JP2003521879A - Single nucleotide polymorphisms in mitochondrial genes segregating with Alzheimer's disease - Google Patents

Abstract

  (57) [Summary] Compositions and methods are provided that are based on the determination of single nucleotide polymorphisms or homoplasmic mtDNA mutations in mtDNA, for detecting the presence or risk of having Alzheimer's disease (AD), and for detecting AD. Useful for identifying agents suitable for treatment. The mtDNA in the sample can be amplified. The invention also provides a method of determining whether an agent causes, contributes to, or may worsen Alzheimer's disease.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates generally to Alzheimer's disease, and more particularly to single nucleotide polymorphisms in mitochondrial DNA.
lymorphysm) and compositions and methods for detecting predisposition to such diseases.

BACKGROUND OF THE INVENTION Many degenerative diseases appear to be caused by or associated with alterations in mitochondrial function. These diseases include Alzheimer's disease, diabetes, Parkinson's disease, Huntington's disease, ataxia, Leber's inherited optic neuropathy, schizophrenia and muscular degenerative disorders (eg, “mitochondrial brain disorders,
Lactic acidosis and seizures "(MELAS), and" myoclonic epilepsy ragged
"red fiber syndrome)" (MERRF)). Other disorders involving altered metabolism or respiration in cells can also be considered as disorders associated with altered mitochondrial function.

Alzheimer's disease (AD) is characterized by neuronal loss and / or atrophy in discrete areas of the brain and is a progressive process involving extracellular deposition of β-amyloid and intracellular accumulation of neurofibrillary tangles. It is a neurodegenerative disorder. It is also a unique disease of humans, affecting more than 13 million people worldwide.
It is also an unparalleled tragic disease. Many normally living individuals (productive lives), as they grow older, are slowly attacked by AD, and the disease gradually deprives them of their memory and other intellectual abilities. Ultimately, they
They stop recognizing their families and loved ones, and they often need ongoing care until they eventually die.

There is evidence to suggest that the genetic basis of at least some of the diseases associated with altered mitochondrial function resides in mitochondrial DNA rather than extra-mitochondrial DNA found in, for example, the nucleus. For example,
Non-Insulin Dependent Diabetes Mellitus (NIDDM) mainly presents a maternal pattern of inheritance and also exists in diseases known to be based on mitochondrial DNA (mtDNA) deficiency. For example, approximately 1.5% of all diabetic individuals harbor mutations at mtDNA3243 in the mitochondrial gene encoding leucyl-tRNA (tRNA ^Leu ). This mutation is known as the MELAS (Mitochondrial Brain Injury, Lactic Acidosis and Stroke) mutation (Gerb
itz et al., Biochim. Biophys. Acta 1271: 253-2
60, 1995). Mutations in mtDNA and other diseases associated with altered mitochondrial function (Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), ataxia, Leber's hereditary optic neuropathy (LHON), schizophrenia) As well as similar theories for similar associations with, but not limited to, myoclonus epilepticus red fiber syndrome (MERRF). The identification of such mutations and their functional significance may provide a target for the development of diagnostic and / or therapeutic agents.

Mitochondria are non-organelles that produce adenosine triphosphate (ATP), which is bioenergetically essential, by oxidative phosphorylation. Functional mitochondria include gene products encoded by mitochondrial genes located in mitochondrial DNA (mtDNA) and gene products encoded by extra-mitochondrial genes not located in the circular mitochondrial genome. This 16
． The 5 kb mtDNA consists of 22 tRNAs, 2 ribosomal RNAs (12s
And 16s rRNA) and electron transport chain (ETC) (exquisite multi-complex mitochondrial assembly, where, for example, respiratory oxidative phosphorylation occurs)
Encodes only 13 enzymes. (For example, Wallace et al., Mitoc
Honda & Free Radicals in Neurogene
rational Diseases, M .; F. Beal, N.M. Howell and I.D. Bodis-Wolner, eds. 1997 Wiley-Liss, In
c. , New York, pp. 283-307, and references cited therein; see, eg, Scheffler, I .; E. , Mitocho
ndria, 1999 Wiley-Liss, Inc. , New York.
Mitochondrial DNA refers to seven subunits of NADH dehydrogenase (ETC complex I (ND1, ND2, ND3, ND4, ND4L, N
D5 and ND6)); one subunit of complex III (ubiquinol: cytochrome c oxidoreductase, Cytb); three cytochrome c oxidase (complex IV) subunits (COX1, COX2 and COX3); And a number of ETC components that include two proton-transferring ATP synthase (complex V) subunits (ATPase 6 and ATPase 8). The overwhelming majority of mitochondrial structural and functional proteins are encoded by extra-mitochondrial genes (probably most often in the nucleus). Thus, mitochondrial and extra-mitochondrial genes refer to gene products and their downstream intermediates (metabolites, degradation products, substrates,
Via precursors, cofactors, etc.), or indirectly. Thus, changes in mitochondrial function (eg, impaired electron transfer activity, incomplete oxidative phosphorylation or increased free radical production) result in incomplete m.
It may occur as a result of tDNA, defective extra-mitochondrial DNA, defective mitochondrial gene product or defective extra-mitochondrial gene product, defective downstream intermediates or combinations of these and other factors.

In the case of AD, attempts to demonstrate an association between mtDNA mutations and disease typically involve preparation of mtDNA, followed by restriction fragment length polymorphism (RFLP).
Or a related analysis (eg, Shoffner et al., 1993 Genomic).
s 17: 171; Petruzzella et al., 1992 Biochem. B
iophys. Res. Commun. 186: 491; Kosel et al., 199.
4 Biochem. Biophys. Res. Commun. 203: 745
: Hutchin et al., 1995 Proc. Nat. Acad. Sci. USA 18: 6892; Brown et al., 1996 Am J .; Med. Genet.
61: 283; Wragg et al., 1995 Neurosci. Lett. 201
: 107; Zsurka et al., 1998 Biol. Psychiatry 44
: 371; Hutchin et al., 1997 Biochem. Biophys. R
es. Commun. 241: 221; Hamblet et al., 1997 Muta.
t. Res. 379: 253; Egensperger et al., 1997 Neur.
opathol. Appl Neurobiol. 23: 315; Lin et al., 1
992 Biochem. Biophys. Res. Commun. 182: 2
38; Tanno et al., 1998 Neurobiol. Of Aging, 19
(1S): S47; see WO94 / 09162), and wild type human m
Sequences of tDNA (eg Anderson et al., 1981 Nature 29
0: 457). However, such an approach relies on the appearance of mutations (eg nucleotide substitutions) at specific positions within the mtDNA sequence such that the restriction fragment pattern profile produced by the selected restriction endonuclease is altered.

Clearly, there is a need for improved compositions and methods for the detection of AD and to identify therapeutic agents that are useful in treating AD. Regardless of whether the underlying defect of AD has a mitochondrial or extra-mitochondrial origin and whether the underlying defect of altered mitochondrial function has been identified, the present invention provides Methods are provided that are useful for determining risk or presence and are useful for identifying agents that are suitable for treating this disease. In particular, as described in detail herein below, the present invention provides for rare single nucleotide polymorphisms or homoplasms.
ic) provides compositions and methods for detecting AD by identification of mtDNA mutations and other related advantages.

SUMMARY OF THE INVENTION Briefly, the present invention relates to compositions and methods useful for detecting AD and for identifying single nucleotide polymorphisms or homoplasmic mutations in mitochondrial DNA (mtDNA). Involved. Accordingly, it is an aspect of the present invention to provide a method for determining the risk or presence of Alzheimer's disease in a first subject suspected of having or at risk of having such a disease. The method is immune from the risk or presence of a disease associated with mitochondrial DNA, a first biological sample obtained from a first subject and altered mitochondrial function. The presence or absence of at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease in each of the first and second biological samples, including a second sample obtained from a second subject known to Determining the presence, wherein at least one Mitco associated with Alzheimer's disease in a first biological sample. The presence of the doria single nucleotide polymorphism and the absence of the mitochondrial single nucleotide polymorphism at the corresponding nucleotide in the second biological sample indicates an increased risk of Alzheimer's disease and then determines the risk or presence of Alzheimer's disease. To do.

In a related embodiment, mitochondrial DN in this first sample
A is amplified and the mitochondrial DNA in the second sample is amplified. In another embodiment, the determining step comprises subjecting each of the first biological sample and the second biological sample to a first biological sample under conditions and for a time sufficient to hybridize the primer to the mitochondrial DNA. Contacting with an oligonucleotide primer having a nucleotide sequence complementary to the sequence present in the sample mitochondrial DNA and a sequence present in the second sample mitochondrial DNA; and a first to produce a first product. Detecting the hybridization and extension of the primer to the mitochondrial DNA of the sample and the hybridization and extension of the primer to the mitochondrial DNA of the second sample to produce a second product distinguishable from the first product, and Comprising at least one step of determining the presence or absence of mitochondrial single nucleotide polymorphism associated with Alzheimer's disease. In certain embodiments, the mitochondrial DNA in the first sample is amplified and the mitochondrial DNA in the second sample is amplified.

In another embodiment, at least one mitochondrial single base associated with Alzheimer's disease and present in the first biological sample and absent in the corresponding nucleotide sequence in the second biological sample. Polymorphism is D-loop,
The mitochondrial tRNA gene, mitochondrial NADH dehydrogenase gene, mitochondrial tRNA gene, mitochondrial cytochrome c oxidase gene, mitochondrial ATP synthase gene, or mitochondrial cytochrome b gene exists in the mitochondrial DNA region. In another embodiment, at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease and present in the first biological sample and absent at the corresponding nucleotide in the second biological sample comprises: D-loop, mitochondrial tRNA gene, mitochondrial NADH dehydrogenase gene, mitochondrial tRN
A gene, mitochondrial cytochrome c oxidase gene, mitochondrial A
It resides in the mitochondrial DNA region where the TP synthase gene or the mitochondrial cytochrome b gene is located. In another embodiment, at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease and present in the first biological sample and absent at the corresponding nucleotide in the second biological sample comprises: The mitochondrial NADH dehydrogenase gene, mitochondrial cytochrome c oxidase gene, mitochondrial ATP synthase gene or mitochondrial cytochrome b gene is located within the mitochondrial DNA region, and its single nucleotide polymorphism is a non-synonymous nucleotide substitution. In another embodiment, is associated with Alzheimer's disease and is present in the first biological sample,
And mitochondrial DNA in which at least one mitochondrial single nucleotide polymorphism not present at the corresponding nucleotide in the second biological sample has mitochondrial NADH dehydrogenase gene, mitochondrial cytochrome c oxidase gene, mitochondrial ATP synthase gene or mitochondrial cytochrome b gene Within the region, and single nucleotide polymorphisms are synonymous nucleotide substitutions.

In a particular embodiment of the invention, at least one mitochondria associated with Alzheimer's disease and present in the first biological sample and absent at the corresponding nucleotide in the second biological sample. Single nucleotide polymorphisms include 72nd, 114th, 146th, 185th, 189th, 199th, 204th, 207th,
228th, 236th, 239th, 456th, 462th, 482th, 489th, 4th
97th, 500th, 516th, 522th, 523nd, 547th, 593th, 66th
9th, 960th, 1007th, 1243th, 1393th, 1719th, 1809th
Position, 2352, 2483, 2702, 2851, 3197, 3333
Position, 3336, 3348, 3394, 3398, 3423, 3505
Position, 3559, 3915, 3992, 4024, 4095, 4216
Position, 4336, 4529, 4727, 4793, 4917, 4991
Position, 5004, 5046, 5228, 5315, 5418, 5426
Position, 5460, 5461, 5516, 5554, 5634, 5656
Position, 5773, 6182, 6221, 6341, 6367, 6371
Position, 6489, 7184, 7325, 7621, 7768, 7787
Position, 7789, 7864, 7895, 7963, 8149, 8251
Position, 8269th, 8276th, 8284th, 8470th, 8485th, 8508th
Position, 8602, 8697, 8752, 8901, 8994, 9123
Position, 9254, 9362, 9380, 9477, 9554, 9708
Position, 9804, 9861, 10034, 10044, 10238, 1
0463th, 10589th, 10978th, 11065th, 11251th, 11th
253rd, 11272th, 11470th, 11527th, 11611th, 116th
74th, 11812th, 11914th, 11947th, 12414th, 1250th
1st, 12609th, 12705th, 12954th, 13111th, 13194th
, 13212, 13368, 13617, 13780, 13966, 14020, 14148, 14178, 14179, 14182,
14212, 14233, 14470, 14582, 14905, 1
5028th, 15043th, 15191th, 15299th, 15380th, 15th
553th, 15607th, 15758th, 15790th, 15808th, 158th
33rd, 15884th, 15924th, 15928th, 16069th, 1608th
6th, 16093th, 16126th, 16129th, 16145th, 16147th
Position, 16172, 16174, 16182, 16183, 16189, 16192, 16193, 16223, 16224, 16234,
16235th, 16239th, 16248th, 16256th, 16261th, 1st
6270th, 16278th, 16290th, 16292th, 16293th, 16th
294, 16298, 16300, 16304, 16309, 163
11th, 16320th, 16355th, 16362th, 16391th, 1648th
It is a mitochondrial single nucleotide polymorphism located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1 at position 2 or 16524.

