Method of detecting inherited equine myopathy
This disclosure describes detecting genetically distinct kinds of inherited myopathies in horses, variously referred to as Polysaccharide Storage Myopathy type 2 (PSSM2), Myofibrillar Myopathy (MFM), or idiopathic myopathy.
1 . A method of mating a horse comprising:
(a) detecting in a biological sample obtained from said horse, the biological sample comprising nucleic acids that include the coding regions for myotilin (MYOT), filamin-C(FLNC) and myozenin-3 (MYOZ3):
1) a guanine (G) or an adenine (A) at nucleotide 1001 of the forward strand of SEQ ID NO:1, wherein detection of the guanine indicates the presence of variant P2;
2) an adenine (A) or a guanine (G) at nucleotide 1001 of SEQ ID NO:2, wherein detection of the adenine indicates the presence of variant P3a,;
3) an adenine (A) or a guanine (G) at nucleotide 1001 of SEQ ID NO:3, wherein detection of the adenine indicates the presence of variant P3b; and
4) an adenine (A) or a guanine (G) at nucleotide 1001 of the forward strand of SEQ ID NO:4, wherein detection of the adenine indicates the presence of variant P4;
wherein in each case the presence of the specified nucleotide can be inferred from detecting the nucleotide present at the complement thereof;
wherein said horse is not homozygous for the presence of P2, P3a, P3b or P4;
wherein and said horse is heterozygous for at least one of P2, P3a, P3b or P4; and
(b) mating said horse with a mare or sire.
2 . The method according to claim 1 wherein said mare or sire is free from variants P2, P3a, P3b and P4.
3 . The method according to claim 1 wherein said mare or sire is heterozygous for variant P2, P3a, P3b or P4.
4 . The method of claim 1 , further comprising:
contacting the nucleic acids of the sample with at least one oligonucleotide probe to form a hybridized nucleic acid; and
amplifying the hybridized nucleic acid.
5 . The method of claim 4 , wherein:
Exon 6 of the equine myotilin coding region (MYOT) or a portion thereof is amplified; Exon 15 of the equine filamin-C coding region (FLNC) or a portion thereof is amplified; exon 21 of the equine filamin-C coding region (FLNC) or a portion thereof is amplified and exon 3 of the equine myozenin-3 coding region (MYOZ3), or a portion thereof is amplified.
6 . The method according to claim 5 , wherein the nucleic acid is genomic DNA.
7 . The method according to claim 5 , wherein the nucleic acid is RNA.
8 . The method according to claim 7 , wherein the method comprises converting the RNA into DNA by reverse transcriptase.
9 . The method of claim 4 , wherein the hybridized nucleic acid is amplified using polymerase chain reaction, strand displacement amplification, ligase chain reaction, or nucleic acid sequence-based amplification.
10 . The method according to claim 9 , wherein the nucleic acid is genomic DNA.
11 . The method according to claim 9 , wherein the nucleic acid is RNA.
12 . The method according to claim 11 , wherein the method comprises converting the RNA into DNA by reverse transcriptase.
13 . The method of claim 4 , wherein said at least one oligonucleotide probe is immobilized on a solid surface or a semisolid surface.
14 . The method according to claim 13 , wherein the nucleic acid is genomic DNA.
15 . The method according to claim 13 , wherein the nucleic acid is RNA.
16 . The method according to claim 15 wherein the method comprises converting the RNA into DNA by reverse transcriptase.
17 . A method according to claim 4 , wherein the nucleic acid is genomic DNA.
18 . The method according to claim 4 , wherein the nucleic acid is RNA.
19 . The method according to claim 18 , wherein the method comprises converting the RNA into DNA by reverse transcriptase.
20 . The method according to claim 1 , wherein the nucleic acid is genomic DNA.
21 . The method according to claim 1 , wherein the nucleic acid is RNA.
22 . The method according to claim 21 , wherein the method comprises converting the RNA into DNA by reverse transcriptase.
23 . The method according to claim 21 , wherein the method comprises
detecting in said nucleic acid:
(1) a cytosine (C) or a thymine (T) at nucleotide position 694 of SEQ ID NO: 5, wherein detection of the cytosine indicates the presence of variant P2;
(2) an adenine (A) for a guanine (G) at nucleotide position 2257 of SEQ ID NO: 6, wherein detection of the adenine indicates the presence of variant P3a;
(3) an adenine (A) for a guanine (G) at nucleotide position 3619 of SEQ ID NO: 6, wherein detection of the adenine indicates the presence of variant P3b, and
(4) a thymine (T) for cytosine (C) at nucleotide position 125 of SEQ ID NO: 8, wherein detection of the thymine indicates the presence of variant P4;
wherein in each case the presence of the nucleotide can be inferred from detecting the nucleotide present at the complement thereof;
wherein said horse is not homozygous for the presence of P2, P3a, P3b or P4;
wherein said horse is heterozygous for at least one of P2, P3a, P3b or P4; and
(b) mating said horse with a mare or sire.
24 . The method according to claim 23 wherein said mare or sire is free from variants P2, P3a, P3b and P4.
25 . The method according to claim 23 wherein said mare or sire is heterozygous for variant P2, P3a, P3b or P4.
26 . A method of mating a horse
(a) detecting in a biological sample obtained from said horse, the biological sample comprising nucleic acids that includes the coding regions for myotilin (MYOT), filamin-C(FLNC) and myozenin-3 (MYOZ3):
(1) a proline (P) or a serine(S) at position 232 of SEQ ID NO:10, wherein detection of proline indicates the presence of variant P2;
(2) a lysine (K) or a glutamate (E) residue at position 753 of SEQ ID NO: 13, wherein detection of proline indicates the presence of variant P3a,
(3) a threonine (T) or an alanine (A) at position 1207 of SEQ ID NO:13, wherein detection of threonine indicates the presence of of variant P3b and
(4) a leucine (L) or a serine(S) at position 42 of SEQ ID NO:16, wherein detection of leucine indicates the presence of variant P4, wherein said horse is not homozygous for the presence of P2, P3a, P3b or P4;
wherein said horse is heterozygous for at least one of P2, P3a, P3b or P4; and
(b) mating said horse with a mare or sire.
27 . The method according to claim 26 wherein said mare or sire is free from variants P2, P3a, P3b and P4.
28 . The method according to claim 26 wherein said mare or sire is heterozygous for variant P2, P3a, P3b or P4.