CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR GENE MUTATIONS

• US 20110082050A1
• Filed: 10/21/2010
• Published: 04/07/2011
• Est. Priority Date: 08/18/2005
• Status: Active Grant

• Current Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Original Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Inventors: Hantash, Feras
• Granted Patent: US 9,422,606 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/172 Haplotypes

Abstract

The present invention provides novel mutations of the CFTR gene related to cystic fibrosis or to conditions associated with cystic fibrosis. The mutations include duplication of exons including duplication of exons 6b through 10. Methods of identifying if an individual contains the exons 6b through 10 duplication are provided as well as nucleic acid fragments that contain the junction site of the duplicated segment. The detection of additional mutations in the CFTR gene are also provided.

Claims

1. A method of assessing whether an individual is predisposed to cystic fibrosis comprising:

(a) determining whether the individual is homozygous for a duplication of exons 6b through 10 in the cystic fibrosis transmembrane regulatory (CFTR) gene in a nucleic acid sample provided from the human, and

(b) identifying the individual as having a predisposition to cystic fibrosis when the human is homozygous.

2. The method of claim 1, wherein the presence of said duplication is determined using genomic DNA or cDNA.

3. The method of claim 1, wherein the presence of said duplication is determined using nucleic acid sequencing.

4. The method of claim 1, wherein the presence of said duplication is determined using allele specific amplification.

5. The method of claim 1, wherein the presence of said duplication is determined using primer extension.

6. The method of claim 1, wherein the presence of said duplication is determined using a detectably labeled probe.

7. The method of claim 1, wherein the detection step further comprises detecting the mutation in the encoded CFTR protein using an antibody with binding specificity for the mutated protein.

8. A method of identifying a human with an increased likelihood of having an offspring predisposed to cystic fibrosis, comprising:

(a) determining the presence or absence of a duplication of exons 6b through 10 in both alleles of the CFTR gene in the nucleic acid of the individual, and

(b) identifying the individual as having an increased likelihood of having an offspring predisposed to cystic fibrosis when the duplication is present in at least one allele.

9. The method of claim 8, wherein the presence of said duplication is determined using genomic DNA or cDNA.

10. The method of claim 8, wherein the presence of said duplication is determined using nucleic acid sequencing.

11. The method of claim 8, wherein the presence of said duplication is determined using allele specific amplification.

12. The method of claim 8, wherein the presence of said duplication is determined using primer extension.

13. The method of claim 8, wherein the presence of said duplication is determined using a detectably labeled probe.

14. The method of claim 8, wherein the detection step further comprises detecting the mutation in the encoded CFTR protein using an antibody with binding specificity for the mutated protein.

15. A method of assessing the cystic fibrosis status of an individual, comprising:

(a) determining the presence or absence of a duplication of exons 6b through 10 in both alleles of the CFTR gene in the nucleic acid of the individual, and

(b) identifying the individual

(i) as having cystic fibrosis or being predisposed to cystic fibrosis when the individual is homozygous for said duplication in the CFTR gene

(ii) as being a cystic fibrosis carrier when the individual is heterozygous for said duplication in the CFTR gene, or

(iii) as having no predisposition or carrier status caused by said duplication in the CFTR gene when said duplication is absent from both alleles of the CFTR gene,

16. The method of claim 15, wherein said determining step is accomplished by:

(i) amplifying nucleic acid from the individual, said amplified nucleic acid consisting of one or more of exon 6b, 7, 8, 9, and 10;

(ii) identifying the amplified exon sequence;

(iii) comparing the amount of each amplified exon sequence to the amount of similarly amplified wildtype exon sequence for a normal CFTR gene, wherein an exon duplication is indicated by a substantial increase in the amount of amplified exon sequence compared with the amount of amplified wildtype exon sequence.

17. The method of claim 16, wherein the presence of said duplication is determined using genomic DNA or cDNA.

18. The method of claim 16, wherein said amplifying step uses oligonucleotide primer pairs that hybridize upstream and downstream of said sequence, and at least one primer of each said primer pair is labeled with a detectable moiety.

19. The method of claim 16, wherein said amplifying step further comprises multiplex ligation-dependent probe amplification or multiplex amplifiable probe hybridization.

20. The method of claim 16, wherein said amplifying step further comprises amplifying across a junction site between exons 10 and 6b resulting from said duplication.

21. The method of claim 20 wherein said junction site is IVS10+2,987/IVS6a+415.