[0012] In another embodiment, at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease and present in the first biological sample and absent at the corresponding nucleotide in the second biological sample. Type is 709, 930
Position, 960, 980, 1189, 1243, 1700, 1719,
1809th, 1811th, 1888th, 2098th, 2158th, 2259.2th
352, 3010, 3197, 669, 789, 793, 870, 980, 1007, 1243, 1393, 1709, 1719,
It is a mitochondrial single nucleotide polymorphism located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1 which is 2156, 2294, 2483, 2581, 2851, 6366 or 12954.

In another particular embodiment, the invention provides a method for determining the risk or presence of Alzheimer's disease in a subject, the method comprising an organism containing mitochondrial DNA derived from the subject. Determining the presence of at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease in the biological sample. In certain embodiments, at least one associated with Alzheimer's disease
The three mitochondrial single nucleotide polymorphisms are D-loop, mitochondrial rRNA gene, mitochondrial NADH dehydrogenase gene, mitochondrial tRNA gene, mitochondrial cytochrome c oxidase gene, mitochondrial ATP.
It resides in the mitochondrial DNA region where the synthase gene or mitochondrial cytochrome b gene is located. In certain other embodiments, the at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease is mitochondrial NAD.
H dehydrogenase gene, mitochondrial cytochrome c oxidase gene,
The mitochondrial ATP synthase gene or mitochondrial cytochrome b gene is located within the mitochondrial DNA region, and its single nucleotide polymorphism is a non-synonymous nucleotide substitution. In certain other embodiments, at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease is mitochondrial NAD.
H dehydrogenase gene, mitochondrial cytochrome c oxidase gene,
The mitochondrial ATP synthase gene or the mitochondrial cytochrome b gene is located within the mitochondrial DNA region, and single nucleotide polymorphisms are synonymous nucleotide substitutions.

In another embodiment, the at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease is 72, 114, 146, 185, 189, 1.
99th, 204th, 207th, 228th, 236th, 239th, 456th, 46th
2nd, 482nd, 489th, 497th, 500th, 516th, 522nd, 523nd
Position, 547th, 593th, 669th, 960th, 1007th, 1243th, 13th
The 93rd, 1719th, 1809th, 2352th, 2483th, 2702th, 28th
51st, 3197th, 3333th, 3336th, 3348th, 3394th, 33rd
98th, 3423th, 3505th, 3559th, 3915th, 3992th, 40th
24th, 4095th, 4216th, 4336th, 4529th, 4727th, 47th
93rd, 4917th, 4991th, 5004th, 5046th, 5228th, 53rd
15th, 5418th, 5426th, 5460th, 5461th, 5516th, 55th
54th, 5634th, 5656th, 5773th, 6182th, 6221st, 63rd
41st, 6367th, 6371st, 6489th, 7148th, 7325th, 76th
21st, 7768th, 7787th, 7789th, 7864th, 7895th, 79th
63rd, 8149th, 8251th, 8269th, 8276th, 8284th, 84th
70th, 8485th, 8508th, 8602th, 8697th, 8752th, 89th
01st, 8994th, 9123th, 9254th, 9362th, 9380th, 94th
77th, 9554, 9708, 9804, 9861, 10034, 1
0044th, 10238th, 10463th, 10589th, 10978th, 11th
065th, 11251th, 11253th, 11272th, 11470th, 115th
27th, 11611th, 11674th, 11812th, 11914th, 1194th
7th, 12414th, 12501th, 12609th, 12705th, 12954th
Position, 13111, 13194, 13212, 13368, 13617, 13780, 13966, 14020, 14148, 14178,
14179th, 14182th, 14212th, 14233th, 14470th, 1st
4582, 14905, 15028, 15043, 15191, 15
299th, 15380th, 15553th, 15607th, 15758th, 157th
90th, 15808th, 15833th, 15884th, 15924th, 1592th
8th, 16069th, 16086th, 16093th, 16126th, 16129th
Position, 16145, 16147, 16172, 16174, 16182, 16183, 16189, 16192, 16193, 16223,
16224th, 16234th, 16235th, 16239th, 16248th, 1st
6256th, 16261st, 16270th, 16278th, 16290th, 16th
292, 16293, 16294, 16298, 16300, 163
04th, 16309th, 16311th, 16320th, 16355th, 1636th
It is a mitochondrial single nucleotide polymorphism located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1, which is position 2, 16391, 16482 or 16524.

[0015] In certain embodiments, the at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease is 709, 930, 960, 980, 1189.
Position, 1243, 1700, 1719, 1809, 1811, 1888
Position, 2098, 2158, 2259, 2352, 3010, 3197
Position, 669th, 789th, 793th, 870th, 980th, 1007th, 124th
3rd, 1393th, 1709th, 1719th, 2156th, 2294th, 248th
It is a mitochondrial single nucleotide polymorphism located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1, which is position 3, 2581, 2851, 6366 or 12954.

In another aspect, the invention provides a method of determining whether an agent causes, contributes to, or may exacerbate Alzheimer's disease, the method comprising: With a candidate agent, and, after contacting the agent, performing an assay of mitochondrial nucleic acid to determine whether the mitochondrial nucleic acid contains one or more single nucleotide polymorphisms, wherein , The nucleic acid is present in or derived from a cell and one or more single nucleotide polymorphisms segregate with Alzheimer's disease.

In another aspect of the invention, there is provided a method of determining whether an agent causes, contributes to, or may exacerbate Alzheimer's disease, the method comprising: Contacting the cells of claim 1 with a candidate drug; incubating the second cell not contacted with the drug and the first cell under equivalent conditions; the mitochondrial nucleic acid contains one or more single nucleotide polymorphisms 1 for deciding whether or not
Carrying out an assay for mitochondrial nucleic acid as described above, wherein said nucleic acid is present in or derived from a cell and a single nucleotide polymorphism segregates with Alzheimer's disease, wherein The presence of one or more mutations in the mitochondrial nucleic acid from the first cell or from a second cell or from the mitochondrial nucleic acid from the second cell. The absence of one or more mutations indicates whether the drug may cause, contribute to, or exacerbate Alzheimer's disease.

In another aspect, the invention provides a nucleic acid array comprising a plurality of isolated nucleic acid molecules immobilized on a solid support, wherein the isolated nucleic acid molecules are Including all or part of the nucleic acid sequence shown in SEQ ID NO: 1, in which there is at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease. In a particular embodiment, the mitochondrial single nucleotide polymorphism is located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1, which is any of the position numbers listed above. In other embodiments, the step of determining determines that each of the first biological sample and the second biological sample is a primer of mitochondrial D.
Contacting with an oligonucleotide primer comprising all or part of the nucleic acid sequence shown in SEQ ID NO: 1 under conditions to allow hybridization to NA and for a time sufficient therefor, In the array
There is at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease; and the amount of hybridization of the oligonucleotide primer to the mitochondrial DNA of the first sample is determined by the hybridization of that primer to the mitochondrial DNA of the second sample. Comparing the amount of hybridization and then determining the presence or absence of at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease. In certain embodiments, the mitochondrial single nucleotide polymorphism is located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1, which is any of the position numbers listed above. In certain other embodiments, the step of determining comprises subjecting each of the first and second biological samples to conditions under which mitochondrial DNA allows hybridization to the isolated nucleic acid molecule, And for a time sufficient therefor, contacting with a nucleic acid array comprising a plurality of isolated nucleic acid molecules immobilized on a solid support, wherein the isolated nucleic acid molecules are , All or part of the nucleic acid sequence shown in SEQ ID NO: 1, in which there is at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease; and
The amount of hybridization of the mitochondrial DNA of the first sample to the nucleic acid array is compared to the amount of hybridization of the mitochondrial DNA of the second sample to the nucleic acid array, and then a fraction of at least one mitochondria associated with Alzheimer's disease. Determining the presence or absence of a nucleotide polymorphism. In a particular embodiment, the mitochondrial single nucleotide polymorphism is located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1, which is any of the position numbers listed above.

These and other aspects of the invention will be apparent with reference to the following detailed description and accompanying drawings. Further, various references are provided herein that describe certain aspects of the invention in more detail, and thus are incorporated by reference in their entireties.

DETAILED DESCRIPTION OF THE INVENTION The present invention generally relates to compositions and methods for diagnosing the risk or presence of Alzheimer's disease (AD), and agents that may be suitable for treating AD. To compositions and methods for the identification of

In accordance with the invention, mitochondrial DNA (mt) as described herein.
Changes in DNA) provide new and useful parameters for diagnosing the risk or presence of AD and for identifying agents that may be suitable for treating this disease. Such changes include, for example, single nucleotide polymorphisms (SNPs) or homoplasmic mtDNA mutations (eg, Scheffler, IE, Mi.
Tochondria, 1999, Wiley-Liss, Inc. New Y
ork, see pages 286-287). Therefore, the present invention provides
Appropriate portions for mutations associated with D include those that in certain embodiments occur at SNPs or homoplasmic mtDNs that occur at specific positions in mtDNA.
Including but not limited to A mutations. In other particular embodiments, the SNP or homoplasmic mtDNA mutation has a subject with or at risk of having AD as compared to a subject known to be not at risk of having AD. In some embodiments, the SNPs or homoplasmic mtDNA mutations occur in AD with altered frequency (eg, in a statistically significant manner, with increasing or decreasing frequency), or in certain other embodiments. Occurs at a varying frequency in a population of patients who have or are at risk of having AD as compared to a population known to have no risk (eg, in a statistically significant manner, With increasing or decreasing frequency).

Biological samples for use in accordance with the invention, including mtDNA, as provided herein, include any tissue or cell population in which mitochondrial-derived nucleic acid (eg, mtDNA) is present. May be included. Compositions and methods useful for obtaining and detecting mtDNA are provided, for example, in US Pat. Nos. 5,565,323 and 5,840,493. The biological sample can be provided by obtaining a blood sample, biopsy sample, tissue explant, organ culture, or any other tissue or cell preparation from the subject or biological source. The subject or biological source can be a human or non-human animal, a primary cell culture or cell line adapted for culture, which includes chromosomally integrated nucleic acid sequences or episomal recombinant nucleic acid sequences. The resulting genetically engineered cell line, an immortalized or immortalizable cell line, a somatic cell hybrid or a cytoplasmic hybrid "cybrid" cell line (eg, US Pat. No. 5,888,498).
No.), differentiated or differentiable cell lines, transformed cell lines, etc., but are not limited thereto. In certain embodiments of the invention, the subject or biological source may be suspected of having or at risk of having a disease associated with altered mitochondrial function (eg, AD). And, in certain embodiments of the invention, the subject or biological source may be known to be free of, or absent of, such disease. For example, according to non-limiting theory, in certain embodiments it may be desirable to use a control individual as the subject or biological source. This control individual is typically tested by any other number of biological, physiological, immunological, pharmaceutical, pathological, neurological, or other biomedical criteria. , Age and / or gender matched individuals, healthy individuals, or individuals suitable as controls for subjects with or suspected of having AD. Those skilled in the art are familiar with the design and selection of such parameters for clinical correlation. For example, in certain embodiments, it may be desirable to identify control individuals who are believed to be free of AD-related signs and symptoms as described below, and in other particular embodiments, control individuals. May share mitochondrial genetic associations with subjects suspected of being at risk for AD, such as the subject's mother or siblings (see, eg, Scheffler, 1999, supra). ). In other particular embodiments of the invention, the subject or biological source is at least 64 years of age, and in other particular embodiments, the subject or biological source is
At least 75 years of age. In another preferred embodiment, the subject or biological source is at least 85 years of age.

