JP2003116576A - Gene detecting method using human mitochondorial dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gene detecting method recognizing amino acid substitution which is advantageous or disadvantageous in regard to long term survival of human, in mitochondorial DNA. SOLUTION: This detecting method for gene using human mitochondorial DNA, detects at least one substitution of a base accompanying an amino acid substitution in the followings; T2A, T2I, H16R, A39T, T47K, T61A, I78T, L82F, Y109H, T158A, D159N, I164V, D171N, A190T, A191T, A193T, F245L, P247A, G251S, N260D, L296M, I300T, I306V, I338V, V343M, S344N, A354T, I369V, I372V, and A380T encoding human cytochrome b in the base sequence of the human mitochondorial DNA.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene detection method for human mitochondrial DNA.

[0002]

2. Description of the Related Art Mitochondria are intracellular organelles present in eukaryotic cells, and most of the energy required for cell activity is supplied by the oxidative phosphorylation system inside them. In this mitochondria, mitochondrial DNA independent of nuclear DNA (hereinafter referred to as "mtDN
It may be abbreviated as "A". ) Has been revealed, and in 1981, human mtDNA 16,
The entire base sequence of 569 base pairs has been determined (Anderso
n, S., Bankier, AT, Barrell, BG, de Bruijn, MH
L., Coulson, AR, Drouin, J., Eperon, IC, Nierlic
h, DP, Ror, BA, Sanger, F., Schreier, PH, Smith,
AHH, Staden, R., & Young, IG: Sequence and orga
nization of the human mitochondrial genome. Nature
290, 457-465, 1981). Within this human mtDNA,
Genes unique to mitochondria exist and their correspondence has been clarified so far (see Fig. 1).

On the other hand, it is becoming clear from the results of molecular biological studies that mutations in mtDNA are associated with specific diseases (Masahiro Tanaka, Molecular biology of enzyme deficiency in mitochondrial electron transport system, biochemistry, 63 (3), 169-187,
1991, Wataru Sato, KiyoshiHayasaka, Yutaka Shoji,
Tsutomu Takahashi, Goro Takada, Masahiro Saito, Osa
mu Fukuwa, and Eiko Wachi: A mitochondrial tRNA mu
tation at 3,256 associated with mitochondrial myop
athy, encephalopathy, lactic acidesis, and stroke-
like episodes (MELAS). BIOCHEMISTRY and MOLECULAR
BIOLOGY INTERNATIONAL 33 (6), 1055-1061, 1994). For example, the inventors of the present invention have thought that there is a correlation between mutations in mtDNA and adult diseases, and as a result of extensive studies, confirmed a certain correlation between specific mutations and adult diseases. However, the invention disclosed in JP-A-11-113597 is made.

[0004]

By the way, Confucius (551-479BC) in "Medium" which is one of the four books
Explains, "Kimiko moderation, dwarf antimoderation." Aristotle (384-322BC) also discusses the concept of such a moderation in Nikomachus ethics. On the other hand, population genetic analysis of the amino acid sequence of the protein defined by mtDNA suggests that most of the isolated amino acid substitutions within the species of an organism are weak deleterious mutations (Weinreich DM , &
Rand DM Contrasting patterns of nonneutral evolu
tion in proteins encoded in nuclear and mitochondr
ial genomes. Genetics 2000; 156: 385-99.).

[0005] However, there are not many data which specifically and specifically analyze the site and frequency of mutation of mtDNA. The present invention has been made in view of the above circumstances, and an object thereof is to provide a gene detection method for confirming amino acid substitution in mtDNA.

[0006]

Means for Solving the Problems, Actions of the Invention, and Effects of the Invention In the following description, all technical and scientific words are used to refer to the technology to which the present invention belongs, unless otherwise defined. It has the same meaning as is well understood by those familiar with it. The present inventor
In order to test this hypothesis, it is hypothesized that there are few weak mitochondrial mutations, that is, those who are 100 years old are genetically moderate. In addition, the nucleotide sequence of the cytochrome b gene in mtDNA was analyzed for each group of 64 people who lived 100 years, 96 patients with Parkinson's disease, and 96 young adults (people aged 20 to 30). The following facts were clarified by making determinations and comparing and examining the positions and frequencies of amino acid substitutions among the groups, and the present invention was basically completed.

