & 2 (Table 2) the presence of the mutation prevents digestion so that fragments having sizes below 260 nucleotide residues do not appear on electrophoresis of the digested DNA (see Table 2). Incomplete digestion can be recognised and corrected for by observing or measuring the intensity of the band at 298.

TABLE 2 Oligonucleotide DNA sequences used to identify, amplify and quantitate 12027t (normal, N) and 12027c( variant, V) in mtDNA samples.

ID Function Oligonucleotid sequence 5'-3'T. (C) Mg" (mM)' Fragment size (bp) Amplification: 1 fwd gagaactctc tgtgctagta 53 1.0 298 2 rev gcctctgttg tcagattcac Digest PCR product: Msel 1 unit/5, Ll/60 min &commat; 370 normal 122,138 variant 260 Alternative method of determining mixed populations: Digest 20 ng genomic DNA sample: MseI 0.5 unit/2,. tI/60 min &commat; 37 , amplify: 3 N+V fwd2 ttgatgacttctagcaagcc 54 1.0 311 4 N+V rev ttacaagagg aaaacccggt 5 V fwd gagaactctc tgtgctagta 252 6 N rev tgtggtgggt gagtgagccc 136,196 Amplification refractory mutation system (ARMS): 7 N fwd ggctcactca cccaccacct 62 3.0 133 8 joint rev3 ttacaagagg aaaacccggt 311 9 joint fwd ttgatgactt ctagcaagcc 10 V rev'cgtgtgaatg agggttttat gttgttcg 225 Differential hybridisation: 11 cccaccacac taacaacata 53-56 Oligonucleotide ligation and primer extension assays: 12 fwd ctcactcacc caccaca 52 1.0 13 rev gtgaatgagg gttttatgtt gtta 14 Antisense atgttgttag tgtggtgggt NOTES 1) Annealing temperature and optimal Mg for the amplification reactions. 2) The fragment of 311 bp is derived from the unhydrolysed portion of the genomic sample and is proportional to (1-h) (12027c+t) where h = the fraction hydrolysed. Similarly the fragment of size 252 bp is derived from the hydrolysis of 12027c and 137+196 bp from hydrolysed 12027t. 3) A duplex format is preferred to avoid differences in amplification between normal and variant which can thus be monitored by the jointly derived 319 bp band 4) The nucleotide at the 3'end can be changed to be complementary to variations other than toc at position 12027 though in fact these have not been observed.

If, (using the nomenclature introduced in the footnotes to Table 1 & 2) the degree of hydrolysis is h the intensity of the band at 298bp will be proportional to (l-h). (c+ t) ; the band at 260 will be proportional to h. c and the bands at 122 & 136 will be proportional to h. t.

Quantitative and objective determination of the proportions of variant and normal forms in a mixture can be carried out by standard image analysis procedures, introducing fluorescent

intensity factors from appropriate controls and solving the simple algebraic expressions to determine the ratio cl (c+t) and h. The determination of the extent of heteroplasmy by conventional PCR-RFLP is attended by a number of difficulties ( (Jacobi et al2001 Mutation Res; 478: 141-151) most notably the formation heteroduplexes during PCR that are difficult to hydrolyse. To overcome this a method is disclosed of digesting genomic mtDNA and then amplifying with a set of primers (Table 2 ID 3,4, 5 & 6) to determine the fragments derived from the normal and variant forms which are then separated and quantitatively analysed as above. Similar results are obtained but at slightly greater cost of time and consumable items.

Amplification refractory mutation (ARMS). In this method the amplification reaction is carried out with oligonucleotide primers designed to discriminate between the normal and mutant DNA such that the extension reaction only proceeds if the primer matches either the normal or the mutant form (Newton et al 1989 Nucleic Acids Research; 17: 2503-2912). By arranging that the mutant and normal primers initiate reactions in opposite directions and the return primers are positioned to yield DNA fragements of different sizes the reaction can be carried out for both mutant and normal sets of primers together in one tube. Examples of sets of primers that have been found to discriminate usefully between the normal and mutant DNA are given (but without implying any delimitation of the invention to these particular oligonucleotides) in Table 2, primer ID 7,8, 9 & 10. This method has been found to be