In certain preferred embodiments, it may be desirable to determine whether a subject or biological source meets clinical parameters that are indicative of Alzheimer's disease (AD). Signs and symptoms of AD that are accepted by those of skill in the art (eg, McKhann et al. (Neurology 34: 939, 1984).
, National Institute of Neurology, Com
unidirectional Disorders and Stroke and
Alzheimer's Disease and Related Diso
rders Association Criteria of Probab
le AD, NINCOS-ADRDA) and the references cited therein, or other procedures known in the art for diagnosing AD involve subject or biological sources. It can be used to specify so. Anderson et al. (SEQ ID NO: 1, 1981, Nature 290:
457; Marzuki et al., 1991 Human Genetics 88:
139 also corresponds to the human mtDNA sequence disclosed by Andrew et al. (1999 Nature Genetics 23:14).
Any mtRNA sequence altered according to 7) or part of a mutated mtDNA sequence, or part or parts thereof, may be useful in these embodiments of the invention. Examples of point mutations in human mtDNA derived from specific mtDNA sequence regions that are useful in these and other embodiments of the invention are listed in Tables 1 through 1.
In 4 and Table 9, wild type and mutant mtDNAs are disclosed according to different nucleotide positions.

A portion of the mtDNA sequence of SEQ ID NO: 1 and a portion of a sample mtDNA sequence from a biological source or subject as provided herein are SEQ ID NO: 1.
"Corresponding" based on the transformation to number the mtDNA nucleic acid positions according to
Seen as nucleic acid sequences, regions, fragments, etc. (Anderson et al., Nat
ure 290: 457, 1981). Here, the sample mtDNA sequence is aligned with the mtDNA sequence of SEQ ID NO: 1 so that at least 70%, preferably at least 80%, and more preferably at least 90% of the nucleotides in a given sequence of at least 20 consecutive sequences. % Are the same. For example,
Part of an mtDNA sequence in a biological sample containing mtDNA from a subject having or suspected of having AD, or as another example, Alzheimer's disease as provided herein. A portion of the mtDNA sequence in mtDNA containing at least one mitochondrial single nucleotide polymorphism (eg, mutated mtDNA) associated with any of the numerous alignment procedures and / or tools known to those of skill in the art (eg, , CLUSTAL W, Thompson et al., 1994 Nucl. Ac. Res. 22: 4673: CAP.
． embnet. org / clustw. html: FASTA / FASTP
Pearson, 1990 Proc. Nat. Acad. Sci. USA
85: 2444, D.I. Hudson, Univ. of Virginia, Ch
(available from Arlottesville, Va.)
It can be aligned with the corresponding part of the tDNA. In certain preferred embodiments, the sample mtDNA sequence is greater than 95% identical to the corresponding mtDNA sequence of SEQ ID NO: 1. In another particular preferred embodiment, the sample mtDNA
The sequence is identical to the corresponding mtDNA sequence of SEQ ID NO: 1. Sequence number 1 mt
Oligonucleotide probes having sequences that are identical in the DNA sequence and in the corresponding regions of the sample mtDNA can be identified and selected according to hybridization target DNA sequence analysis to confirm the absence of mutations.

According to the present invention and as is known in the art, the term “haplotype” refers to a specific combination of genetic markers in defined regions of the mitochondrial chromosome. Such genetic markers include, for example, RFLP and SNP. RELP (restriction fragment polymorphism) is a DNAs known as a restriction enzyme.
It results from a change in the recognition site (often a palindrome) that is specifically cleaved by e in a site-specific manner. A SNP (single nucleotide polymorphism) is a change (eg, deletion, insertion, or substitution) in any single nucleotide base in a region of the genome of interest. In a particularly preferred embodiment provided by the present disclosure, the genome of interest is the mitochondrial genome. Since SNPs vary from individual to individual, these are useful markers for studying genomic associations. Moreover, since they are more frequent than other markers such as RFLP, S
Analysis of NPs is not surprising to generate "high resolution" maps of segregation of disease-related genetic markers (Weiss, Genome Res. 8: 691-697, 199).
8; Gelbert and Gregg, Curr. Opin. Biotechn
ol. 8: 669-674, 1997).

The term “haplogroup” refers to a group of haplotypes found in association with each other. Several mitochondrial DNA haplotypes and haplogroups are known in the art, including 10 European mtDNA haplogroups and distinct Asian, Native American and African mtDNA haplogroups, each of which Was identified based on the presence or absence of one or more specific restriction endonuclease recognition sites (
For example, Wallace et al., 1999 Gene 238: 211; Torro.
ni et al., 1996 Genetics 144: 1835).

Nucleic acid sequences within the scope of the present invention include mtDNA nucleotide sequences under conditions of moderate or high stringency, including mtDNA sequences disclosed herein or fragments thereof, and their complements. Included are isolated DNA and RNA sequences that specifically hybridize to. As used herein, conditions of moderate stringency are as known to those of skill in the art, and in Sambrook et al., Molecular Cloning: A Labo.
rally Manual, Second Edition, Volume 1, 1.101-104, Col.
d Spring Harbor Laboratory Press (19
89), for example, a pre-wash solution of 5 × SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) for a starting nucleic acid-immobilized nitrocellulose filter. , 50% formamide at 42 ° C., 6 × SSC (or other similar hybridization solution) hybridization conditions, and 0.5 × SSC at about 50-60 ° C., 0.1% SDS.
Including use as a cleaning condition. Conditions of high stringency were defined as hybridization conditions as above, and were 60-68 ° C, 0.2x.
A wash of SSC, 0.1% SDS is used. In another embodiment, mtDNA
Hybridization with nucleotide sequences can be at normal stringency, which is about 25-30 ° C. below the Tm of the native duplex (eg, 5 × SSPE, 0.5% SDS, 5 × Denhardt's solution, 50% formamide, 42 ° C. or equivalent conditions). For low stringency hybridization, conditions of about 40 ° C below Tm are used, or for high stringency hybridization, conditions of about 10 ° C below Tm are used. Those skilled in the art will appreciate that the temperature, salt concentration, and chaotrope composition of the hybridization and wash solutions can be adjusted as necessary according to factors such as probe length and nucleotide base composition. (For example, Ausubel et al., Current
Protocols in Molecular Biology, Green
See also, Publishing, 1987). Thus, the desired variation in stringency of hybridization conditions is achieved by varying the time, temperature, and / or concentration of solution used for the prehybridization, hybridization, and wash steps. obtain. Thus, it is understood that appropriately stringent conditions can be readily selected without undue experimentation, wherein the desired probe selectivity is hybridized to one or more particular starting sequence. Identified based on the ability to soy but not hybridize to other particular starting sequences.

“Isolated nucleic acid molecule” refers to a polynucleotide molecule in the form of separate fragments, or as a component of a large nucleic acid construct, that is its source cell (usually there From existing chromosomes), at least once, preferably in substantially pure form. An isolated nucleic acid molecule can be a nucleic acid having a particular disclosed nucleotide sequence, or can be a region, portion or fragment thereof. One of skill in the art will appreciate that an isolated nucleic acid molecule having the complete nucleotide sequence, or any portion of a particular isolated nucleic acid molecule, when provided with the appropriate nucleic acid sequence information as disclosed herein. Can be prepared. Nucleic acid molecules can be composed of a wide range of nucleotides, including
A, RNA, nucleotide analogs (eg phosphorothioate or peptide nucleic acids) or other analogs well known to those skilled in the art, or some combination thereof.

The present invention provides mtDNA sequences and isolated mtDNA nucleic acid molecules, as described herein. mtDNA is a well-known methodology (eg, the methodology described in US Pat. No. 5,840,493, which is incorporated by reference in its entirety).
Can be isolated from cellular DNA according to.

Where it is advantageous to use oligonucleotide primers according to the invention, such primers are from 10 to 60 nucleotides in length, preferably 15 to 3 nucleotides.
It may be 5 nucleotides long, and even more preferably 18-30 nucleotides long. The primer may be a mtDNA mutation (as described herein) by any of a variety of techniques well known to those of skill in the art for determining the amount of a particular nucleic acid target sequence present in a sample based on the specific hybridization of the primer to the target sequence. (Including single nucleotide polymorphisms or homoplasmy mtDNA mutations provided therein) can be useful in the present invention. If necessary, in these particular technologies,
Hybridization occurs prior to nucleotide polymerase-catalyzed extension of the primer using a strand containing the target sequence as a template and / or ligation of oligonucleotides that are hybridized to adjacent target sequences, and primer extension of the present invention using primer extension. The embodiment is particularly preferred.

As an example of a reference to such a quantitative detection technique, a nucleotide insertion, substitution or deletion in a part of the mtDNA sequence site near the oligonucleotide primer target hybridization site corresponding to a part of the wild type mtDNA sequence. Loss (disclosed in Anderson et al. (1981 Nature 290: 457, SEQ ID NO: 1)) or a mutated site such as may be created by any of the mtDNA point mutations disclosed herein. Mention may be made of detection techniques which may be used, and in addition techniques involving primer extension. All of which are incorporated by reference, U.S.P. S. See 5,706,205 and references cited therein. For example, Botstein et al. (Am. J. Hu
m. Gen. 32: 314, 1980), Gibbs et al. (Nucl. Ac. Re).
s. 17: 2437, 1989), Newton et al. (Nucl. Ac. Res.
17: 2503, 1989), Grossman et al. (Nucl. Ac. Res.
22: 4527, 1994), and Saiki et al. (Proc. Nat. Aca).
d. Sci. 86: 6230, 1989) (all of these are
Incorporated as a reference). A particularly useful method for this purpose is the primer extension assay (Fahy et al. (Nucl. Acids Res. 25: 3102, 1997) and Ghos, both incorporated by reference in their entireties.
h et al. (Am. J. Hum. Genet. 58: 325, 1996),
Kook et al. (Also disclosed in Hum. Molec. Genet. 1: 391, 1995). These teach modified methods of primer extension reactions to detect multiple nucleotide substitutions, insertions, deletions or other mutations. Other examples of techniques useful for quantifying the presence of a particular nucleic acid target sequence in a sample include, but are not limited to, the following: the target nucleic acid is first part from other nucleic acids present in the sample. Of labeled probes to target nucleic acid sequences with or without selective separation.

Examples of other useful techniques for determining the amount of a particular nucleic acid target sequence present in a sample based on the hybridization of primers to this target sequence include: Specific amplification and quantification of amplification products.
This specific amplification and quantification of amplification products includes, but is not limited to, the polymerase chain reaction (PCR, Gibbs et al., Nucl. Ac).
． Res. 17: 2437, 1989), transcription amplification system, strand displacement amplification (stran
d displacement amplification) and autonomous sequence replication (3SR, Ghosh et al., Molecular Methods fo).
r Virus Detection, 1995 Academic Pres
s, NY, 287-314) (the references cited therein are incorporated by reference in their entirety). Examples of other useful techniques include: ligase chain reaction, single chain conformation polymorphism analysis, Q-β replicase assay, restriction fragment length polymorphism (RFLP, Botstein et al., Am. J. Hum.
． Gen. 32: 314, 1980) Analyzed and cycled probe technology, and other useful methods known to those of skill in the art.

In certain preferred embodiments of the invention, primer extension is used to quantify mtDNA present in a biological sample (Ghosh et al., Am. J. Hum.
． Genet. 58: 325, 1996). This embodiment uses wild-type mtDNA
Certain advantages may be provided by allowing both mutated and mutated mtDNA to be quantified simultaneously with a single oligonucleotide primer capable of hybridizing to a complementary nucleic acid target sequence. This complementary nucleic acid target sequence is a wild-type mtD
It is located in a defined region of NA and in the corresponding region of the mutated mtDNA sequence. Without wishing to be bound by theory, using a single primer to quantify wild-type and mutated mtDNA was associated with a potential imbalance in the relative hybridization properties of multiple primers. It is believed to avoid uncertainty and may offer other advantages. Such a target sequence has a mutated mtDNA nucleotide sequence position (corresponding wild type mtDN
Primer extension assay, the oligonucleotide extension product of the primer that hybridizes to the mutated mtDNA is of the primer that hybridizes to the wild-type mtDNA. As it has a different length than the oligonucleotide extension product,
Can be designed. Thus, the amount of mutated mtDNA in a sample and the amount of wild-type mtDNA in a sample can be determined by quantification of different extension products that are separable based on sequence length or molecular weight.