(1) For many individuals (group of 100 people of 100 years of life, 75 young adults, 70 patients with Parkinson's disease),
The so-called "Revised Cambridge Standard Array" (Andrews R.
M., Kubacka I., Chinnery PF, Lightowlers RN, Tu
rnbull DM, Howell N. Reanalysis and revision of
the Cambridge reference sequence for human mitocho
ndrial DNA. Nat Genet 1999; 23: 147.),
It had no amino acid substitutions. (2) However, in the centenarian group, 9 different amino acid substitutions (H16R, A39T, I78T, I164V, A191T, N260D,
I306V, V343M, and I369V) were confirmed. In the present specification, a single amino acid substitution is meant with two letters between numbers. In that case,
The first letter shows the amino acid before substitution, and the latter letter shows the amino acid after substitution. Also, the numbers indicate the positions where amino acid substitutions have been made by the positions in the amino acid sequence of cytochrome b. For example, I306V is 3
It means that the 06th isoleucine is a mutation in which valine is substituted. In the Parkinson's disease patient group, 21 different amino acid substitutions (T2A, T2I, T47K,
T61A, I78T, T158A, I164V, D171N, A190T, A193T, F24
5L, P247A, G251S, N260D, I300T, I306V, I338V, S34
4N, A354T, I369V, I372V) was confirmed. In the young adult group, 15 different amino acid substitutions (T2A, I78
T, L82F, Y109H, D159N, I164V, A193T, G251S, N260D,
L296M, I306V, I338V, I369V, I372V, A380T) were confirmed. Of these, 5 amino acid substitutions (I78T, I164V, N
260D, I306V, I369V) was commonly confirmed in all groups.

(3) T2A + I338V and A193T + G251S + were amino acid substitutions that were not detected in the 100-year-old group and were commonly observed in the young adult group and the Parkinson's disease patient group.
It was I372V (note that multiple amino acid substitutions were "+")
When linked by, it means that those amino acid substitutions are concentrated in one human. )
Amino acid substitutions found only in patients with Parkinson's disease were T2I, T47K, T61A, T158A, D171N, A190T, F245
L, P247A, I300T, S344N, and A354T. (4) N260D was found at a frequency of 6.25% (4/64) in the 100-year-old group, while 1.04% in both the young adult group (1/96) and the Parkinson's disease group (1/96). Found in frequency. In addition, the frequency of N260D in the group of 100 people (4/6
4) was significantly higher than the frequency (2/192) in the other two groups (odds ratio = 6.33, p = 0.036, Fisher's direct method).

(5) In contrast, the frequency of G251S substitution in the Parkinson's disease patient group (6/96) was significantly higher than that in the 100-year-old group (0/64) (p = 0.044, Fisher
Direct method). The frequency of G251S replacement in other disease control groups (19/593, 3.2% in patients with heart disease) was intermediate between the frequency in patients with Parkinson's disease (6.9%) and that in centenarians (0.0%). From these results, the first invention for achieving the above object is a gene detection method using human mitochondrial DNA, which comprises human mitochondrial DN.
It is characterized in that the base sequence of A is detected to be replaced with a base accompanied by amino acid substitution in the protein encoded by the base sequence.

In the present invention, the term "base sequence" often determines the base sequence of mtDNA as it is,
It is also possible to determine the base sequence of mtDNA as a template by determining the base sequence of complementary DNA or mRNA. It is also known to those skilled in the art that the genetic code of mtDNA includes a non-universal code (for example, UGA, which is a common stop codon, encodes tryptophan, AUA (in many cases, isoleucine). Note that it contains methionine, etc.). In the present invention, “base substitution” means that a specific base is substituted as compared with the so-called “revised Cambridge standard sequence”. However, it should be noted that base substitution does not necessarily involve amino acid substitution. In the genetic code, multiple types of triplets (three consecutive base sequences) form one type of amino acid (or stop codon).
This is because the triplet after substitution may encode the same amino acid as before even if base substitution occurs. In addition, in the present specification, in principle, the one-letter code is used for amino acids.

A second invention is the same as the first invention, wherein said base sequence encodes cytochrome b (SEQ ID NO: 1) and said amino acid substitution is T2A, T2I, H16R, A3.
9T, T47K, T61A, I78T, L82F, Y109H, T158A, D159N, I1
64V, D171N, A190T, A191T, A193T, F245L, P247A, G251
S, N260D, L296M, I300T, I306V, I338V, V343M, S344
It is characterized by being at least one of N, A354T, I369V, I372V, and A380T. The amino acid substitutions described here are all the amino acid substitutions confirmed by the present inventor in the above three groups. Third
In the first invention, in the first invention, the nucleotide sequence encodes cytochrome b (SEQ ID NO: 1), and the amino acid substitution is T2A, T2I, T47K, T61A, L82F, Y109H,
T158A, D159N, D171N, A190T, A193T, F245L, P247A, G
251S, L296M, I300T, I338V, S344N, A354T, I372V,
And at least one of A380T. The amino acid substitutions described here exclude all nine amino acid substitutions, except for the nine types of amino acid substitutions found in the 100-year-old group.

