U.S. patent application number 10/601692 was filed with the patent office on 2004-01-29 for mitochondrial dosimeter.
This patent application is currently assigned to Johns Hopkins University. Invention is credited to Fliss, Makiko, Jen, Jin, Kinzler, Kenneth W., Polyak, Kornelia, Sidransky, David, Vogelstein, Bert.
Application Number | 20040018538 10/601692 |
Document ID | / |
Family ID | 24095095 |
Filed Date | 2004-01-29 |
United States Patent
Application |
20040018538 |
Kind Code |
A1 |
Fliss, Makiko ; et
al. |
January 29, 2004 |
Mitochondrial dosimeter
Abstract
Mitochondrial mutations occur as a product of contact of a
person with an environmental pollutant. Mitochondrial mutations are
readily detectable in body fluids. Measurement of mitochondrial
mutations in body fluids can be used as a dosimeter to monitor
exposure to the environmental pollutant. Mitochondrial mutations
can also be detected in cancer patients. Probes and primers
containing mutant mitochondrial sequences can be used to monitor
patient condition.
Inventors: |
Fliss, Makiko; (Columbia,
MD) ; Sidransky, David; (Baltimore, MD) ; Jen,
Jin; (Brookville, MD) ; Polyak, Kornelia;
(Brookline, MA) ; Vogelstein, Bert; (Baltimore,
MD) ; Kinzler, Kenneth W.; (BelAir, MD) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
Johns Hopkins University
Baltimore
MD
|
Family ID: |
24095095 |
Appl. No.: |
10/601692 |
Filed: |
June 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10601692 |
Jun 24, 2003 |
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09525906 |
Mar 15, 2000 |
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6605433 |
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09525906 |
Mar 15, 2000 |
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09377856 |
Aug 20, 1999 |
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6344322 |
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60097307 |
Aug 20, 1998 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12Q 1/6883 20130101; C12Q 1/6886 20130101; C12Q 2600/156 20130101;
C12Q 1/6809 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Goverment Interests
[0002] The U.S. Government retains certain rights in this invention
due to funding as provided by grant CA43460 awarded by the National
Institutes of Health.
Claims
1. A method of monitoring exposure of a person to an environmental
pollutant, comprising the step of: determining at two or more time
points the presence of one or more mutations in mitochondrial DNA
(mtDNA) in a body fluid of a person exposed to an environmental
pollutant; comparing the amount of mutations in mtDNA at different
time points, wherein the amount of mutations correlates with amount
of exposure to the environmental pollutant.
2. A method of monitoring exposure of a person to an environmental
pollutant, comprising the step of: measuring prevalence of one or
more mutations in mitochondrial DNA (mtDNA) in a body fluid of a
person exposed to an environmental pollutant, wherein a measured
prevalence of one or more mutations in mtDNA of greater than 1%
indicates clonal expansion of cells which harbor the one or more
mutations in the person.
3. A method of monitoring exposure of a person to an environmental
pollutant, comprising the step of: measuring one or more mutations
in a D-loop of mitochondrial DNA (mtDNA) in a body fluid of a
person exposed to an environmental pollutant, wherein the number of
mutations in mtDNA correlates with exposure to the environmental
pollutant.
4. The method of claim 1, 2, or 3 wherein the body fluid is
urine.
5. The method of claim 1, 2, or 3 wherein the body fluid is
saliva.
6. The method of claim 1, 2, or 3 wherein the body fluid is
sputum.
7. The method of claim 1, 2, or 3 wherein the body fluid is
brouchoalveolar lavage.
8. The method of claim 1, 2, or 3 wherein mutations in mtDNA are
measured with reference to mtDNA isolated from a normal tissue of
the human.
9. The method of claim 8 wherein the normal tissue is
paraffin-embedded.
10. The method of claim 1, 2, or 3 wherein mutations in mtDNA are
measured with reference to mtDNA isolated from a blood, serum, or
plasma sample of the human.
11. The method or claim 1 or 2 wherein mutations in mtDNA are
measured by analysis of a gene encoding NADH dehydrogenase 4.
12. The method of claim 1 or 2 wherein mutations in mtDNA are
measured by analysis of a gene encoding 16S rRNA.
13. The method of claim 2 wherein prevalence of one or more
mutations in mtDNA in the body fluid is measured over time, whereby
clonal expansion of cells can be monitored.
14. The method of claim 1 or 2 wherein mutations in mtDNA are
measured by analysis of a cytochrome b gene.
15. The method of claim 1, 2, or 3 wherein the mutated mtDNA is
found to bear a silent mutation.
16. The method of claim 1 or 2 wherein the mutated mtDNA is
measured by analysis of non-coding regions.
17. The method of claim 1, 2, or 3 wherein the mutated mtDNA is
measured by amplifying mtDNA segments of about 10 bp to about 4
kb.
18. The method of claim 1, 2, or 3 wherein the mutated mtDNA is
measured by amplifying mtDNA segments of about 10 bp to about 2
kb.
19. The method of claim 1, 2, or 3 wherein the mutated mtDNA is
measured by amplifying mtDNA segments of about 2 kb to about 4
kb.
20. The method of claim 1, 2, or 3 wherein the mutated mtDNA is
measured by an oligonucleotide mismatch ligation assay.
21. The method of claim 1, 2, or 3 wherein the environmental
pollutant is cigarette smoke.
22. The method of claim 1, 2, or 3 wherein the environmental
pollutant is a biological toxin.
23. The method of claim 1, 2, or 3 wherein the environmental
pollutant is radiation.
24. The method of claim 1, 2, or 3 wherein the environmental
pollutant is industrial waste.
25. The method of claim 1, 2, or 3 wherein the environmental
pollutant is chemical.
26. The method of claim 1, 2, or 3 wherein the environmental
pollutant is water-borne.
27. The method of claim 1, 2, or 3 wherein the environmental
pollutant is air-borne.
28. The method of claim 1, 2, or 3 wherein the environmental
pollutant is a drug.
29. A kit for monitoring exposure of a person to environmental
pollutants, comprising: one or more primers which hybridize to a
mitochondrial D-loop for making a primer extension product; and
written material identifying mutations which are found in the
D-loop as a result of exposure to one or more environmental
pollutants.
30. The kit of claim 29 further comprising a buffer.
31. The kit of claim 29 further comprising nucleic acid probes for
hybridization to the extension product.
32. The kit of claim 29 wherein the primers are for PCR.
33. The kit of claim 29 wherein the primers are for a ligation
assay.
34. The method of claim 3 wherein the mutation is selected from the
group consisting of: T.fwdarw.C at nucleotide 114; .DELTA.C at
nucleotide 302; C.fwdarw.A at nucleotide 386; insert T at
nucleotide 16189; A.fwdarw.C at nucleotide 16265; A.fwdarw.T at
nucleotide 16532; C.fwdarw.T at nucleotide 150; T.fwdarw.C at
nucleotide 195; .DELTA.C at nucleotide 302; C.fwdarw.A at
nucleotide 16183; C.fwdarw.T at nucleotide 16187; T.fwdarw.C at
nucleotide 16519; G.fwdarw.A at nucleotide 16380; G.fwdarw.A at
nucleotide 75; insert C at nucleotide 302; insert CG at nucleotide
514; T.fwdarw.C at nucleotide 16172; C.fwdarw.T at nucleotide
16292; and A.fwdarw.G at nucleotide 16300.
35. The kit of claim 29 wherein the written material identifies a
mutation selected from the group consisting of: T.fwdarw.C at
nucleotide 114; .DELTA.C at nucleotide 302; C.fwdarw.A at
nucleotide 386; insert T at nucleotide 16189; A.fwdarw.C at
nucleotide 16265; A.fwdarw.T at nucleotide 16532; C.fwdarw.T at
nucleotide 150; T.fwdarw.C at nucleotide 195; .DELTA.C at
nucleotide 302; C.fwdarw.A at nucleotide 16183; C.fwdarw.T at
nucleotide 16187; T.fwdarw.C at nucleotide 16519; G.fwdarw.A at
nucleotide 16380; G.fwdarw.A at nucleotide 75; insert C at
nucleotide 302; insert CG at nucleotide 514; T.fwdarw.C at
nucleotide 16172; C.fwdarw.T at nucleotide 16292; and A.fwdarw.G at
nucleotide 16300.
36. The method of claim 11 wherein the mutation is selected from
the group consisting of: A.fwdarw.G at nucleotide 10792, C.fwdarw.T
at nucleotide 10793, C.fwdarw.T at nucleotide 10822, A.fwdarw.G at
nucleotide 10978, A.fwdarw.G at nucleotide 11065, G.fwdarw.A at
nucleotide 11518, C.fwdarw.T at nucleotide 12049, T.fwdarw.C at
nucleotide 10966, and G.fwdarw.A at nucleotide 11150.
37. The method of claim 12 wherein the mutation is selected from
the group consisting of: G.fwdarw.A at nucleotide 2056, T.fwdarw.C
at nucleotide 2445, T.fwdarw.C at nucleotide 2664, and G.fwdarw.A
at nucleotide 3054.
38. The method of claim 14 wherein the mutation is .DELTA.7 amino
acids at nucleotide 15642.
39. An oligonucleotide probe comprising a sequence of at least 10
contiguous nucleotides of a human mitochondrial genome, wherein the
oligonucleotide comprises a mutation selected from the group
consisting of: a mutation selected from the group consisting of:
T.fwdarw.C at nucleotide 114; .DELTA.C at nucleotide 302;
C.fwdarw.A at nucleotide 386; insert T at nucleotide 16189;
A.fwdarw.C at nucleotide 16265; A.fwdarw.T at nucleotide 16532;
C.fwdarw.T at nucleotide 150; T.fwdarw.C at nucleotide 195;
.DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 16183;
C.fwdarw.T at nucleotide 16187; T.fwdarw.C at nucleotide 16519;
G.fwdarw.A at nucleotide 16380; G.fwdarw.A at nucleotide 75; insert
C at nucleotide 302; insert CG at nucleotide 514; T.fwdarw.C at
nucleotide 16172; C.fwdarw.T at nucleotide 16292; A.fwdarw.G at
nucleotide 16300; A.fwdarw.G at nucleotide 10792; C.fwdarw.T at
nucleotide 10793; C.fwdarw.T at nucleotide 10822; A.fwdarw.G at
nucleotide 10978; A.fwdarw.G at nucleotide 11065; G.fwdarw.A at
nucleotide 11518; C.fwdarw.T at nucleotide 12049; T.fwdarw.C at
nucleotide 10966; G.fwdarw.A at nucleotide 11150; G.fwdarw.A at
nucleotide 2056; T.fwdarw.C at nucleotide 2445; T.fwdarw.C at
nucleotide 2664; T.fwdarw.C at nucleotide 10071; T.fwdarw.C at
nucleotide 10321; T.fwdarw.C at nucleotide 12519; .DELTA.7 amino
acids at nucleotide 15642; G.fwdarw.A at nucleotide 5521;
G.fwdarw.A at nucleotide 12345; G.fwdarw.A at nucleotide 3054;
T.fwdarw.C substitution at position 710; T.fwdarw.C substitution at
position 1738; T.fwdarw.C substitution at position 3308; G.fwdarw.A
substitution at position 8009; G.fwdarw.A substitution at position
14985; T.fwdarw.C substitution at position 15572; G.fwdarw.A
substitution at position 9949; T.fwdarw.C substitution at position
10563; G.fwdarw.A substitution at position 6264; A insertion at
position.12418; T.fwdarw.C substitution at position 1967; and
T.fwdarw.A substitution at position 2299.
40. An oligonucleotide primer comprising a sequence of at least 10
contiguous nucleotides of a human mitochondrial genome, wherein the
oligonucleotide comprises a mutation selected from the group
consisting of: a mutation selected from the group consisting of:
T.fwdarw.C at nucleotide 114; .DELTA.C at nucleotide 302;
C.fwdarw.A at nucleotide 386; insert T at nucleotide 16189;
A.fwdarw.C at nucleotide 16265; A.fwdarw.T at nucleotide 16532;
C.fwdarw.T at nucleotide 150; T.fwdarw.C at nucleotide 195;
.DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 16183;
C.fwdarw.T at nucleotide 16187; T.fwdarw.C at nucleotide 16519;
G.fwdarw.A at nucleotide 16380; G.fwdarw.A at nucleotide 75; insert
C at nucleotide 302; insert CG at nucleotide 514; T.fwdarw.C at
nucleotide 16172; C.fwdarw.T at nucleotide 16292; A.fwdarw.G at
nucleotide 16300; A.fwdarw.G at nucleotide 10792; C.fwdarw.T at
nucleotide 10793: C.fwdarw.T at nucleotide 1 0822; A.fwdarw.G at
nucleotide 10978; A.fwdarw.G at nucleotide 11065; G.fwdarw.A at
nucleotide 11518; C.fwdarw.T at nucleotide 12049; T.fwdarw.C at
nucleotide 10966; G.fwdarw.A at nucleotide 11150; G.fwdarw.A at
nucleotide 2056; T.fwdarw.C at nucleotide 2445; T.fwdarw.C at
nucleotide 2664; T.fwdarw.C at nucleotide 10071; T.fwdarw.C at
nucleotide 10321; T.fwdarw.C at nucleotide 12519; .DELTA.7 amino
acids at nucleotide 15642; G.fwdarw.A at nucleotide 5521;
G.fwdarw.A at nucleotide 12345; G.fwdarw.A at nucleotide
3054;T.fwdarw.C substitution at position 710; T.fwdarw.C
substitution at position 1738; T.fwdarw.C substitution at position
3308; G.fwdarw.A substitution at position 8009; G.fwdarw.A
substitution at position 14985; T.fwdarw.C substitution at position
15572; G.fwdarw.A substitution at position 9949; T.fwdarw.C
substitution at position 10563; G.fwdarw.A substitution at position
6264; A insertion at position 1241 8; T.fwdarw.C substitution at
position 1967; and T.fwdarw.A substitution at position 2299.
