An EHPB1L1 Nonsense Mutation Associated with Congenital Dyserythropoietic Anemia and Polymyopathy in Labrador Retriever Littermates

Abstract

In this report, we describe a novel genetic basis for congenital dyserythropoietic anemia and polymyopathy in Labrador Retriever littermates characterized by incidental detection of marked microcytosis, inappropriate metarubricytosis, pelvic limb weakness and muscle atrophy. A similar syndrome has been described in English Springer Spaniel littermates with an early onset of anemia, megaesophagus, generalized muscle atrophy and cardiomyopathy. Muscle histopathology in both breeds showed distinctive pathological changes consistent with congenital polymyopathy. Using whole genome sequencing and mapping to the CanFam4 (Canis lupus familiaris reference assembly 4), a nonsense variant in the EHBP1L1 gene was identified in a homozygous form in the Labrador Retriever littermates. The mutation produces a premature stop codon that deletes approximately 90% of the protein. This variant was not present in the English Springer Spaniels. Currently, EHPB1L1 is described as critical to actin cytoskeletal organization and apical-directed transport in polarized epithelial cells, and through connections with Rab8 and a BIN1-dynamin complex generates membrane vesicles in the endocytic recycling compartment. Furthermore, EHBP1L1 knockout mice die early and develop severe anemia. The connection of EHBP1L1 to BIN1 and DMN2 functions is particularly interesting due to BIN1 and DMN2 mutations being causative in forms of centronuclear myopathy. This report, along with an independent study conducted by another group, are the first reports of an association of EHBP1L1 mutations with congenital dyserythropoietic anemia and polymyopathy.

Keywords: anemia; animal model; dog; muscle; myopathy; whole genome sequencing.

Figure 1

Review of histopathological changes in red blood cells, bone marrow and muscle in Labrador Retrievers with an EHBP1L1 variant. (A) Representative photomicrograph of peripheral blood. Note the marked poikilocytosis characterized by schistocytes, microcytes and acanthocytes. A single metarubricyte is shown. (B) Representative photomicrograph of bone marrow findings displaying cytoplasmic bridging (arrow). (C) Representative photomicrograph of bone marrow displaying atypical mitotic figure (arrow). (D) Representative photomicrograph of bone marrow aspirate displaying rubriphagocytosis (arrow) and increased proportion of metarubricytes. Wrights Giemsa Stain used for (A–D). Bar = 10 µm for (A–D). Cryosections from the triceps were stained with the mitochondrial specific reactions cytochrome C oxidase (E) and succinic dehydrogenase (F). These images show variability in myofiber size, central accumulations of dark brown or dark blue reactivity (arrowheads, arrows) and central nuclei.

Figure 2

Structure of the EHBP1L1 gene and site of the mutation. All 19 exons are filled in gray or blue. The premature stop codon mutation in exon 5 is marked by a red line. The wild-type protein will contain 19 exons (i.e., all blue and gray exons), while the mutant protein, if produced, would contain only exons 1–4 and part of exon 5 (i.e., the blue ones). The locations of the predicted homology domains, in reference to the human EHBP1L1 protein, are also indicated. A comparative amino acid sequence analysis determined a 70.3% identity between the canine and human orthologues.

Figure 3

Examples of haplotypes encompassing the EHBP1L1 gene. Haplotypes were generated from 144 SNPs within 1 Mb flanking the EHBP1L1 gene. The gene location is denoted by a bolded yellow box at the top. Each row represents a haplotype obtained from the dog identified on the left. Each cell represents a SNP and the CFA18 positions of the 5′ and 3′ SNPs are indicated at the top; the mutation position is also indicated. The haplotype (highlighted in orange) that contains the EHBP1L1 mutation (highlighted in red) is present as two copies (i.e., two rows) in the case. An identical 144 SNP haplotype was found as a single copy in twelve of the controls. These haplotypes were subsequently shown to differ from that in the case by having a reference allele (denoted as blue). Several other haplotypes containing a segment of the affected haplotype were also observed in the controls. These haplotypes also contained the reference allele.