The sequence length or molecular weight of the primer extension assay product can be determined using any known method for characterizing the size of nucleic acid sequences, which is familiar to one of skill in the art. In a preferred embodiment, the primer extension product is characterized by gel electrophoresis. In another preferred embodiment, the primer extension product is characterized by mass spectrometry (MS). Mass spectrometry may further include matrix assisted laser desorption / ionization / time of flight (MALDI-TOF) analysis or other MS techniques known to those of skill in the art. For example, U. S. 5,622,824, U.S.P. S. 5,60
5,798 and U.S.P. S. 5, 547, 835 (all of these are
The whole is incorporated as a reference). In another preferred embodiment, the primer extension product is characterized by liquid chromatography or gas chromatography. These chromatographies may further include high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) or other well known chromatographic methodologies.

In another particular preferred embodiment of the invention, the DNA in the biological product containing mtDNA is first amplified by methodologies well known in the art such that this amplification product is present in the sample. Single nucleotide polymorphism or homoplasmy mtDNA
It can be used as a template in a method for detecting mutations. Therefore,
It is desirable to use oligonucleotide primers that are identical in wild-type mutated DNA and mutant mtDNA and are complementary to a common target sequence (eg, Fahy et al. (Nucl. Acids Res., 25: 3102, 1997.
) And Davis et al. (Proc. Nat. Acad. Sci. USA 94.
: 4526, 1997; Hirano et al., Proc. Nat. Acad. Sci
． USA 94: 14894, 1997 and Wallace et al., Proc. N
at. Acad. Sci. USA 94: 14900, 1997) also prepared PCR amplification template and primers).

In certain other preferred embodiments, the mtDNA mutation is a plurality of different mt
Can be efficiently detected, screened, and / or quantified by high-throughput hybridization methodologies directed to independently probe DNA, or multiple different oligonucleotide primers provided herein. . These are immobilized as a nucleic acid array on a solid support. Typically, the solid support can be silica, quartz or glass or known in the art and allow the appropriate hybridization, washing and detection steps provided herein. It can be any other material that can leave the nucleic acid immobilized in a manner. In a preferred embodiment, the solid phase nucleic acid array is precisely spaced and positioned at specific locations, eg, as described in US Pat. No. 5,800,992 (eg, WO95 / 21944; Schena).
Et al., 1995 Science 270: 467-470, 1995; Peas.
e et al., 1994 Proc. Nat. Acad. Sci. USA 91: 502
2; Lipshutz et al., 1995 Biotechniques 19:44.
See also 2-447).

Detection of hybridized (eg, double-stranded) nucleic acids on a nucleic acid array is performed according to any known procedure, eg, by spectroscopy or potentiometry (eg, MALDI-MS). obtain. In certain preferred embodiments, the array comprises oligonucleotides less than 50 bp in length. For high throughput screening of nucleic acid arrays, formats can be analyzed, preferably based on automation. For example, automated devices for use in accordance with the high throughput screening embodiments of the present invention are preferably under the control of a computer or other programmable controller. The controller can continuously monitor the results of each step of nucleic acid deposition, washing, hybridization, detection and related processes, and automatically change the test paradigm in response to these results.

The present invention also provides compositions and methods that are useful in pharmacogenomics for classifying and / or stratifying a subject or patient population. Such stratification can be performed, for example, with one or more particular traits in a subject (and optionally with an indication of responsiveness to a particular therapeutic treatment or efficacy of this therapeutic treatment), Correlations with single nucleotide polymorphisms or homoplasmy mtDNA mutations provided herein are included. In one aspect of the present invention, a single nucleotide polymorphism or homoplasmy mtDN separated from AD derived from a subject
Detection of the A mutation in a biological sample is accomplished by detecting the apolipoprotein E (A
POE) in combination with genotyping, Alzheimer's disease (A
The risk of D) and the presence of this disease are determined. The apolipoprotein E4 type allele (APOE-ε4 allele) is a genetic susceptibility factor for sporadic AD and confers a two-fold risk for AD (Corder et al., Science).
e 261: 921, 1993; "National Institute o.
n Aging / Alzheimer's Association Work
ing Group Consensus Statement ", Lance
See also t 347: 1091, 1996 and references cited therein, all of which are incorporated by reference in their entireties. Thus, in a preferred embodiment of the invention, the method for determining the risk of AD and the presence of AD in a subject by detecting single nucleotide polymorphisms or homoplasmy mtDNA point mutations according to the present disclosure is AD Further comprising determining the APOE genotype of the subject suspected of being at risk for. By using a combination of methods for determining mtDNA point mutations and methods known in the art for genotyping APOE, as disclosed herein, the relative risk for AD. The enhanced ability to detect L., along with other related advantages is provided by the present invention. Similarly, where APOE genotype and risk for AD are correlated, the present invention provides an advantageous method for identifying suitable agents for treating AD, where such The effect of a drug can be correlated with the detection of one or more specific single nucleotide polymorphisms or homoplasmy mtDNA mutations in a biological sample.

As described herein, certain single nucleotide polymorphisms or homoplasmy mt
Determination of DNA mutations can be used to stratify the AD patient population. Therefore,
In another preferred embodiment of the invention, the determination of such mutations in a biological sample from an AD subject may provide a useful indicator of correlation for the subject. AD subjects so stratified on the basis of one or more specific mutations can then be monitored using the AD clinical parameters mentioned above. As a result, the correlation between specific mtDNA mutations and any tube floor clinical score used to assess AD can be monitored. For example, AD according to at least one single nucleotide polymorphism or homoplasmy mtDNA mutation provided herein.
Stratification of the patient population can provide useful markers that correlate the efficacy of any candidate therapeutic agent used in AD subjects. In a further preferred embodiment of this aspect of the invention, one or more specific mtDNA mutations coordinated with APOE genotyping of AD subjects may be useful, as described above. These and related advantages will be appreciated by those skilled in the art.

In a particularly preferred embodiment oligonucleotide primers are used,
This primer allows specific detection of single nucleotide polymorphisms or homoplasmy mtDNA point mutations disclosed in Tables 1-4 and Table 9. Here, mitochondrial genes (eg, include: 12S rRNA, 16S rRNA
, Some tRNAs, COX1, COX2, COX3, cytochrome b, AT
Specific genes in Pase 8, ATPase 6, ND1, ND2, ND4 and ND5) and many mutations in the mtDNA loop region are also disclosed. Each mutation listed in Tables 1-4 and Table 9 is represented as follows: (i) nucleotide identity at specific nucleotide positions according to the wild-type human mtDNA sequence (Anderson et al., 198).
1 Nature 290: 457; Andrews et al., 1999 Nature.
e Genetics 23: 147 and references cited therein), (ii) nucleotide position numbers according to the convention of Anderson et al. (1981) and (iii) identified as disclosed herein. The identity of the mutated nucleotides at different positions. Thus, for example, wild type mtDNA 12S
Nucleotide T (thymine), located at position 1189 of the rRNA gene, is a nucleotide C (in mtDNA analyzed from a substantial number of patients diagnosed with AD.
Cytosine) (see Tables 1, 2, and 4). Table 9 also shows the specific AD
The number of samples in which the related SNP was detected and the AD-related SNP was first identified m
Represents the mitochondrial haplogroup of tDNA sample donors (see also Examples below).

As shown in Tables 2-4 and disclosed herein, mitochondrial single nucleotide polymorphisms or homoplasmy mtDNA point mutations (Anderson et al. (198
MtD against the "wild type" human mtDNA sequence (CRS) disclosed by 1).
(Including deviations in the identity of nucleotide bases located at specific positions in the NA sequence) can fall into at least one of the following categories: "error"
Is a sequencing error in the human mtDNA sequence reported by Anderson et al. (1981) (Andrews et al. (1999 Nature Gen).
etics 23: 147)). The "polymorphisms" in Tables 2-4 are not associated with a particular human disease but are detected and described as a result of naturally occurring variations in the identity of the nucleotide bases located at a given position in the human mtDNA sequence. Known polymorphism in the human mtDNA sequence (see, for example, "Mitomap", Emory University).
School of Medicine, http: // www. gen. e
more. Available on edu). "Rare polymorphism" in Tables 2-4 means Ande
The Cambridge Reference Seque of Rson et al. (1981).
ence (CRS), but different from the base located at the corresponding position, but upon subsequent accumulation of human mtDNA sequence data from multiple subjects (and CRS).
(As opposed to the reliability of a single donor mtDNA for producing Anderson et al.) To a larger sample population, referring to mtDNA nucleotides suggesting the presence of a low frequency allele in the CRS donor (Andrew).
s et al., 1999 Nature Genetics 23: 147 and references cited therein). Particularly useful mutations isolated from AD in accordance with the present invention include: non-error homoplasmy mtDNA point mutations (eg, single nucleotide polymorphisms) shown in Tables 2-4, the polymorphism described immediately above. Or rare polymorphisms, further homoplasmy mtDNA point mutations shown in Table 9 (eg single nucleotide polymorphisms).

[0042]

[Table 1]

[0043]

[Table 2]

[0044]

[Table 3]

[0045]

[Table 4] The following examples are provided for purposes of illustration and not limitation.

Example 1 DNA Isolation from Blood and Brain Samples Venous blood samples were obtained from donor individuals and vacutainers containing EDTA.
Collected in a tube. The white blood cell fraction was obtained by centrifugation at 2,500 rpm for 30 minutes at 4 ° C. The white blood cell layer was collected and 5 ml of sterile TE buffer (10 mM Tri
s-HCl, pH 7.4, 1 mM EDTA) and diluted at 2,500 rpm to 1
Centrifuge for 0 minutes at 4 ° C. The cell pellet is then 1% SD in TE buffer.
Lysis was achieved by the addition of 10 ml lysis buffer containing S and 400 μg / ml proteinase K. The cells were incubated for 4 hours at 37 ° C. in an orbital shaker with shaking at 200 rpm. All cellular DNA was purified by two extractions with phenol / chloroform and two extractions with chloroform. DNA was precipitated by adding 1/10 volume 5M NaCl and 2x volume 100% ethanol and placed at -20 ° C. The DNA was pelleted by centrifugation, washed with 70% ethanol and resuspended in TE buffer.

All cellular DNA was also lysed with a lysis buffer (50 mM Tris-HCl, pH 7.9, 100 mM EDTA, using a Duns glass homogenizer).
Isolated from frozen brain tissue by homogenizing the tissue in 0.1 M NaCl, 0.03 M DDT, 1% SDS, 1 mg / ml proteinase K). Homogenized brain tissue was incubated for 30-60 minutes at 45-50 ° C. The DNA was purified by two extractions with phenol / chloroform and two extractions with chloroform. 1/10 volume of 5M Na
Precipitated by adding Cl and 2x volume 100% ethanol and placed at -20 ° C. The DNA was pelleted by centrifugation, washed with 70% ethanol and resuspended in TE buffer.

All cellular DNA was also isolated from human SH-SY5Y neuroblastoma cells by first removing the cells from tissue culture flasks by treatment with 0.5 mM EDTA in Dulbecco's PBS. did. Then the cells for 10 minutes 2,
Pelletized by centrifugation at 000 g and the DNA was isolated according to the manufacturer's recommendations with the DNAzol reagent (Molecular Research Center, I.
nc. , Clincinnati, OH).

[0049]   DNA concentration was determined by UV absorption at 260 nm.

