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Last Updated: March 28, 2024

Claims for Patent: 6,270,963


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Summary for Patent: 6,270,963
Title: Method for testing for mutations in DNA from a patient sample
Abstract:A hierarchy of at least two assay techniques is utilized in testing for disease-associated mutations. The first assay in the hierarchy is selected to provide a highly specific test for the existence of the disease-associated mutation, although the accuracy of the test need not be high. The final assay in the hierarchy is selected to provide a highly accurate and highly specific test for the existence of the disease associated mutation. Intermediate tests of progressively greater accuracy may also be included in the hierarchy. Once the hierarchy has been selected for a given mutation-associated disease, a patient sample is analyzed the patient sample using the first, lowest accuracy assay in the hierarchy. If the result of the first assay is negative for the presence of a disease-associated mutation, then the next assay in the hierarchy is performed. This process is repeated until the final assay has been performed on all samples which gave negative results when tested by all less-accurate assays in the hierarchy. The test may be used for diagnosis and targeted screening for p53 mutations and mutations in the RB1 gene.
Inventor(s): Stevens; John K. (Toronto, CA), Dunn; James M. (Scarborough, CA), Capatos; Denis (Waterloo, CA), Matthews; David E. (Kitchener, CA)
Assignee: Visible Genetics Inc. (Toronto, CA)
Application Number:08/750,232
Patent Claims:1. A method for testing a plurality of patients for a disease-associated mutation in a gene of interest comprising the steps of:

(a) performing an immunoassay on samples obtained from each of the plurality of patients by combining a portion of sample with an antibody which forms an immunological reaction product by binding to a protein gene product of the gene of interest and monitoring for formation of the immunological reaction product, said antibody being selected to provide fewer than 1% false results indicating the presence of a mutation when combined with the protein gene product of the gene of interest, whereby the formation of an immunological product is indicative of the presence or absence of a mutation in the gene of interest;

(b) performing a probe-based assay on each patient sample for which the results of the immunoassay did not indicate the presence of a mutation, but not on those samples for which the immunoassay indicated the presence of the mutation by combining a second portion of each patient sample with a nucleic acid probe which specifically and selectively hybridizes with the gene of interest in its mutated or wild-type form, whereby the formation of a duplex nucleic acid hybrid containing the nucleic acid probe is indicative of the presence or absence, respectively, of the mutation in the gene of interest; and

(c) determining the sequence of DNA in at least one selected region of the gene of interest on each patient sample for which the results of the probe-based assay did not indicate the presence of the mutation and comparing the sequence determined with known sequences of normal or mutant forms of the gene of interest,

wherein the immunoassay and probe-based assay used are selected by a method comprising the steps of:

identifying a plurality of immunoassays and probe-based assays for the disease-associated mutation;

determining the level of false results for each assay by comparing results from assays on a statistically significant plurality of samples against results from assays on the same samples using a test protocol accepted as having essentially no false results;

defining an outcome mapping matrix containing a plurality of diagnostic algorithms each comprising a combination of assays to be evaluated and assessing the significance of all possible combinations of negative and positive results for the algorithms in the matrix;

determining overall sensitivity overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result for at least some of the algorithms in the outcome matrix;

determining the estimated cost of testing for those combinations where the overall sensitivity overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result meet a predefined threshold; and

selecting a diagnostic algorithm of assays based upon the low estimated cost of testing.

2. A method for testing a plurality of patients for a disease-associated mutation in a gene of interest comprising the steps of:

(a) performing an immunoassay on samples obtained from each of the plurality of patients by combining a portion of sample with an antibody which forms an immunological reaction product by binding to a protein gene product of the gene of interest and monitoring for formation of the immunological reaction product, said antibody being selected to provide fewer than 1% false results indicating the presence of a mutation when combined with the protein gene product of the gene of interest, whereby the formation of an immunological product is indicative of the presence or absence of the mutation in the gene of interest; and

