Screening for Arthrogryposis Multiplex in Bovines

• US 20110151440A1
• Filed: 12/18/2009
• Published: 06/23/2011
• Est. Priority Date: 12/18/2009
• Status: Active Grant

• Current Assignee: Agrigenomics, Inc.
• Original Assignee: Agrigenomics, Inc.
• Inventors: BEEVER, Jonathan Edward, MARRON, Brandy Michele
• Granted Patent: US 8,431,346 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 2600/124 Animal traits, i.e. production traits, including athletic performance or the like

Abstract

Provided are methods, materials and kits for analyzing DNA samples from bovine to determine whether the animal is a recessive carrier of a genetic mutation that is associated with arthrogryposis multiplex (AM). DNA-containing samples are analyzed by genetic testing to determine whether or not a deletion mutation is present in one of the alleles that are responsible for the AM genetic mutation. In an aspect the deletion encompasses the entirety of the ISG15 ubiquitin-like modifier (ISG15) gene. In an aspect the deletion further encompasses one or both of the 5′ regulatory region of the hairy and enhancer split 4 (HES4) and of the agrin (AGRN) gene and of the first two exons of the AGRN gene.

Claims

1. A method of diagnosing a mutation in a bovine genome, wherein said mutation is associated with Arthrogryposis Multiplex (AM), said method comprising:

obtaining a DNA sample from the bovine; and

analyzing said DNA sample to determine the presence or absence of a deletion mutation for one or more of an AGRN gene, ISG15 gene or HES4 gene,

said DNA having an upstream region, a downstream region, and a middle region that separates said upstream region from said downstream region indicates absence of the deletion mutation; and

said DNA having said upstream region and said downstream region in a contiguous configuration is identified as having the deletion mutation.

2. The method of claim 1, wherein said deletion mutation results in loss of function of one or more of the AGRN, ISG15 or HES4 gene products.

3. The method of claim 1, wherein said middle region comprises the sequence of SEQ ID NO:

3.

4. The method of claim 3, wherein said middle region is SEQ ID NO:

4 or SEQ ID NO;

5.

5. The method of claim 1, wherein said upstream region comprises the sequence from base 1 to 6491 of SEQ ID NO:

1 (cross-referenced as SEQ ID NO;

6), from base 1 to 6507 of SEQ ID NO;

1 (cross-referenced as SEQ ID NO;

7), from base 1872 to base 6491 of SEQ ID NO;

1 (cross-referenced as SEQ ID NO;

16), or from base 1872 to base 6507 of SEQ ID NO;

1 (cross-referenced as SEQ ID NO;

17).

6. The method of claim 1, wherein said downstream region comprises the sequence from base 29871 to 35035 of SEQ ID NO:

1 (cross-referenced as SEQ ID NO;

8), from base 29855 to 35035 of SEQ ID NO;

1 (cross-referenced as SEQ ID NO;

9), or from base 29871 to 30234 of SEQ ID NO;

1 (cross-referenced as SEQ ID NO;

19).

7. The method of claim 1, wherein the step of analyzing the DNA sample is by polymerase chain reaction (PCR).

8. The method of claim 7, wherein the step of PCR further comprises:

providing a forward primer which binds specifically to a first selected DNA upstream region, said first selected DNA upstream region corresponding to between bases 3000 and 6491 of SEQ ID NO;

1;

providing a reverse primer which binds specifically to a second selected DNA downstream region, said selected DNA downstream region corresponding to between bases 29871 and 35035 of SEQ ID NO;

1; and

performing PCR amplification such that said forward and reverse primer generate an amplified DNA product only in the presence of a DNA sample comprising the deletion mutation associated with AM.

9. The method of claim 8, wherein the forward primer has a sequence that is SEQ ID NO:

11 and the reverse primer has a sequence that is a reverse complement to SEQ ID NO;

12 (cross-referenced as SEQ ID NO;

13).

10. The method of claim 8 further comprising providing a third primer, said third primer capable of specific binding to at least a portion of the middle region of DNA, said middle region corresponding to a DNA sequence encompassed by bases 6507 and 7500 of SEQ ID NO:

1, wherein DNA that contains said middle region results in amplification of a first DNA product by said forward primer and said third primer, and wherein DNA that does not contain said middle region results in amplification of a second DNA product by said forward primer and said reverse primer.

11. The method of claim 10, wherein the third primer has a sequence that is a reverse complement of SEQ ID NO:

14 (cross-referenced as SEQ ID NO;

15).

