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US 11,001,890 B2Granted
Mitochondrial health parameters as clinical predictors of Parkinson's disease
AssigneeUniversity of Pittsburgh—Of the Commonwealth System of Higher Education
InventorsLaurie H. Andolina, John T. Greenamyre, Sruti Shiva
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Abstract
Methods are disclosed for detecting peripheral mitochondrial DNA damage and dysfunction. Methods are also disclosed that utilize blood samples to detect a neurodegenerative disease, such as Parkinson's disease, and determine the efficacy of therapy.
Claims27
A method of diagnosing Parkinson's disease, comprising contacting a biological sample comprising mitochondrial DNA with a first forward primer comprising the nucleic acid sequence set forth as SEQ ID NO:
- 1, and a reverse primer comprising the nucleic acid sequence set forth as SEQ ID NO:
- 2, wherein the first forward primer and the reverse primer amplify a large mitochondrial DNA product of about 8900 nucleotides in length, wherein the biological sample comprises white blood cells;
- amplifying the mitochondrial DNA in a quantitative polymerase chain reaction comprising a fluorescent dye, wherein the fluorescent dye has the formula
- wherein all of A1, A2, A3, and A4 are H or one of A1, A2, A3, and A4 is a halogenyl and the others are H;
- B is selected from the group consisting of S, O, N—R, and C—(R)2 wherein R is C1-C6 alkyl;
- D is an unsubstituted or substituted C1-C6 alkyl;
- X is H or a methoxy group;
- Y is selected from the group consisting of S, O·N—R wherein R is C1-C6 alkyl, and Z1-C═C—Z2 wherein Z1 and Z2 independently are H or a methoxy group;
- L is CH3 or phenyl;
- and
- M is phenyl or a substituted or unsubstituted C1-C18 amino-alkyl
- to produce the large mitochondrial DNA product,
- wherein the quantitative PCR is performed for 40 cycles or less, and wherein the fluorescent dye is bound to the large DNA product during amplification;
- quantitating the amount of the fluorescence emitted by the large DNA product at multiple points during the quantitative PCR reaction to establish the linear emission range for the biological sample;
- and
- determining the lesion frequency of mitochondrial DNA in the biological sample in the linear emission range,
- wherein an increase in the lesion frequency in the linear emission range as compared to a control lesion frequency in a control linear emission range indicates that the subject has Parkinson's disease.
14 more elements
A method of diagnosing Parkinson's disease in a subject, comprising
- a) contacting a biological sample comprising mitochondrial DNA from the subject with a first forward primer comprising the nucleic acid sequence set forth as SEQ ID NO:
- 1, and a reverse primer comprising the nucleic acid sequence set forth as SEQ ID NO:
- 2, wherein the first forward primer and the reverse primer amplify a large mitochondrial DNA product of about 8900 nucleotides in length;
- amplifying the mitochondrial DNA in a quantitative polymerase chain reaction comprising a fluorescent dye, wherein the fluorescent dye has the formula
- wherein all of A1, A2, A3, and A4 are H or one of A1, A2, A3, and A4 is a halogenyl and the others are H;
- B is selected from the group consisting of S, O, N—R, and C—(R)2 wherein R is C1-C6 alkyl;
- D is an unsubstituted or substituted C1-C6 alkyl;
- X is H or a methoxy group;
- Y is selected from the group consisting of S, O, N—R wherein R is C1-C6alkyl, and Z1-C═C—Z2 wherein Z1 and Z2 independently are H or a methoxy group;
- L is CH3 or phenyl;
- and
- M is phenyl or a substituted or unsubstituted C1-C18 amino-alkyl to produce the large mitochondrial DNA product,
- wherein the quantitative PCR is performed for 40 cycles or less, and wherein the fluorescent dye is bound to the large DNA product during amplification;
- quantitating the amount of the fluorescence emitted by the large DNA product at multiple points during the quantitative PCR reaction to establish the linear emission range for the biological sample;
- and
- determining the lesion frequency of mitochondrial DNA in the biological sample in the linear emission range, wherein an increase in the lesion frequency in the linear emission range as compared to a control lesion frequency in a control linear emission range indicates that the subject has Parkinson's disease;
- and
- b) performing a bioenergetics assay on a white blood cell or a platelet sample from the subject,
- thereby diagnosing Parkinson's disease in the subject.