(b) performing a probe-based assay on each patient sample for which the results of the immunoassay did not indicate the presence of a mutation, but not for those samples for which the immunoassay indicated the presence of the mutation by combining a second portion of each patient sample with a nucleic acid probe which specifically and selectively hybridizes with the gene of interest in its mutated or wild-type form, whereby the formation of a duplex nucleic acid hybrid containing the nucleic acid probe is indicative of the presence or absence, respectively, of the mutation in the gene of interest,

wherein the immunoassay and probe-based assay used are selected by a method comprising the steps of:

identifying a plurality of immunoassays and probe-based assays for the disease-associated mutation;

determining the level of false results for each assay by comparing results from assays on a statistically significant plurality of samples against results from tests on the same samples using a test protocol accepted as having essentially no false results;

defining an outcome mapping matrix containing a plurality of diagnostic algorithms each comprising a combination of tests to be evaluated and assessing the significance of all possible combinations of negative and positive results for the algorithms in the matrix;

determining overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result for at least some of the algorithms in the outcome matrix;

determining the estimated cost of testing for those combinations where the overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result meet the predefined threshold; and

selecting a diagnostic algorithm of tests based upon the low estimated cost of testing.

3. A method for testing a plurality of patients for a disease-associated mutation in a gene of interest comprising the steps of:

(a) performing a probe-based assay on each patient sample by combining a second portion of each patient sample with a nucleic acid probe which specifically and selectively hybridizes with the gene of interest in its mutant or wild-type form said probe-based assay providing essentially no false indications of the presence of a mutation, whereby the formation of a duplex nucleic acid hybrid containing the nucleic acid probe is indicative of the presence or absence, respectively, of the mutation in the gene of interest; and

(b) determining the sequence of DNA in at least a selected region of the gene of interest on each patient sample for which the results of the probe-based assay did not indicate the presence of a mutation, but not on those samples for which the probe-based assay indicated the presence of the mutation and comparing the sequence determined with known sequences of normal or mutant forms of the gene of interest,

wherein the probe-based assay used is selected by a method comprising the steps of:

identifying a plurality of probe-based assays for the disease-associated mutation;

determining the level of false results for each assay by comparing results from assays on a statistically significant plurality of samples against results from assays on the same samples using a test protocol accepted as having essentially no false results;

defining an outcome mapping matrix containing a plurality of diagnostic algorithms each comprising a combination of tests to be evaluated and assessing the significance of all possible combinations of negative and positive results for the algorithms in the matrix;

determining overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result for at least some of the algorithms in the outcome matrix;

determining the estimated cost of testing for those combinations where the overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result meet the predefined threshold; and

selecting a diagnostic algorithm of assays based upon the low estimated cost of testing.

4. A method for testing a patient sample for the presence or absence of a disease-associated mutation in a gene of interest in a patient sample, comprising the steps of:

(a) selecting a hierarchy of molecular assay techniques comprising at least a first molecular assay and a final molecular assay, said first molecular assay being selected to provide a test for the existence of the disease-associated mutation with essentially no false results indicating the presence of a mutation and said final molecular assay being selected to provide a test for the existence of the disease associated mutation with essentially no false results indicating the presence or absence of a mutation;

(b) analyzing the patient sample using the first assay; and

(c) if the result of the first assay did not unambiguously indicate the presence of a disease-associated mutation, analyzing the patient sample using the final assay, wherein at least one assay in the hierarchy comprises the steps of

quantitatively amplifying one or more exons of the gene of interest in the sample using primers complementary to intron regions flanking each amplified exon; and

determining the lengths of the amplification products for each amplified sample exon and comparing that length to the length of amplification products obtained when a wild-type gene is amplified using the same primers, whereby differences in length between an amplified sample exon and the corresponding amplified wild-type exon reflect the occurrence of an insertion or deletion mutation in the gene of interest in the sample,

and wherein the assays used are selected by a method comprising the steps of:

identifying a plurality of assays for the disease-associated mutation;

determining the level of false results for each assay by comparing results from assays on a statistically significant plurality of samples against results from assays on the same samples using a test protocol accepted as having essentially no false results;

defining an outcome mapping matrix containing a plurality of diagnostic algorithms each comprising a combination of tests to be evaluated and assessing the significance of all possible combinations of negative and positive results for the algorithms in the matrix;

determining overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result for at least some of the algorithms in the outcome matrix;

determining the estimated cost of testing for those combinations where the overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result meet the predefined threshold; and

selecting a diagnostic algorithm of assays based upon the low estimated cost of testing.