41. A method to aid in detecting the presence of tumor cells in a
patient, comprising: determining the presence of a single basepair
mutation in a mitochondrial genome of a cell sample of a patient,
wherein the mutation is found in a tumor of the patient but not in
normal tissue of the patient, wherein the tumor is not a colorectal
tumor; and identifying the patient as having a tumor if one or more
single basepair mutations are determined in the mitochondrial
genome of the cell sample of the patient.
42. A method to aid in detecting the presence of tumor cells in a
patient, comprising: determining the presence of a mutation in a
D-loop of a mitochondrial genome of a cell sample of a patient,
wherein the mutation is found in a tumor of the patient but not in
normal tissue of the patient; and identifying the patient as having
a tumor if one or more single basepair mutations are determined in
the mitochondrial genome of the cell sample of the patient.
43. The method of claim 42 wherein the mutation is selected from
the group consisting of: T.fwdarw.C at nucleotide 114; .DELTA.C at
nucleotide 302; C.fwdarw.A at nucleotide 386; insert T at
nucleotide 16189; A.fwdarw.C at nucleotide 16265; A.fwdarw.T at
nucleotide 16532; C.fwdarw.T at nucleotide 150; T.fwdarw.C at
nucleotide 195; .DELTA.C at nucleotide 302; C.fwdarw.A at
nucleotide 16183; C.fwdarw.T at nucleotide 16187; T.fwdarw.C at
nucleotide 16519; G.fwdarw.A at nucleotide 16380; G.fwdarw.A at
nucleotide 75; insert C at nucleotide 302; insert CG at nucleotide
514; T.fwdarw.C at nucleotide 16172; C.fwdarw.T at nucleotide
16292; and A.fwdarw.G at nucleotide 16300.
44. A method to aid in detecting the presence of tumor cells in a
patient, comprising: determining the presence of a single basepair
mutation in a mitochondrial genome of a cell sample of a patient,
wherein the mutation is found in a cancer of the patient but not in
normal tissue of the patient, wherein the cancer is selected from
the group of cancers consisting of: lung, head and neck, bladder,
brain, breast, lymphoma, leukaemia, skin, prostate, stomach,
pancreas, liver, ovarian, uterine, testicular, and bone; and
identifying the patient as having a tumor if one or more single
basepair mutations are determined in the mitochondrial genome of
the cell sample of the patient.
45. A method to aid in detecting the presence of tumor cells in a
patient, comprising: step for determining the presence of a single
basepair mutation in a mitochondrial genome of a cell sample of a
patient, wherein the mutation is found in a tumor of the patient
but not in normal tissue of the patient, wherein the cancer is
selected from the group of cancers consisting of: lung, head and
neck, and bladder; and identifying the patient as having a tumor if
one or more single basepair mutations are determined in the
mitochondrial genome of the cell sample of the patient.
46. A method to aid in detecting the presence of tumor cells in a
patient, comprising: determining the presence of a mutation in a
mitochondrial genome of a cell sample of a patient, wherein the
mutation is selected from the group consisting of: T.fwdarw.C at
nucleotide 114; .DELTA.C at nucleotide 302; C.fwdarw.A at
nucleotide 386; insert T at nucleotide 16189; A.fwdarw.C at
nucleotide 16265; A.fwdarw.T at nucleotide 16532; C.fwdarw.T at
nucleotide 150; T.fwdarw.C at nucleotide 195; .DELTA.C at
nucleotide 302; C.fwdarw.A at nucleotide 16183; C.fwdarw.T at
nucleotide 16187; T.fwdarw.C at nucleotide 16519; G.fwdarw.A at
nucleotide 16380; G.fwdarw.A at nucleotide 75; insert C at
nucleotide 302; insert CG at nucleotide 514; T.fwdarw.C at
nucleotide 16172; C.fwdarw.T at nucleotide 16292; A.fwdarw.G at
nucleotide 16300; A.fwdarw.G at nucleotide 10792; C.fwdarw.T at
nucleotide 10793; C.fwdarw.T at nucleotide 10822; A.fwdarw.G at
nucleotide 10978; A.fwdarw.G at nucleotide 11065; G.fwdarw.A at
nucleotide 11518; C.fwdarw.T at nucleotide 12049; T.fwdarw.C at
nucleotide 10966; G.fwdarw.A at nucleotide 11150; G.fwdarw.A at
nucleotide 2056; T.fwdarw.C at nucleotide 2445; T.fwdarw.C at
nucleotide 2664; T.fwdarw.C at nucleotide 10071; T.fwdarw.C at
nucleotide 10321; T.fwdarw.C at nucleotide 12519; .DELTA.7 amino
acids at nucleotide 15642; G.fwdarw.A at nucleotide 5521;
G.fwdarw.A at nucleotide 12345; and G.fwdarw.A at nucleotide 3054;
and identifying the patient as having a tumor if one or more
mutations are determined in the mitochondrial genome of the cell
sample of the patient.
47. The method of claim 41, 42, 44, 45, or 46 wherein the cell
sample is from blood.
48. The method of claim 41, 42, 44, 45, or 46 wherein the cell
sample is from urine.
49. The method of claim 41, 42, 44, 45, or 46 wherein the cell
sample is from sputum.
50. The method of claim 41, 42, 44, 45, or 46 wherein the cell
sample is from saliva.
51. The method of claim 41, 42, 44, 45, or 46 wherein the cell
sample is from feces.
52. The method of claim 41, 42, 44, 45, or 46 wherein the step for
determining comprises amplifying mitochondrial DNA.
53. The method of claim 41, 42, 44, 45, or 46 wherein the step for
determining comprises sequencing mitochondrial DNA.
54. The method of claim 41, 42, 44, 45, or 46 wherein the step for
determining comprises hybridization of DNA amplified from the
mitochondrial genome of the cell sample to an array of
oligonucleotides which comprises matched and mismatched sequences
to human mitochondrial genomic DNA.
55. The method of claim 41, 42, 44, 45, or 46 wherein the mutation
is a substitution mutation.
56. The method of claim 41, 42, 44, 45, or 46 wherein the mutation
is a one basepair insertion.
57. The method of claim 41, 42, 44, 45, or 46 wherein the mutation
is a one basepair deletion.
58. The method of claim 41, 42, 44, 45, or 46 wherein the mutation
is a transition mutation.
59. The method of claim 41, 42, 44, 45, or 46 wherein the mutation
is a homoplasmic mutation.
60. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C substitution at position
710.
61. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C substitution at position
1738.
62. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C substitution at position
3308.
63. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A substitution at position
8009.
64. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A substitution at position
14985.
65. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C substitution at position
15572.
66. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A substitution at position
9949.
67. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C substitution at position
10563.
68. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A substitution at position
6264.
69. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is an A insertion at position 12418.
70. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C substitution at position
1967.
71. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.A substitution at position
2299.
72. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 10071.
73. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 10321.
74. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 12519.
75. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A at nucleotide 5521.
76. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A at nucleotide 12345.
77. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 114.
78. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a .DELTA.C at nucleotide 302.
79. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a C.fwdarw.A at nucleotide 386.
80. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is an insert T at nucleotide 16189.
81. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a A.fwdarw.C at nucleotide 16265.
82. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a A.fwdarw.T at nucleotide 16532.
83. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a C.fwdarw.T at nucleotide 150.
84. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 195.
85. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a .DELTA.C at nucleotide 302.
86. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a C.fwdarw.A at nucleotide 16183.
87. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a C.fwdarw.T at nucleotide 16187.
88. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 16519.
89. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A at nucleotide 16380.
90. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A at nucleotide 75
91. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is an insert C at nucleotide 302.
92. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is an insert CG at nucleotide 514.
93. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 16172.
94. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a C.fwdarw.T at nucleotide 16292.
95. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is an A.fwdarw.G at nucleotide 16300.
96. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is an A.fwdarw.G at nucleotide 10792.
97. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a C.fwdarw.T at nucleotide 10793.
98. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a C.fwdarw.T at nucleotide 10822.
99. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a A.fwdarw.G at nucleotide 10978.
100. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a A.fwdarw.G at nucleotide 11065.
101. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A at nucleotide 11518.
102. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a C.fwdarw.T at nucleotide 12049.
103. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 10966.
104. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A at nucleotide 11150.
105. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A at nucleotide 2056.
106. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 2445.
107. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 2664.
108. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 10071.
109. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 10321.
110. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a T.fwdarw.C at nucleotide 12519.
111. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a .DELTA.7 amino acids at nucleotide
15642.
112. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A at nucleotide 5521.
113. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A at nucleotide 12345.
114. The oligonucleotide probe of claim 39 or primer of claim 40
wherein the mutation is a G.fwdarw.A at nucleotide 3054.
115. The method of claim 2 wherein the mutation was identified
previously in a tumor of the patient.
116. The method of claim 115 wherein the patient has received
anti-cancer therapy and the step for determining is performed at
least three times to monitor progress of the anti-cancer
therapy.
117. The method of claim 1 further comprising a step for testing a
normal tissue of the patient to determine the absence of the
mutation.
Description
[0001] This application is a continuation-in-part of application
Ser. No. 09/377,856 filed Aug. 20, 1999, which claims priority to
provisional application Serial No. 60/097,307 filed Aug. 20, 1998.
The disclosure of these prior applications is expressly
incorporated herein.
TECHNICAL FIELD OF THE INVENTION
[0003] This invention is related to the field of environmental
toxicology, in particular to methods for measuring the effects of
environmental toxins.
BACKGROUND OF THE INVENTION
[0004] The human mitochondrial (mt) genome is small (16.5 kb) and
encodes 13 respiratory chain subunits, 22 tRNAs and two rRNAs.
Mitochondrial DNA is present at extremely high levels
(10.sup.3-10.sup.4 copies per cell) and the vast majority of these
copies are identical (homoplasmic) at birth (1). Expression of the
entire complement of mt genes is required to maintain proper
function of the organelle, suggesting that even slight alterations
in DNA sequences could have profound effects (2). It is generally
accepted that mtDNA mutations are generated endogenously during
oxidative phosphorylation via pathways involving reactive oxygen
species (ROS), but they can also be generated by external
carcinogens or environmental toxins. These mutations may accumulate
partially because mitochondria lack protective histones and highly
efficient DNA repair mechanisms as seen in the nucleus (3).
Recently several mtDNA mutations were found specifically in human
colorectal cancer (4).
SUMMARY OF THE INVENTION
[0005] It is an object of the present invention to provide methods
of monitoring exposure of a person to an environmental
pollutant.
[0006] It is another object of the present invention to provide a
kit for monitoring exposure of a person to environmental
pollutants.
[0007] It is an object of the invention to provide methods to aid
in the detection of cancer or metastasis.
[0008] It is an object of the invention to provide probes and
primers for detecting mitochondrial mutations.
[0009] It is an object of the invention to provide a method to aid
in detecting the presence of tumor cells in a patient.
[0010] These and other objects of the invention are achieved by
providing one or more of the embodiments described below. In one
embodiment a method is provided for monitoring exposure of a person
to an environmental pollutant. The presence of one or more
mutations in mitochondrial DNA (mtDNA) in a body fluid of a person
exposed to an environmental pollutant is determined at two or more
time points. The amounts of mutations in mtDNA at different time
points are compared. The amount of mutations correlates with amount
of exposure to the environmental pollutant.
[0011] According to another embodiment another method is provided
for monitoring exposure of a person to an environmental pollutant.
The prevalence of one or more mutations in mitochondrial DNA
(mtDNA) in a body fluid of a person exposed to an environmental
pollutant is measured. A measured prevalence of one or more
mutations in mtDNA of greater than 1% indicates clonal expansion of
cells which harbor the one or more mutations in the person.