A fourth invention is the same as the first invention, wherein the base sequence encodes cytochrome b (SEQ ID NO: 1), and the amino acid substitution is T2I, T47K, T61A, T.
It is characterized by being at least one of 158A, D171N, A190T, F245L, P247A, I300T, S344N, and A354T. The amino acid substitution described here is an amino acid substitution specifically found only in the Parkinson's disease patient group. A fifth invention is the same as the first invention, wherein said base sequence encodes cytochrome b (SEQ ID NO: 1).
Wherein the amino acid substitution is A193T, G251S, and I372
It is characterized in that it is at least one of V. The amino acid substitutions described here are characteristic among the amino acid substitutions commonly found in the young adult group and the Parkinson's disease patient group. According to the invention, mtD
It is possible to detect whether or not there is a base substitution for substituting the amino acid sequence at the specific site of the cytochrome b gene in NA.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Next, one embodiment of the present invention will be described in detail with reference to the drawings, but the technical scope of the present invention is not limited to the following embodiments, and The present invention can be implemented with various modifications without changing the gist. Moreover, the technical scope of the present invention extends to an equivalent range. Mt by polymerase chain reaction method (hereinafter referred to as "PCR method")
The determination of the nucleotide sequence of DNA is carried out, for example, by Masashi Tanaka,
Mika Hayakawa, and Takayuki Ozawa, Automated Seque
ncing of Mitochondrial DNA, Methods in Enzymology,
This can be done according to the method shown in Volume 264, 407-421, 1996. In this method, after performing the PCR method twice as shown in FIG. 2, the second PCR product is directly sequenced by the PCR method.

In the first PCR method, mtDNA was used as a template for L13901 (SEQ ID NO: 3: TCTCCAACAT).
ACTCGGATTC) and H609 (SEQ ID NO: 4: GCCCGTCTAAAC)
About 3000b using two kinds of primers (ATTTTCAG)
Amplify the p DNA. The first-time PCR product contains the gene for cytochrome b. next,
FL2 (FL
14559 (SEQ ID NO: 5: GTAAAACGACGGCCAGTCGACCACAC
CGCTAACAATC), FL14837 (SEQ ID NO: 6 GTAAAAC
GACGGCCAGTTGAAACTTCGGCTCACTCCT), FL15126
(SEQ ID NO: 7: GTAAAACGACGGCCAGTCCTTCATAGGCTATGTCCT
C), FL15405 (SEQ ID NO: 8: GTAAAACGACGGCCAGT
TCCACCCTTACTACACAATC), FL15696 (SEQ ID NO: 9: GTAAAACGACGGCCAGTTTCGCCCACTAAGCCAATCA), FL
15948 (SEQ ID NO: 10: GTAAAACGACGGCCAGTAGGACAA
ATCAGAGAAAAAG)) and H2 (H15162 (SEQ ID NO: 11: TACTGTGGCCCCTCAGAATG), H15340 (SEQ ID NO: 12: ATCCCGTTTCGTGCAAGAAT), H15755 (SEQ ID NO: 13: ACTGGTTGTCCTCCGATTCA), H16016 (SEQ ID NO: 14: CCCATGAAAGAACAGAGAAT), H81 (SEQ ID NO: 14). ) About 500 bp to about 1 by performing a second PCR method using the two kinds of primers
Amplify 000 bp of DNA. Finally, the second P
The PCR method for determining the nucleotide sequence is performed using the CR product as a template. The combination of primers and the base sequence of the primer DNA when performing the PCR method are shown in Examples described later.

The obtained nucleotide sequences are compared and analyzed by, for example, a computer. In many cases, multiple nucleotide substitutions are confirmed in the nucleotide sequences of mtDNA of centenarians, young adults, and Parkinson's disease patients. However, not all of those base substitutions are accompanied by amino acid substitutions. Therefore, in particular, only base substitutions accompanied by amino acid substitutions are extracted and summarized for each group of centenarians, young adults, and Parkinson disease patients. Furthermore, the site in which amino acid substitutions occur remarkably in each group is specified. In addition, after the base substitution site that causes a specific amino acid substitution is identified, known methods such as Southern blotting and F
ISH method, RIGS method, CGH method, LOH method, direct sequence method, PCR-SSCP method, MASA method, ASO
Gene detection can be performed by using a probe method, a PCR method, an OPA method, or the like (BIOCLinica 10
(9), 1995, 18-23).

