Association of the <i>MKLN1</i>:c.400+3A>C genotype with the LAD phenotype.

<p>Association of the <i>MKLN1</i>:c.400+3A>C genotype with the LAD phenotype.</p

Sanger confirmation of the <i>MKLN1</i>:c.400+3A>C variant.

<p><b>(A)</b> Electropherograms from dogs with the three different genotypes. <b>(B)</b> Wildtype and mutant allele compared to the consensus sequence for the human U2 GT-AG type 5’-splice sites [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007264#pgen.1007264.ref013" target="_blank">13</a>]. Subscript numbers in the consensus sequence indicate the percentage of the respective conserved nucleotide in 183,682 investigated human 5’-splice site motifs of the U2 GT-AG type. The additional difference to the optimal consensus in the U1 spliceosomal RNA recognition site in the mutant allele is highlighted in red. In human 5’-splice sites the most frequent base at position 3 is an A (60%). G is also common at this position (35%), while C and T are both rare (<3%) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007264#pgen.1007264.ref013" target="_blank">13</a>].</p

Association of the <i>MKLN1</i>:c.400+3A>C genotype with the LAD phenotype.

<p>Association of the <i>MKLN1</i>:c.400+3A>C genotype with the LAD phenotype.</p

LAD phenotype.

<p><b>(A)</b> Inflammatory skin lesions in the face of an affected Bull Terrier. <b>(B)</b> Similar lesions in the inguinal region. <b>(C)</b> LAD affected puppy in the middle of two non-affected littermates. A pronounced growth delay and a subtle coat color dilution are visible. <b>(D, E)</b> Fore paws of an LAD affected Bull Terrier puppy at necropsy. Symmetrical scaling and crusting of the skin including interdigital areas and foot pads is visible <b>(F, G)</b> Histopathological micrographs of the junction of interdigital haired skin and digital pad from an affected Bull Terrier puppy <b>(F)</b> and a control dog <b>(G)</b>. Marked thickening of the epidermis, excessive layers of non-cornifying epithelium and a large pustule are evident in the affected dog. Hematoxylin-eosin, bar = 400 µm.</p

Experimental verification of the <i>MKLN1</i> splice defect.

<p><b>(A)</b> The genomic organization of the <i>MKLN1</i> gene. Exons 2–6 are enlarged and the position of the primers used for RT-PCR is indicated. <b>(B)</b> RT-PCR was performed using skin cDNA from a control and an LAD affected Bull Terrier. The picture shows a Fragment Analyzer gel image of the experiment. In the control animal, only the expected 366 bp product is visible. In the LAD affected dog, a 277 bp product representing a transcript lacking exon 4 is visible. The identity of the bands was verified by Sanger sequencing. Thus, the <i>MKLN1</i>:c.400+3A>C variant leads to complete skipping of exon 4 (<i>MKLN1</i>:r.312_400del89).</p