Claims for Patent: 6,013,436

Summary for Patent: 6,013,436

Title:	Compositions and methods for diagnosis of mutation in the von Hippel-Lindau tumor suppressor gene
Abstract:	The instant invention relates to methods and products for the diagnosis of mutations in the von Hippel-Lindau tumor suppressor gene. More specifically, the products are DNA oligonucleotides for use in amplifying and sequencing genomic DNA and kits including these oligonucleotides. The method of the invention relates to a hierarchical system for cost-effective diagnosis of von Hippel-Lindau tumor suppressor disease-associated mutations.
Inventor(s):	Hui; May (Toronto, CA), Dunn; James M. (Scarborough, CA), Stevens; John K. (Toronto, CA), Capatos; Denis (Waterloo, CA), Matthews; David E. (Kitchener, CA)
Assignee:	Visible Genetics, Inc. (Toronto, CA)
Application Number:	08/699,628
Patent Claims:	1. A method for testing a plurality of patients for a disease-associated mutation in the VHL tumor suppressor gene, comprising the steps of: a) selecting a hierarchical system of assays comprising at least a first and final assay, said first assay being selected to provide a test for the existence of the disease-associated mutation with less than 1% false results indicating the presence of a disease associated mutation and said final assay being selected to provide a test for the existence of the disease associated mutation with less than 1% false results indicating the presence of a disease associated mutation and less than 1% false results indicating the absence of the disease associated mutation; b) analyzing a body sample from each of the plurality of patients using the first assay; and, if the result of the first assay is negative for the presence of a disease-associated mutation; c) analyzing the body sample using the final assay. 2. A method according to claim 1 wherein said first assay is a DNA analysis. 3. A method according to claim 1 wherein said final assay is DNA sequencing of at least one exon of the VHL tumor suppressor gene. 4. A method according to claim 1 wherein the hierarchical system of assay techniques is selected from among those techniques which use electrophoretic analysis. 5. A method according to claim 4 wherein the electrophoretic analysis has a resolution of one nucleotide. 6. A method according to claim 1, wherein the hierarchy of assay techniques is selected by a method comprising the steps of: a) identifying a plurality of tests for the VHL disease-associated mutation; b) determining the sensitivity and specificity 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 a sensitivity and specificity of 100 percent; c) defining a outcome mapping matrix containing a plurality of diagnostic test combinations each comprising a combination of tests to be evaluated and assessing the significance of all possible combinations of negative and positive results for the diagnostic test combinations in the matrix; d) determining overall sensitivity, overall specificity, overall predictive value of a positive test combination result and overall predictive value of a negative test combination result for at least some of the test combinations in the outcome matrix; e) determining the estimated cost of testing for those combinations where the overall sensitivity, overall specificity, overall predictive value of a positive test combination result and overall predictive value of a negative test combination result meet a predefined threshold, said predefined threshold defining test combinations with sufficiently high sensitivity and specificity; and f) selecting a diagnostic test combination which has a low estimated cost of testing. 7. A method for testing a plurality of patients for a disease-associated mutation in the Von Hippel-Lindau tumor suppressor gene, comprising the steps of: a) quantitatively amplifying at least one exon of the Von Hippel-Lindau tumor suppressor gene from a body sample of each of the plurality of patients to produce amplified fragments and comparing the length and quantity of the amplification fragments to standard values based upon the fragments produced by amplification of the same exon in a non-mutant VHL gene; and b) performing a DNA analysis or the disease-associated mutation having a lower level of false results indicating the absence of a mutation than the test of step a on a body sample from each of the plurality of patients that demonstrates the standard values for the amplified exon in the test of step a. 8. A method for testing a plurality of patients for a disease-associated mutation in the Von Hippel-Lindau tumor suppressor gene, comprising the steps of: a) quantitatively amplifying, from a body sample from each of the plurality of patients, at least one exon of the Von Hippel-Lindau tumor suppressor gene using primers complementary to intron regions flanking each amplified exon; b) comparing the length of the amplification products for each amplified exon to the length of the amplification products obtained when a wild-type VHL tumor suppressor 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 sample Von Hippel-Lindau tumor suppressor gene; c) determining the nucleic acid sequence of each exon identified in step (b) to