[0016]

The present invention will be described below with reference to examples.
The present invention is not limited to these examples. Example 1: Extraction of total DNA sample As the total DNA, "Gen Torukun" from Takara Shuzo Co., Ltd. was used. A brief description of the method is as follows. To a 1.5 ml microtube, 500 μl of GenTLE solution I in the kit and 100 μl of blood were added and immediately stirred for a few seconds. After standing at room temperature for 10 minutes or more, 12,000 at room temperature
Centrifuge at xg or more for 5 minutes. Remove the supernatant and deposit 1 ml
GenTLE solution II in the kit of. The microtube was mixed by inversion and then centrifuged at 12,000 xg or more for 2 minutes at room temperature to remove the supernatant. 500 μl G in the kit for precipitation
enTLE solution III was added and stirred for 10 seconds. 1 at room temperature
After centrifugation at 2,000 xg or more for 5 minutes, the supernatant was transferred to a new microtube. After adding 500 μl of isopropanol to the supernatant and mixing by inversion, the mixture was centrifuged at 4 ° C. and 12,000 × g for 5 minutes, the supernatant was removed, the precipitate was washed with 70% ethanol, and dried in a vacuum dryer. Total DNA was dissolved in 50 μl of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA).

Example 2: Confirmation of nucleotide sequence of gene encoding cytochrome b in mtDNA of centenarians, young adults and Parkinson's disease patients Centenarians (64 persons), young adults (96 persons) and Parkinson's disease patients ( The nucleotide sequence of the gene encoding cytochrome b was confirmed in mtDNA (96 persons). For centenarians, we visited centenarians residing in Tokyo, Aichi, and Gifu, collected blood cells or buccal mucosa cells from the centenarians who obtained consent, extracted the total DNA, and then extracted the corresponding bases. The sequence was confirmed. DNA sequences are described in Methods in En
Based on the method reported in zymology, Volume 264, 407-421, 1996, it carried out as follows. Table 1 shows combinations of primer DNAs used in the first and second PCR methods.

[0018]

[Table 1]

In the first PCR method, all DNA was used as a template, and L1 and H1 were used as primers. The composition of the reaction solution is as follows. 1 μl of total DNA (10 n
g / μl), 5 μl L1 (10 μM), 5 μl H1 (10 μM
M), 4 μl of 2.5 mM dNTPs, 0.25 μl of 5 units / μl Taq
DNA polymerase, 5 μl of 10x PCR buffer (100 mM Tr
29.75μ in is-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl ₂ )
0.2 μl MicroAmp with 50 μl of purified water
Mix in tube. PC of this reaction solution by GeneAmp PCR system Model 9600 manufactured by Perkin Elmer
The R method was performed. Reaction conditions are denaturation at 94 ℃ for 15 seconds, 60 ℃
40 cycles were repeated, with annealing for 15 seconds and extension reaction at 72 ° C. for 185 seconds as one cycle. After adding 5 μl of 3 M sodium acetate (pH 7.4) and 100 μl of ethanol to the reaction solution, leave it on ice for 10 minutes and then at 1 ℃ x 13,000 xg.
It was centrifuged at 10 minutes. The precipitate was washed with 150 μl of 70% ethanol and then centrifuged at 13,000 xg for 5 minutes at 4 ° C. After drying the precipitate for 10 minutes, 50 μl of purified water or TE (10 mM Tris-HCl,
pH 8.0, 1 mM EDTA).

The second PCR method is the above-mentioned first PC
The R product was used as a template and FL2 and H2 were used as primers. The composition of the reaction solution is as follows. 1 μl of the first PCR product solution, 2 μl of FL2 (10
μM), 2 μl H2 (10 μM), 1.6 μl 2.5 mM dNT
P, 0.1 μl of 5 units / μl rTaq DNA polymerase, 2
Add 11.3 μl purified water to μl 10x PCR buffer to bring the total volume to 2
Mix in 0.2-ml MicroAmp tubes as 0 μl.
The reaction conditions of the PCR method were 40 cycles of heat denaturation (94 ° C.-15 seconds), annealing (60 ° C.-15 seconds) and extension reaction (72 ° C.-3 minutes) as one cycle. 72 ° C for the final extension reaction
It was set to -10 minutes. 1.5 μl of 3 M sodium acetate in the reaction solution
After adding (pH 7.4) and 30 μl of ethanol, the mixture was allowed to stand on ice for 10 minutes and centrifuged at 13,000 × g for 10 minutes at 4 ° C. Precipitation 45
After washing with μl of 70% ethanol, centrifugation was performed at 13,000 xg for 5 minutes at 4 ° C. The precipitate was dried for 10 minutes and then dissolved in 10 μl of purified water.

