CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR GENE MUTATIONS

• US 20080153088A1
• Filed: 12/22/2006
• Published: 06/26/2008
• Est. Priority Date: 12/22/2006
• Status: Active Grant

• Current Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Original Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Inventors: Buller, Arlene, Peng, Mei, McGinniss, Matthew J., Fenwick, Raymond, Huang, Donghui, Quan, Franklin, Sun, Weimin
• Granted Patent: US 7,794,937 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 2800/382 Cystic fibrosis
• CPC Class Codes: G01N 2800/50 Determining the risk of developing a disease
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention provides novel mutations of the CFTR gene related to cystic fibrosis or to conditions associated with cystic fibrosis. Also provided are probes for detecting the mutant sequences. Methods of identifying if an individual has a genotype containing one or more mutations in the CFTR gene are further provided.

Claims

1. A method of determining if a cystic fibrosis transmembrane regulatory (CFTR) gene contains one or more mutations selected from the group consisting of 3443A>

T, 2443delA (A at position 2443 is deleted), 2777insTG (TG are inserted at position

     2777), 3123-3125delGTT (GTT at positions 3123-3125 are deleted), 4177delG (G at position 4177 is deleted), 630delG (G at position 630 is deleted), 2068G>

T, 1342−

2A>

G (A in the splice acceptor site of intron 8, 2 nucleotides upstream of position 1342, is substituted with G), 297−

1G>

A (G in the splice acceptor site of intron 2, 1 nucleotide upstream of position 297, is substituted with A) 3500−

2A>

T (A in the splice acceptor site of intron 17b, 2 nucleotides upstream of position 3500, is substituted with T), 4375−

2A>

G (A in the splice acceptor site of intron 23, 2 nucleotides upstream of position 4375, is substituted with G), 3172-3174delTAC (TAC at positions 3172 to 3174 are deleted), 2902G>

C, 4115T>

C, 4185G>

C, 520C>

G, 842A>

C, 4528G>

T, 448A>

G, 574A>

T, 3704T>

C, 1248+5T>

C (T in the splice donor site of intron 7, 5 nucleotides downstream of position 1248, is substituted with C), 296+12T>

G (T in intron 2, 12 nucleotides downstream of position 296, is substituted with G), 3849+3G>

A (G in the splice donor site of intron 19, 3 nucleotides downstream of position 3849, is substituted with A), 497A>

G, −

141C>

A, 2875G>

C, 2689A>

G, 3039A>

G, 405G>

C, 886G>

A, 4445G>

A, −

228G>

C, −

295C>

T, −

379delC (C at position −

379 is deleted), and −

540A>

G, comprising determining whether CFTR nucleic acid contains one or more of said mutations.

5. The method of claim 1 wherein said mutations selected from the group consisting of 3443A>

T, 2443delA (A at position 2443 is deleted), 2777insTG (TG are inserted at position

     2777), 3123-3125delGTT (GTT at positions 3123-3125 are deleted), 4177delG (G at position 4177 is deleted), 630delG (G at position 630 is deleted), 2068G>

T, 1342−

2A>

G (A in the splice acceptor site of intron 8, 2 nucleotides upstream of position 1342, is substituted with G), 297−

1G>

A (G in the splice acceptor site of intron 2, 1 nucleotide upstream of position 297, is substituted with A) 3500−

2A>

T (A in the splice acceptor site of intron 17b, 2 nucleotides upstream of position 3500, is substituted with T), 4375−

2A>

G (A in the splice acceptor site of intron 23, 2 nucleotides upstream of position 4375, is substituted with G), and 3172-3174delTAC (TAC at positions 3172 to 3174 are deleted).

6. The method of claim 1 wherein said mutations selected from the group consisting of 2902G>

C, 4115T>

C, 4185G>

C, 520C>

G, 842A>

C, 4528G>

T, 448A>

G, 574A>

T, 37Q4T>

C, 1248+5T>

C (T in the splice donor site of intron 7, 5 nucleotides downstream of position 1248, is substituted with C), 296+12T>

G (T in intron 2, 12 nucleotides downstream of position 296, is substituted with G), 3849+3G>

A (G in the splice donor site of intron 19, 3 nucleotides downstream of position 3849, is substituted with A), 497A>

G, and −

141C>

A.

7. The method of claim 1 wherein both alleles in the individual are evaluated for said one or more mutations.

8. The method of claim 1 wherein genomic DNA is tested for the presence of said one or more mutations.

9. The method of claim 1 wherein a cDNA copy of the CFTR coding sequence is tested for the presence of said one more mutations.

10. The method of claim 1 wherein sequence from the CFTR gene is amplified by the polymerase chain reaction and the amplified sequence is tested for the presence of said one or more mutations.

11. The method of claim 1 wherein the presence of said one or more mutations is determined by nucleic acid sequencing.

12. The method of claim 1 wherein the presence of said one or more mutations is determined by restriction fragment length polymorphism analysis following treatment of CFTR gene sequence with at least one endonuclease restriction enzyme.

