Claims for Patent: 6,265,157

Summary for Patent: 6,265,157

Title:	Compositions and methods for detecting altered COL1A1 gene sequences
Abstract:	Compositions and methods useful for determining whether a subject has an alteration in a gene encoding a protein chain of Type I or Type IX collagen are described. Novel intronic sequences of five human genes, COL1A1, COL1A2, COL9A1, COL9A2, and COL9A3 are described. Methods of determining the existence in a subject of a pathological condition associated with an altered gene encoding a Type I or Type IX collagen protein chain are provided, wherein such pathological conditions include diseases and disorders which are known to be associated with an altered gene encoding a Type I or Type IX collagen protein chain. Primers, probes, and methods of detecting a genetic predisposition of a subject for a pathological condition associated with an altered gene encoding a Type I or Type IX collagen protein chain are provided. Diseases and disorders for which the methods and compositions of the invention are useful for diagnostic or prognostic purposes include, but are not limited to osteoporosis, osteoarthritis, chondrodysplasia, multiple epiphyseal dysplasia, osteogenesis imperfecta, shortness of stature, scoliosis, low bone density, and degenerative joint disease.
Inventor(s):	Prockop; Darwin J. (Philadelphia, PA), Spotila; Loretta D. (Haddonfield, NJ), Deltas; Constantinos D. (Nicosia, CY), Sereda; Larisa (Philadelphia, PA), Westerhausen Larson; Andrea (Forrest Hills, PA), Pack; Michael (Philadelphia, PA), Colige; Alain (Philadelphia, PA), Early; James (Upper Darby, PA), Korkko; Jarmo (Philadelphia, PA), Ala-Kokko; Leena (Oulu, FI), Annunen; Susanna (Oulu, FI), Pihlajamaa; Tero (Oulu, FI), Vuoristo; Mirko (Oulu, FI), Paassilta; Petteri (Oulu, FI)
Assignee:	Allegheny University of the Health Sciences (Philadelphia, PA) Thomas Jefferson University (Philadelphia, PA) University of Oulu (Oulu, FI)
Application Number:	08/943,731
Patent Claims:	1. A method of detecting a collagen gene alteration associated with a pathological condition in a human subject, the method comprising obtaining from the subject a sample nucleic acid comprising a portion which consists of at least fifteen consecutive nucleotides of a segment of the gene, wherein the portion comprises at least one intronic nucleotide, a first site, and a second site; determining the nucleotide sequence of the portion; and comparing the nucleotide sequence of the portion with the corresponding consensus nucleotide sequence of the gene, whereby a difference between the nucleotide sequence of the portion and the corresponding consensus nucleotide sequence indicates the presence of the collagen gene alteration in the subject, wherein the segment is the segment of the COL1A1 gene extending in the 5'- to 3'-direction from and including the 78 nucleotides of intron 27 located adjacent exon 28 through the 3'-end of intron 51 wherein the consensus nucleotide sequence of the COL1A1 gene is SEQ ID NO: 1. 2. The method of claim 1, further comprising contacting the sample nucleic acid with a first intronic primer prior to determining the nucleotide sequence of the portion, the first intronic primer being either substantially complementary to or substantially homologous with the first site. 3. The method of claim 2, wherein the first intronic primer has a sequence selected from the group consisting of SEQ ID NOs: 273-299 and 301-319. 4. The method of claim 1, further comprising contacting the sample nucleic acid with a first intronic primer homologous with the first site, contacting the sample nucleic acid with a second primer complementary to the second site, and amplifying the portion to obtain an amplified polynucleotide prior to determining the nucleotide sequence of the portion, wherein determining the nucleotide sequence of the portion comprises determining the nucleotide sequence of the amplified polynucleotide. 5. The method of claim 4, further comprising performing conformation-sensitive gel electrophoresis (CSGE) after amplifying the portion and prior to determining the nucleotide sequence of the portion, the CSGE comprising denaturing the amplified polynucleotide, annealing the amplified polynucleotide, and determining whether the amplified polynucleotide forms a heteroduplex. 6. The method of claim 4, wherein the first intronic primer is either substantially complementary to or substantially homologous with a part of a non-coding region of the segment that has a sequence selected from the group consisting of SEQ ID NOs: 6, 7, 24-57. 7. The method of claim 4, wherein the first intronic primer has a sequence selected from the group consisting of SEQ ID NOs: 273-299 and 301-319. 8. The method of claim 4, wherein the first primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 9. The method of claim 4, wherein the sample nucleic acid is contacted with a plurality of pairs of primers and a plurality of portions of the segment are amplified to obtain a plurality of amplified polynucleotides. 10. The method of claim 9, further comprising performing CSGE after amplifying the portions and prior to determining the nucleotide sequence of the portions, the CSGE comprising denaturing the amplified polynucleotides, annealing the amplified polynucleotides, and determining whether any of the amplified polynucleotides forms a heteroduplex. 11. The method of claim 10, wherein each of the primers is either substantially complementary to or substantially homologous with a part of a non-coding region of the segment. 12. The method of claim 9, wherein the length of each amplified polynucleotide is between about two hundred and about five hundred nucleotides. 