The sequence reaction was carried out as follows. (1) Purification of template Primers and dNTPs were removed by an ultrafiltration method using PCR Screen (for Millipore 96-well). Second PC
Add 280 μl of sterilized water to the R product (20 μl) to make 300 μL, and add M
Dispensed in ultiScreen-PCR Plate. Attach the PCR Plate to the multi-screen vacuum manifold and set it to 24 inch.
Ultrafiltration was performed by suctioning with es Hg for 10 minutes. 100 μL of sterilized water was dispensed to the PCR plate, shaken vigorously for 5 minutes with a mixer, and then transferred to a new PCR tube. (2) Sequencing reaction premix fluorescence Terminator (DNA Sequencing Kit, Bi
gDye ^TM Terminator Cycle Sequencing Ready Reaction,
ABI PRISM, PE Applied Biosystems, Japan) was used. 2 μl of the second PCR product after ultrafiltration was mixed with 2 μl of P
remix 4 μl, -21M13 Forward Primer (0.8 pmol / μl)
Add 4 μl, 5 × Sequence Buffer 2 μl, sterile water 8 μl,
The total volume was 20 μl. After the first heat denaturation (96 ℃ -10 minutes), heat denaturation (96 ℃ -10 seconds), annealing (50 ℃ -5 minutes)
Second) and extension reaction (60 ° C.-4 minutes) as one cycle, and this was repeated 25 cycles. (3) Removal of Dye Terminator Gel filtration (for 96-well) Dry Sephadex G50 Superfine
Using a 45 μL column loader.
The well of the V plate was filled. To swell Sephadex, 300 μL of sterilized water was added to the well and left at room temperature for 3 hours. A multiscreen HV plate was placed on the 96-well plate and centrifuged at 740 xg for 6 minutes. Sequence reaction solution w
I added it to the center of ell quietly. A multiscreen HV plate was placed on top of 8 GeneAmp Strip Tubes (8-tubes / strip) lined up and centrifuged at 740 xg for 6 minutes. 8 GeneAmp St
The rip tubes were placed in a centrifugal vacuum dryer and dried for 50 minutes. The dried sample was protected from light with an aluminum wheel or the like and stored frozen. (4) DNA Sequencing 20 μl of Template Suppression Reagent (TSR) is used for Seque
The reaction mixture was added to 8 GeneAmp Strip Tubes containing the reaction product and left at room temperature for 10 minutes. Thoroughly applied a Vortex mixer and spun down. After heating at 95 ° C for 2 minutes, the sample was immediately quenched with ice (10 minutes). 8 GeneAmp
ABI PRISM ^TM 310 Genetic Analyzer for samples in tube
Then, the base sequence was determined.

Example 3: Analysis of amino acid substitution in each group of centenarians, young adults and patients with Parkinson's disease After determining the nucleotide sequence of the gene encoding cytochrome b in mtDNA by the method described in Example 2. , Its base substitution and amino acid substitution were analyzed. Tables 2 and 3 show the presence or absence of confirmed base substitutions and amino acid sequence substitutions. The meaning of the table is as follows. For example, in the substitution on the first line in Table 2, the 14750th adenine (A) of mitochondrial DNA is guanine (G).
And the base substitution means that the second amino acid of cytochrome b, threonine, is replaced with alanine (that is, the above-mentioned “T2A”).

[0023]

[Table 2]

[Table 3]

Further, FIGS. 3 and 4 visually show what kind of amino acid substitution is carried out in each group. Of these, FIG. 3 shows the amino acid substitutions confirmed in each of all groups, whereas FIG. 4 shows the amino acid substitutions commonly confirmed in the three groups in the population. . Therefore, it is convenient to refer to FIG. 4 to confirm the amino acid substitution characteristic of each group. Of these, as shown in FIG. 3 (A), nine different amino acid substitutions were found in the 100-year-old group, whereas the young adult group (FIG. 3 (B)) and the Parkinson's disease patient group ( In FIG. 3 (C), 15 and 21 types of amino acid substitutions were found, respectively. The majority of individuals (group 100
Of 64/75, 75/96 of young adult group, 70/96 of Parkinson's disease group) did not have amino acid substitutions compared to the so-called “revised Cambridge standard sequence” .

Further, 5 amino acid substitutions (I78T, I164V,
N260D, I306V, I369V) were commonly found in the 3 groups. In the 100-year-old group, H16R, A39T, A191T, and V343M were found individually. In the young adult group, seven amino acid substitutions that were not detected in the centenarian group were found. T2A + I338
V, Y109H, D159N, L296M and A380T in 1 individual, A193T + G251S + L82F in 1 individual, A193T + G251S + I372V
Was found in 2 individuals. In order to quantify the deviation from the standard amino acid sequence, the value reported by Grantham (Grantham R. Amino acid difference formula to hel
p explain protein evolution. Science 1974; 185: 86
According to 2-4.), The total physicochemical difference between the standard amino acid and the changed amino acid was calculated for each individual. However, amino acid substitutions commonly found in the 3 groups were excluded from this calculation. Degree of deviation from the standard amino acid sequence in the 100-year-old group (Grantham value: 58, 58, 29, 21 is 1 individual each,
0 for 60 individuals is the deviance (1
43, 143, 136, 87, 83, 58, 23, 15 were statistically significantly smaller than 0 for 1 and 88 individuals (p <0.
0001, Wald-Wolfowitz run test).