12. The method of claim 10, wherein said amplified first DNA product and second DNA product have lengths that are at least 10% different, said method further comprising:

determining the length of the amplified DNA product; and

identifying the presence or absence of the AM mutation based on said determined length.

13. The method of claim 7 further comprising providing a forward primer and a reverse primer, wherein said forward primer is capable of specific binding to said middle region of the DNA, and said reverse primer is capable of specific binding to said middle region of the DNA, wherein said binding regions are different and the primers are capable of generating an amplification DNA product only if the DNA sample does not comprise a mutation associated with AM.

14. The method of claim 1 further comprising

providing a probe or primer that specifically binds to the sample of DNA having said upstream region and said downstream region in a contiguous configuration;

and

identifying the sample as containing a mutation gene associated with AM for DNA samples that hybridize with said probe or primer.

15. The method of claim 1, wherein the analyzing the DNA sample further comprises providing a DNA probe or primer, wherein said DNA probe or primer specifically binds to said middle region.

16. The method of claim 1, wherein said analyzing the DNA sample comprises DNA sequencing.

17. The method of claim 1, wherein said analyzing the DNA sample comprises providing a probe or primer comprising a purified oligonucleotide, wherein the oligonucleotide has a length between about 15 to 50 nucleotides and specifically binds to a target sequence of SEQ ID NO:

3 or complement thereof without binding to SEQ ID NO;

7 or SEQ ID NO;

9.

18. The method of claim 1, wherein said analyzing the DNA of the bovine subject comprises providing a probe or primer comprising a purified oligonucleotide, wherein the oligonucleotide has a length between about 15 to 50 nucleotides and specifically binds a breakpoint mutation region in the bovine genome, said breakpoint located between bases T429 and G430, 445A and 446C, or both of SEQ ID NO:

2.

19. The method of claim 1, wherein the bovine is an Angus or Angus composite.

20. The method of claim 1, wherein the DNA sample is obtained from blood or semen.

21. A method for screening a bovine to determine if said bovine carries a mutation for AM, said method comprising:

providing a biological sample which was removed from said bovine to be screened; and

conducting a biological assay to determine the presence of a mutation in the gene responsible for AM, wherein the mutation comprises a deletion of bases 6507 to 29870 in SEQ ID NO;

1 (cross-referenced as SEQ ID NO;

3).

22. An oligonucleotide for diagnosing heterozygous carriers of AM, wherein the oligonucleotide has a length of 15 to 80 nucleotides, and having a region, wherein the region is identical or a reverse complement to a sequence of an at least fifteen-nucleotide segment of SEQ ID NO:

1 or SEQ ID NO;

2.

23. The oligonucleotide of claim 22, wherein said oligonucleotide is DNA.

24. The oligonucleotide of claim 22, wherein the region is identical to the portion of SEQ ID NO:

2 comprising bases 429 and 430 or bases 445 and 446.

25. A kit for assaying for the presence of an AM marker in a bovine DNA, said kit comprising oligonucleotide sequences of claim 22 for PCR priming or probing for the presence or absence of a deletion mutation in the bovine genome associated with AM.

26. The kit of claim 25 wherein the oligonucleotide sequences comprise:

a first primer that specifically binds an upstream region, wherein said upstream region comprises at least a portion of bases 5000 to 6491 of SEQ ID NO;

1, and wherein said upstream region has an ending at base number 6491 or 6507 of SEQ ID NO;

1; and

a second primer that specifically binds a downstream region, wherein said downstream region comprises at least a portion of bases 29,871 to 31,000 of SEQ ID NO;

1, and wherein said downstream region has a beginning corresponding to base 29,855 or 29,871 of SEQ ID NO;

1,

wherein generation of an amplified DNA product by hybridization of the first primer and the second primer to said DNA indicates presence of an AM marker.

27. The kit of claim 26, further comprising:

a third primer that specifically binds a middle region, wherein said middle region comprises at least a portion of bases 6507 to 29,886 or 6491 to 29,870 of SEQ ID NO;

1.

28. The kit of claim 25 further comprising a positive control and a negative control, wherein said positive control comprises a functional fragment of the DNA sequence of SEQ ID NO:

2, said sequence comprising a breakpoint region located between bases 429 and 430 or between bases 445 and 446 of SEQ ID NO;

2, and wherein said negative control comprises a functional fragment of the DNA sequence of SEQ ID NO;

1, said fragment comprising bases 6491 to 29,871 of SEQ ID NO;

1.