13. The method of claim 12, wherein the length of the polynucleotide amplified using each pair of primers is different than the length of the polynucleotides amplified using every other pair of primers. 14. The method of claim 9, wherein the plurality of pairs of primers comprises pairs of primers sufficient to amplify substantially all exons and exon flanking regions of the gene. 15. The method of claim 14, wherein the length of each amplified polynucleotide is between about two hundred and about five hundred nucleotides and is different from the length of every other amplified polynucleotide. 16. The method of claim 15, further comprising performing CSGE after amplifying the portions and prior to determining the nucleotide sequence of the portions, the CSGE comprising denaturing the amplified polynucleotides, annealing the amplified polynucleotides, and determining whether any of the amplified polynucleotides forms a heteroduplex. 17. The method of claim 16, wherein the segment is the segment of the COL1A1 gene extending in the 5'- to 3'-direction from and including the 78 nucleotides of intron 27 located adjacent exon 28 through the 3'-end of intron 51, and wherein each pair of primers comprises a first intronic primer and a second primer, the first intronic primer and second primer being selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 18. The method of claim 17, wherein the pathological condition is selected from the group consisting of osteoporosis, multiple epiphyseal dysplasia, osteogenesis imperfecta, shortness of stature, and low bone density. 19. An isolated nucleic acid comprising at least about fifteen consecutive nucleotides, wherein the isolated nucleic acid is either completely complementary to or completely homologous with at least fifteen consecutive nucleotides of a segment of a human collagen gene, including at least one nucleotide of the segment located in a non-coding region of the gene, and wherein the segment is the segment of the COL1A1 gene (SEQ ID NO: 1) extending in the 5'- to 3'-direction from and including the 78 nucleotides of intron 27 located adjacent exon 28 through the 3'-end of intron 51. 20. The isolated nucleic acid of claim 19, wherein the nucleic acid is completely complementary to the segment. 21. The isolated nucleic acid of claim 19, wherein the nucleic acid is completely homologous to the segment. 22. The isolated nucleic acid of claim 19, wherein the isolated nucleic acid is complementary to or homologous with at least three nucleotides of the segment located in the non-coding region. 23. The isolated nucleic acid of claim 19, wherein the segment comprises only nucleotides located in the non-coding region and wherein the non-coding region has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 24-57. 24. The isolated nucleic acid of claim 19, having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 273-299 and 301-319. 25. A kit for detecting an alteration in a segment of a human collagen gene, the kit comprising a consensus sequence of the gene, a first intronic primer, and a second primer, wherein each of the first intronic primer and the second primer comprises at least about fifteen consecutive nucleotides, wherein the segment comprises at least one nucleotide located in a non-coding region of the gene, a first site, and a second site, the first intronic primer being substantially homologous with the first site and the second primer being substantially complementary to the second site, wherein the segment is the segment of the COL1A1 gene extending in the 5'- to 3'-direction from and including the 78 nucleotides of intron 27 located adjacent exon 28 through the 3'-end of intron 51 wherein the consensus nucleotide sequence of the COL1A1 gene is SEQ ID NO: 1. 26. The kit of claim 25, wherein the length of the segment is between about two hundred and about five hundred nucleotides. 27. The kit of claim 25, further comprising at least one sequencing primer for determining the nucleotide sequence of at least a portion of the segment. 28. The kit of claim 25, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 29. The kit of claim 28, comprising a plurality of the pairs of primers. 30. The method of claim 8, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 31. The method of claim 8, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 32. The method of claim 8, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 33. The method of claim 8, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 34. The method of claim 8, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 35. The method of claim 8, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 36. The kit of claim 28, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 37. The kit of claim 28, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 38. The kit of claim 28, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 39. The kit of claim 28, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 40. The kit of claim 28, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table: 41. The kit of claim 28, wherein the first intronic primer and the second primer are selected such that when the first intronic primer has a sequence listed in Column 1 of the following table, the second primer has the sequence listed on the same line in Column 2 of the following table:

Details for Patent 6,265,157

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		2011-12-03
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2011-12-03
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2011-12-03