Amino acid substitutions that were not detected in the 100-year-old group and were commonly observed in the young adult group and the Parkinson's disease patient group were T2A + I338V and A193T + G251S + I372V.
Met. Amino acid substitutions found only in Parkinson's disease patients were T2I, T47K, T61A, T158A, D171N, A
They were 190T, F245L, P247A, I300T, S344N, and A354T. Degree of deviation from standard amino acid sequence in Parkinson's disease patient group (Grantham value: 143 is 5 individuals, 114, 89, 8
9, 87, 78 1 each, 58 58, 46, 29, 27, 27, 2
3, 22 was 1 for each and 0 for 75) was statistically significantly higher than the deviance in the 100-year-old group (p <0.0001,
Wald-Wolfowitz run test and p = 0.0058, Mann-Whitn
ey's U test), and was greater than the deviance in the young adult group (p <0.0001, Wald-Wolfowitz's run test and
p = 0.0097, Mann-Whitney U test).

Next, the amino acid substitutions found at a frequency of 5% or more in each group were statistically analyzed. N260D was found at a frequency of 6.25% (4/64) in the centenarian group, whereas it was found at a frequency of 1.04% in both young adults (1/96) and Parkinson's disease group (1/96). It was The frequency of N260D (4/64) in the 100-year-old group was significantly higher than that in the other 2 groups (2/192) (odds ratio = 6.33, p = 0.036, Fisher's direct method). These observations suggest that having the N260D substitution leads to longevity, but its functional impact needs further investigation. In contrast, the frequency of G251S substitution in the Parkinson's disease patient group (6/96) was higher than that in the 100-year-old group (0
/ 64) (p = 0.044, Fisher's direct method). The frequency of G251S replacement in other disease control groups (19/593, 3.2% in patients with heart disease) was intermediate between the frequency in patients with Parkinson's disease (6.9%) and that in centenarians (0.0%).

Deviations from the canonical amino acid sequence, such as the G251S substitution, are associated with increased production of reactive oxygen species from mitochondria, which can lead to age-related diseases. The amino acid residue Gly251 is highly conserved in mammalian species. Gly251 is located at the outer binding site (Qo site) of ubiquinone in cytochrome b protein, and plays an important role in binding to ubiquinone.
Near the residue. When Gly251 is replaced by Ser, Ser may form a hydrogen bond with Glu271.
It is assumed that this limits the movement of Glu271 and changes the binding of ubiquinone to the Qo site. These findings suggest that G251S substitution is disadvantageous for long-term survival.

The present inventor has already reported that the genotype Mt5178A is found at a high frequency in the Japanese centenarians (Tanaka M, Gong JS, Zhang J, Yoneda M, Yagi K.
Mitochondrial genotype associated with longevity.
Lancet 1998; 351: 185-6.). Meanwhile, Ivanova et al. (Ivano
va R, Lepage V, Charron D, Schachter F. Mitochondr
ial genotype associated with French Caucasian cent
enarians. Gerontology 1998; 44: 349.) is genotype M
Reported that t9055A was found at high frequency by French centenarians. The small deviation from the standard amino acid sequence may be a phenomenon common to centenarians in Asia and Europe with different genotypes. In conclusion, gaining modestness at least for the amino acid sequence of cytochrome b can be said to be an important genetic factor for longevity.

What can be inferred from the work herein is that
At least the following two points. First, the diverse amino acid substitutions found in the young adult population imply that these substitutions have little effect on individual survival to maturity and transmission of the genome to the next generation. ing. Second, the absence of certain amino acid substitutions in centenarians and in Parkinson's disease patients has the effect that these amino acid substitutions are detrimental to long-term survival and predispose to adult-onset disease. Indicates that it has.