particularly useful since the mutation discrimination step can be incorporated into the previously described DNA amplification step and in addition polymerisation activated fluorphors (Livak et al 1995 PCR Methods Applic; 4: 357-362, Ye et al 1998 Nucleic Acids Research; 26: 3614-3615) can be incorporated into the ARMS primers so that the presence of the mutation can be monitored by fluoroimagery during the amplification reaction without the necessity for a further electrophoretic separation. This invention allows the detection of the mutation at 12027 along with others that might be thought to be relevant simultaneously in one sample from each patient by the inclusion of all four sets of ARMS primers in the reaction mixture. Multiplexed mutation detection. If each mutant detection primer is labelled with a polymerisation sensitive fluorophore each of the 4 having a different emission wavelength then the method permits the simultaneous detection of all four mutations in a one step process which moreover can be done in conjunction with a fluoroimager in microtitre plate batches of 96 or 384 samples. The throughput afforded by this invention allows the possibilty of mass screening. Another utility of the ARMS procedure disclosed herein is that by changing the nucleotide at the 3'end of primer ID 10 to either a or c the method will recognise and discriminate substitutions at position 12027 alternative to the commonly observed t-c. A representative sample has not however yielded any examples.

There are several other methods of mutation detection that may be mentioned as within the spirit of this invention. These include; direct sequencing of the products of the amplification reaction by any of the various methods and machinery currently available commercially or described in the prior art (Sanger et al (1977) PNAS 74 5463-5467), the oligonucleotide ligation (Grossmann et al Nucleic Acids Research 1994; 22: 4527-4534). and primer extension assay (Thalainen et al J. Clin. Pathol 1994 ; 47: 1082-1084). The use of denaturing

gel elctrophoresis to recognise mutations and also situations where heteroplasmy exists (Orita et al PNAS 1989; 86: 2766-2770) and differential hybridisation on solid media using fluorescently or immunoactive labelled probes can be utilised for mass screening in the context of this invention.

Separation of DNA fragments Standard electrophoretic procedures either on agarose gels or polyacrylamide gels are suitable for separating on the basis of their size the various fragments of DNA generated by the above methods. Where multiple samples of DNA must be examined the MADGE format enables 96 or 384 samples of mtDNA in microtitre plate format to be separated simultaneously (Day et al 1999 Genetic Analysis Bimolecular Engineering: 14, 197-204). All the procedures described in this invention generate fragments in the size range 100-450 BP so that agarose gels of concentration 1-2% or polyacrylamide gels of 4-6% are required. Fragments on the gel in amounts of 50-500 ng can be readily visulaised by staining with ethidium bromide. For lesser amounts fluorescent methods in which fluorescent nucleotides may be incorporated into the primers with modification to ensure that they fluoresce only when incorporated into the amplified product to to enable the identification and measurement of amplified product by fluorescence or flouroimagery. Radioactive nucleotides may be used in the extension reaction so that the amplified product may be identified and measured by phosphoimagery or other suitable techniques for measuring radioactivity.

Primers may be labelled with biotin so that the bands can on electrophoresis can be recognised by any one of the many immunological procedures for recognising biotin with streptavidin conjugates. Primers, probes and target mtDNA described in this invention can be attached to solid supports such as glass, silicon or nitrocellulose membrane for hybridisation and amplification in microarrays for mass screening.

Methods of determining the biochemical effects of the mutations. The normal function of NADH-Ubiquinone reductase can be measured as the enzymic transfer of electrons from NADH to ubiquinone or the artificial substrate 2,4-dichlorindophenol which is conveniently carried out spectrophotrometrically at 340,280 or 650 nm respectively. The production of superoxide can be estimated by its reaction with nitro-tetrazolium blue which absorbs light at 460 nm. The methods for estimating NADH-Ubiquinone reductsase and superoxide production are given in detail in (Whatley et al Molecular Psychiatry 1998; 3: 227-237).

It is disclosed in this invention (Table 3) that there is an increased superoxide production in cells carrying mitochondria with the 12027 mutation. Neuroleptic drugs are known to cause a decrease in the expression of Complex I which results in some decrease in superoxide production as well as less ATP for use by the cell. The above methods allow the disadvantageous production of superoxide in tissues having mutation at 12027 to be monitored and enable procedures to be developed by which the effects of superoxide may be overcome without reducing the overall activity of Complex I and therefore jeopardising ATP production.

Since the exact position of the amino-acid change in the NADH-ubiquinone reductase protein is disclosed by this invention it makes possible the design by those practised in such procedures of antigenic peptides to which antibodies capable of recognising the mutation can be raised. This enables the immunochemical recognition of the mutation which has many applications in the development of diagnostic methods and biochemical monitoring of treatment.