13. The method of claim 1 wherein the presence of said one or more mutations is determined by allele specific amplification.

14. The method of claim 1 wherein the presence of said one or more mutations is determined by primer extension.

15. The method of claim 1 wherein the presence of said one or more mutations is determined by oligonucleotide ligation.

16. The method of claim 1 wherein the presence of said one or more mutations is determined by hybridization with a detectably labeled probe containing the mutant sequence.

17. The method of claim 1 wherein the presence of said one or more mutations is determined by detecting the mutation in the encoded CFTR protein using an antibody with binding specificity for the mutated CFTR protein.

2. A method of identifying an individual that has one or more mutations in the cystic fibrosis transmembrane regulatory (CFTR) gene comprising determining if nucleic acid from the individual has one or more mutations in one or more CFTR genes, said mutations selected from the group consisting of 3443A>

T, 2443delA (A at position 2443 is deleted), 2777insTG (TG are inserted at position

     2777), 3123-3125delGTT (GTT at positions 3123-3125 are deleted), 4177delG (G at position 4177 is deleted), 630delG (G at position 630 is deleted), 2068G>

T, 1342−

2A>

G (A in the splice acceptor site of intron 8, 2 nucleotides upstream of position 1342, is substituted with G), 297−

1 G>

A (G in the splice acceptor site of intron 2, 1 nucleotide upstream of position 297, is substituted with A) 3500−

2A>

T (A in the splice acceptor site of intron 17b, 2 nucleotides upstream of position 3500, is substituted with T), 4375−

2A>

G (A in the splice acceptor site of intron 23, 2 nucleotides upstream of position 4375, is substituted with G), 3172-3174delTAC (TAC at positions 3172 to 3174 are deleted), 2902G>

C, 4115T>

C, 4185G>

C, 520C>

G, 842A>

C, 4528G>

T, 448A>

G, 574A>

T, 3704T>

C, 1248+5T>

C (T in the splice donor site of intron 7, 5 nucleotides downstream of position 1248, is substituted with C), 296+12T>

G (T in intron 2, 12 nucleotides downstream of position 296, is substituted with G), 3849+3G>

A (G in the splice donor site of intron 19, 3 nucleotides downstream of position 3849, is substituted with A), 497A>

G, −

141C>

A, 2875G>

C, 2689A>

G, 3039A>

G, 405G>

C, 886G>

A, 4445G>

A, −

228G>

C, −

295C>

T, −

379delC (C at position −

379 is deleted), and −

540A>

G.

3. (canceled)

4. (canceled)

18. A substantially purified nucleic acid comprising 8-20 nucleotides fully complementary to a segment of the cystic fibrosis transmembrane regulatory (CFTR) gene that is fully complementary to a portion of the CFTR gene and encompasses a mutant CFTR sequence selected from the group consisting of 3443A>

T, 2443delA (A at position 2443 is deleted), 2777insTG (TG are inserted at position

     2777), 3123-3125delGTT (GTT at positions 3123-3125 are deleted), 4177delG (G at position 4177 is deleted), 630delG (G at position 630 is deleted), 2068G>

T, 1342−

2A>

G (A in the splice acceptor site of intron 8, 2 nucleotides upstream of position 1342, is substituted with G), 297−

1 G>

A (G in the splice acceptor site of intron 2, 1 nucleotide upstream of position 297, is substituted with A) 3500−

2A>

T (A in the splice acceptor site of intron 17b, 2 nucleotides upstream of position 3500, is substituted with T), 4375−

2A>

G (A in the splice acceptor site of intron 23, 2 nucleotides upstream of position 4375, is substituted with G), 3172-3174delTAC (TAC at positions 3172 to 3174 are deleted), 2902G>

C, 4115T>

C, 4185G>

C, 520C>

G, 842A>

C, 4528G>

T, 448A>

G, 574A>

T, 3704T>

C, 1248+5T>

C (T in the splice donor site of intron 7, 5 nucleotides downstream of position 1248, is substituted with C), 296+12T>

G (T in intron 2, 12 nucleotides downstream of position 296, is substituted with G), 3849+3G>

A (G in the splice donor site of intron 19, 3 nucleotides downstream of position 3849, is substituted with A), 497A>

G, −

141C>

A, 2875G>

C, 2689A>

G, 3039A>

G, 405G>

C, 886G>

A, 4445G>

A, −

228G>

C, −

295C>

T, −

379delC (C at position −

379 is deleted), and −

540A>

G, or a complementary nucleic acid sequence thereof, wherein the purified nucleic acid is no more than 50 nucleotides in length.

19. The substantially purified nucleic acid of claim 18 wherein said nucleic acid is labeled with a detectable label.

20. The substantially purified nucleic acid of claim 19 wherein said detectable label is selected from the group consisting of a radioisotope, a dye, a fluorescent molecule, a hapten and a biotin molecule.