[0012] According to still another embodiment of the invention a
method is provided for monitoring exposure of a person to an
environmental pollutant. One or more mutations in a D-loop of
mitochondrial DNA (mtDNA) in a body fluid of a person exposed to an
environmental pollutant are measured. The number of mutations in
mtDNA correlates with exposure to the environmental pollutant.
[0013] According to yet another embodiment of the invention a kit
is provided. The kit comprises one or more primers which hybridize
to a mitochondrial D-loop for making a primer extension product. In
addition, the kit contains written material identifying mutations
which are found in the D-loop as a result of exposure to one or
more environmental pollutants.
[0014] According to another embodiment of the invention an
oligonucleotide probe is provided. The probe comprises a sequence
of at least 10 contiguous nucleotides of a human mitochondrial
genome. The probe can optionally contain at least 12, 14, 16, 18,
20, 22, 24, 26, or 30 such contiguous nucleotides. The
oligonucleotide comprises a mutation selected from the group
consisting of: a mutation selected from the group consisting of:
T.fwdarw.C at nucleotide 114; .DELTA.C at nucleotide 302;
C.fwdarw.A at nucleotide 386; insert T at nucleotide 16189;
A.fwdarw.C at nucleotide 16265; A.fwdarw.T at nucleotide 16532;
C.fwdarw.T at nucleotide 150; T.fwdarw.C at nucleotide 195;
.DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 16183;
C.fwdarw.T at nucleotide 16187; T.fwdarw.C at nucleotide 16519;
G.fwdarw.A at nucleotide 16380; G.fwdarw.A at nucleotide 75; insert
C at nucleotide 302; insert CG at nucleotide 514; T.fwdarw.C at
nucleotide 16172; C.fwdarw.T at nucleotide 16292; A.fwdarw.G at
nucleotide 16300; A.fwdarw.G at nucleotide 10792; C.fwdarw.T at
nucleotide 10793; C.fwdarw.T at nucleotide 10822; A.fwdarw.G at
nucleotide 10978; A.fwdarw.G at nucleotide 11065; G.fwdarw.A at
nucleotide 11518; C.fwdarw.T at nucleotide 12049; T.fwdarw.C at
nucleotide 10966; G.fwdarw.A at nucleotide 11150; G.fwdarw.A at
nucleotide 2056; T.fwdarw.C at nucleotide 2445; T.fwdarw.C at
nucleotide 2664; T.fwdarw.C at nucleotide 10071; T.fwdarw.C at
nucleotide 10321; T.fwdarw.C at nucleotide 12519; .DELTA.7 amino
acids at nucleotide 15642; G.fwdarw.A at nucleotide 5521;
G.fwdarw.A at nucleotide 12345; T.fwdarw.C substitution at position
710; T.fwdarw.C substitution at position 1738; T.fwdarw.C
substitution at position 3308; G.fwdarw.A substitution at position
8009; G.fwdarw.A substitution at position 14985; T.fwdarw.C
substitution at position 15572; G.fwdarw.A substitution at position
9949; T.fwdarw.C substitution at position 10563; G.fwdarw.A
substitution at position 6264; A insertion at position 12418;
T.fwdarw.C substitution at position 1967; T.fwdarw.A substitution
at position 2299; and G.fwdarw.A at nucleotide 3054.
[0015] According to another aspect of the invention an
oligonucleotide primer is provided. It comprises a sequence of at
least 10 contiguous nucleotides of a human mitochondrial genome.
The primer can optionally contain at least 12, 14, 16, 18, 20, 22,
24, 26, or 30 such contiguous nucleotides. The oligonucleotide
comprises a mutation selected from the group consisting of: a
mutation selected from the group consisting of: T.fwdarw.C at
nucleotide 114; .DELTA.C at nucleotide 302; C.fwdarw.A at
nucleotide 386; insert T at nucleotide 16189; A.fwdarw.C at
nucleotide 16265; A.fwdarw.T at nucleotide 16532; C.fwdarw.T at
nucleotide 150; T.fwdarw.C at nucleotide 195; .DELTA.C at
nucleotide 302; C.fwdarw.A at nucleotide 16183; C.fwdarw.T at
nucleotide 16187; T.fwdarw.C at nucleotide 16519; G.fwdarw.A at
nucleotide 16380; G.fwdarw.A at nucleotide 75; insert C at
nucleotide 302; insert CG at nucleotide 514; T.fwdarw.C at
nucleotide 16172; C.fwdarw.T at nucleotide 16292; A.fwdarw.G at
nucleotide 16300; A.fwdarw.G at nucleotide 10792; C.fwdarw.T at
nucleotide 10793; C.fwdarw.T at nucleotide 10822; A.fwdarw.G at
nucleotide 10978; A.fwdarw.G at nucleotide 11065; G.fwdarw.A at
nucleotide 11518; C.fwdarw.T at nucleotide 12049; T.fwdarw.C at
nucleotide 10966; G.fwdarw.A at nucleotide 11150; G.fwdarw.A at
nucleotide 2056; T.fwdarw.C at nucleotide 2445; T.fwdarw.C at
nucleotide 2664; T.fwdarw.C at nucleotide 10071; T.fwdarw.C at
nucleotide 10321; T.fwdarw.C at nucleotide 12519; .DELTA.7 amino
acids at nucleotide 15642; G.fwdarw.A at nucleotide 5521;
G.fwdarw.A at nucleotide 12345; T.fwdarw.C substitution at position
710; T.fwdarw.C substitution at position 1738; T.fwdarw.C
substitution at position 3308; G.fwdarw.A substitution at position
8009; G.fwdarw.A substitution at position 14985; T.fwdarw.C
substitution at position 15572; G.fwdarw.A substitution at position
9949; T.fwdarw.C substitution at position 10563; G.fwdarw.A
substitution at position 6264; A insertion at position 12418;
T.fwdarw.C substitution at position 1967; T.fwdarw.A substitution
at position 2299; and G.fwdarw.A at nucleotide 3054.
[0016] Another aspect of the invention is a method to aid in
detecting the presence of tumor cells in a patient. The presence of
a single basepair mutation is detected in a mitochondrial genome of
a cell sample of a patient. The mutation is found in a tumor of the
patient but not in normal tissue of the patient. The tumor is not a
colorectal tumor. The patient is identified as having a tumor if
one or more single basepair mutations are determined in the
mitochondrial genome of the cell sample of the patient.
[0017] Yet another embodiment of the invention is provided by
another method to aid in detecting the presence of tumor cells in a
patient. The presence of a mutation is determined in a D-loop of a
mitochondrial genome of a cell sample of a patient. The mutation is
found in a tumor of the patient but not in normal tissue of the
patient. The patient is identified as having a tumor if one or more
single basepair mutations are determined in the mitochondrial
genome of the cell sample of the patient.
[0018] According to still another aspect of the invention a method
is provided to aid in detecting the presence of tumor cells in a
patient. The presence of a single basepair mutation is determined
in a mitochondrial genome of a cell sample of a patient. The
mutation is found in a cancer of the patient but not in normal
tissue of the patient. The cancer is selected from the group of
cancers consisting of: lung, head and neck, bladder, brain, breast,
lymphoma, leukaemia, skin, prostate, stomach, pancreas, liver,
ovarian, uterine, testicular, and bone. The patient is identified
as having a tumor if one or more single basepair mutations are
determined in the mitochondrial genome of the cell sample of the
patient.
[0019] According to still another aspect of the invention a method
is provided to aid in detecting the presence of tumor cells in a
patient. The presence of a single basepair mutation is determined
in a mitochondrial genome of a cell sample of a patient. The
mutation is found in a tumor of the patient but not in normal
tissue of the patient. The cancer is selected from the group of
cancers consisting of: lung, head and neck, and bladder. The
patient is identified as having a tumor if one or more single
basepair mutations are determined in the mitochondrial genome of
the cell sample of the patient.
[0020] Another embodiment of the invention provides a method to aid
in detecting the presence of tumor cells in a patient. The presence
of a mutation in a mitochondrial genome of a cell sample of a
patient is determined. The mutation is selected from the group
consisting of: T.fwdarw.C at nucleotide 114; .DELTA.C at nucleotide
302; C.fwdarw.A at nucleotide 386; insert T at nucleotide 16189;
A.fwdarw.C at nucleotide 16265; A.fwdarw.T at nucleotide 16532;
C.fwdarw.T at nucleotide 150; T.fwdarw.C at nucleotide 195;
.DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 16183;
C.fwdarw.T at nucleotide 16187; T.fwdarw.C at nucleotide 16519;
G.fwdarw.A at nucleotide 16380; G.fwdarw.A at nucleotide 75; insert
C at nucleotide 302; insert CG at nucleotide 514; T.fwdarw.C at
nucleotide 16172; C.fwdarw.T at nucleotide 16292; A.fwdarw.G at
nucleotide 16300; A.fwdarw.G at nucleotide 10792; C.fwdarw.T at
nucleotide 10793; C.fwdarw.T at nucleotide 10822; A.fwdarw.G at
nucleotide 10978; A.fwdarw.G at nucleotide 11065; G.fwdarw.A at
nucleotide 11518; C.fwdarw.T at nucleotide 12049; T.fwdarw.C at
nucleotide 10966; G.fwdarw.A at nucleotide 11150; G.fwdarw.A at
nucleotide 2056; T.fwdarw.C at nucleotide 2445; T.fwdarw.C at
nucleotide 2664; T.fwdarw.C at nucleotide 10071; T.fwdarw.C at
nucleotide 10321; T.fwdarw.C at nucleotide 12519; .DELTA.7 amino
acids at nucleotide 15642; G.fwdarw.A at nucleotide 5521;
G.fwdarw.A at nucleotide 12345; T.fwdarw.C substitution at position
710; T.fwdarw.C substitution at position 1738; T.fwdarw.C
substitution at position 3308; G.fwdarw.A substitution at position
8009; G.fwdarw.A substitution at position 14985; T.fwdarw.C
substitution at position 15572; G.fwdarw.A substitution at position
9949; T.fwdarw.C substitution at position 10563; G.fwdarw.A
substitution at position 6264; A insertion at position 12418;
T.fwdarw.C substitution at position 1967; T.fwdarw.A substitution
at position 2299; and G.fwdarw.A at nucleotide 3054. The patient is
identified as having a tumor if one or more mutations are
determined in the mitochondrial genome of the cell sample of the
patient.
[0021] These and other embodiments provide the art with
non-invasive tools for monitoring exposure to and the effects of
environmental pollutants on the human body as well as early
detection methods for cancer and metastasis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1. Schematic representation of a linearized mt genome.
Hatched bars indicate the regions sequenced in this study and solid
bars indicate the positions of tRNAs (transfer RNAs).
rRNA=ribosomal RNA, ND=NADH dehydrogenase, COX=cytochrome c
oxidase, Cyt b=cytochrome b, ATPase=ATP synthase.
[0023] FIG. 2. Sequence detection of mutated mtDNAs in samples from
tumors and bodily fluids. (FIG. 2A) The mt mutation was analyzed by
direct sequencing of the tumor (T), normal (N), and corresponding
urine (U) DNAs of bladder cancer patient #799. The arrow indicates
a single nucleotide change (G(A) at 2056 np in the 16S rRNA gene.
(FIG. 2B and FIG. 2C) Examples of somatic mutations in head and
neck cancers. Both mutations at 16172 np (B) and 10822 np (C) were
detected from saliva (S) samples from patients #1680 and #1708,
respectively. (FIG. 2D) Mutated mtDNA at 2664 np was not detected
by sequence analysis in the paired BAL fluid (B), obtained from
lung cancer patient #898.
[0024] FIG. 3. Oligonucleotide-mismatch ligation assay (22) to
detect mtDNA mutations in BAL. The arrows identify mutated mt
sequences at 12345 np within tRNA (FIG. 3A) and at 2664 np (FIG.
3B) within 16S rRNA in the tumor DNA. More dilute signals are seen
in the corresponding BAL (B) samples with no detectable signal from
the paired normal (N) tissue.
[0025] FIG. 4. Highly enriched mutated mtDNA in BAL samples from
lung cancer patients. Oligonucleotide (oligo) specific
hybridization detected .about.2000 plaques containing WT p53 clones
in the BAL from patient #1113, and only two plaques (2/2000=0.1%)
with the p53 gene mutation (FIG. 4A) were found in the primary
tumor. The same BAL sample demonstrated a much greater enrichment
of mutated mtDNA; 445 plaques contained mtDNA mutations (FIG. 4A)
at 16159 np (445/2000=22.3%; 220-fold) compared to approximately
1500 WT clones. A similar enrichment was seen in patient #1140
where oligo specific hybridization detected 12 p53 mutant plaques
among 437 WT clones (2.7%, FIG. 4B), while mutant mtDNA at 16380 np
(FIG. 4B) represented over 50% of the plaques (52.3%, 460/880;
19-fold) amplified from mtDNA.