TABLE 3 Biochemical pharmacological effects of mt DNA mutation 12027

NADH-ubiquinone reductase' Electron donor Electron transfer3 Superoxide4 (NADH oxidation) (to CoQ, DCIP) production Brain tissue: samples N S c/ (c+t) 0- 0. 5 (normal) 6 1 4. 77+. 53 1. 88. 54 1. 70. 03" c/ (c+t) 0. 5 1 (variant) 3 4 4. 62+. 68 2. 24+. 50 2. 18. 03" = = = = = = = = = = = as % control (variant) = = = = = = = = = = Superoxide5 in vitro quenching neuroleptic treated 986~10 108~24 51~6* 72~2** + 10 M quercetin 62~8* 81 88 43 - 3-rutoside +10 M quercetin 78~7* 93 55 44 + 10JlM ferredoxin 84 114 113 5 + 1004trans-3, 4, 5- 125 116 123 41 trihydroxystilbene + 100 M trolox7 70 62 137 NOTES : (1) Given as nmol/min. mg protein measured spectrophotometrically as generally described in Whatley et al 1998 ; 3: 227-237. Statistic given is the standard error of multiple measurements on each sample (2) by rotenone sensitive decrease of optical adsorption (OD) at 340 mu in presence of CoQ. (3) as decrease in OD of CoQ at 280 mit, or cytochrome c at 550 m in presence of cyanide or dichlorindophenol at 600 mfL. (4) Given as pmol/min. mg protein measured as tetrazolium blue formazan precipitated by scanning or spectrophotometrically at 460 mll. (5) Measured as the quenching of superoxide produced from solid potassium superoxide in vitro. (6) cis-flupenthixol 100 M. (7) Water soluble form of vitamin E (*)p < .01(**)p < .001.

Applications of the invention for therapy The methods disclosed in this invention can reveal and quantitate a heteroplasmic mutation which confers increased liability to a wide variety of illnesses and conditions in addition to that of schizophrenia. Accordingly it is part of this invention to exploit the information thus obtained to alleviate the effects in patients of the mutation.

Modification ofmtDNA. The introduction into cells of DNA to replace that containing the mutation is possible by several procedures well known to practitioners. These include Ca++ treatment, electroporation, and the insertion of retroviral vectors or cyhrid mitochondria containing the normal mtDNA (Templeton & Lasic eds Gene Therapy 2000 Marcel Dekker Inc NY). A preferred embodiment of this invention is to circumvent the problem of targeting the DNA to the mitochoindria by inserting a DNA construct into the cell which can be expressed in the nucleus and whose protein product has the necessary N-terminal amino-acid sequence to"lead"it into the mitochondria. These procedures have all been used with varying degrees of success. The DNA construct must contain the ND4 gene in its normal form, an appropriate control and promoter sequence and the N-terminal sequence rich in positively charged and hydroxylated amino-acids that"leads"the polypeptide formed in the nucleus or through the mitochondrial membrane at specific entry ports (Siebel et al (1995) Nucleic Acids Research 1995; 23: 10-17).

Preventing the expression of the mutant mtDNA. The introduction of normal mtDNA must be complemented by the destruction or disablement of mutant DNA. The use of antisense nucleotides targeted at the region of mutation will prevent the expression of that

region specifically. Antisense nucleotides are short oligonucleotides complementary to the target (mutant) sequence which attach to the mtDNA in the region of the mutation and prevent its expression by impeding the RNA polymerase and thus preventing the synthesis of the mRNA and so the enzymic protein. This invention enables the construction of oligonucleotides that will specifically bind to the mutant region examples being given in Table 2. To enable the the easy penetration of such oligonucleotides into the mitochondria it is useful to couple the antisense oligonucleotides to a signal peptide sequence as described in the previous section. Alternatively the antisense oligonucleotide can be presented as a sequence complementary peptide nucleic acid (Nielsen & Egholm et al, Peptide Nucleic Acids 1999; Horizon Scientific Press, Wymondham UK). Peptide nucleic acids are nucleobases covalently bound to a polyamide backbone. They are known to bind to complementary DNA or RNA and appear to enter the mitochondrion efficiently and prevent transcription and translation of the mitochondrial genes to which they are complementary.

Immunological intervention. Antibodies designed to bind to the abberant threonine at position 423 in the ND4 subunit introduced by the mutation at 12027 can be expected to modify its deleterious effects on the NADH-ubiquinone reductase of which it is a component. Such antibodies along perhaps with replacement proteins can be encapsulated into liposomes whose membranes contain mannosylated proteins that ensure their passage of the blood brain barrier and uptake into mitochondria (Chomyn (1991) Mol Cell Biol 1991; 11: 2236-2244, Przyrembel (1987) J inherit Metab Dis; 10: 120-146).