Example 2 PCR Amplification, Sequencing, and Sequence Analysis All cellular DNA prepared from leukocytes, brain tissue and SH-SY5Y cells as described in Example 1 was treated with mtDNA light. Chain nucleotide sequence (Table 5, "L
Primer) and a set of oligonucleotide primers specific for the indicated regions of the mtDNA heavy chain nucleotide sequence (Table 5 "H"), or mtDNA
A set of primers complementary to a portion of the D loop region (this is the mitochondrial D
It was used for amplification (see Wallace et al., Mitochondria & Free), which can amplify essentially the entire region of the NA molecule (Table 6).
Radicals in Neurodegenerative Diseases
es, M .; F. Beal, N.M. Howell and I.D. Bodis-Wolln
er, 1997 Wiley-Liss, Inc. , Pp. 283-307, and references cited therein). Amplification was performed using 0.5-1.0 μg of DNA, 200 ng of each light and heavy chain or forward primer (Table 6 “
F ") and reverse primers (Table 6," R "), 200 μM dNT each.
P, 10 mM Tris-HCl, pH 8.3, 50 mM KCL, 2 mM M
gCl ₂ and 1 unit of AmpliTaq DNA polymerase (Perkin
-Elmer, Norwalk, CT) in 50 μl reaction volumes.
Gene Amp PCR System 9600 Thermal Cycler (Perki
n-Elmer) was used to amplify as follows: 95 ° C., 10 seconds,
30 cycles of 95 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 1 minute, and 1
Cycle 72 ° C for 4 minutes. Amplicon with QIAquick PCR Puri
Purified by fication Kit (Quiagen, Chatsworth, CA).

[0051]

[Table 5]

[0052]

[Table 6] Sequencing of the purified PCR product was performed using the same primers and BigDye Terminator Cycle Sequ as previously used for amplification.
It was carried out using an encoding kit (Perkin-Elmer). Sequencing reaction products were processed by ethanol precipitation or by CentriSep spin columns (Princeton Separations, Adelphia, NJ).
And purified using Applied Biosystems Model 373.
A DNA Sequencing System (Applied Bio
systems Division, Perkin-Elmer, Foster
Electrophoresis in City, CA). In addition to the CAP and ALIGN sequence analysis programs, Sequence Navigator software (
Applied Biosystems Division, Perkin-E
lmer) was used for the analysis of sequence data and the mutations were published to human mt
Sequence of DNA (Anderson et al., 1981 Nature 290: 457.
) And identified.

Example 3 Single Nucleotide Polymorphism (SNP) in Mitochondrial Ribosomal RNA Separated with Alzheimer's Disease Mitochondrial DNA (mtDNA) was analyzed according to Examples 1 and 2 in 24 controls (12 partial confirmation). Disease control, 2 biopsy-confirmed normal controls, and 10 surviving controls) and 33 AD cases (2
Were isolated and sequenced from brain and blood samples from 0 biopsy-validated AD and 13 surviving AD patients). Changed mitochondrial rRN
The A sequence was detected in 82% of AD samples vs. 50% of controls (Table 1).
And Figure 1). If this analysis is limited to the part confirmation (ac) case, the rRN
A sequence changes were detected in 85% AD and 43% control samples.

Approximately half of the mtDNA changes are in haplogroups T, U, and H (familial strains)
Related to. However, considering only the mutations in mtDNA encoding mitochondrial rRNA not associated with the haplogroup, 49% AD vs. 21% control samples retained sequence changes (Table 1 and Figure 1). The specific correlation of AD with non-haplogroups associated with mutations in mtDNA encoding mitochondrial rRNA is higher when only the confirmed cases are included. Mutations in mtDNA encoding mitochondrial rRNA are detected in 40% AD vs. 7% biopsy-validated control cases.

Example 4 (Homoplasmic Single Nucleotide Polymorphism in Mitochondrial DNA of a Subject Diagnosed with Alzheimer's Disease) This example describes a subject diagnosed with Alzheimer's disease (D-1). Detection of mtDNA single nucleotide polymorphisms in DNA of cybrid cell lines prepared using white blood cells derived from E. coli and platelet-derived mtDNA from the same subject.

Venous blood samples were taken from human donor subjects diagnosed with AD (D-1) (
McKhann et al., Neurology 34: 939, 1984, Natio.
nal Institute of Neurology, Communica
live Disorders and Stroke and Alzhei
mer's Disease and Related Disorders
Association Criteria of Probable AD,
NINCDS-ADRDA) from vacutainer tubes containing EDTA and maintained at 0-4 ° C. for preparation of leukocyte fractions or containing citrate / glucose and maintained at room temperature for preparation of platelet fractions. (Becton-D
ickinson, Inc. , San Jose, CA).

To prepare the leukocyte fraction, blood samples were overlaid on Histopaque®-1077 (Sigma, St. Louis, MO), 2500
Centrifuge at 4 ° C. for 30 minutes at rpm. The white blood cell layer was collected, diluted with 5 ml sterile TE buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) and centrifuged at 2500 rpm for 10 minutes at 4 ° C. The cell pellet was then mixed with 10 ml lysis buffer (1% SDS and 400 μg / ml proteinase K).
TE), and dissolved in an orbital shaker set at 200 rpm for 4 hours at 37 ° C. All cellular DNA was purified by two extractions with phenol / chloroform and two extractions with chloroform. DNA was precipitated by adding 1/10 volume 5M NaCl and 2x volume 100% ethanol and incubated at -20 ° C. The DNA was pelleted by centrifugation, washed with 70% ethanol and resuspended in TE buffer.

Preparation of platelets and use of platelets (as a source of mtDNA) for fusion to ρ ⁰ SH-SY5Y neuroblastoma to produce a cybrid cell line is described by Miller et al. (1996 J. Neurochem). .67: 1897-1
907). Obtained cybrid cell line (AD
-1) is a mitochondrial complex IV (cytochrome c oxidase, COX,
It had a stable deletion in CO) activity (described below). Expanded cultures of AD-1 cybrid cells were treated with Dulbecco's PBS (GIBCO-BRL, G
were removed from the tissue culture flasks by exposing them to 0.5 mM EDTA in rand Island, NY). Harvested AD-1 cells
Pellet by centrifugation (2000 xg, 10 min) and collect all cellular DNA according to the manufacturer's instructions with DNAzol reagent (Molecular).
Research Center, Inc. , Clincinnati, OH).

DNA concentration was determined by UV absorption at 260 nm. AD leukocytes and A
All cellular DNA prepared from D cybrid cells was used as a template for DNA amplification by polymerase chain reaction (PCR) with a set of L and H chain oligonucleotide primers over the entire mtDNA molecule (Table 7). ). Amplification was performed using 0.5-1.0 μg template DNA, 200 ng each of L and H chain primers or forward and reverse primers, 200 μM dNTP, 10 mM Tris-HC, respectively.
pH 8.3, 50 mM KCl, 2 MM MgCl ₂ and 1 unit of Am
pliTaq DNA Polymerase (Perkin-Elmer, Norway)
k, CT) in a 50 μl reaction volume. Gene Amp ^™ PCR
Amplification was performed using a System 9600 thermocycler (Perkin-Elmer) as follows: 95 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C 1
Min, 30 cycles, followed by 1 cycle of 72 ° C for 4 minutes. Amplification products were purified by horizontal agar gel electrophoresis, band excision, elution of DNA from agar, and ethanol precipitation. Alternatively, amplicons were purified by the QIAquick ^™ PCR Purification Kit (Qiagen, Chatsworth, CA).

The PCR product was subjected to TA-Cloning ^™ kit (Invitrogen, I.
nc. , Carlsbad, CA) and XL2Blue ^™ or XL2Bl.
ueMRF ^™ competent cells (Stratagene, Inc., La Jo)
(Ila, CA). All followed the supplier's recommendations. Recombinant colonies were selected and the plasmid DNA was labeled with Wizard ^™ Series 9
600 DNA Purification System (Promega, Inc., Madison, W
Purified using I).

[0061]

[Table 7] Sequencing of the purified PCR product was performed as described in Example 2
~ 12 clones were sequenced for each cloned PCR product. Mutation,
As noted above, polymorphisms were reported for human mtDNA, identified by comparison to published sequences containing related correlations (Anderson et al., 1981 Nat.
ure 290: 457; Andrews et al., 1999 Nature Gen.
etics 23: 147; Mitomap, www. gen. emory. e
du). The same homoplasmon mtDNA mutation was detected in DNA samples from D-1 leukocytes and DNA from AD-1 cybrid cells. Table 2
Rare polymorphisms and sequence "errors" for CRS (as described above) were detected, as indicated by the polymorphisms in. In addition, three novel homoplasmon single nucleotide polymorphisms were detected as mtDNA mutations (Table 2): T1189C was observed in the 12S rRNA gene, G6366A was present in the CO1 (COX1) gene, and T12954C. Was found in the ND5 gene. Sequence analysis
The G6366A mutation causes a valine to isoleucine substitution (missense) at amino acid position 155 of the COX1 subunit of complex IV, and in contrast, T12954C reveals a silent mutation in the ND5 subunit of complex I. I showed that.

Example 5 Expression of AD Donor-Induced Mitochondrial DNA in Cybrid Cell Lines This example was constructed using mtDNA from a donor diagnosed with AD as described in Example 4. In an established AD-1 cybrid cell line
Expression of donor-derived mtDNA sequences is shown.

Competitive primer extension assays are essentially (Fahy et al., 1997 Nucl. Ac.) Except that the homoplasmon G6366A pair mutation detected in AD-1 cybrid cells is developed (as described in Example 4). Res. 25: 3102
). Template DNA was prepared from white blood cells and cybrid cells as described in Example 4, and parent SH-SY5.
Y neuroblastoma cells and pooled control cybrid cells (ie, except re-growth with mtDNA from subjects diagnosed as having no AD, Miller et al. (1996 J. Neurochem. 67: 1897-19).
(Cybrid cells constructed as described in 07)). mtDNA
To evaluate the expression of the encoded COX1 (COI) gene, all cellular RNA
To the Trizol ^™ reagent (Life Technologies, Inc., G.
Isomersburg, MD) and isolated from cybrid cells using Superscript according to the manufacturer's instructions to generate cDNA.
Reverse transcription was performed using t ^™ reverse transcriptase.

The following 5'-FAM labeled oligonucleotide primers were prepared according to standard methods: 5'-TGATGAAATTTGATGGCCCCTAAGATAGAGGAG.
A--3 '[SEQ ID NO:].

Using a nucleotide mixture comprising dTTP, ddATP and ddCTP,
As a result, the wild-type (ie, CRS) mtDNA template directs extension of the primer bearing the ddC residue, while D-1 (ie, AD-induced) mtD
The NA template resulted in extension of the primer with dT and ddA residues. As described by Fahy et al. (1997), the primer extension reaction product is electrophoresed under conditions that allow the determination of differentially extended (eg, by one or two nucleotides) fluorescent primer products. Is divided into As shown in FIG. 2, the template DNA or cDNA derived from AD-1 cybrid cells (lanes 5 and 7) and D-1 (AD) leukocytes (lane 4) is a primer extended by two nucleotide residues. Homoplasmon G6366 in mtDNA from these cells that is directed to the exclusive production of extension products
Consistent with the presence of the A mutation. In contrast, template DNA or cDNA from pooled control cybrids or parental SH-SY5Y cells (
Lanes 6, 8 and 9) direct only the production of primer extension products with a single nucleotide extension, indicating the presence of wild-type DNA in these cells.

Example 6 Mitochondrial Electron Transfer Chain Enzymes in AD-1 Cybrid Cells This example describes the expression and activity of mitochondrial electron transfer chain enzymes in a cybrid cell model of Alzheimer's disease.

AD-1 cybrid cells were prepared as described in Example 4. A control cybrid cell line was prepared as described (Miller et al. 1996 J.
． Neurochem. 67: 1897), ρ ⁰ SH-SY5Y neuroblastoma cells and PEG fusion of platelets from 3 age-matched cognitively normal subjects. Control cybrid cells had normal Complex I and IV activity when compared to parental SH-SY5Y cells when assayed 32-37 days after fusion (see below). Subsequently, equal numbers of cells from each control cybrid cell line were pooled and this mixed control culture was maintained. The activity of complex I (ND) and complex IV (COX) was determined as described (
Miller et al., 1996 J. Neurochem. 67: 1897) determined. And reported as min-1mg-1 whole cell protein. Reactive oxygen species (ROS) were detected using the dichlorofluorescein diacetate (DCF-DA) assay (Miller et al. 1996). The activities of catalase, total superoxide dismutase (SOD), Mn SOD, Cu / Zn SOD, glutathione peroxidase and glutathione reductase were as previously described (Cassarino et al., 1997 Biochim. Bioph.
ys. Acta 1362: 77) determined. 8-hydroxyguanosine (8O
For the determination of (H-dG) levels, DNA samples were prepared by hypotonic lysis of cybrid cells, followed by proteolysis at 95 ° C with a thermostable alkaline protease. The DNA was digested with exonuclease PI and endonuclease III and analyzed for 8OH-dG using electrochemical detection. Hydroxyl radicals using thiobarbituric acid (S
attler et al., 1998 Meths. Mol. Biol. 110: 167-
191) Assayed.