[0026] FIG. 5. Pseudoclonal selection of mtDNA. A mitochondrial
genome gains some replicative advantage due to a somatic mutation
(such as in the D-loop region), leading to a dominant mitochondrial
genotype (step 1). This mitochondrion can gain additional
replicative advantage through nuclear influences: for example, a
mutated sequence gains a higher binding affinity to nuclear-encoded
mitochondrial trans-acting factors (step 2). Due to its stochastic
segregation together with the clonal expansion of a neoplastic cell
driven by nuclear mutations, mutated mitochondria overtake the
entire population of tumor cells (step 3).
BRIEF DESCRIPTION OF THE TABLES
[0027] Table 1 provides a summary of mutations in mitochondria of
colorectal tumors.
[0028] Table 2 provides a summary of mutations in mitochondria of
bladder, lung, head and neck tumors.
[0029] Table 3 provides a summary of new polymorphisms in
mitochondria of bladder, lung, head and neck tumors.
DETAILED DESCRIPTION
[0030] It is a discovery of the present inventors that
mitochondrial DNA mutations can be monitored non-invasively and
sensitively and used as an indicator of environmental pollutants.
It is shown below that these mutations are more prevalent in body
samples than nuclear mutations, and thus are detected more
sensitively. Mitochondrial mutations can be monitored over time to
detect changes in the amount of exposure to pollutants. In
addition, the prevalence of the mitochondrial mutation in the
sample indicates whether clonal proliferation has occurred.
Finally, the D-loop has been identified as a hotspot of mutations
within the mitochondrial genome.
[0031] Mitochondrial mutations are determined with reference to
wild-type human mitochondrial sequence. Sequence information can be
found at the website http://www.gen.emory.edu/mitomap.html and at
SEQ ID NO: 1. However, some differences between a sample sequence
and a documented wild-type sequence can be polymorphisms, not
mutations. Table 3 provides a number of new polymorphisms. Other
polymorphisms can be found in references 2 and 8. Polymorphisms can
be distinguished from somatic mutations by comparing the sequence
in the sample to the corresponding sequence in a normal body tissue
of the same person. If the same variant sequence is found in the
sample as in the normal body tissue it is a polymorphism. Normal
tissues can be paraffin-embedded. It has been found by the present
inventors that mitochondrial DNA which is paraffin-embedded remains
more highly intact and amplifiable than genomic DNA. Amplifiable
regions of mitochondrial DNA may be from 10 bp to about 4 kb,
desirably 2 kb to 4 kb or 10 bp to about 2 kb. Other suitable
sources of reference mtDNA are blood, serum, or plasma of the human
being tested.
[0032] Suitable bodily fluids for testing according to the present
invention include saliva, sputum, urine, and bronchoalveolar lavage
(BAL). These can be collected as is known in the art. People who
are prime candidates for testing and supplying such bodily fluids
are those who have been episodically, periodically or chronically
exposed to environmental pollutants. These include without
limitation cigarette smoke, biological toxins, such as aflatoxin,
cholera toxin, and botulinum toxin, radiation including UV
irradiation, industrial wastes, chemicals, water-borne or air-borne
pollutants, and drugs. The environmental pollutant can be known,
suspected, or unidentified, as the assay depends on the effect and
not on the identity of the pollutant.
[0033] The inventors have found that there are certain
characteristics of the mutations which are found in mitochondrial
DNA. Many mutations are found in sequences which do not encode
proteins. These include the D-loop region (i.e., nucleotides
16024-526), the 16S RNA gene, and the tRNA genes.
[0034] Furthermore, even where the mutations do occur in protein
coding regions, they often result in silent mutations which do not
affect the encoded amino acids. Other regions frequently affected
include the genes for NADH dehydrogenase 4, NADH dehydrogenase 3,
NADH dehydrogenase 5, and cytochrome B.
[0035] Mutation detection can be done according to any methodology
which is known in the art for determining mutations. These include
without limitation, nucleotide sequencing, hybridization,
amplification, PCR, oligonucleotide mismatch ligation assays,
primer extension assays, heteroduplex analysis, allele-specific
amplification, allele-specific primer extension, SCCP, DGGE, mass
spectroscopy, high pressure liquid chromatography, and combinations
of these techniques.
[0036] Prevalence of a particular mutation according to the present
invention can be used to monitor clonal expansion. Mutations which
are present in greater than 1% of the mitochondrial DNA present in
a sample have may have conferred a growth advantage on the cells
harboring them. Even if no growth advantage is conferred by the
mutation itself, the mutation serves as a marker for a clone which
is expanding relative to the population of cells in the sample.
Clonal expansion can be measured over time to monitor the growth of
the clone or to monitor the efficacy of anti-proliferative agents
which can be considered environmental pollutants, according to the
present invention.
[0037] The inventors have also found that the presence of subtle
mutations in the mitochondrial genome can be used as a means to
trace the presence, spread, metastasis, growth, or recurrence of a
tumor in a patient. Such subtle mutations include single basepair
substitutions, single basepair insertions, and single basepair
deletions. Single basepair substitutions can be either transitions
or transversions, although the former are more frequent. Detection
of such mutations can be useful to screen for the initial
appearance of a tumor as well as the recurrence of a previously
identified tumor. The methods are particularly suited to monitor
anti-cancer therapy, recurrence, metastasis, and completeness of
surgical removals.
[0038] A single basepair substitution is the substitution of a
single nucleotide base with a different nucleotide base at the same
position, with the corresponding substitution of the complementary
base on the other strand of the DNA. While any single basepair
substitution is conceivable within the scope of the invention, the
most frequently encountered substitutions are those which are
consistent with endogenous oxidative damage, such as T to C or G to
A transitions, or which are consistent with a variety of external
carcinogens which cause a variety of types of mutations. The
mutations can appear in protein coding or non-coding regions or in
regions which encode ribosomal or transfer RNAs.
[0039] The homoplasmic or near homoplasmic property of most mutant
mitochondrial genomes from tumors permits the ready detection of
such mutations within a sample of mitochondrial DNA from a patient.
Homoplasmic mutations are those which appear in essentially all of
the copies of the mitochondrial genome within a given cell or
tissue. However, heteroplasmic mutations, which are those appearing
in only a fraction of the mitochondrial genomes of a cell or
tissue, are also suitable for use with the invention.
[0040] Any cell sample can be tested from a patient who has cancer
or is suspected of having cancer. Suitable cell samples include,
but are not limited to, tissue from a growth suspected or known to
be cancerous, tissue adjacent to a resection of a tumor, and tissue
distant from the site of a tumor, such as lymph nodes which are
suspected of bearing metastatic cells. Cells can also be obtained
from bodily fluids or secretions, e.g., blood, urine, sputum,
saliva, or feces, which may contain cancerous cells or metastatic
cells. Cell samples can also be collected from other bodily
secretions and tissues as is known in the art. A cell sample can be
collected from suspected or known cancerous tissue or from bodily
fluids or secretions harboring cancer cells as well as from
suspected or known normal tissue or bodily fluids or secretions
harboring normal cells.
[0041] In order to detect mutations of the mitochondrial genome
from a cell sample of a patient, mitochondrial DNA can be isolated
from the cell sample using any method known in the art. One way of
identifying subtle mutations involves sequencing the mitochondrial
DNA. This can be done according to any method known in the art. For
example, isolated mitochondrial DNA can be cleaved using
endonucleases into overlapping fragments of appropriate size for
sequencing, e.g., about 1-3 kilobases in length, followed by
polymerase chain reaction (PCR) amplification and sequencing of the
fragments. Examples of DNA sequencing methods are found in Brumley,
R. L. Jr., and Smith, L. M., 1991, Rapid DNA sequencing by
horizontal ultrathin gel electrophoresis, Nucleic Acids Res.
19:4121-4126 and Luckey, J. A., Drossman, H., Kostihka, T.; and
Smith, L. M., 1993, High-speed DNA sequencing by capillary gel
electrophoresis, Methods Enzymol. 218:154-172. Amplification
methods such as PCR can be applied to samples as small as a single
cell and still yield sufficient DNA for complete sequence analysis.
The combined use of PCR and sequencing of mitochondrial DNA is
described in Hopgood, R., Sullivan, K. M., and Gill, P., 1992,
Strategies for automated sequencing of human mitochondrial DNA
directly from PCR products, Biotechniques 13:82-92 and Tanaka, M.,
Hayakawa, M., and Ozawa, T., 1996, Automated sequencing of
mitochondrial DNA, Methods Enzymol. 264:407-21.
[0042] Mutations can first be identified by comparison to sequences
present in public databases for human mitochondrial DNA, e.g., at
http://www.gen.emory.edu/mitomap.html and at SEQ ID NO: 1. Any
single basepair substitution identified in the sample DNA compared
to a normal sequence from a database can be confirmed as being a
somatic mutation as opposed to a polymorphic variant by comparing
the sample mitochondrial DNA or sequences obtained from it to
control cell mitochondrial DNA from the same individual or
sequences obtained from it. Control cells are isolated from other
apparently normal tissues, i.e., tissues which are phenotypically
normal and devoid of any visible, histological, or immunological
characteristics of cancer tissue. A difference between the sample
and the control identifies a somatic mutation which is associated
with the tumor.
[0043] An alternative to serially sequencing the entire
mitochondrial genome in order to identify a single basepair
substitution is to use hybridization of the mitochondrial DNA to an
array of oligonucleotides. Hybridization techniques are available
in the art which can rapidly identify mutations by comparing the
hybridization of the sample to matched and mismatched sequences
which are based on the human mitochondrial genome. Such an array
can be as simple as two oligonucleotide probes, one of whose
sequence matches the wild-type or mutant region containing the
single base substitution (matched probe) and another whose sequence
includes a single mismatched base (mismatch control probe). If the
sample DNA hybridizes to the matched probe but not the mismatched
probe, it is identified as having the same sequence as the matched
probe. Larger arrays containing thousands of such
matched/mismatched pairs of probes on a glass slide or microchip
("microarrays" or "gene chips") are available which are capable of
sequencing the entire mitochondrial genome very quickly. Such
arrays are commercially available. Review articles describing the
use of microarrays in genome and DNA sequence analysis and links to
their commercial suppliers are available at www.gene-chips.com.
[0044] The invention can be used to screen patients suspected of
having cancer for the presence of tumor cells. A cell sample is
first obtained from a suspected tumor of the patient, or is
obtained from another source such as blood or lymph tissue, for
example, if metastasis is suspected. The cell sample is tested to
determine the presence of a single basepair mutation in
mitochondrial DNA from the cell sample using the techniques
outlined above. Optionally, a cell sample from normal,
non-cancerous cells or tissue of the patient is also obtained and
is tested for the presence or absence of a single basepair mutation
in mitochondrial DNA. If a single basepair mutation is determined
which is not present in a cell sample from normal tissue of the
patient, then the mutation is a somatic mutation and the presence
of tumor cells in the patient is indicated. If one or more single
basepair mutations are determined in the mitochondrial genome of
the cell sample of the patient, then the patient is identified as
having a tumor. As in any diagnostic technique for cancer, to
confirm or extend the diagnosis, further diagnostic techniques may
be warranted. For example, conventional histological examination of
a biopsy specimen can be performed to detect the presence of tumor
cells, or analysis of a tumor-specific antigen in a blood or tissue
sample can be performed.
[0045] The method outlined above can be practiced either in the
situation where the somatic mutation is previously known or
previously unknown. The method can be practiced even in the absence
of prior knowledge about any particular somatic mutation. The
method can also be carried out subsequent to the discovery of a
somatic mutation in a mitochondrial genome of a cell of the patient
or of another patient. In this case, a previous association of the
somatic mutation with the presence of a tumor in the patient or in
another patient strongly indicates the presence of tumor cells in
the patient. It may also indicate the recurrence of a tumor or the
incomplete prior removal of cancerous tissue from the patient.
[0046] The effectiveness of therapy can be evaluated when a tumor
has already been identified and found to contain a single basepair
substitution in the mitochondrial genome. Once a single basepair
mutation has been identified in the mitochondrial DNA of a tumor of
the patient, further tumor cells can be detected in tissue
surrounding a resection or at other sites, if metastasis has
occurred. Using the methods outlined above, the recurrence of the
tumor or its incomplete removal can be assessed. Similarly, if a
tumor has been treated using a non-surgical method such as
chemotherapy or radiation, then the success of the therapy can be
evaluated at later times by repeating the analysis. The step for
determining the presence of a single basepair mutation in a
mitochondrial genome of a cell sample of a patient can be performed
1, 2, 3, 4, 5, 6, 8, 10, or more times in order to monitor the
development or regression of a tumor or to monitor the progress or
lack of progress of therapy undertaken to eliminate the tumor.
[0047] Upon repeated analyses, the step for determining the
presence of a single basepair mutation is simplified, because only
a well defined and limited region of the genome need be sequenced.
