Precision medicine using whole genome sequencing identifies a novel dystrophin ( DMD ) variant for X-linked muscular dystrophy in a cat

Figures

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  FIGURE 1. A 1‐year‐old male domestic shorthaired cat with suspected muscular dystrophy. The cat had a 6‐month history of difficulty walking, prominent hypertrophy of the neck muscles (left), progressive dysphagia and a thickened tongue (right). The cat also could not close its mouth.

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  FIGURE 2. Immunofluorescent staining of cryosections from the trapezius muscle of the dystrophic cat and archived control vastus lateralis muscle was performed for localization of dystrophy‐associated proteins. Antibodies against dystrophy‐associated proteins included those against the rod (DYS1) and carboxy terminus (DYS2) of dystrophin, utrophin (DRP2), developmental myosin heavy chain (dMHC) to demonstrate regenerating fibers, and against laminin α2 and collagen 6. Protein localization for both DYS1 and DYS2 could not be detected and utrophin was increased compared to archived control muscle. Regenerating fibers were highlighted by staining for dMHC. Staining for both laminin α2 and collagen 6 was like control tissue. Bar in lower right image equals 50 μm for all images.

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  FIGURE 3. Electropherograms for DMD Sanger sequencing. 1: Control cat—Fcat_4406 coding strand; 2: Control cat—Fcat_4406 complementary strand; 3: Control cat—Fcat_12682 coding strand; 4: Control cat—Fcat_12682 complementary strand; 5: Case cat—Fcat_23286 coding strand; 6: Case cat—Fcat_23286 complementary strand; 7: Sibling cat—Fcat_24202 coding strand. Box indicates region where the case cat is hemizygous for the c.4849C>T variant. Cytosine (C) are blue peaks and thymine (T) are red peaks.

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  FIGURE 4. Multispecies alignment of Felis catus DMD exon 35. The alignments indicate the position of the DMD stop gain LoF variant (XM_023249210.1, ENSFCAT00000068370:c.4849C>T; p.Gln1617Ter) at chromosome position X:28201541 Felis_catus_9.0 at the beginning of exon 35. (A) Nucleotide alignment for diverse species (DMD affected feline case, normal feline, human, canine, bovine, porcine, murine, and zebrafish) indicating the 100% conservation of the nucleotide and the high conservation of the exon 35, lower histogram. Nucleotides are represented by the different colors. (B) Protein alignments for the same species depicting the termination in exon 35 for the affected cat. The alanine that is 2 amino acids upstream of the amber termination codon is the 5′ start of exon 35. The mammals have nearly 100% amino acid conservation within this region.