Erythrocytic pyruvate kinase mutations causing hemolytic anemia, osteosclerosis, and secondary hemochromatosis in dogs

Abstract

Background: Erythrocytic pyruvate kinase (PK) deficiency, first documented in Basenjis, is the most common inherited erythroenzymopathy in dogs.

Objectives: To report 3 new breed-specific PK-LR gene mutations and a retrospective survey of PK mutations in as mall and selected group of Beagles and West Highland White Terriers (WHWT).

Animals: Labrador Retrievers (2 siblings, 5 unrelated), Pugs (2 siblings, 1 unrelated), Beagles (39 anemic, 29 other),WHWTs (22 anemic, 226 nonanemic), Cairn Terrier (n = 1).

Methods: Exons of the PK-LR gene were sequenced from genomic DNA of young dogs (<2 years) with persistent highly regenerative hemolytic anemia.

Results: A nonsense mutation (c.799C>T) resulting in a premature stop codon was identified in anemic Labrador Retriever siblings that had osteosclerosis, high serum ferritin concentrations, and severe hepatic secondary hemochromatosis. Anemic Pug and Beagle revealed 2 different missense mutations (c.848T>C, c.994G>A, respectively) resulting in intolerable amino acid changes to protein structure and enzyme function. Breed-specific mutation tests were developed. Among the biased group of 248 WHWTs, 9% and 35% were homozygous (affected) and heterozygous, respectively, for the previously described mutation (mutant allele frequency 0.26). A PK-deficient Cairn Terrier had the same insertion mutation as the affected WHWTs. Of the selected group of 68 Beagles, 35% were PK-deficient and 3% were carriers (0.37).

Conclusions and clinical importance: Erythrocytic PK deficiency is caused by different mutations in different dog breeds and causes chronic severe hemolytic anemia, hemosiderosis, and secondary hemochromatosis because of chronic hemolysis and, an as yet unexplained osteosclerosis. The newly developed breed-specific mutation assays simplify the diagnosis of PK deficiency.

Fig 1

Histopathology of liver and hepatic lymph node from PK-deficient Labrador (Lab 2). (A) Perl’s iron stain of hepatic parenchyma at 20× and (B) at 40×, shows accumulation of dark blue granules of hemosiderin within Kupffer cells and blue tint of iron within hepatocytes. (C) Hematoxylin and eosin of hepatic lymph node (100×), with medullary cords expanded by large numbers of macrophages that contain coarse, dark brown refractile granules (hemosiderin) and intracytoplasmic fragments of erythrocytes (erythrophagocytosis). Scattered foci of extramedullary hematopoiesis are also present.

Fig 2

Sequence tracings of gDNA regions in exons 7 and 8 of the canine PK-LR gene. The wild-type sequence is compared with sequences from a PK-deficient Labrador (A), Pug (B), and Beagle (C). Mutation positions are indicated with an arrow on the coding strand scale and the codon with the mutation is underlined for each breed.

Fig 2

Sequence tracings of gDNA regions in exons 7 and 8 of the canine PK-LR gene. The wild-type sequence is compared with sequences from a PK-deficient Labrador (A), Pug (B), and Beagle (C). Mutation positions are indicated with an arrow on the coding strand scale and the codon with the mutation is underlined for each breed.

Fig 2

Sequence tracings of gDNA regions in exons 7 and 8 of the canine PK-LR gene. The wild-type sequence is compared with sequences from a PK-deficient Labrador (A), Pug (B), and Beagle (C). Mutation positions are indicated with an arrow on the coding strand scale and the codon with the mutation is underlined for each breed.

Fig 3

Amino acid sequence homology among mammals and the sites of canine R-PK mutations. The analysis shows that the R-PK amino acid sequences are highly conserved among dog, human, chimpanzee, cow, mouse, and rat at 3 mutation positions (267-Labrador, 283-Pug, and 332-Beagle). The conserved areas around the mutations are shaded, and the active sites are shown in bold capital letters. The polymorphism [isoleucine (I)] at position 283 for the mouse and rat, is a tolerated amino acid change by the SIFT protein prediction analysis.

Fig 4

Labrador and Pug mutations analyzed by restriction enzyme digestion followed by electrophoresis on polyacrylamide gel (6%). Lanes: M: 100 bp DNA marker: UL and UP: uncut fragment from affected Labrador (Lab 1) and Pug (Pug 1), respectively; 1 and 12: healthy control Labrador; 2 and 13: affected Lab 1 (proband); 3: affected Lab 2; 4 and 11: healthy control of different breed; 5 and 8: healthy control Pug; 6 and 9: affected Pug 1 (proband); 7 and 10: carrier Pug; B: blank. Lanes UL and UP at 188 bp are the PCR amplification products of exon 7 from affected Labrador and Pug dogs before digestion. Products are digested with MwoI for Labradors, which cuts the wild-type allele twice producing 3 fragments of 96, 46, and 46 bp (lanes 1). Because of the 3 base 3′ overhangs, the center 46 bp fragment has 3 bp overhangs on both sides thus migrates at slower pace and appears slightly larger. The affected Lab 1 and Lab 2 are cut only once by MwoI resulting in 2 DNA fragments of sizes 142 and 46 bp (lanes 2, 3). The digestion of the PCR products with PFlFI for the Pug produces two bands of 141 and 47 bp for the normal allele (lane 8), however, the mutant allele is missing the restriction site and does not get cleaved, thus showing the 188 bp uncut DNA band (lane 9). The carrier Pug shows both mutant and wild-type alleles and thus has 3 bands (lane 10). Amplification products from different breeds of dogs (lanes 4,11) show only the wild-type allele digestion pattern. Digesting the PCR products from Labradors with PFlFI and vice-versa for the Pug validates the specificity of the restriction enzyme digests: Pug samples (lanes 5, 6, 7) for Labrador digest and Labrador samples (lanes 12, 13) for Pug digest.

Fig 5

Amplification products from healthy and PK-deficient Beagles analyzed by restriction enzyme digestion followed by electrophoresis on polyacrylamide gel (6%). Lanes: M: 100 bp DNA marker; 1 and 2: Normal and affected controls, respectively; 3 and 4: anemic Beagles in duplicates: N: Normal: A: Affected. The restriction digest with NgoMIV cuts the 109 bp wild-type allele into 90 and 19 bp fragments; however, the mutant allele is not digested thus only shows the uncut band at 109 bp. The 19 bp fragment is indistinguishable from primers and therefore is not shown in this figure.

Fig 6

Structure of the canine PK-LR gene, transcripts of liver (NM_001256018.1) and erythrocyte (NM_001256262.1) splicing variants and the known breed specific PK mutation sites. Although the PK-LR gene has 12 exons, the different tissue-specific splicings reduce the exon count to 11 for both L-PK and R-PK. Exons and introns are drawn to approximate scale. Exons are numbered and depicted by rectangles. The 5′ and 3′ gray regions in the L-PK transcript are untranslated regions of that splice variant as per NCBI. Arrows indicate the locations of 5 mutations associated with PK deficiency, including the single bp deletion in exon 5 in the Basenji, and the 6 bp insertion in exon 10 in the WHWT and Cairn terrier, and the newly identified nonsense mutation in the Labrador (c.799C>T) and missense mutations in Pug (c.848T>C) and Beagle (c.994G>A).