[0031]                                SEQUENCE LISTING        <110> Masashi, TANAKA <120> Gene Diagnosis Method using Human Mitochondrial DNA <130> human_mtDNA_cytB <140> P-01024TAN <141> 2001-10-17 <160> 15 <170> PatentIn Ver. 2.1 <210> 1 <211> 1134 <212> DNA <213> Homo sapiens <400> 1 atgaccccaa tacgcaaaac taacccccta ataaaattaa ttaaccactc attcatcgac 60 ctccccaccc catccaacat ctccgcatga tgaaacttcg gctcactcct tggcgcctgc 120 ctgatcctcc aaatcaccac aggactattc ctagccatgc actactcacc agacgcctca 180 accgcctttt catcaatcgc ccacatcact cgagacgtaa attatggctg aatcatccgc 240 taccttcacg ccaatggcgc ctcaatattc tttatctgcc tcttcctaca catcgggcga 300 ggcctatatt acggatcatt tctctactca gaaacctgaa acatcggcat tatcctcctg 360 cttgcaacta tagcaacagc cttcataggc tatgtcctcc cgtgaggcca aatatcattc 420 tgaggggcca cagtaattac aaacttacta tccgccatcc catacattgg gacagaccta 480 gttcaatgaa tctgaggagg ctactcagta gacagtccca ccctcacacg attctttacc 540 tttcacttca tcttgccctt cattattgca gccctagcag cactccacct cctattcttg 600 cacgaaacgg gatcaaacaa ccccctagga atcacctccc attccgataa aatcaccttc 660 cacccttact acacaatcaa agacgccctc ggcttacttc tcttccttct ctccttaatg 720 acattaacac tattctcacc agacctccta ggcgacccag acaattatac cctagccaac 780 cccttaaaca cccctcccca catcaagccc gaatgatatt tcctattcgc ctacacaatt 840 ctccgatccg tccctaacaa actaggaggc gtccttgccc tattactatc catcctcatc 900 ctagcaataa tccccatcct ccatatatcc aaacaacaaa gcataatatt tcgcccacta 960 agccaatcac tttattgact cctagccgca gacctcctca ttctaacctg aatcggagga 1020 caaccagtaa gctacccttt taccatcatt ggacaagtag catccgtact atacttcaca 1080 acaatcctaa tcctaatacc aactatctcc ctaattgaaa acaaaatact caaa 1134    <210> 2 <211> 378 <212> PRT <213> HOMO SAPIENS <400> 2 Met Thr Pro Met Arg Lys Thr Asn Pro Leu Met Lys Leu Ile Asn His   1 5 10 15 Ser Phe Ile Asp Leu Pro Thr Pro Ser Asn Ile Ser Ala Trp Trp Asn              20 25 30 Phe Gly Ser Leu Leu Gly Ala Cys Leu Ile Leu Gln Ile Thr Thr Gly          35 40 45 Leu Phe Leu Ala Met His Tyr Ser Pro Asp Ala Ser Thr Ala Phe Ser      50 55 60 Ser Ile Ala His Ile Thr Arg Asp Val Asn Tyr Gly Trp Ile Ile Arg  65 70 75 80 Tyr Leu His Ala Asn Gly Ala Ser Met Phe Phe Ile Cys Leu Phe Leu                  85 90 95 His Ile Gly Arg Gly Leu Tyr Tyr Gly Ser Phe Leu Tyr Ser Glu Thr             100 105 110 Trp Asn Ile Gly Ile Ile Leu Leu Leu Ala Thr Met Ala Thr Ala Phe         115 120 125 Met Gly Tyr Val Leu Pro Trp Gly Gln Met Ser Phe Trp Gly Ala Thr     130 135 140 Val Ile Thr Asn Leu Leu Ser Ala Ile Pro Tyr Ile Gly Thr Asp Leu 145 150 155 160 Val Gln Trp Ile Trp Gly Gly Tyr Ser Val Asp Ser Pro Thr Leu Thr                 165 170 175 Arg Phe Phe Thr Phe His Phe Ile Leu Pro Phe Ile Ile Ala Ala Leu             180 185 190 Ala Ala Leu His Leu Leu Phe Leu His Glu Thr Gly Ser Asn Asn Pro         195 200 205 Leu Gly Ile Thr Ser His Ser Asp Lys Ile Thr Phe His Pro Tyr Tyr     210 215 220 Thr Ile Lys Asp Ala Leu Gly Leu Leu Leu Phe Leu Leu Ser Leu Met 225 230 235 240 Thr Leu Thr Leu Phe Ser Pro Asp Leu Leu Gly Asp Pro Asp Asn Tyr                 245 250 255 Thr Leu Ala Asn Pro Leu Asn Thr Pro Pro His Ile Lys Pro Glu Trp             260 265 270 Tyr Phe Leu Phe Ala Tyr Thr Ile Leu Arg Ser Val Pro Asn Lys Leu         275 280 285 Gly Gly Val Leu Ala Leu Leu Leu Ser Ile Leu Ile Leu Ala Met Ile     290 295 300 Pro Ile Leu His Met Ser Lys Gln Gln Ser Met Met Phe Arg Pro Leu 305 310 315 320 Ser Gln Ser Leu Tyr Trp Leu Leu Ala Ala Asp Leu Leu Ile Leu Thr                 325 330 335 Trp Ile Gly Gly Gln Pro Val Ser Tyr Pro Phe Thr Ile Ile Gly Gln             340 345 350 Val Ala Ser Val Leu Tyr Phe Thr Thr Ile Leu Ile Leu Met Pro Thr         355 360 365 Ile Ser Leu Ile Glu Asn Lys Met Leu Lys     370 375    <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: L13901 <400> 3 tctccaacat actcggattc 20    <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: H609 <400> 4 gcccgtctaa acattttcag 20    <210> 5 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: FL14559 <400> 5 gtaaaacgac ggccagtcga ccacaccgct aacaatc 37    <210> 6 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: FL14837 <400> 6 gtaaaacgac ggccagttga aacttcggct cactcct 37    <210> 7 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: FL15126 <400> 7 gtaaaacgac ggccagtcct tcataggcta tgtcctc 37    <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: FL15405 <400> 8 gtaaaacgac ggccagttcc acccttacta cacaatc 37    <210> 9 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: FL15696 <400> 9 gtaaaacgac ggccagtttc gcccactaag ccaatca 37    <210> 10 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: FL15948 <400> 10 gtaaaacgac ggccagtagg acaaatcaga gaaaaag 37    <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: H15162 <400> 11 tactgtggcc cctcagaatg 20    <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: H15340 <400> 12 atcccgtttc gtgcaagaat 20    <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: H15755 <400> 13 actggttgtc ctccgattca 20    <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: H16016 <400> 14 cccatgaaag aacagagaat 20    <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: H81 <400> 15 cagcgtctcg caatgctatc 20