5. A method for testing a patient sample for a disease-associated mutation in a gene of interest, comprising the steps of:

(a) selecting a hierarchy of molecular assay techniques comprising at least a first molecular assay and a final molecular assay, said first molecular assay being selected to provide a test for the existence of the disease-associated mutation with essentially no false results indicating the presence of a mutation, and said final molecular assay being selected to provide a test for the existence of the disease associated mutation with essentially no false results indicating the presence or absence of a mutation;

(b) analyzing the patient sample using the first molecular assay; and, if the result of the first molecular assay did not unambiguously indicate the presence of the disease-associated mutation,

(c) analyzing the patient sample using the final molecular assay,

wherein the hierarchy of tests is selected by a method comprising the steps of:

identifying a plurality of tests for the disease-associated mutation;

determining the level of false results for each test by comparing results from tests on a statistically significant plurality of samples against results from tests on the same samples using a test protocol accepted as having essentially no false results;

defining a outcome mapping matrix containing a plurality of diagnostic algorithms each comprising a combination of tests to be evaluated and assessing the significance of all possible combinations of negative and positive results for the algorithms in the matrix;

determining overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result for at least some of the algorithms in the outcome matrix;

determining the estimated cost of testing for those combinations where the overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result meet the predefined threshold; and

selecting a diagnostic algorithm of tests based upon the low estimated cost of testing.

6. A method according to claim 5, wherein the first assay is an immunoassay in which the sample is combined with an antibody that forms an immunological reaction product by binding to a protein gene product of the gene of interest.

7. A method according to claim 5, wherein the nucleic acid sequence of at least one region of the gene of interest is determined in the course of performing the final assay.

8. A method according to claim 7, wherein at least one assay in the hierarchy comprises the steps of

quantitatively amplifying one or more exons of the gene of interest in the sample using primers complementary to intron regions flanking each amplified exon; and

determining the lengths of the amplification products for each amplified sample exon and comparing that length to the length of amplification products obtained when a wild-type gene is amplified using the same primers, whereby differences in length between an amplified sample exon and the corresponding amplified wild-type exon reflect the occurrence of an insertion or deletion mutation in the gene of interest in the sample.

9. A method according to claim 8, wherein the length of the amplification products is determined by electrophoresis on a sequencing gel.

10. A method according to claim 9, wherein the sequencing gel has a resolution of one base pair.

11. A method according to claim 10, wherein the sequencing gel is a polyacrylamide gel.

12. A method according to claim 8, wherein the primers are each coupled to a detectable label.

13. A method according to claim 12, wherein the detectable label is fluorescein.

14. A method according to claim 8, wherein a plurality of exons of the gene of interest are coamplified in a single amplification reaction.

15. A method according to claim 14, wherein each coamplified exon is amplified using a primer pair, and wherein the primer pairs are selected to have a common melting temperature and to produce amplification products having differing lengths.

16. A method according to claim 1, wherein the antibody binds to the gene product of the wild-type gene of interest, and the observation of a reduced level of immunological product is indicative of the presence of a mutation.

17. A method according to claim 1, wherein the antibody binds to a mutant gene product of the gene of interest, and the observation of an immunological product is indicative of the presence of a mutation.

18. A method according to claim 1, wherein the sequences of a plurality of regions in the gene of interest are determined.

19. A method according to claim 18, wherein the sequences of the plurality of regions are determined in at least two phases in which mutations having a greater incidence in a relevant population to which the patient belongs are tested before mutations having a lower incidence.

20. A method according to claim 5, wherein the cost per sample for performing the first assay is less than the cost per sample for performing the final assay.

21. A method according to claim 5, wherein the hierarchy includes an intermediate molecular assay which yields fewer false results indicating the absence of a mutation than the first molecular assay.

22. A method according to claim 21, wherein the first assay is an immunoassay and the intermediate assay is a nucleic acid hybridization probe-based assay.

23. A method according to claim 5, wherein the estimated cost of testing, E[C.sub.A ], is determined using the equation ##EQU5##

where .rho..sub.A,r is the probability that a given test (the r-th test) will have to be performed to achieve an unambiguous determination concerning the presence of a mutation and C.sub.(j) is the cost of the j-th test.

Details for Patent 6,270,963

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 06/04/1986 ⤷  Try a Trial 2014-07-08
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 ⤷  Try a Trial 2014-07-08
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b Injection 103132 ⤷  Try a Trial 2014-07-08
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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