Using the hybridization method, for example, it is possible to
evaluate the presence of the mutation with only a single
matched/mismatched pair of oligonucleotide probes in the array. In
the event that a mixture of genotypes is observed, it is possible
to obtain quantitative information about the relative amount of
each mitochondrial genotype using techniques known to the art,
e.g., hybridization. Quantitative analysis can reveal changes in
the relative proportion of tumor to normal cells in a tissue over
time or in response to therapy.
[0048] The following examples are provided to demonstrate certain
aspects of the invention but they do not define the scope of the
invention.
EXAMPLE 1
[0049] This example demonstrates detection of mt mutations in
tissue samples.
[0050] To determine whether mt mutations could be identified in
cancer other than colorectal cancer, we studied primary bladder
(n=14), head and neck (n=13), and lung (n=14) tumors (5). Eighty
percent of the mt genome of all the primary tumor samples was
PCR-amplified (6) and sequenced manually (FIG. 1). Tumor mtDNA was
compared to mtDNA from paired blood samples in all cases, and mtDNA
from corresponding normal tissue when available (7). Of the 292
sequence variants detected, 196 were previously recorded
polymorphisms (2, 8), while 57 were novel polymorphisms (Table 3).
The remaining 39 variants were acquired (somatic) mutations
identified in 64% (9/14) of the bladder cancer, 46% (6/13) of the
head and neck cancer, and 43% (6/14) of the lung cancer patients
(Table 2). Most of these mutations were T-to-C and G-to-A base
transitions, indicating possible exposure to ROS-derived mutagens
(9). Similar to the previous observation by Polyak et al. (Table 1;
4), the majority of the somatic mutations identified here were also
homoplasmic in nature. In addition, several of the bladder and head
and neck cancers studied here (Table 2) had multiple mutations
implying possible accumulation of mtDNA damage.
[0051] In the bladder tumors, mutation hot spots were primarily in
the NADH dehydrogenase subunit 4 (ND4) gene (35%), and in the
displacement-loop (D-loop) region (30%). The D-loop region is a
critical site for both replication and expression of the mt genome
since it contains the leading-strand origin of replication and the
major promoters for transcription (10). Many (73%) of the mutations
identified within protein-coding regions were silent, except for a
(Val.fwdarw.Ala) substitution in the NADH dehydrogenase subunit 3
(ND3) and a 7-amino-acid deletion in cytochrome b (Cyt b). The
D-loop region was also commonly mutated in head and neck cancer
(67%). Two of the head and neck tumors (22%) contained mutations in
the ND4 gene at nucleotide pairs (nps) 10822 and 11150, resulting
in amino acid substitutions of Thr.fwdarw.Met and Ala.fwdarw.Thr,
respectively. A similar tendency was observed in lung cancers,
demonstrating a high concentration of mutations in the D-loop
region (70%).
EXAMPLE 2
[0052] This example demonstrates detection of mt mutations in
bodily fluids.
[0053] We hypothesized that the homoplasmic nature of these
mutations would make them readily detectable in paired bodily
fluids. To test this, we extracted and directly amplified mtDNA
from urine samples from patients diagnosed with bladder cancer. All
three corresponding urine samples available in this study contained
the mutant mtDNA derived from tumor tissues. For example, the mtDNA
from a urine sample of bladder cancer patient #799 showed the same
nucleotide transition (G.fwdarw.A) as seen in the tumor (FIG. 2A).
In all cases, the urine sample contained a relatively pure
population of tumor-derived mtDNA, comparable to that of the
micro-dissected tumor sample. Consistent with this observation,
saliva samples obtained from head and neck cancer patients
contained no detectable wild-type (WT) signals (FIG. 2B, and 2C).
By sequence analysis alone, we were able to detect mtDNA mutations
in 67% (6/9) of saliva samples from head and neck cancer patients.
In lung cancer cases, we were initially unable to identify mutant
bands from paired bronchoalveolar lavage (BAL) fluids because of
the significant dilution of neoplastic cells in BAL fluid (11),
(FIG. 2D). We, thus, applied a more sensitive
oligonucleotide-mismatch ligation assay to detect mutated mtDNA. As
shown in FIG. 3A and 3B, both lung cancer mutations (arrows) were
confirmed in tumor mtDNA with more dilute signals in the
corresponding BAL samples, and no signal in the corresponding
normal tissues. Again, we detected the majority of mtDNA mutations
(8/10) in BAL fluids with the exception of two cases where the
ligation assays were not feasible due to the sequence compositions
(16183 and 302 nps) adjacent to the mutations.
EXAMPLE 3
[0054] This example demonstrates the enrichment of mitochondrial
mutant DNA in samples relative to nuclear mutant DNA.
[0055] To quantitate this neoplastic DNA enrichment, we compared
the abundance of mt gene mutations to that of nuclear-encoded p53
mutations in bodily fluids using a quantitative plaque assay.
Nuclear and mt fragments that contained a mutated sequence were
PCR-amplified and cloned for plaque hybridization (12). Two BAL
samples from lung cancer patients were chosen for analysis because
they had mutations in both the mt and nuclear genomes. For p53
mutations, the percentages of neoplastic cells among normal cells
for patients #1113 and #1140 were 0.1 and 3.0%, respectively.
Remarkably, the abundance of the corresponding mutated mtDNA (MT)
was 22% and 52% when compared to the wild-type (WT) mt sequence
(FIG. 4). This enrichment of mtDNA is presumably due to the
homoplasmic nature of these mutations and the high copy number of
mt genomes in cancer cells. Enrichment was further suggested by our
observations in head and neck paraffin samples where we were able
to PCR-amplify 2-3 kb fragments of mtDNA, whereas we were unable to
amplify nuclear p53 gene fragments of over 300 bp.
[0056] A role for mitochondria in tumorigenesis was implicated when
tumor cells were found to have an impaired respiratory system and
high glycolytic activity (13,14). Recent findings elucidating the
role of mitochondria in apoptosis (15) and the high incidence of
mtDNA mutations in colon cancer (4) further support the original
hypothesis of mitochondrial participation in the initiation and
progression of cancer. Although further investigation is needed to
define the functional significance of mt mutations, our data
clearly show that those mutations are frequent and present at high
levels in all of the tumor types examined.
[0057] The homoplasmic nature of the mutated mitochondria remains
puzzling. It is estimated that each cell contains several
hundred-to-thousands of mitochondria and that each mitochondrion
contains 1-10 genomes (16). Conceivably, certain mutated mtDNAs may
gain a significant replicative advantage. For example, mutations in
the D-loop regulatory region might alter the rate of DNA
replication by modifying the binding affinity of important
trans-acting factors. Mitochondria that undergo the most rapid
replication are likely to acquire more DNA damage, leading to an
accumulation of mutational events. Although the mechanism may vary
for other mutations (such as silent mutations in the ND4 gene), the
accumulation of a particular mtDNA mutation may become more
apparent during neoplastic transformation. Even subtle mtDNA
mutations may also gain significant replicative advantage, perhaps
through interactions with important nuclear factors. Homoplasmic
transformation of mtDNA was observed in small populations of cells
in other non-neoplastic, but diseased tissues (17), sometimes
associated with aging (18). We hypothesize that, in contrast to
classic clonal expansion, the process may occur as "pseudoclonal"
selection where stochastic segregation of mitochondria (16)
together with neoplastic clonal expansion driven by nuclear
mutations lead to a homogeneous population of a previously
"altered" mitochondrion (FIG. 5).
[0058] The large number of mt polymorphisms identified here and
elsewhere (2) likely reflects the high mutation rate of mtDNA,
which is thought to be caused mainly by high levels of ROS (19). In
agreement with this, our data imply that constitutive hypervariable
areas such as the D-loop region represent somatic mutational hot
spots. As further mutations are tabulated in primary V tumors,
DNA-chip technology can be harnessed to develop high-throughput
analyses with sufficient sensitivity (20, 21). Due to its high copy
number, mtDNA may provide a distinct advantage over other nuclear
genome based methods for cancer and environmental pollutant
detection.
Literature Cited
[0059] 1. R. N. Lightowlers, P. F. Chinnery, D. M. Turnbull, N.
Howell, Trends Genet 13, 450 (1997).
[0060] 2. MITOMAP: A Human Mitochondrial Genome Database. Center
for Molecular Medicine, Emory University, Atlanta, Ga., USA.
http://www.gen.emory.edu/mitomap.html
[0061] 3. D. L. Croteau and V. A. Bohr, J. Biol. Chem. 272, 25409
(1997).
[0062] 4. K. Polyak et al., Nature Genet. 20, 291 (1998).
[0063] 5. Paired normal and tumor specimens along with blood and
bodily fluids were collected following surgical resections with
prior consent from patients in The Johns Hopkins University
Hospital. Tumor specimens were frozen and micro-dissected on a
cryostat so that the tumor samples contained greater than 70%
neoplastic cells. DNA from tumor sections was digested with 1%
SDS/Proteinase K, extracted by phenol-chloroform, and ethanol
precipitated. Control DNA from peripheral lymphocytes, matched
normal tissues, from urine, saliva, and BAL fluid were processed in
the same manner as described in (11).
[0064] 6. Mitochondrial DNAs were amplified using overlapping
primers (4) in PCR buffer containing 6% DMSO. Approximately 5-20 ng
of genomic DNA was subjected to the step-down PCR protocol:
94.degree. C. 30 sec, 64.degree. C. 1 min, 70.degree. C. 3 min, 3
cycles, 94.degree. C. 30 sec, 61.degree. C. 1 min, 70.degree. C. 3
min, 3 cycles, 94.degree. C. 30 sec, 58.degree. C. 1 min,
70.degree. C. 3.5 min 15 cycles, 94.degree. C. 30 sec, 1 min,
70.degree. C. 3.5 min, 15 cycles, and a final extension at
70.degree. C. for 5 min. PCR products were gel-purified using a
Qiagen gel extraction kit (Qiagen) and sequence reactions were
performed with Thermosequenase (Perkin-Elmer) using the cycle
conditions (95.degree. C. 30 sec, 52.degree. C. 1 min, and
70.degree. C. 1 min for 25 cycles).
[0065] 7. Corresponding normal tissues from 4 patients (#874, #915,
#1684, and #1678) were available and DNA was extracted from
paraffin samples as described previously (9).
[0066] 8. R. M. Andrew et al., Nature Genet. 23 147, (1999)
[0067] 9. J. Cadet, M. Berger, T. Douki, J. L. Ravanat, Rev.
Physiol. Biochem. Pharmacol. 131, 1 (1997).
[0068] 8. J. W. Taanman, Biochimica. et. Biophysica. Acta. 1410,
103 (1999).
[0069] 9. S. A. Ahrendt et al., J. Natl. Cancer Inst. 91, 332
(1999).
[0070] 10. Subcloning of PCR fragments into phage vector was
performed according to the manufacturer's instructions
(Stratagene). Titered plaques were plated and subjected to
hybridization using tetramethylammonium chloride (TMAC) as a
solvent. Positive signals were confirmed by secondary screenings.
Oligonucleotides (Oligos) used for this assay were as follows; for
patient #1113, p53 and mtDNA sequence alterations were detected
using oligos containing either WT-(p53:
5'-GTATTTGGATGTCAGAAACACTT-3' (SEQ ID NO: 2)/mtDNA:
5'-ACTTCAGGGTCATAAAGCC-3' (SEQ ID NO: 3)) or MT (p53:
5'-GTATTTGGATGTCAGAAACACTT-3' (SEQ ID NO:
4)/mtDNA:5'-ACTTCAGGGCCATAAAGCC- -3'(SEQ ID NO: 5)) sequences,
respectively. For patient #1140, oligos 5'-ACCCGCGTCCGCGCCATGGCC-3'
(SEQ ID NO: 6) and 5'-ACCCGCGTCCTCGCCATGGCC-3- ' (SEQ ID NO: 7)
were used to detect WT and MT sequences, respectively.
[0071] 11. O. Warburg, Science 123, 309 (1956).
[0072] 12. J. W. Shay and H. Werbin, Mut. Res. 186, 149 (1987).
[0073] 13. D. R. Green and J. C. Reed, Science 281, 1309
(1999).
[0074] 14. D. C. Wallace, Annu. Rev. Biochem. 61, 1175 (1992).
[0075] 15. D. C. Wallace, Proc. Natl. Acad. Sci. USA 91, 8746
(1994).
[0076] 16. K. Khrapko et al., N.A.R. 27, 2434 (1999).
[0077] 17. C. Richter, J. W. Park, B. N. Ames, Proc. Natl. Acad.
Sci. U S A 17, 6465 (1988).
[0078] 18. M. Chee et al., Science 274, 610 (1996).
[0079] 19. S. A. Ahrendt et al., Proc. Natl. Acad. Sci. USA 96,
7382 (1999).