The activity of complex I in AD-1 cybrid cells was essentially the same as the activity detected in mixed control cybrid cells. In contrast, complex IV (COX) activity is AD-1 compared to mixed control cybrid cells.
It was remarkably suppressed in cybrid cells. And this deficiency remained stable over an extended culture period (FIG. 3). The reduced complex IV activity in AD-1 cybrids was due to the reduced amount of COX protein in these cells (
For example, reduced C associated with the G6366A mutation in the COX1 subunit
OX production), the expression levels of COX subunits 1, 2 and 4 were compared at the polypeptide level by Western immunoblot analysis. Established procedures (Ausubel et al., Current Pr)
otocols in Molecular Biology, Greene
Publishing, 1987), detergent lysates of AD-1 cybrid cells, mixed control cybrid cells and SH-SY5Y neuroblastoma cell lines were electrophoretically separated and blotted to nitrocellulose (bl).
), and mouse anti-human COX subunit-specific antibody (Molecular Probes, Inc., Eugene, OR).
I searched in. Blots were HRP-conjugated anti-mouse immunoglobulin (Amersham, Inc., Arlington Heights) according to the supplier's instructions.
, IL) and ECL chemiluminescence detection (Amersham). As shown in FIG. 4, AD-1 cybrid cells, mixed control cybrid cells and parental SH-SY5Y neuroblastoma cell lines all expressed comparable levels of COX subunits. Without attempting to be bound by theory, these results are
Catalytic deficiency of COX (and an unreduced level of COX biosynthesis) in the ability of the COX subunits to bind to each other or to be localized to the appropriate subcellular location results in G6366A in the COX1 subunit of AD-1 cybrid cells. It suggests that it may be the result of mutation.

AD-1 cybrid cells also had elevated levels of reactive oxygen species (ROS) products compared to mixed control cybrid cells, with an apparent taper in relative levels of ROS that was elevated over time. Was shown (Table 8). To determine whether the compensatory mechanism is associated with this progressive decline, ROS by the DCF-DA method
Prior to determination, add AD-1 and mixed control cybrid cells for 2 hours, 3 hours.
Preloaded with 0 μM DCF-DA, rinsed and then treated with either 50 μM ethacrynic acid (reduced glutathione scavenger) or 2.5 mM aminotriazole (inhibitor of the radical scavenging enzyme catalase) for 30 minutes. As shown in Figure 5, interference of radical scavengers by treatment with either ethacrynic acid or aminotriazole resulted in elevated ROS levels in AD-1 cells compared to ROS levels in mixed control cybrid cells. . It was also determined that oxidative buffering activity levels of multiple specific radical scavenger enzymes were also elevated in AD-1 cybrid cells compared to mixed control cybrids (FIG. 6). Gene expression in AD-1 cybrids compared to control cybrids was investigated for two of these enzymes (Cu / Zn SOD and Mn SOD). As shown in FIG. 6, elevated activity levels for these enzymes were achieved with elevated expression levels. This suggests a possible compensatory mechanism associated with the apparent oxidative deficiency in AD-1 cybrids.

[0070]

[Table 8] (Example 7) (Homoplasmic single nucleotide polymorphism in mitochondrial DNA of a subject diagnosed with Alzheimer's disease) This example shows a second subject (D-2) diagnosed with Alzheimer's disease.
We describe the detection of mtDNA single nucleotide polymorphisms in a cybrid cell line prepared using DNA obtained from the white blood cells of erythrocytes and mtDNA from platelets from the same subject. Materials and methods are described in Example 4, except that a different donor (D-2) was the source of leukocytes and platelets and that a second AD cybrid cell line (AD-2) was constructed. Is the same as The results are shown in Table 3. A new homoplasmic mtDNA mutation was identified in the mitochondrial gene (T980C) 1
It was identified in the 2S rRNA coding region.

Example 8 Single Nucleotide Polymorphism in Mitochondrial Ribosomal RNA Gene This example describes the analysis of mtDNA sequences for homoplasmic mutations in the 12S and 16S rRNA genes. Brain and / or blood samples from 13 normal survival control subjects, 41 autopsy-confirmed normal neurological disease control (ie, non-AD) subjects, 13 surviving AD subjects and 45 Were obtained from autopsy-confirmed AD and LBV (Lewy body variant) subjects.

Isolation of DNA from brain and blood samples has been performed on mitochondria of several brain samples using Mecocci et al. (1994 Ann. Neurol. 3).
6: 747) except that it was initially produced and the mtDNA was extracted, essentially as described in Examples 1 and 4, respectively. Oligonucleotide primers specific for the 12S rRNA and 16S rRNA genes are listed in Tables 5 and 7. These were used for PCR amplification, cloning and sequencing as described above.

The ten fragments generated by PCR amplification with primer set 110-119 were amplified, cloned and sequenced as described in Examples 1 and 4. Table 4 shows the homoplasmic single nucleotide polymorphisms detected in these mtDNA regions and the frequency of occurrence of these mutations. this is,"
It includes mutations that are neither "polymorphic" nor "rare polymorphic", which is also not "error" (these terms are mentioned above in the text of Tables 2-4). These include G709A, G
930A, T980C, T1189C, T1243C, C1700T, G171
9A, T1809C, A1811G, G1888A, G2098A, T2158
C, C2259T, T2352C, G3010A, T3197C and base 96
0 deletions, and T669C, T789C, C870T, T980C,
G1007A, T1243C, G1393A, G1709A, G1719A, T
Also included is a T insertion at the bases at positions 2156C, A2294G, T2483C, A2581G, A2851G and 793.

The analysis of the data presented in Table 4 as a function of age shows that mtD when comparing AD and control subjects over 85 years of age in the group of subjects studied.
It is shown that the difference in loading of NA 12S / 16S rRNA region homoplasmic single nucleotide polymorphisms is most obvious (FIG. 7). A for control subjects
A trend towards large single nucleotide polymorphisms in the mitochondrial rRNA gene in D cases was detectable in all age groups analyzed (64-74 years, 75-84 and 85 years and older).

Example 9 (Single nucleotide polymorphism in mitochondrial DNA that separates Alzheimer's disease (S
NP)) Mitochondrial DNA (mtDNA) was isolated and sequenced from brain and blood samples from 24 control and 24 autopsy-confirmed AD cases.
The confirmed AD samples consisted of 11 frontal bone samples, 3 parietal cortex samples and 10 blood samples. Control samples were 12 autopsy-confirmed brain samples (average age 79.3 years: 5 normal controls, whole skull; 5 diffuse Lewy body dementia (DLBD) samples, whole skull; 2 supranuclear paralysis ( par
It was composed of asupranular palsy (PSP) samples, whole skull) and 12 blood samples from healthy body, normal volunteers without a family history of AD (mean age 88.8 years).

Isolation of DNA by organic extraction after proteinase K / SDS solubilization was described in Example 1. PCR amplification was performed using 68 different PCR-generated fragments (crosslinking the complete mtDNA molecule with the oligonucleotide primers shown in Table 10 (
Each fragment was made as described in Example 2 except that it produced about 50% sequence overlap with the surrounding production fragment). This strategy allows direct mtDNA sequencing in both the forward and reverse directions with 4-fold degeneracy in the identification of each nucleotide base, which results in error-free sequencing. Therefore, for each patient sample, approximately 68,000 nucleotides were sequenced and demonstrated analysis of homoplasmic mutations.

[0077]

[Table 10] Sequencing was performed according to the manufacturer's instructions with a Pe equipped with a 96 capillary array.
Performed using the rkin-Elmer Model 3700 DNA Analyzer, and data analysis (performed essentially as described in Example 2) also included classification of the sample sequence according to various parameters including: Source of tissue sample, parental clinical status (eg, AD or control), parental haplogroup, mtDNA gene region (identified SNP is present) and protein-encoding mtDNA gene (AD-related SPN identified) Is a SNP a synonymous substitution (ie, no change in the amino acid sequence of the encoded protein) or a non-synonymous substitution (ie, a different amino acid sequence in the encoded protein).

The AD-related SNPs are shown in Table 9. 8-14 show the results of quantitative data analysis. Here, AD-related SNPs were classified according to the selected parameters. Therefore,
FIG. 8 shows the profile of the samples analyzed in this example according to the mtDNA haplogroup. In FIG. 9, the distribution of AD-related SNPs (ie loci) from Table 9 that affect non-homoplasmic nucleotide substitutions according to the mtDNA protein coding region (analyzed with one or more SNPs per indicated gene region). Sample percentage). Here, the SNP detected in the AD group is compared with the SNP detected in all non-AD control groups.

[0079]

[Table 9] In FIG. 10, the distribution of AD-related SNPs (ie, loci) from Table 9 that interfere with non-homoplasmic nucleotide substitutions according to the mtDNA protein coding regions (analyzed with one or more SNPs per gene region shown) was analyzed. Sample percentage). Here, the SNP detected in the AD group is
That is, compared to the SNP detected in the non-AD control group that is further classified as a healthy body) or a disease control.

FIG. 11 shows the distribution of AD-related SNPs (ie loci) from Table 9 that interfere with homoplasmic nucleotide substitutions according to the mtDNA protein coding region (
The percentage of analyzed samples with more than one SNP per indicated gene region is shown. Here, the SNPs detected in the AD group are compared to the SNPs detected in the non-AD control group, which is further classified as a normal (ie, healthy) or disease control.

FIG. 12 shows the distribution of AD-associated SNPs (ie loci) from Table 9 (indicated gene regions) that affect nucleotide substitutions in the mitochondrial tRNA gene according to each of the 22 known human mitochondrial tRNA protein coding regions. Percentage of analyzed samples with one or more SNPs per). Here, the SNPs detected in the AD group are compared to the SNPs detected in the non-AD control group, which is further classified as a normal (ie, healthy) or disease control.

FIG. 13 shows the distribution of AD-related SNPs (percentage of analyzed samples with more than one SNP per gene region shown) from Table 9 that interferes with nucleotide substitutions in the indicated DNA regions. To summarize. Here, the SNPs detected in the AD group are compared to the SNPs detected in the corresponding mtDNA region in samples from normal healthy controls.

FIG. 14 summarizes the distribution of AD-related SNPs (number of SNPs detected per patient) from Table 9, which interferes with nucleotide substitutions in the indicated DNA regions. Here, the SNPs detected in the AD group are compared to the SNPs detected in the corresponding mtDNA region in samples from normal healthy controls.

From the foregoing, although particular embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. it is obvious. Accordingly, the invention is, except as by the appended claims,
Not limited.

[Brief description of drawings]

[Figure 1]   Figure 1 shows mitochondrial rRNA gene mutations associated with AD.

FIG. 2 shows oligonucleotide primer extension reactions obtained from AD cells and control cybrid cells.

FIG. 3 shows mitochondrial ETC complex IV activity in AD cells and control cybrid cells.

FIG. 4 shows AD cells and control cybrid cells, and SH-SY.
3 shows Western immunoblot analysis of COX subunits in 5Y neuroblastoma cells.

FIG. 5 illustrates the effect of agents that interfere with radical scavenging enzymes on ROS production in AD cells and control cybrid cells.

FIG. 6 shows the oxidative buffering capacity of radical scavenging enzymes in AD-1 cybrid cells compared to mixed control cybrid cells, and also 2
The relative levels of gene expression for two enzymes, Cu / Zn SOD and Mn SOD, are shown.

FIG. 7 shows the difference between AD and control subjects in mtDNA single nucleotide polymorphisms in the mitochondrial rRNA gene as a function of age.