[Brief description of drawings]

Figure 1: Genetic map of mtDNA

FIG. 2 is a conceptual diagram showing a mtDNA sequencing method.

FIG. 3 shows a molecular phylogenetic network of amino acid substitutions for cytochrome b in centenarians, young adults, and Parkinson's disease patients. Lightly filled circles indicate the number of individuals having a standard amino acid sequence or common amino acid substitutions. Open circles indicate amino acid substitutions found in centenarians, and filled circles indicate amino acid substitutions detected in Parkinson's disease patients or young adults. The area of the circle is proportional to the number of individuals (shown in brackets) and the length of the bar is Gr
It is proportional to the value of antham.

FIG. 4 shows a molecular phylogenetic network of amino acid substitutions of cytochrome b in centenarians, young adults, and patients with Parkinson's disease. Compared to FIG. 3, those having the same amino acid substitution in the three groups have (A thin circle)
Indicated as. Others are the same as in FIG.

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4B024 AA01 AA11 BA80 CA03 CA20                       HA11                 4B063 QA13 QA17 QA19 QQ42 QQ43                       QR08 QR32 QR35 QR40 QR42                       QR62 QS16 QS25 QS28 QS36                       QS39 QX10

Claims (5)

[Claims] 1. A method for detecting a gene using human mitochondrial DNA, which comprises detecting that the nucleotide sequence of human mitochondrial DNA is substituted with a nucleotide accompanied by amino acid substitution in a protein encoded by the nucleotide sequence. . 2. The nucleotide sequence encoding cytochrome b (SEQ ID NO: 1), wherein the amino acid substitution is
T2A, T2I, H16R, A39T, T47K, T61A, I78T, L82F, Y109
H, T158A, D159N, I164V, D171N, A190T, A191T, A193
T, F245L, P247A, G251S, N260D, L296M, I300T, I306
The human mitochondrial DNA according to claim 1, which is at least one of V, I338V, V343M, S344N, A354T, I369V, I372V, and A380T.
A method for detecting a gene using. 3. The nucleotide sequence encoding cytochrome b (SEQ ID NO: 1), wherein the amino acid substitution is
T2A, T2I, T47K, T61A, L82F, Y109H, T158A, D159N, D
171N, A190T, A193T, F245L, P247A, G251S, L296M, I3
The gene detection method using human mitochondrial DNA according to claim 1, which is at least one of 00T, I338V, S344N, A354T, I372V, and A380T. 4. The nucleotide sequence encoding cytochrome b (SEQ ID NO: 1), wherein the amino acid substitution is
T2I, T47K, T61A, T158A, D171N, A190T, F245L, P247
The method for detecting a gene using human mitochondrial DNA according to claim 1, which is at least one of A, I300T, S344N, and A354T. 5. The nucleotide sequence encoding cytochrome b (SEQ ID NO: 1), wherein the amino acid substitution is
The gene detection method using human mitochondrial DNA according to claim 1, which is at least one of A193T, G251S, and I372V.