[0080] 20. Fragments containing mutations were PCR-amplified and
then ethanol precipitated. For each mutation, discriminating
oligonucleotides that contained the mutated base at the 3' end were
designed (TAACCATA-3' (SEQ ID NO: 8) for patient #915 and
TCTCTTACC-3' (SEQ ID NO: 9) for patient #898). Immediately adjacent
[.sup.32P] end-labeled 3' sequences (5'-CACACTACTA-3' (SEQ ID NO:
10) for patient #915 and 5'-TTTAACCAG-3' (SEQ ID NO: 11) for
patient #898) were used as substrate together with discriminating
oligonucleotides for the ligation reaction. After a denaturing step
of 95.degree. C. for 5', the reactions were incubated for 1 hr at
37.degree. in the presence of T4 DNA ligase (Life Technologies), in
a buffer containing 50 mM Tris-Cl, 10 mM MgCl.sub.2, 150 mM NaCl, 1
mM Spermidine, 1 mM ATP, 5 mM DTT, and analyzed on denatured 12%
polyacrylamide gels. [Jen et al., Cancer Res. 54, 5523 (1994)].
1TABLE 1 Summary of mtDNA mutations Tumor* Position DNA Protein
Gene C478 710 T .fwdarw. C -- 12S rRNA " 1738 T .fwdarw. C -- 16S
rRNA " 3308 T .fwdarw. C M1T ND1 V429 8009 G .fwdarw. A V142M COX
subunit II " 14985 G .fwdarw. A R80H CYT b " 15572 T .fwdarw. C
F276L CYT b V441 9949 G .fwdarw. A V2481 COX subunit III V456 10563
T .fwdarw. C C32R ND4L V425 6264 G .fwdarw. A G121trun COX subunit
I " 12418 insA K28frameshift ND5 V451 1967 T .varies.3 C -- 16S
rRNA V410 2299 T .fwdarw. A -- 16S rRNA *All the mutations were
homoplasmic except V451 T11967C and V410 T2299A, which were present
in .about.50% of the mitochondrial DNA molecules.
[0081]
2TABLE 2 Summary of mitochondrial mutations in primary tumors.
Patient# Location Sequence Protein Gene Bladder Cancer (9/14, 57%)
1124 114 T .fwdarw. C N/C D-loop 580 302 Del C N/C D-loop 580 386 C
.varies.3 A N/C D-loop 799 2058 G .fwdarw. A N/C 16SrRNA 716 2445 T
.varies.3 C N/C 16SrRNA 1127 3054 G .fwdarw. A N/C 16SrRNA 884
10071 T .fwdarw. C L-L ND3 884 10321 T .fwdarw. C V-A ND3 884 10792
A .fwdarw. G L-L ND4 884 10793 C .fwdarw. T L-L ND4 899 10822 C
.fwdarw. T H-H ND4 716 10978 A .fwdarw. G L-L ND4 870 11065 A
.fwdarw. G L-L ND4 870 11518 G .fwdarw. A L-L ND4 884 12049 C
.fwdarw. T F-F ND4 874 12519 T .fwdarw. C V-V ND5 580 15642 Del 7aa
Cyt b 899 16189 Ins T N/A D-loop 1124 16265 A .fwdarw. C N/A D-loop
1127 16532 A .fwdarw. T N/A D-loop Lung Cancer (6/15, 40%) 1174 150
C .fwdarw. T N/C D-loop 1174 195 T .fwdarw. C N/C D-loop 902 302
Del C N/C D-loop 898 2664 T .fwdarw. C N/C 16sRNA 915 5521 G
.fwdarw. A N/C tRNATrp 915 12345 G .fwdarw. A N/C tRNALeu 915 16183
C .fwdarw. A N/C D-loop 915 16187 C .varies.3 T N/C D-loop 1113
16519 T .fwdarw. C N/C D-loop 1140 16380 G .fwdarw. A N/C D-loop
Head and Neck Cancer (6/13, 46%) 1637 75 G .fwdarw. A N/C D-loop
1680 302 Ins C N/C D-loop 1565 514 Ins CG N/C D-loop 1708 10966 T
.fwdarw. C T .varies.3 T ND 4 1678 11150 G .varies.3 A A .varies.3
T ND 4 1680 16172 T .varies.3 C N/C D-loop 1680 16292 C .fwdarw. T
N/C D-loop 1680 16300 A .varies.3 G N/C D-loop Only D-loop region
was analyzed for lung patients #1113, #1140, and #1174
[0082]
3TABLE 3 New mtDNA polymorphisms (n = 57) found in this study.
Sequence change Tumor Position Gene DNA Protein B 633 tRNA Phe A
.fwdarw. G -- B 723 12S rRNA A .fwdarw. G -- B, L, HNC 1738 16S
rRNA T .fwdarw. C -- B 1872 16S rRNA T .fwdarw. C -- L 2308 16S
rRNA A .fwdarw. G -- B, L 2395 16S rRNA Del A -- HNC 2712 16S rRNA
G .fwdarw. A -- HNC 2758 16S rRNA G .fwdarw. A -- L 2765 16S rRNA A
.fwdarw. G -- HNC 2768 16S rRNA A .fwdarw. C -- HNC 3148 16S rRNA C
.fwdarw. T -- B, L, HNC 3308 ND1 T .fwdarw. C M .fwdarw. T B 4823
ND2 T .fwdarw. C V .fwdarw. V B 4917 ND2 A .fwdarw. G N .fwdarw. D
B 5509 ND2 T .fwdarw. C L .fwdarw. S B 5567 tRNA Trp T .fwdarw. C
-- B 5580 NCN T .fwdarw. C -- B 5899 NCN Del C -- B 6149 CoxI A
.fwdarw. G L .fwdarw. L B 6150 CoxI G .fwdarw. A V .fwdarw. I B
6253 CoxI T .fwdarw. C M .fwdarw. T B 6261 CoxI G .fwdarw. A A
.fwdarw. T B 6302 CoxI A .fwdarw. G A .fwdarw. A B 7966 CoxII C
.fwdarw. T F .fwdarw. F B 8037 CoxII G .fwdarw. A R .fwdarw. H B
8248 CoxII A .fwdarw. G M .fwdarw. M B 8655 ATPase6 C .fwdarw. T I
.fwdarw. I B 8877 ATPase6 T .fwdarw. C F .fwdarw. F B 9072 ATPase6
A .fwdarw. G S .fwdarw. S B 9093 ATPase6 A .fwdarw. G T .fwdarw. T
B 9266 CoxIII G .fwdarw. A G .fwdarw. G B 9497 CoxIII T .fwdarw. C
F .fwdarw. F L 10321 ND3 T .fwdarw. C V .fwdarw. A HNC 10403 ND3 A
.fwdarw. G E .fwdarw. E B, L, HNC 10688 ND 4L G .fwdarw. A V
.fwdarw. V B, L, HNC 10810 ND 4 T .fwdarw. C L .fwdarw. L B 11164
ND 4 A .fwdarw. G R .fwdarw. R L 11257 ND 4 C .fwdarw. T Y .fwdarw.
Y HNC 11339 ND 4 T .fwdarw. C L .fwdarw. L L 11899 ND 4 T .fwdarw.
C S .fwdarw. S L 12519 ND 5 T .fwdarw. C V .fwdarw. V B, L 14769
Cyt b A .fwdarw. G N .fwdarw. S B 14992 Cyt b T .fwdarw. C L
.fwdarw. L L 15139 Cyt b T .fwdarw. C Y .fwdarw. Y L 15514 Cyt b T
.fwdarw. C Y .fwdarw. Y L 15586 Cyt b T .fwdarw. C I .fwdarw. I B
15601 Cyt b T .fwdarw. C P .fwdarw. P L 15670 Cyt b T .fwdarw. C H
.fwdarw. H B 15672 Cyt b T .fwdarw. C M .fwdarw. T B 15787 Cyt b T
.fwdarw. C F .fwdarw. F HNC 15941 tRNA Thr T .fwdarw. C -- L 15942
tRNA Thr T .fwdarw. C -- B 16130 D-Loop G .fwdarw. A -- L 16170
D-Loop A .fwdarw. G -- L 16204 D-Loop G .fwdarw. C -- L 16211
D-Loop C .fwdarw. T -- B 16225 D-Loop C .fwdarw. T -- B = Bladder
cancer; L = Lung cancer; HNC = Head and neck cancer; NCN =
non-coding nucleotide.
[0083]
Sequence CWU 1
1
11 1 16568 DNA Homo sapiens 1 gatcacaggt ctatcaccct attaaccact
cacgggagct ctccatgcat ttggtatttt 60 cgtctggggg gtatgcacgc
gatagcattg cgagacgctg gagccggagc accctatgtc 120 gcagtatctg
tctttgattc ctgcctcatc ctattattta tcgcacctac gttcaatatt 180
acaggcgaac atacttacta aagtgtgtta attaattaat gcttgtagga cataataata
240 acaattgaat gtctgcacag ccactttcca cacagacatc ataacaaaaa
atttccacca 300 aaccccccct cccccgcttc tggccacagc acttaaacac
atctctgcca aaccccaaaa 360 acaaagaacc ctaacaccag cctaaccaga
tttcaaattt tatcttttgg cggtatgcac 420 ttttaacagt caccccccaa
ctaacacatt attttcccct cccactccca tactactaat 480 ctcatcaata
caacccccgc ccatcctacc cagcacacac acaccgctgc taaccccata 540
ccccgaacca accaaacccc aaagacaccc cccacagttt atgtagctta cctcctcaaa
600 gcaatacact gaaaatgttt agacgggctc acatcacccc ataaacaaat
aggtttggtc 660 ctagcctttc tattagctct tagtaagatt acacatgcaa
gcatccccgt tccagtgagt 720 tcaccctcta aatcaccacg atcaaaagga
acaagcatca agcacgcagc aatgcagctc 780 aaaacgctta gcctagccac
acccccacgg gaaacagcag tgattaacct ttagcaataa 840 acgaaagttt
aactaagcta tactaacccc agggttggtc aatttcgtgc cagccaccgc 900
ggtcacacga ttaacccaag tcaatagaag ccggcgtaaa gagtgtttta gatcaccccc
960 tccccaataa agctaaaact cacctgagtt gtaaaaaact ccagttgaca
caaaatagac 1020 tacgaaagtg gctttaacat atctgaacac acaatagcta
agacccaaac tgggattaga 1080 taccccacta tgcttagccc taaacctcaa
cagttaaatc aacaaaactg ctcgccagaa 1140 cactacgagc cacagcttaa
aactcaaagg acctggcggt gcttcatatc cctctagagg 1200 agcctgttct
gtaatcgata aaccccgatc aacctcacca cctcttgctc agcctatata 1260
ccgccatctt cagcaaaccc tgatgaaggc tacaaagtaa gcgcaagtac ccacgtaaag
1320 acgttaggtc aaggtgtagc ccatgaggtg gcaagaaatg ggctacattt
tctaccccag 1380 aaaactacga tagcccttat gaaacttaag ggtcgaaggt
ggatttagca gtaaactaag 1440 agtagagtgc ttagttgaac agggccctga
agcgcgtaca caccgcccgt caccctcctc 1500 aagtatactt caaaggacat
ttaactaaaa cccctacgca tttatataga ggagacaagt 1560 cgtaacatgg
taagtgtact ggaaagtgca cttggacgaa ccagagtgta gcttaacaca 1620
aagcacccaa cttacactta ggagatttca acttaacttg accgctctga gctaaaccta
1680 gccccaaacc cactccacct tactaccaga caaccttagc caaaccattt
acccaaataa 1740 agtataggcg atagaaattg aaacctggcg caatagatat
agtaccgcaa gggaaagatg 1800 aaaaattata accaagcata atatagcaag
gactaacccc tataccttct gcataatgaa 1860 ttaactagaa ataactttgc
aaggagagcc aaagctaaga cccccgaaac cagacgagct 1920 acctaagaac
agctaaaaga gcacacccgt ctatgtagca aaatagtggg aagatttata 1980
ggtagaggcg acaaacctac cgagcctggt gatagctggt tgtccaagat agaatcttag
2040 ttcaacttta aatttgccca cagaaccctc taaatcccct tgtaaattta
actgttagtc 2100 caaagaggaa cagctctttg gacactagga aaaaaccttg
tagagagagt aaaaaattta 2160 acacccatag taggcctaaa agcagccacc
aattaagaaa gcgttcaagc tcaacaccca 2220 ctacctaaaa aatcccaaac
atataactga actcctcaca cccaattgga ccaatctatc 2280 accctataga
agaactaatg ttagtataag taacatgaaa acattctcct ccgcataagc 2340
ctgcgtcaga ttaaaacact gaactgacaa ttaacagccc aatatctaca atcaaccaac
2400 aagtcattat taccctcact gtcaacccaa cacaggcatg ctcataagga
aaggttaaaa 2460 aaagtaaaag gaactcggca aatcttaccc cgcctgttta
ccaaaaacat cacctctagc 2520 atcaccagta ttagaggcac cgcctgccca
gtgacacatg tttaacggcc gcggtaccct 2580 aaccgtgcaa aggtagcata
atcacttgtt ccttaaatag ggacctgtat gaatggctcc 2640 acgagggttc