contain an insertion or deletion mutation, or in the event no insertion or deletion mutations are identified, the nucleic acid sequence of a least one exon of the Von Hippel-Lindau tumor suppressor gene. 9. A method according to claim 8, further comprising a step before step (c) of determining the quantity of the amplification product and comparing this quantity to that of the corresponding amplified wild-type exon, whereby differences in quantity between an amplified sample exon and the corresponding amplified wild-type exon reflect the occurrence of a mutation in the sample Von Hippel-Lindau tumor suppressor gene. 10. A method according to claim 8, wherein at least two of the exons of the sample Von Hippel-Lindau tumor suppressor gene are coamplified in a single reaction. 11. A method according to claim 10, wherein the primers for the coamplified exons of the Von Hippel-Lindau tumor suppressor gene are selected to provide amplified fragments of different lengths. 12. A method according to claim 8 wherein at least part of exon 1 of the VHL tumor suppressor gene is quantitatively amplified, and wherein the primers used are SEQ ID NO:6 and SEQ ID NO:7. 13. A method according to claim 8 wherein at least part of exon 1 of the VHL tumor suppressor gene is quantitatively amplified, and wherein the primers used are SEQ ID NO:8 and SEQ ID NO:9. 14. A method according to claim 8 wherein at least exon 2 of the Von Hippel-Lindau tumor suppressor gene is quantitatively amplified, and wherein the primers used are SEQ ID NO:10 and SEQ ID NO:11. 15. A method according to claim 8 wherein at least exon 3 of the Von Hippel-Lindau tumor suppressor gene is quantitatively amplified, and wherein the primers used are SEQ ID NO:12 and SEQ ID NO:13. 16. A method according to claim 8 wherein the sequence of the exon is obtained using a primer consisting of a DNA sequence selected from among: SEQ ID NO:14 through SEQ ID NO:21. 17. A method for genetic screening of family members of an individual diagnosed as having Von Hippel-Lindau disease, comprising the steps of: a) obtaining a body sample from the diagnosed individual; b) amplifying at least one exon of the Von Hippel-Lindau tumor suppressor gene from the body sample using primers complementary to intron regions flanking each amplified exon; c) determining the length of the amplification product for each exon amplified and comparing that length to the length or amount of amplification product obtained when a wild-type Von Hippel-Lindau tumor suppressor 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 inherited insertion or deletion mutation in the Von Hippel-Lindau tumor suppressor gene of the diagnosed individual; and d) if an inheritable mutation is identified, obtaining body samples from the familial relations of the diagnosed individual; amplifying the exon of the Von Hippel-Lindau tumor suppressor gene found to contain an insertion or deletion mutation in the diagnosed individual in said body samples from the familial relations using the same primers used to amplify the exon in the diagnosed individual; and determining the length of the amplification product for the exon in the amplified familial relation sample and comparing that length to the length of amplification products obtained when the patient sample was amplified, wherein a correlation between the length of the amplified product obtained from the patient sample and the length of the amplified product of a sample from a familial relation is indicative that the relation is a carrier of the mutation. 18. A method according to claim 17, wherein the amplification step is quantitative and further comprising the step of determining the quantity of the amplification products and comparing this quantity to that of the corresponding amplified wild-type exon. 19. A method according to claim 17, further comprising the step of determining the sequence of the exon containing the inherited mutation. 20. A method according to claim 17 wherein at least part of exon 1 of the Von Hippel-Lindau tumor suppressor gene is quantitatively amplified, and wherein the primers used are SEQ ID NO:6 and SEQ ID NO:7. 21. A method according to claim 17 wherein at least part of exon 1 of the Von Hippel-Lindau tumor suppressor gene is quantitatively amplified, and wherein the primers used are SEQ ID NO:8 and SEQ ID NO:9. 22. A method according to claim 17 herein at least exon 2 of the Von Hippel-Lindau tumor suppressor gene is quantitatively amplified, and wherein the primers used are SEQ ID NO:10 and SEQ ID NO:11. 23. A method according to claim 17 wherein at least exon 3 of the Von Hippel-Lindau tumor suppressor gene is quantitatively amplified, and wherein the primers used are SEQ ID NO:12 and SEQ ID NO:13. 24. A method according to claim 6, 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) in the diagnostic test combination, A, will have to be performed to achieve an unambiguous diagnostic result and C.sub.(j) is the cost of the j-th test.

Details for Patent 6,013,436

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		2014-07-08
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2014-07-08
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2014-07-08