Pharmacological intervention. The mutation at 12027 increases the transfer of electrons to oxygen peoducing the toxic superoxide. The therapeutic effects of the neuroleptic drugs involve reduction of the superoxide levels by reducing the electron transfer activity of the energy producing NADH-ubiquinone reductase. (Table 3) Thus their therapeutic effect on superoxide concentration is compromised by their interference with the essential energy transfer activities. It is disclosed by this invention that the flavonoid quercetin and various glycosidic derivaatives are effective neutralisers of toxic superoxide while not affecting the essential energy transfer activity of the reductase (Table 3). Being water soluble and the constituents of many edible plant products they commend themselves for inclusion in the diet and as therapeutic agents for topical application to the relevant tissues to counteract local oxidative stress.

Claims

Claims What is claimed is: Claim 1. A method of estimating the risk of illnesses associated with oxidative stress before the onset of clinical symptoms and alleviating their effects in patients by determining the presence of a mutation and its biochemical consequences at position 12027 in the sequence of human mitochondrial DNA from the patient.
Claim 2. The method according to claim 1 wherein the estimation of risk comprises (though not necessarily exclusively) the correlation of the proportion of mutated to normal DNA at position 12027 in extracts of human mitochondrial DNA from lymphocytes, skin punch, cheek swab or other relevant biological tissue from the patient.
Claim 3. The method of claim 1 wherein the illness associated with oxidative stress is schizophrenia or related psychotic conditions.
Claim 4. The method of claim 1 wherein the illnesses associated with oxidative stress include; cardiovascular disorders, cancer, auto-immune disorders, neurological and mental disorders such as Alzheimer's disease, Parkinson's disease, manic depressive disorder, the results of ageing particularly as it effects bone (osteoporosis), skin (wrinkling and hyper-keratinisation), and eyes (cataract & glaucoma).

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Claim 5. The method of claim I wherein the mutation at position 12027 in human mitochondrial DNA is the substitution of thymidine usually but not exclusively by cytidine which results in the change of an amino acid from isoleucine to usually but not exclusively threonine at position 423 of the amino-acid sequence of the ND4 subunit of NADH-Ubiquinone reductase.
Claim 6. The method as in claim 1 wherein among the biochemical consequences of the mutation that are to be alleviated in patients are included the production of excessive toxic superoxide and dysfunctional electron transfer by NADH-Ubiquinone reductase in tissues containing a high proportion of mitochondrial DNA mutated at position 12027.
Claim 7. Methods whereby the effects of the mutation as in claim 5 may be neutralised by antisense nucleotides or the mitochondrial DNA replaced so that the ailments according to claims 3 and 4 may be alleviated.
Claim 8. Methods whereby the biochemical consequences of the mutation as in claim 6 may be may be rendered innocuous by removing excess superoxide without compromising the normal function of the NADH-Ubiquinone reductase so that the ailments according to claim 3 and 4 may be alleviated.
Claim 9. A method whereby the mutation according to claim 5 is recognised and estimated by the change it causes to a DNA sequence specifically recognised for cleavage by a restriction enzyme.

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Claim 10. A method according to claim 9 where the restriction enzyme is Mse I.
Claim 11. A method whereby the mutation according to claim 5 is recognised and estimated by measuring the differential change in the hybridisation between the normal and mutated mtDNA sequence to an oligonucleotide or antibody probe.
Claim 12. A method whereby the mutation according to claim 5 is recognised and estimated by oligonucleotide sequences specific to the normal or mutated mtDNA acting as primers for the polymerase mediated amplification reaction.
Claim 13. Methods whereby the mutation according to claim 5 is recognised and estimated by direct sequencing, the oligonucleotide ligation assay or the primer extension assay.
Claim 14. A method whereby the DNA regions containing the mutation according to claim 5 before or after treatments according to claims 9 or 10 or 11 or 13 are specifically amplified by the polymerase mediated extension of suitable oligonucleotide primers.
Claim 15. Methods whereby mtDNA fragments containing or derived according to claims 9 or 10 from the mutation according to claim 5 before or after treatment according to claims 11 or 12 or 13 and amplified according to claim 14 are separated by an applied electric field on a substrate of agarose and, or polymerised acrylamide in a format suitable for the simultaneous separation of multiple DNA samples.