agctgtctct tacttttaac cagtgaaatt gacctgcccg tgaagaggcg 2700
ggcataacac agcaagacga gaagacccta tggagcttta atttattaat gcaaacagta
2760 cctaacaaac ccacaggtcc taaactacca aacctgcatt aaaaatttcg
gttggggcga 2820 cctcggagca gaacccaacc tccgagcagt acatgctaag
acttcaccag tcaaagcgaa 2880 ctactatact caattgatcc aataacttga
ccaacggaac aagttaccct agggataaca 2940 gcgcaatcct attctagagt
ccatatcaac aatagggttt acgacctcga tgttggatca 3000 ggacatcccg
atggtgcagc cgctattaaa ggttcgtttg ttcaacgatt aaagtcctac 3060
gtgatctgag ttcagaccgg agtaatccag gtcggtttct atctacttca aattcctccc
3120 tgtacgaaag gacaagagaa ataaggccta cttcacaaag cgccttcccc
cgtaaatgat 3180 atcatctcaa cttagtatta tacccacacc cacccaagaa
cagggtttgt taagatggca 3240 gagcccggta atcgcataaa acttaaaact
ttacagtcag aggttcaatt cctcttctta 3300 acaacatacc catggccaac
ctcctactcc tcattgtacc cattctaatc gcaatggcat 3360 tcctaatgct
taccgaacga aaaattctag gctatataca actacgcaaa ggccccaacg 3420
ttgtaggccc ctacgggcta ctacaaccct tcgctgacgc cataaaactc ttcaccaaag
3480 agcccctaaa acccgccaca tctaccatca ccctctacat caccgccccg
accttagctc 3540 tcaccatcgc tcttctacta tgaacccccc tccccatacc
caaccccctg gtcaacctca 3600 acctaggcct cctatttatt ctagccacct
ctagcctagc cgtttactca atcctctgat 3660 cagggtgagc atcaaactca
aactacgccc tgatcggcgc actgcgagca gtagcccaaa 3720 caatctcata
tgaagtcacc ctagccatca ttctactatc aacattacta ataagtggct 3780
cctttaacct ctccaccctt atcacaacac aagaacacct ctgattactc ctgccatcat
3840 gacccttggc cataatatga tttatctcca cactagcaga gaccaaccga
acccccttcg 3900 accttgccga aggggagtcc gaactagtct caggcttcaa
catcgaatac gccgcaggcc 3960 ccttcgccct attcttcata gccgaataca
caaacattat tataataaac accctcacca 4020 ctacaatctt cctaggaaca
acatatgacg cactctcccc tgaactctac acaacatatt 4080 ttgtcaccaa
gaccctactt ctaacctccc tgttcttatg aattcgaaca gcataccccc 4140
gattccgcta cgaccaactc atacacctcc tatgaaaaaa cttcctacca ctcaccctag
4200 cattacttat atgatatgtc tccataccca ttacaatctc cagcattccc
cctcaaacct 4260 aagaaatatg tctgataaaa gagttacttt gatagagtaa
ataataggag cttaaacccc 4320 cttatttcta ggactatgag aatcgaaccc
atccctgaga atccaaaatt ctccgtgcca 4380 cctatcacac cccatcctaa
agtaaggtca gctaaataag ctatcgggcc cataccccga 4440 aaatgttggt
tatacccttc ccgtactaat taatcccctg gcccaacccg tcatctactc 4500
taccatcttt gcaggcacac tcatcacagc gctaagctcg cactgatttt ttacctgagt
4560 aggcctagaa ataaacatgc tagcttttat tccagttcta accaaaaaaa
taaaccctcg 4620 ttccacagaa gctgccatca agtatttcct cacgcaagca
accgcatcca taatccttct 4680 aatagctatc ctcttcaaca atatactctc
cggacaatga accataacca atactaccaa 4740 tcaatactca tcattaataa
tcataatagc tatagcaata aaactaggaa tagccccctt 4800 tcacttctga
gtcccagagg ttacccaagg cacccctctg acatccggcc tgcttcttct 4860
cacatgacaa aaactagccc ccatctcaat catataccaa atctctccct cactaaacgt
4920 aagccttctc ctcactctct caatcttatc catcatagca ggcagttgag
gtggattaaa 4980 ccaaacccag ctacgcaaaa tcttagcata ctcctcaatt
acccacatag gatgaataat 5040 agcagttcta ccgtacaacc ctaacataac
cattcttaat ttaactattt atattatcct 5100 aactactacc gcattcctac
tactcaactt aaactccagc accacgaccc tactactatc 5160 tcgcacctga
aacaagctaa catgactaac acccttaatt ccatccaccc tcctctccct 5220
aggaggcctg cccccgctaa ccggcttttt gcccaaatgg gccattatcg aagaattcac
5280 aaaaaacaat agcctcatca tccccaccat catagccacc atcaccctcc
ttaacctcta 5340 cttctaccta cgcctaatct actccacctc aatcacacta
ctccccatat ctaacaacgt 5400 aaaaataaaa tgacagtttg aacatacaaa
acccacccca ttcctcccca cactcatcgc 5460 ccttaccacg ctactcctac
ctatctcccc ttttatacta ataatcttat agaaatttag 5520 gttaaataca
gaccaagagc cttcaaagcc ctcagtaagt tgcaatactt aatttctgta 5580
acagctaagg actgcaaaac cccactctgc atcaactgaa cgcaaatcag ccactttaat
5640 taagctaagc ccttactaga ccaatgggac ttaaacccac aaacacttag
ttaacagcta 5700 agcaccctaa tcaactggct tcaatctact tctcccgccg
ccgggaaaaa aggcgggaga 5760 agccccggca ggtttgaagc tgcttcttcg
aatttgcaat tcaatatgaa aatcacctcg 5820 gagctggtaa aaagaggcct
aacccctgtc tttagattta cagtccaatg cttcactcag 5880 ccattttacc
tcacccccac tgatgttcgc cgaccgttga ctattctcta caaaccacaa 5940
agacattgga acactatacc tattattcgg cgcatgagct ggagtcctag gcacagctct
6000 aagcctcctt attcgagccg agctgggcca gccaggcaac cttctaggta
acgaccacat 6060 ctacaacgtt atcgtcacag cccatgcatt tgtaataatc
ttcttcatag taatacccat 6120 cataatcgga ggctttggca actgactagt
tcccctaata atcggtgccc ccgatatggc 6180 gtttccccgc ataaacaaca
taagcttctg actcttacct ccctctctcc tactcctgct 6240 cgcatctgct
atagtggagg ccggagcagg aacaggttga acagtctacc ctcccttagc 6300
agggaactac tcccaccctg gagcctccgt agacctaacc atcttctcct tacacctagc
6360 aggtgtctcc tctatcttag gggccatcaa tttcatcaca acaattatca
atataaaacc 6420 ccctgccata acccaatacc aaacgcccct cttcgtctga
tccgtcctaa tcacagcagt 6480 cctacttctc ctatctctcc cagtcctagc
tgctggcatc actatactac taacagaccg 6540 caacctcaac accaccttct
tcgaccccgc cggaggagga gaccccattc tataccaaca 6600 cctattctga
tttttcggtc accctgaagt ttatattctt atcctaccag gcttcggaat 6660
aatctcccat attgtaactt actactccgg aaaaaaagaa ccatttggat acataggtat
6720 ggtctgagct atgatatcaa ttggcttcct agggtttatc gtgtgagcac
accatatatt 6780 tacagtagga atagacgtag acacacgagc atatttcacc
tccgctacca taatcatcgc 6840 tatccccacc ggcgtcaaag tatttagctg
actcgccaca ctccacggaa gcaatatgaa 6900 atgatctgct gcagtgctct
gagccctagg attcatcttt cttttcaccg taggtggcct 6960 gactggcatt
gtattagcaa actcatcact agacatcgta ctacacgaca cgtactacgt 7020
tgtagcccac ttccactatg tcctatcaat aggagctgta tttgccatca taggaggctt
7080 cattcactga tttcccctat tctcaggcta caccctagac caaacctacg
ccaaaatcca 7140 tttcactatc atattcatcg gcgtaaatct aactttcttc
ccacaacact ttctcggcct 7200 atccggaatg ccccgacgtt actcggacta
ccccgatgca tacaccacat gaaacatcct 7260 atcatctgta ggctcattca
tttctctaac agcagtaata ttaataattt tcatgatttg 7320 agaagccttc
gcttcgaagc gaaaagtcct aatagtagaa gaaccctcca taaacctgga 7380
gtgactatat ggatgccccc caccctacca cacattcgaa gaacccgtat acataaaatc
7440 tagacaaaaa aggaaggaat cgaacccccc aaagctggtt tcaagccaac
cccatggcct 7500 ccatgacttt ttcaaaaagg tattagaaaa accatttcat
aactttgtca aagttaaatt 7560 ataggctaaa tcctatatat cttaatggca
catgcagcgc aagtaggtct acaagacgct 7620 acttccccta tcatagaaga
gcttatcacc tttcatgatc acgccctcat aatcattttc 7680 cttatctgct
tcctagtcct gtatgccctt ttcctaacac tcacaacaaa actaactaat 7740
actaacatct cagacgctca ggaaatagaa accgtctgaa ctatcctgcc cgccatcatc
7800 ctagtcctca tcgccctccc atccctacgc atcctttaca taacagacga
ggtcaacgat 7860 ccctccctta ccatcaaatc aattggccac caatggtact
gaacctacga gtacaccgac 7920 tacggcggac taatcttcaa ctcctacata
cttcccccat tattcctaga accaggcgac 7980 ctgcgactcc ttgacgttga
caatcgagta gtactcccga ttgaagcccc cattcgtata 8040 ataattacat
cacaagacgt cttgcactca tgagctgtcc ccacattagg cttaaaaaca 8100
gatgcaattc ccggacgtct aaaccaaacc actttcaccg ctacacgacc gggggtatac
8160 tacggtcaat gctctgaaat ctgtggagca aaccacagtt tcatgcccat
cgtcctagaa 8220 ttaattcccc taaaaatctt tgaaataggg cccgtattta
ccctatagca ccccctctac 8280 cccctctaga gcccactgta aagctaactt
agcattaacc ttttaagtta aagattaaga 8340 gaaccaacac ctctttacag
tgaaatgccc caactaaata ctaccgtatg gcccaccata 8400 attaccccca
tactccttac actattcctc atcacccaac taaaaatatt aaacacaaac 8460
taccacctac ctccctcacc aaagcccata aaaataaaaa attataacaa accctgagaa
8520 ccaaaatgaa cgaaaatctg ttcgcttcat tcattgcccc cacaatccta
ggcctacccg 8580 ccgcagtact gatcattcta tttccccctc tattgatccc
cacctccaaa tatctcatca 8640 acaaccgact aatcaccacc caacaatgac
taatcaaact aacctcaaaa caaatgataa 8700 ccatacacaa cactaaagga
cgaacctgat ctcttatact agtatcctta atcattttta 8760 ttgccacaac
taacctcctc ggactcctgc ctcactcatt tacaccaacc acccaactat 8820
ctataaacct agccatggcc atccccttat gagcgggcac agtgattata ggctttcgct
8880 ctaagattaa aaatgcccta gcccacttct taccacaagg cacacctaca
ccccttatcc 8940 ccatactagt tattatcgaa accatcagcc tactcattca
accaatagcc ctggccgtac 9000 gcctaaccgc taacattact gcaggccacc
tactcatgca cctaattgga agcgccaccc 9060 tagcaatatc aaccattaac
cttccctcta cacttatcat cttcacaatt ctaattctac 9120 tgactatcct
agaaatcgct gtcgccttaa tccaagccta cgttttcaca cttctagtaa 9180
gcctctacct gcacgacaac acataatgac ccaccaatca catgcctatc atatagtaaa
9240 acccagccca tgacccctaa caggggccct ctcagccctc ctaatgacct
ccggcctagc 9300 catgtgattt cacttccact ccataacgct cctcatacta
ggcctactaa ccaacacact 9360 aaccatatac caatgatggc gcgatgtaac
acgagaaagc acataccaag gccaccacac 9420 accacctgtc caaaaaggcc
ttcgatacgg gataatccta tttattacct cagaagtttt 9480 tttcttcgca
ggatttttct gagcctttta ccactccagc ctagccccta ccccccaatt 9540
aggagggcac tggcccccaa caggcatcac cccgctaaat cccctagaag tcccactcct
9600 aaacacatcc gtattactcg catcaggagt atcaatcacc tgagctcacc
atagtctaat 9660 agaaaacaac cgaaaccaaa taattcaagc actgcttatt
acaattttac tgggtctcta 9720 ttttaccctc ctacaagcct cagagtactt
cgagtctccc ttcaccattt ccgacggcat 9780 ctacggctca acattttttg
tagccacagg cttccacgga cttcacgtca ttattggctc 9840 aactttcctc