FIG. 8 shows AD-related and control SNPs according to the haplogroup.

FIG. 9 shows AD-related and control non-synonymous SNPs according to the mtDNA locus. ND; NADH dehydrogenase; CO, cytochrome c oxidase; AT, ATP synthase; CYB, cytochrome b.

FIG. 10 shows AD-related and control non-synonymous SNPs according to the mtDNA locus. It is an abbreviated figure of FIG.

FIG. 11 shows AD-related and control nonsynonymous SNPs according to the mtDNA locus. It is an abbreviated figure of FIG.

FIG. 12 shows amino acid or mitochondrial genetic code specificity (F, V, LUU).
R, I, Q, M, W, A, N, C, Y, SUCN, D, K, G, R, H, SAG
Y, LCUR, E, T, P; for more information on the mitochondrial genetic code, see, for example, Steele et al., 1996 Proc. Nat. Acad
． Sci. USA 93: 5223 and references cited therein) showing AD-related and control non-synonymous SNPs according to each of the 22 mitochondrial tRNA genes of mtDNA.

FIG. 13. AD-related and control SNPs according to mtDNA region.
To summarize.

FIG. 14: AD-related and control SNPs according to mtDNA region
The frequency per subject of is summarized.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 37/00 102 A61P 25/28 // A61K 45/00 C12N 15/00 ZNAF A61P 25/28 F (81 ) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW) ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN , IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F terms (reference) 4B024 AA01 AA11 CA04 CA05 CA09 CA12 HA08 HA09 HA11 HA12 HA13 HA14 HA19 4B029 AA07 BB20 CC03 FA15 4B063 QA01 QA13 QA17 QA19 QQ02 QQ03 QQ08 QQ12 QQ43 QQ44 QQ53 QQ54 QQ59 QQ61 QQ62 QQ89 QR01 QR08 QR14 QR20 QR31 QR32 QR36 QR41 QR42 QR56 QR58 QR62 QR63 QR66 QR72 QR80 QX02 4C084 AA06 AA07 AA17 NA20 ZA161 ZC781

Claims (24)

