Centronuclear myopathy in labrador retrievers: a recent founder mutation in the PTPLA gene has rapidly disseminated worldwide

Centronuclear myopathies (CNM) are inherited congenital disorders characterized by an excessive number of internalized nuclei. In humans, CNM results from ~70 mutations in three major genes from the myotubularin, dynamin and amphiphysin families. Analysis of animal models with altered expression of these genes revealed common defects in all forms of CNM, paving the way for unified pathogenic and therapeutic mechanisms. Despite these efforts, some CNM cases remain genetically unresolved. We previously identified an autosomal recessive form of CNM in French Labrador retrievers from an experimental pedigree, and showed that a loss-of-function mutation in the protein tyrosine phosphatase-like A (PTPLA) gene segregated with CNM. Around the world, client-owned Labrador retrievers with a similar clinical presentation and histopathological changes in muscle biopsies have been described. We hypothesized that these Labradors share the same PTPLA<sup>cnm</sup> mutation. Genotyping of an international panel of 7,426 Labradors led to the identification of PTPLA<sup>cnm</sup> carriers in 13 countries. Haplotype analysis demonstrated that the PTPLA<sup>cnm</sup> allele resulted from a single and recent mutational event that may have rapidly disseminated through the extensive use of popular sires. PTPLA-deficient Labradors will help define the integrated role of PTPLA in the existing CNM gene network. They will be valuable complementary large animal models to test innovative therapies in CNM

A 3.8-Mb haplotype is highly associated with CNM.

<p>The acrocentric region of the <i>PTPLA</i> locus within canine chromosome 2 (CFA2) is depicted. Positions of genotyped SNPs are indicated. The short and long haplotypes associated with CNM are shown in green and red, respectively. For each SNP, the allele detected in the CNM associated haplotype is indicated and represented as a grey box. The alternative allele is represented as a white box. For each SNP, the minor allele frequency (MAF) in the healthy population of Labradors is given. The <i>PTPLA^cnm</i> allele is represented by a black dot (•) and the wild-type <i>PTPLA⁺</i> allele by a “+”. Frequencies of long 3.8-Mb haplotypes in each population of CNM or healthy dogs are given below each haplotype. For haplotypes with frequencies >10%, width of haplotypes is proportional to its frequency. Haplotypes with frequencies below 3% have been omitted and are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046408#pone.0046408.s003" target="_blank">Figure S3</a>.</p

Number of genotyped dogs used in this study.

<p>The whole panel includes all dogs for which samples were received for testing purposes. The initial confirmation panel includes dogs with an early diagnosis of HLMR or phenotypically similar myopathies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046408#pone.0046408.s004" target="_blank">Table S1</a>).</p

<i>PTPLA</i> mutation in the initial confirmation panel of CNM dogs.

<p>(A) Wild-type (wt = 610 bp) and <i>PTPLA^cnm</i> (<i>cnm</i> = wt+238 bp) alleles in a healthy carrier (FR-1) and an affected (FR-2) Labrador from the French experimental pedigree. (B) Segregation of the <i>PTPLA^cnm</i> allele in a four-generation pedigree of a client-owned US proband female (arrow). (C) Genotypes of client-owned Labradors from several countries, diagnosed with CNM-related myopathies (asterisks; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046408#pone.0046408.s004" target="_blank">Table S1</a>). US-6 is a champion known to have produced CNM pups; DE-5 is a control affected by a neuropathy and FR-2 was reloaded for size comparison.</p

Percentage of wild-type homozygous (+/+), healthy carriers (+/<i>cnm</i>) and CNM affected (<i>cnm</i>/<i>cnm</i>) Labradors tested for medical or breeding purposes.

<p>The total number of dogs for each period is indicated above histograms. Additional dog samples from Australia (n = 15), New-Zealand (n = 2), Puerto Rico (n = 1) and Argentina (n = 1) were tested; they were all homozygous for the wild-type allele (+/+).</p

Hierarchical clustering from the 81 dogs at k = 8.

<p>The analysis was based on genotypes obtained for ten loci (SNP 20687 to SNP 24518). Hubert Gamma values are indicated for k≥2 on the top left panel. The scale on the right axis represents the genetics distances calculated by PLINK software. In the dendogram, each vertical line represents a dog and colors reflect the eight clusters obtained by the analysis. Grey dash lines indicate common ancestors inferred from the analysis. Below the dendogram, dogs are named by their unique identifier. The “CNM_” prefix was added to the name of affected Labradors.</p

Client-owned US Labradors share similar morphological and histopathological features with French CNM dogs from the experimental pedigree.

<p>French CNM (A–C) and US HMLR (D–F) affected dogs have atrophic skeletal muscles, the most affected being those of pelvic limbs (e.g. <i>biceps femoris</i> muscle, arrows in A,D). B,C,E,F are Hematoxylin-Eosin-stained transverse sections of the <i>biceps femoris</i> muscle from 6-month-old (B, FR-4; E, US-18) or 10-year-old (C,F) affected Labradors. Early signs include groups of atrophic fibers, surrounded by endomysial (e) and perimysial (p) fibrosis. In older dogs, increased internalized or centralized nuclei (asterisks) and fatty infiltration (f) are observed. Scale bar = 50 µm.</p