actatctgct tcatccgcca actaatattt cactttacat ccaaacatca 9900
ctttggcttc gaagccgccg cctgatactg gcattttgta gatgtggttt gactatttct
9960 gtatgtctcc atctattgat gagggtctta ctcttttagt ataaatagta
ccgttaactt 10020 ccaattaact agttttgaca acattcaaaa aagagtaata
aacttcgcct taattttaat 10080 aatcaacacc ctcctagcct tactactaat
aattattaca ttttgactac cacaactcaa 10140 cggctacata gaaaaatcca
ccccttacga gtgcggcttc gaccctatat cccccgcccg 10200 cgtccctttc
tccataaaat tcttcttagt agctattacc ttcttattat ttgatctaga 10260
aattgccctc cttttacccc taccatgagc cctacaaaca actaacctgc cactaatagt
10320 tatgtcatcc ctcttattaa tcatcatcct agccctaagt ctggcctatg
agtgactaca 10380 aaaaggatta gactgaaccg aattggtata tagtttaaac
aaaacgaatg atttcgactc 10440 attaaattat gataatcata tttaccaaat
gcccctcatt tacataaata ttatactagc 10500 atttaccatc tcacttctag
gaatactagt atatcgctca cacctcatat cctccctact 10560 atgcctagaa
ggaataatac tatcgctgtt cattatagct actctcataa ccctcaacac 10620
ccactccctc ttagccaata ttgtgcctat tgccatacta gtctttgccg cctgcgaagc
10680 agcggtgggc ctagccctac tagtctcaat ctccaacaca tatggcctag
actacgtaca 10740 taacctaaac ctactccaat gctaaaacta atcgtcccaa
caattatatt actaccactg 10800 acatgacttt ccaaaaaaca cataatttga
atcaacacaa ccacccacag cctaattatt 10860 agcatcatcc ctctactatt
ttttaaccaa atcaacaaca acctatttag ctgttcccca 10920 accttttcct
ccgaccccct aacaaccccc ctcctaatac taactacctg actcctaccc 10980
ctcacaatca tggcaagcca acgccactta tccagtgaac cactatcacg aaaaaaactc
11040 tacctctcta tactaatctc cctacaaatc tccttaatta taacattcac
agccacagaa 11100 ctaatcatat tttatatctt cttcgaaacc acacttatcc
ccaccttggc tatcatcacc 11160 cgatgaggca accagccaga acgcctgaac
gcaggcacat acttcctatt ctacacccta 11220 gtaggctccc ttcccctact
catcgcacta atttacactc acaacaccct aggctcacta 11280 aacattctac
tactcactct cactgcccaa gaactatcaa actcctgagc caacaactta 11340
atatgactag cttacacaat agcttttata gtaaagatac ctctttacgg actccactta
11400 tgactcccta aagcccatgt cgaagccccc atcgctgggt caatagtact
tgccgcagta 11460 ctcttaaaac taggcggcta tggtataata cgcctcacac
tcattctcaa ccccctgaca 11520 aaacacatag cctacccctt ccttgtacta
tccctatgag gcataattat aacaagctcc 11580 atctgcctac gacaaacaga
cctaaaatcg ctcattgcat actcttcaat cagccacata 11640 gccctcgtag
taacagccat tctcatccaa accccctgaa gcttcaccgg cgcagtcatt 11700
ctcataatcg cccacgggct tacatcctca ttactattct gcctagcaaa ctcaaactac
11760 gaacgcactc acagtcgcat cataatcctc tctcaaggac ttcaaactct
actcccacta 11820 atagcttttt gatgacttct agcaagcctc gctaacctcg
ccttaccccc cactattaac 11880 ctactgggag aactctctgt gctagtaacc
acgttctcct gatcaaatat cactctccta 11940 cttacaggac tcaacatact
agtcacagcc ctatactccc tctacatatt taccacaaca 12000 caatggggct
cactcaccca ccacattaac aacataaaac cctcattcac acgagaaaac 12060
accctcatgt tcatacacct atcccccatt ctcctcctat ccctcaaccc cgacatcatt
12120 accgggtttt cctcttgtaa atatagttta accaaaacat cagattgtga
atctgacaac 12180 agaggcttac gaccccttat ttaccgagaa agctcacaag
aactgctaac tcatgccccc 12240 atgtctaaca acatggcttt ctcaactttt
aaaggataac agctatccat tggtcttagg 12300 ccccaaaaat tttggtgcaa
ctccaaataa aagtaataac catgcacact actataacca 12360 ccctaaccct
gacttcccta attcccccca tccttaccac cctcgttaac cctaacaaaa 12420
aaaactcata cccccattat gtaaaatcca ttgtcgcatc cacctttatt atcagtctct
12480 tccccacaac aatattcatg tgcctagacc aagaagttat tatctcgaac
tgacactgag 12540 ccacaaccca aacaacccag ctctccctaa gcttcaaact
agactacttc tccataatat 12600 tcatccctgt agcattgttc gttacatggt
ccatcataga attctcactg tgatatataa 12660 actcagaccc aaacattaat
cagttcttca aatatctact catcttccta attaccatac 12720 taatcttagt
taccgctaac aacctattcc aactgttcat cggctgagag ggcgtaggaa 12780
ttatatcctt cttgctcatc agttgatgat acgcccgagc agatgccaac acagcagcca
12840 ttcaagcaat cctatacaac cgtatcggcg atatcggttt catcctcgcc
ttagcatgat 12900 ttatcctaca ctccaactca tgagacccac aacaaatagc
ccttctaaac gctaatccaa 12960 gcctcacccc actactaggc ctcctcctag
cagcagcagg caaatcagcc caattaggtc 13020 tccacccctg actcccctca
gccatagaag gccccacccc agtctcagcc ctactccact 13080 caagcactat
agttgtagca ggaatcttct tactcatccg cttccacccc ctagcagaaa 13140
atagcccact aatccaaact ctaacactat gcttaggcgc tatcaccact ctgttcgcag
13200 cagtctgcgc ccttacacaa aatgacatca aaaaaatcgt agccttctcc
acttcaagtc 13260 aactaggact cataatagtt acaatcggca tcaaccaacc
acacctagca ttcctgcaca 13320 tctgtaccca cgccttcttc aaagccatac
tatttatgtg ctccgggtcc atcatccaca 13380 accttaacaa tgaacaagat
attcgaaaaa taggaggact actcaaaacc atacctctca 13440 cttcaacctc
cctcaccatt ggcagcctag cattagcagg aatacctttc ctcacaggtt 13500
tctactccaa agaccacatc atcgaaaccg caaacatatc atacacaaac gcctgagccc
13560 tatctattac tctcatcgct acctccctga caagcgccta tagcactcga
ataattcttc 13620 tcaccctaac aggtcaacct cgcttcccca cccttactaa
cattaacgaa aataacccca 13680 ccctactaaa ccccattaaa cgcctggcag
ccggaagcct attcgcagga tttctcatta 13740 ctaacaacat ttcccccgca
tcccccttcc aaacaacaat ccccctctac ctaaaactca 13800 cagccctcgc
tgtcactttc ctaggacttc taacagccct agacctcaac tacctaacca 13860
acaaacttaa aataaaatcc ccactatgca cattttattt ctccaacata ctcggattct
13920 accctagcat cacacaccgc acaatcccct atctaggcct tcttacgagc
caaaacctgc 13980 ccctactcct cctagaccta acctgactag aaaagctatt
acctaaaaca atttcacagc 14040 accaaatctc cacctccatc atcacctcaa
cccaaaaagg cataattaaa ctttacttcc 14100 tctctttctt cttcccactc
atcctaaccc tactcctaat cacataacct attcccccga 14160 gcaatctcaa
ttacaatata tacaccaaca aacaatgttc aaccagtaac tactactaat 14220
caacgcccat aatcatacaa agcccccgca ccaataggat cctcccgaat caaccctgac
14280 ccctctcctt cataaattat tcagcttcct acactattaa agtttaccac
aaccaccacc 14340 ccatcatact ctttcaccca cagcaccaat cctacctcca
tcgctaaccc cactaaaaca 14400 ctcaccaaga cctcaacccc tgacccccat
gcctcaggat actcctcaat agccatcgct 14460 gtagtatatc caaagacaac
catcattccc cctaaataaa ttaaaaaaac tattaaaccc 14520 atataacctc
ccccaaaatt cagaataata acacacccga ccacaccgct aacaatcaat 14580
actaaacccc cataaatagg agaaggctta gaagaaaacc ccacaaaccc cattactaaa
14640 cccacactca acagaaacaa agcatacatc attattctcg cacggactac
aaccacgacc 14700 aatgatatga aaaaccatcg ttgtatttca actacaagaa
caccaatgac cccaatacgc 14760 aaaactaacc ccctaataaa attaattaac
cactcattca tcgacctccc caccccatcc 14820 aacatctccg catgatgaaa
cttcggctca ctccttggcg cctgcctgat cctccaaatc 14880 accacaggac
tattcctagc catgcactac tcaccagacg cctcaaccgc cttttcatca 14940
atcgcccaca tcactcgaga cgtaaattat ggctgaatca tccgctacct tcacgccaat
15000 ggcgcctcaa tattctttat ctgcctcttc ctacacatcg
ggcgaggcct atattacgga 15060 tcatttctct actcagaaac ctgaaacatc
ggcattatcc tcctgcttgc aactatagca 15120 acagccttca taggctatgt
cctcccgtga ggccaaatat cattctgagg ggccacagta 15180 attacaaact
tactatccgc catcccatac attgggacag acctagttca atgaatctga 15240
ggaggctact cagtagacag tcccaccctc acacgattct ttacctttca cttcatcttg
15300 cccttcatta ttgcagccct agcaacactc cacctcctat tcttgcacga
aacgggatca 15360 aacaaccccc taggaatcac ctcccattcc gataaaatca
ccttccaccc ttactacaca 15420 atcaaagacg ccctcggctt acttctcttc
cttctctcct taatgacatt aacactattc 15480 tcaccagacc tcctaggcga
cccagacaat tataccctag ccaacccctt aaacacccct 15540 ccccacatca
agcccgaatg atatttccta ttcgcctaca caattctccg atccgtccct 15600
aacaaactag gaggcgtcct tgccctatta ctatccatcc tcatcctagc aataatcccc
15660 atcctccata tatccaaaca acaaagcata atatttcgcc cactaagcca
atcactttat 15720 tgactcctag ccgcagacct cctcattcta acctgaatcg
gaggacaacc agtaagctac 15780 ccttttacca tcattggaca agtagcatcc
gtactatact tcacaacaat cctaatccta 15840 ataccaacta tctccctaat
tgaaaacaaa atactcaaat gggcctgtcc ttgtagtata 15900 aactaataca
ccagtcttgt aaaccggaga tgaaaacctt tttccaagga caaatcagag 15960
aaaaagtctt taactccacc attagcaccc aaagctaaga ttctaattta aactattctc
16020 tgttctttca tggggaagca gatttgggta ccacccaagt attgactcac
ccatcaacaa 16080 ccgctatgta tttcgtacat tactgccagc caccatgaat
attgtacggt accataaata 16140 cttgaccacc tgtagtacat aaaaacccaa
tccacatcaa aaccccctcc ccatgcttac 16200 aagcaagtac agcaatcaac
cctcaactat cacacatcaa ctgcaactcc aaagccaccc 16260 ctcacccact
aggataccaa caaacctacc cacccttaac agtacatagt acataaagcc 16320
atttaccgta catagcacat tacagtcaaa tcccttctcg tccccatgga tgacccccct
16380 cagatagggg tcccttgacc accatcctcc gtgaaatcaa tatcccgcac
aagagtgcta 16440 ctctcctcgc tccgggccca taacacttgg gggtagctaa
agtgaactgt atccgacatc 16500 tggttcctac ttcagggtca taaagcctaa
atagcccaca cgttcccctt aaataagaca 16560 tcacgatg 16568 2 23 DNA Homo
sapiens 2 gtatttggat gtcagaaaca ctt 23 3 19 DNA Homo sapiens 3
acttcagggt cataaagcc 19 4 23 DNA Homo sapiens 4 gtatttggat
gtcagaaaca ctt 23 5 19 DNA Homo sapiens 5 acttcagggc cataaagcc 19 6
21 DNA Homo sapiens 6 acccgcgtcc gcgccatggc c 21 7 21 DNA Homo
sapiens 7 acccgcgtcc tcgccatggc c 21 8 8 DNA Homo sapiens 8
taaccata 8 9 9 DNA Homo sapiens 9 tctcttacc 9 10 10 DNA Homo
sapiens 10 cacactacta 10 11 9 DNA Homo sapiens 11 tttaaccag 9
* * * * *
References