Claims for Patent: 6,518,016

Summary for Patent: 6,518,016

Title:	Method for diagnosing an increased risk for thrombosis or a genetic defect causing thrombosis and kit for use with the same
Abstract:	Method for screening for the presence of a genetic defect associated with thrombosis and/or poor anticoagulant response to activated protein C (APC). The method is directed at detecting one or more mutations at one or more of the cleavage and/or binding sites for APC of Factor V and/or Factor Va or at Factor VIII and/or Factor VIIIa at either nucleic acid or protein level or both.
Inventor(s):	Bertina; Rogier Maria (Leiden, NL), Reitsma; Pieter Hendrik (Leiden, NL)
Assignee:	Rijks Universiteit Leiden (Leiden, NL)
Application Number:	08/454,353
Patent Claims:	1. A method for diagnosing an increased risk for thrombosis or a genetic defect causing thrombosis comprising the steps of: (A) obtaining, from a test subject, test nucleic acid comprising codon 506 within EXON 10 of the human Factor V gene; and (B) assaying for the presence of a point mutation in the nucleotides of codon 506 within EXON 10 of the human Factor V gene, wherein said point mutation correlates to a decrease in the degree of inactivation of human Factor V and/or human Factor Va by activated protein C, wherein the presence of said point mutation in said test nucleic acid indicates an increased risk for thrombosis or a genetic defect causing thrombosis. 2. The method of claim 1, wherein said point mutation is in the second nucleotide (G) of said codon 506 within EXON 10 of the human Factor V gene. 3. The method of claim 2, wherein said point mutation is a G to A point mutation. 4. The method of claim 1, wherein said assaying in step (B) is carried out by hybridizing said test nucleic acid with a nucleic acid probe for said point mutation. 5. The method of claim 4, wherein said probe is selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:13. 6. The method of claim 1, wherein said assaying in step (B) is carried out by subjecting said test nucleic acid to restriction enzyme digestion. 7. The method of claim 6, wherein said assaying in step (B) is carried out by subjecting said test nucleic acid to MnlI restriction enzyme digestion, and assaying for the loss of a MlnI restriction site within EXON 10 of the human Factor V gene, wherein the loss of said restriction site indicates the presence of said point mutation. 8. The method of claim 1, wherein said assaying in step (B) is carried out by sequencing said test nucleic acid. 9. The method of claim 1, wherein prior to step (B), said test nucleic acid is subjected to amplification using a forward and a reverse primer that border or encode said codon 506 within EXON 10 of the Factor V gene. 10. The method of claim 9, wherein said forward primer is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9 and SEQ ID NO:11, and said reverse primer is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10 and SEQ ID NO:14. 11. The method of claim 10, wherein said forward primer and said reverse primer are SEQ ID NO:4 and SEQ ID NO:5, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; or SQ. ID NO:11 and SEQ ID NO:10, respectively. 12. The method of claim 9, wherein said reverse primer borders or is within INTRON 10 of the human Factor V gene. 13. The method of claim 9, wherein said amplification is selected from the group consisting of nucleic acid sequence based amplification (NASBA), polymerase chain reaction (PCR), ligase chain reaction (LCR) and repair chain reaction (RCR). 14. The method of claim 9, wherein said assaying in step (B) is carried out by hybridizing the resulting amplified nucleic acid with a nucleic acid probe for said mutation. 15. The method of claim 14, wherein said probe is selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:13. 16. The method of claim 9, wherein said assaying in step (B) is carried out by subjecting the resulting amplified nucleic acid to restriction enzyme digestion. 17. The method of claim 16, wherein said assaying in step (B) is carried out by subjecting said test nucleic acid to MnlI restriction enzyme digestion, and assaying for the loss of a MlnI restriction site within EXON 10 of the human Factor V gene, wherein the loss of said restriction site indicates the presence of said point mutation. 18. The method of claim 9, wherein said assaying in step (B) is carried out by sequencing the resulting amplified nucleic acid. 19. A kit for diagnosing an increased risk for thrombosis or a genetic defect causing thrombosis comprising a forward and a reverse primer that are capable of amplifying EXON 10 of the human Factor V gene. 20. The kit of claim 19, wherein said forward primer is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9 and SEQ ID NO:11, and said reverse primer is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10 and SEQ ID NO:14. 21. The kit of claim 20, wherein said forward primer and said reverse primer are SEQ ID NO:4 and SEQ ID NO:5, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; or SEQ ID NO:1 and SEQ ID NO:10, respectively. 22. The kit of claim 19, wherein said kit additionally comprises a nucleic acid probe for detecting a point mutation in codon 506 within EXON 10 of the human Factor V gene. 23. The kit of claim 22, wherein said point mutation is in the second nucleotide (G) of said codon 506 within EXON 10 of the human Factor V gene. 24. The kit of claim 23, wherein said point mutation is a G to A point mutation. 25. The kit of claim 22, wherein said probe is selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:13. 26. The kit of claim 19, wherein said kit additionally comprises a restriction endonuclease which is capable of being used to assay for a point mutation in codon 506 within EXON 10 of the human Factor V gene. 27. The kit of claim 26, wherein said restriction endonuclease is MnlI. 28. An isolated nucleic acid molecule consisting of SEQ ID NO:14 or its complementary strand. 29. An isolated nucleic acid molecule consisting of a nucleic acid sequences selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, or the complementary strand thereof.

Details for Patent 6,518,016

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		2020-02-11
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2020-02-11
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2020-02-11