[Claims] 1. A method for determining the risk or presence of Alzheimer's disease in a first subject suspected of having, or at risk of having Alzheimer's disease, the method comprising: Determining the presence or absence of at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease in each of the first and second biological samples containing mitochondrial DNA, the first organism Scientific sample
A step obtained from the first subject and the second sample obtained from a second subject known to be neither at risk nor present for a disease associated with altered mitochondrial function. Where the presence of at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease in the first biological sample and the mitochondrial single nucleotide polymorphism at the corresponding nucleotide in the second biological sample. The method, wherein the absence of the type indicates an increased risk of Alzheimer's disease, and the step of determining the risk or presence of Alzheimer's disease therefrom. 2. The mitochondrial DNA in the first sample is amplified,
The method of claim 1, wherein the mitochondrial DNA in the second sample is then amplified. 3. The determining step comprises the following steps: sequence of each of the first and second biological samples present in mitochondrial DNA of the first sample and mitochondrial DNA of the second sample.
Contacting with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in, under conditions and for a time sufficient to allow hybridization of the primer to the mitochondrial DNA; and a first product. Hybridization and extension of a primer to mitochondrial DNA of the first sample, which produces, and hybridization and extension of a primer to mitochondrial DNA of the second sample, which produces a second product that is distinguishable from the first product. Detecting the presence or absence of at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease, the method comprising the steps of: 4. The mitochondrial DNA in the first sample is amplified,
The method of claim 3, wherein the mitochondrial DNA in the second sample is then amplified. 5. At least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease, present in said first biological sample and absent from the corresponding nucleotide in said second biological sample. The type is present in the mitochondrial DNA region selected from the group consisting of D-loop, mitochondrial rRNA gene, mitochondrial NADH dehydrogenase gene, mitochondrial tRNA gene, mitochondrial cytochrome c oxidase gene, mitochondrial ATP synthase gene and mitochondrial cytochrome b gene, The method according to claim 3. 6. At least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease, present in said first biological sample and absent at the corresponding nucleotide in said second biological sample. The type is present in the mitochondrial DNA region selected from the group consisting of D-loop, mitochondrial rRNA gene, mitochondrial NADH dehydrogenase gene, mitochondrial tRNA gene, mitochondrial cytochrome c oxidase gene, mitochondrial ATP synthase gene and mitochondrial cytochrome b gene, The method of claim 1. 7. At least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease, present in said first biological sample and absent at the corresponding nucleotide in said second biological sample. Type is mitochondrial NA
A DH dehydrogenase gene, a mitochondrial cytochrome c oxidase gene, a mitochondrial ATP synthase gene, and a mitochondrial cytochrome b gene, present in a mitochondrial DNA region, wherein the single nucleotide polymorphism is a non-synonymous nucleotide substitution. 7. The method of claim 6, wherein 8. At least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease, present in said first biological sample and absent at the corresponding nucleotide in said second biological sample. Type is mitochondrial NA
The DH dehydrogenase gene, the mitochondrial cytochrome c oxidase gene, the mitochondrial ATP synthase gene and the mitochondrial cytochrome b gene are present in the mitochondrial DNA region, wherein the single nucleotide polymorphism is a synonymous nucleotide substitution. The method of claim 6, wherein the method is: 9. At least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease, present in said first biological sample and absent at the corresponding nucleotide in said second biological sample. The mold is 72nd, 114th,
146th, 185th, 189th, 199th, 204th, 207th, 228th, 2nd
36th, 239th, 456th, 462th, 482nd, 489th, 497th, 50th
0th, 516th, 522nd, 523nd, 547th, 593th, 669th, 960th
Position, 1007, 1243, 1393, 1719, 1809, 2352
Position, 2483, 2702, 2851, 3197, 3333, 3336
Position, 3348, 3394, 3398, 3423, 3505, 3559
Position, 3915, 3992, 4024, 4095, 4216, 4336
Position, 4529, 4727, 4793, 4917, 4991, 5004
Position, 5046, 5228, 5315, 5418, 5426, 5460
Position, 5461, 5516, 5554, 5634, 5656, 5773
Rank, 6182 rank, 6221 rank, 6321 rank, 6341 rank, 6367 rank, 6371 rank, 6489 rank
Position, 7184, 7325, 7621, 7768, 7787, 7789
Position, 7864th, 7895th, 7963th, 8149th, 8251st, 8269th
No. 8276-8284, 8470, 8485, 8508, 8602, 8697, 8752, 8901, 8994, 9123, 9254, 9362, 9380, 9477, 9554, 9708. Position, 9804, 9861, 10034, 10044, 10238, 10463, 1
0589th, 10978th, 11065th, 11251th, 11253th, 11th
272th, 11470th, 11527th, 11611th, 11674th, 118th
12th, 11914, 11947, 12414, 12501, 1260
9th, 12705th, 12954th, 13111th, 13194th, 13212th
Position, 13368, 13617, 13780, 13966, 14020, 14148, 14178, 14179, 14182, 14212,
14233rd, 14470th, 14582th, 14905th, 15028th, 1
5043th, 15191th, 15299th, 15380th, 15553th, 15th
607th, 15758th, 15790th, 15808th, 15833th, 158th
84th, 15924th, 15928th, 16069th, 16086th, 1609th
3rd, 16126th, 16129th, 16145th, 16147th, 16172th
Position, 16174, 16182, 16183, 16189, 16192, 16193, 16223, 16224, 16234, 16235,
16239th, 16248th, 16256th, 16261th, 16270th, 1st
6278th, 16290th, 16292th, 16293th, 16294th, 16th
298th, 16300th, 16304th, 16309th, 16311th, 163rd
20th, 16355th, 16362th, 16391th, 16482nd and 16th
The method according to claim 6, which is a mitochondrial single nucleotide polymorphism located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1 selected from the group consisting of position 524. 10. At least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease, present in said first biological sample and absent at the corresponding nucleotide in said second biological sample. The type is a mitochondrial single nucleotide polymorphism located at a nucleotide corresponding to the nucleotide position of SEQ ID NO: 1, and the nucleotide positions are 709 position, 930 position, 960 position, 980 position, 1189 position, 1243 position, 1700 position, 1719th, 1809th, 1811th, 1888th, 2098th, 2158th, 2158th, 2259th, 2352th, 3010th, 3197th, 669th, 789th, 793th, 870th, 980th, 1007th, 1243
Position, 1393, 1709, 1719, 2156, 2294, 2483
7. The method of claim 6, selected from the group consisting of positions 2581, 2851, 6366 and 12954. 11. A method for determining the risk or presence of Alzheimer's disease in a subject, comprising: in a biological sample containing mitochondrial DNA from the subject,
Determining the presence of at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease. 12. The at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease has a D-loop, mitochondrial rRNA gene, mitochondrial NADH dehydrogenase gene, mitochondrial tRNA gene, mitochondrial cytochrome c oxidase gene, mitochondrial ATP synthase gene and mitochondrial cytochrome. The method according to claim 11, which is present in a mitochondrial DNA region selected from the group consisting of b genes. 13. Mitochondrial DNA wherein at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease is selected from the group consisting of mitochondrial NADH dehydrogenase gene, mitochondrial cytochrome c oxidase gene, mitochondrial ATP synthase gene and mitochondrial cytochrome b gene. 12. The method of claim 11, wherein the method is present in a region and wherein the single nucleotide polymorphism is a non-synonymous nucleotide substitution. 14. Mitochondrial DNA wherein at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease is selected from the group consisting of mitochondrial NADH dehydrogenase gene, mitochondrial cytochrome c oxidase gene, mitochondrial ATP synthase gene and mitochondrial cytochrome b gene. 12. The method of claim 11, wherein the method is present in a region, and wherein said single nucleotide polymorphism is a synonymous nucleotide substitution. 15. The at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease has positions 72, 114, 146, 185, 189, 199.
No. 204, No. 207, No. 228, No. 236, No. 239, No. 456, No. 462, No. 482, No. 489, No. 497, No. 500, No. 516, No. 522, No. 523,
547th, 593th, 669th, 960th, 1007th, 1243th, 1393
Position, 1719, 1809, 2352, 2483, 2702, 2851
Position, 3197, 3333, 3336, 3348, 3394, 3398
Position, 3423, 3505, 3559, 3915, 3992, 4024
Position, 4095th, 4216th, 4336th, 4529th, 4727th, 4793th
Position, 4917, 4991, 5004, 5046, 5228, 5315
Position, 5418, 5426, 5460, 5461, 5516, 5554
Position, 5634, 5656, 5773, 6182, 6221, 6341
Position, 6367, 6371, 6489, 7184, 7325, 7621
Position, 7768, 7787, 7789, 7864, 7895, 7963
Positions, 8149, 8251, 8269, 8276-8284, 8470, 8485, 8508, 8602, 8697, 8752, 8901, 8994, 9123, 9254, 9362, 9380. Position, 9477, 9554, 9708, 9804, 9861, 10034, 1004
4th, 10238th, 10463th, 10589th, 10978th, 11065
Positions, 11251, 11253, 11272, 11470, 11527, 11611, 11674, 11812, 11914, 11947,
12414, 12501, 12609, 12705, 12954, 1
3111th, 13194th, 13212th, 13368th, 13617th, 13th
780th, 13966th, 14020th, 14148th, 14178th, 141th
79th, 14182th, 14212th, 14233th, 14470th, 1458th
2nd, 14905, 15028, 15043, 15191, 15299
Position, 15380, 15553, 15607, 15758, 15790, 15808, 15833, 15884, 15924, 15928,
16069th, 16086th, 16093th, 16126th, 16129th, 1
6145th, 16147th, 16172th, 16174th, 16182th, 16th
183rd, 16189th, 16192th, 16193th, 16223th, 162th
24th, 16234th, 16235th, 16239th, 16248th, 1625th
6th, 16261, 16270, 16278, 16290, 16292
Position, 16293, 16294, 16298, 16300, 16304, 16309, 16311, 16320, 16355, 16362,
The method according to claim 11, which is a mitochondrial single nucleotide polymorphism located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1 selected from the group consisting of positions 16391, 16482 and 16524. 16. At least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease is a mitochondrial single nucleotide polymorphism located at a nucleotide corresponding to the nucleotide position of SEQ ID NO: 1, and the nucleotide positions are 709 and 930.
Position, 960, 980, 1189, 1243, 1700, 1719,
1809th, 1811th, 1888th, 2098th, 2158th, 2259th,
2352nd, 3010th, 3197th, 669th, 789th, 793th, 870th
Position, 980, 1007, 1243, 1393, 1709, 1719, 2156, 2294, 2483, 2581, 2851, 6366 and 12954. 11. The method according to 11. 17. A method of determining whether a drug is likely to cause Alzheimer's disease, contribute to the pathology of Alzheimer's disease, or exacerbate Alzheimer's disease, the method comprising an organism containing cells. A biological sample with a candidate agent, performing an assay for mitochondrial nucleic acid to determine whether the mitochondrial nucleic acid contains one or more single nucleotide polymorphisms after being contacted with the agent. Wherein the nucleic acid is present in or derived from the cell and the one or more single nucleotide polymorphisms segregate with Alzheimer's disease. 18. A method of determining whether a drug is likely to cause Alzheimer's disease, contribute to the pathology of Alzheimer's disease, or exacerbate Alzheimer's disease, the method comprising the steps of: a) contacting the first cell with a candidate drug; (b) incubating the second cell not contacted with the drug and the first cell under the same conditions; (c) one A step of performing the above-described assay of mitochondrial nucleic acid to determine whether the mitochondrial nucleic acid contains one or more single nucleotide polymorphisms, wherein the nucleic acid is present in the cell. , Or is derived from the cell, and the single nucleotide polymorphism is separated together with Alzheimer's disease, wherein the step is present in the first cell, or derived from the first cell, The Mito The presence of one or more of said single nucleotide polymorphisms in a ndria nucleic acid and one or more of said single nucleotide polymorphisms in said mitochondrial nucleic acid present in or derived from said second cell. Of the presence of the agent indicates that the agent is likely to cause Alzheimer's disease, contribute to the pathology of Alzheimer's disease, or exacerbate Alzheimer's disease. 19. A nucleic acid array comprising a plurality of isolated nucleic acid molecules immobilized on a solid support, wherein said isolated nucleic acid molecules are at least one associated with Alzheimer's disease. Sequence number 1 in which mitochondrial single nucleotide polymorphism exists
A nucleic acid array comprising all or part of the nucleic acid sequence shown in. 20. The mitochondrial single nucleotide polymorphisms are 72, 114, 1
46th, 185th, 189th, 199th, 204th, 207th, 228th, 23rd
6th, 239th, 456th, 462th, 482nd, 489th, 497th, 500
No. 516, 522, 523, 547, 593, 669, 960, 1007, 1243, 1393, 1719, 1809, 2352, 2483, 2702, 2851, 3197th, 3333th, 3336th, 3348th, 3394th, 3398th, 3398th, 3423th, 3505th, 3559th, 3559th, 3915th, 3992th, 4024th, 4095th, 4216th, 4336th, 4529th, 4727th , 4793, 4917, 4991, 5004, 5046, 5228, 5315, 5418, 5426, 5460, 5461, 5516, 5554, 5634, 5656, 5773, 6182. Position, 6221, 6341, 6367, 6371, 6489, 7184, 73 5, 7621 place, 7768 place, 7787 place, 7789 place, 7864 place, 7895 place, 7963 place, 8149 place, 8251 place, 8269 positions, 8276-8284 place, 8470 place, 8485 place, 8508 place, 8602 position,
8697th, 8752th, 8901th, 8994th, 9123th, 9254th,
9362, 9380, 9477, 9554, 9708, 9804,
9861th, 10034th, 10044th, 10238th, 10463th, 10th
589th, 10978th, 11065th, 11251th, 11253th, 112th
72nd, 11470th, 11527th, 11611th, 11674th, 1181th
2nd, 11914, 11947, 12414, 12501, 12609
Position, 12705, 12954, 13111, 13194, 13212, 13368, 13617, 13780, 13966, 14020,
14148th, 14178th, 14179th, 14182th, 14212th, 1st
4233rd, 14470th, 14582th, 14905th, 15028th, 15th
043, 15191, 15299, 15380, 15553, 156
07th, 15758th, 15790th, 15808th, 15833th, 1588th
4th, 15924, 15928, 16069, 16086, 16093
No. 16126, 16129, 16145, 16147, 16172, 16174, 16182, 16183, 16189, 16192,
16193, 16223, 16224, 16234, 16235, 1
6239th, 16248th, 16256th, 16261th, 16270th, 16th
278th, 16290th, 16292th, 16293th, 16294th, 162th
98th, 16300th, 16304th, 16309th, 16311th, 1632
0th, 16355th, 16362th, 16391th, 16482th, 16524th
Position, 709, 930, 960, 980, 1189, 1243, 17
00th, 1719th, 1809th, 1811th, 1888th, 2098th, 21st
58th, 2259th, 2352th, 2350th, 3010th, 3197th, 669th, 789th
1st, 793th, 870th, 980th, 1007th, 1243th, 1393th, 1st
709th, 1719th, 2156th, 2294th, 2483th, 2581th, 2nd
20. The nucleic acid array of claim 19, located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1 selected from the group consisting of positions 851, 6366 and 12954. 21. The determining step comprises: SEQ ID NO: 1 wherein each of the first and second biological samples is present in at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease. Contacting with an oligonucleotide primer containing all or part of the nucleic acid sequence described above under conditions and for a time sufficient to allow hybridization of the primer to the mitochondrial DNA; and to mitochondrial DNA of the first sample. The amount of hybridization of the oligonucleotide primer was determined by measuring the mitochondrial D of the second sample.
Comparing the amount of hybridization of the primer to NA to determine the presence or absence of at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease. The method described in. 22. The mitochondrial single nucleotide polymorphisms are in positions 72, 114, 1
46th, 185th, 189th, 199th, 204th, 207th, 228th, 23rd
6th, 239th, 456th, 462th, 482nd, 489th, 497th, 500
No. 516, 522, 523, 547, 593, 669, 960, 1007, 1243, 1393, 1719, 1809, 2352, 2483, 2702, 2851, 3197th, 3333th, 3336th, 3348th, 3394th, 3398th, 3398th, 3423th, 3505th, 3559th, 3559th, 3915th, 3992th, 4024th, 4095th, 4216th, 4336th, 4529th, 4727th , 4793, 4917, 4991, 5004, 5046, 5228, 5315, 5418, 5426, 5460, 5461, 5516, 5554, 5634, 5656, 5773, 6182. Position, 6221, 6341, 6367, 6371, 6489, 7184, 73 5, 7621 place, 7768 place, 7787 place, 7789 place, 7864 place, 7895 place, 7963 place, 8149 place, 8251 place, 8269 positions, 8276-8284 place, 8470 place, 8485 place, 8508 place, 8602 position,
8697th, 8752th, 8901th, 8994th, 9123th, 9254th,
9362, 9380, 9477, 9554, 9708, 9804,
9861th, 10034th, 10044th, 10238th, 10463th, 10th
589th, 10978th, 11065th, 11251th, 11253th, 112th
72nd, 11470th, 11527th, 11611th, 11674th, 1181th
2nd, 11914, 11947, 12414, 12501, 12609
Position, 12705, 12954, 13111, 13194, 13212, 13368, 13617, 13780, 13966, 14020,
14148th, 14178th, 14179th, 14182th, 14212th, 1st
4233rd, 14470th, 14582th, 14905th, 15028th, 15th
043, 15191, 15299, 15380, 15553, 156
07th, 15758th, 15790th, 15808th, 15833th, 1588th
4th, 15924, 15928, 16069, 16086, 16093
No. 16126, 16129, 16145, 16147, 16172, 16174, 16182, 16183, 16189, 16192,
16193, 16223, 16224, 16234, 16235, 1
6239th, 16248th, 16256th, 16261th, 16270th, 16th
278th, 16290th, 16292th, 16293th, 16294th, 162th
98th, 16300th, 16304th, 16309th, 16311th, 1632
0th, 16355th, 16362th, 16391th, 16482th, 16524th
Position, 709, 930, 960, 980, 1189, 1243, 17
00th, 1719th, 1809th, 1811th, 1888th, 2098th, 21st
58th, 2259th, 2352th, 2350th, 3010th, 3197th, 669th, 789th
1st, 793th, 870th, 980th, 1007th, 1243th, 1393th, 1st
709th, 1719th, 2156th, 2294th, 2483th, 2581th, 2nd
22. The method of claim 21, located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1 selected from the group consisting of positions 851, 6366 and 12954. 23. A nucleic acid array comprising a plurality of isolated nucleic acid molecules immobilized on a solid support for each of the first and second biological samples, the mitochondrial DNA comprising: Contacting under conditions and for a time sufficient to allow hybridization to the isolated nucleic acid molecule,
Wherein the isolated nucleic acid molecule comprises all or part of the nucleic acid sequence set forth in SEQ ID NO: 1 in which at least one mitochondrial single nucleotide polymorphism associated with Alzheimer's disease is present; The amount of hybridization of the mitochondrial DNA of one sample to the nucleic acid array is compared to the amount of hybridization of the mitochondrial DNA of the second sample to the nucleic acid array from which at least one Alzheimer's disease-related Determining the presence or absence of a mitochondrial single nucleotide polymorphism. 24. The mitochondrial single nucleotide polymorphisms are 72, 114, 1
46th, 185th, 189th, 199th, 204th, 207th, 228th, 23rd
6th, 239th, 456th, 462th, 482nd, 489th, 497th, 500
No. 516, 522, 523, 547, 593, 669, 960, 1007, 1243, 1393, 1719, 1809, 2352, 2483, 2702, 2851, 3197th, 3333th, 3336th, 3348th, 3394th, 3398th, 3398th, 3423th, 3505th, 3559th, 3559th, 3915th, 3992th, 4024th, 4095th, 4216th, 4336th, 4529th, 4727th , 4793, 4917, 4991, 5004, 5046, 5228, 5315, 5418, 5426, 5460, 5461, 5516, 5554, 5634, 5656, 5773, 6182. Position, 6221, 6341, 6367, 6371, 6489, 7184, 73 5, 7621 place, 7768 place, 7787 place, 7789 place, 7864 place, 7895 place, 7963 place, 8149 place, 8251 place, 8269 positions, 8276-8284 place, 8470 place, 8485 place, 8508 place, 8602 position,
8697th, 8752th, 8901th, 8994th, 9123th, 9254th,
9362, 9380, 9477, 9554, 9708, 9804,
9861th, 10034th, 10044th, 10238th, 10463th, 10th
589th, 10978th, 11065th, 11251th, 11253th, 112th
72nd, 11470th, 11527th, 11611th, 11674th, 1181th
2nd, 11914, 11947, 12414, 12501, 12609
Position, 12705, 12954, 13111, 13194, 13212, 13368, 13617, 13780, 13966, 14020,
14148th, 14178th, 14179th, 14182th, 14212th, 1st
4233rd, 14470th, 14582th, 14905th, 15028th, 15th
043, 15191, 15299, 15380, 15553, 156
07th, 15758th, 15790th, 15808th, 15833th, 1588th
4th, 15924, 15928, 16069, 16086, 16093
No. 16126, 16129, 16145, 16147, 16172, 16174, 16182, 16183, 16189, 16192,
16193, 16223, 16224, 16234, 16235, 1
6239th, 16248th, 16256th, 16261th, 16270th, 16th
278th, 16290th, 16292th, 16293th, 16294th, 162th
98th, 16300th, 16304th, 16309th, 16311th, 1632
0th, 16355th, 16362th, 16391th, 16482th, 16524th
Position, 709, 930, 960, 980, 1189, 1243, 17
00th, 1719th, 1809th, 1811th, 1888th, 2098th, 21st
58th, 2259th, 2352th, 2350th, 3010th, 3197th, 669th, 789th
1st, 793th, 870th, 980th, 1007th, 1243th, 1393th, 1st
709th, 1719th, 2156th, 2294th, 2483th, 2581th, 2nd
24. The method of claim 23, wherein the method is located at the nucleotide corresponding to the nucleotide position of SEQ ID NO: 1 selected from the group consisting of positions 851, 6366 and 12954.