16 more elements
A method of determining if an agent is effective for treating a subject with Parkinson's disease, comprising
- contacting a biological sample comprising mitochondrial DNA from a subject treated with the agent with a first forward primer comprising the nucleic acid sequence set forth as SEQ ID NO:
- 1, and a reverse primer comprising the nucleic acid sequence set forth as SEQ ID NO:
- 2, wherein the first forward primer and the reverse primer amplify a large mitochondrial DNA product of 8900 nucleotides in length, wherein the biological sample comprises white blood cells;
- amplifying the mitochondrial DNA in a quantitative polymerase chain reaction comprising a fluorescent dye, wherein the fluorescent dye has the formula
- wherein all of A1, A2, A3, and A4 are H or one of A1, A2, A3, and A4 is a halogenyl and the others are H;
- B is selected from the group consisting of S, O, N—R, and C—(R)2 wherein R is C1-C6 alkyl;
- D is an unsubstituted or substituted C1-C6 alkyl;
- X is H or a methoxy group;
- Y is selected from the group consisting of S, O, N—R wherein R is C1-C6 alkyl, and Z1-C═C—Z2 wherein Z1 and Z2 independently are H or a methoxy group;
- L is CH3 or phenyl;
- and
- M is phenyl or a substituted or unsubstituted C1-C18 amino-alkyl
- to produce the large mitochondrial DNA product,
- wherein the quantitative PCR is performed for 40 cycles or less, and wherein the fluorescent dye is bound to the large DNA product during amplification;
- quantitating the amount of the fluorescence emitted by the large DNA product at multiple points during the quantitative PCR reaction to establish the linear emission range for the biological sample;
- and
- determining the lesion frequency of mitochondrial DNA in the biological sample in the linear emission range,
- wherein a decrease in the lesion frequency in the linear emission range as compared to a control lesion frequency in a control linear emission range indicates that the agent is effective for treating the subject.
15 more elements
A method of determining if an agent is effective for treating a subject with Parkinson's disease, comprising
- a) contacting a biological sample comprising mitochondrial DNA from the subject with Parkinson's disease treated with the agent with a first forward primer comprising the nucleic acid sequence set forth as SEQ ID NO:
- 1, and a reverse primer comprising the nucleic acid sequence set forth as SEQ ID NO:
- 2, wherein the first forward primer and the reverse primer amplify a large mitochondrial DNA product of 8900 nucleotides in length
- amplifying the mitochondrial DNA in a quantitative polymerase chain reaction comprising a fluorescent dye, wherein the fluorescent dye has the formula
- wherein all of A1, A2, A3, and A4 are H or one of A1, A2, A3, and A4 is a halogenyl and the others are H;
- B is selected from the group consisting of S, O, N—R, and C—(R)2 wherein R is C1-C6 alkyl;
- D is an unsubstituted or substituted C1-C6 alkyl;
- X is H or a methoxy group;
- Y is selected from the group consisting of S, O, N—R wherein R is C1-C6alkyl, and Z1-C═C—Z2 wherein Z1 and Z2 independently are H or a methoxy group;
- L is CH3 or phenyl;
- and
- M is phenyl or a substituted or unsubstituted C1-C18 amino-alkyl
- to produce the large mitochondrial DNA product,
- wherein the quantitative PCR is performed for 40 cycles or less, and wherein the fluorescent dye is bound to the large DNA product during amplification;
- quantitating the amount of the fluorescence emitted by the large DNA product at multiple points during the quantitative PCR reaction to establish the linear emission range for the biological sample;
- and
- determining the lesion frequency of mitochondrial DNA in the biological sample in the linear emission range, wherein a decrease in the lesion frequency in the linear emission range as compared to a control lesion frequency in a control linear emission range indicates that the agent is effective for treating the subject;
- and
- b) performing a bioenergetics assay on a white blood cell or platelet sample from the subject,
- thereby determining that the agent is effective for treating the subject with Parkinson's disease.
17 more elements
The method of 7, wherein the subject is elderly.
Dependencies are inferred from the claim text.