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Claim 16. Methods whereby mtDNA fragments containing or derived according to claims 9 or 10 from the mutation according to claims 5 and, or the primers and probes according to claims 11 or 12 or 13 or 14 are immobilised on nitrocellulose, silica or other suitable solid support before or after treatment.
Claim 17. Methods whereby the mtDNA fragments containing or derived acording to claims 9 or 10 from the mutation according to claim 5 and, or the primers and probes according to claims 11 or 12 or 13 or 14 are recognised by treatment with ethidium bromide or labelling them with fluorescent or polymerisation activated fluorescent or radioactive nucleotides or biotin coupled nucleotides or antibodies.
Claim 18. Methods whereby the mtDNA fragments as in claim 17 are quantitated by autoradiography, phosphoimagery, fluoroimagery or densitometry of streptavidin linked horseradish peroxidase product.
Claim 19. A method whereby the results of quantitation of the DNA according to claim 18 are used to calculate values as in claim 2 for the proportion of mutated to normal DNA at position 12027 in extracts of human mitochondrial DNA.

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Claim 20. Methods whereby the use of multiple sets of oligonucleotide primers or probes according to claim 11 or 12 are labelled with fluorescent or polymerisation activated fluorescent nucleotides with different emission spectra according to claim 17 so that estimations can be carried out simultaneously on one DNA sample to determine the presence or absence of other mutations in addition to that according to claim 5.
Claim 21. Methods whereby the reagents required to determine the mutation according to claims 5 and their proportions estimated according to claim 2 are combined in a kit so multiple DNA samples may be conveniently estimated alone or in conjunction with the determination of other mutations.
Claim 22. A method whereby antibodies recognising the changed amino-acid sequence of the NADH-Ubiquinone reductase caused by the mutation as in claim 5 are raised for use to assist diagnosis or therapy as in claim 8.
Claim 23. Methods whereby the excessive superoxide caused in tissues containing increased proportions of the mutation as in claim 6 can be measured spectrophotometrically by the increased rate of reduction of nitro-tetrazolium blue.
Claim 24. Methods whereby the dysfunctional electron transfer by NADH-Ubiquinone reductase in tissues containing a high proportion of the mutation as in claim 6 can be measured spectrophotometrically by the reduced rate of reduction of 2: 4 dichlorindophenol.

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Claim 25. Methods whereby the reagents according to claims 22 or 23 or 24 to determine the biochemical consequences of the mutation according to claim 6 can be combined in a kit form in order to facilitate estimation of multiple samples.
Claim 26. A method of utilising antisense nucleotide sequences encompassing the mutation site according to claim 5 with or without coupling to peptides or other substances to improve penetration of mitochondria thus enabling the inhibition of transcription or translation of mutated sequences in claim 7.
Claim 27. A method to import mtDNA into cells to replace that containing the mutation as in claim 7 by fabricating a DNA construct carrying the correct amino-acid sequence of the NADH-ubiquinone reductase coupled to a peptide leader sequence and DNA promoter the whole incorporated into a suitable vector in order to incorporate the replacement DNA into affected tissues.
Claim 28. A method whereby cybrid mitochondria are constructed with DNA containing the mutation according to claim 5 for use as suitable test systems to test pharmaceutical methods of nullifying the undesirable biochemical sequelae of the mutation as in claim 8.

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Claim 29. A method of using the flavonoid compounds 2- (3, 4-Dihydroxyphenyl)- 3,5, 7-trihydroxy-4H- I-benzopyran-4-one ; 3, 3'. 4', 5, 7-pentahydroxyflavone (quercetin) and its 3- [ [6-0- (6-Deoxy-0-l-mannopyranosyl)-6-D-glucopyranosylJoxy] derivative (rutin) and other related glycosides to absorb the superoxide formed by the mitochondria without compromising the electron transfer functions of the NADH-Ubiquinone reductase as in claim 8.
Claim 30. Methods for the preparation of pharmaceuticals according to claims 22 or 26 and, or 27 or 29 with the addition of adjuvants, penetrants and preservatives including their encapsulation within liposomes for general, local or topical application to affected tissues according to claims 7 or 8.
Claim 31. Methods for the formulation of dietary regimes containing the compounds according to claim 29 or foods containing these compounds for therapeutic use according to claim 8.
Claim 32. Cosmetic preparations containing the compounds according to claims 22 or 26 and, or 27 or 29 in addition to emollient, odorant, penetrant, preservative compounds and including their encapsulation within liposomes for use according to claims 7 or 8.