Veterinary Clinical Pathology ISSN 0275-6382
BRIEF COMMUNICATION
A novel point mutation in the b1-tubulin gene in asymptomatic macrothrombocytopenic Norfolk and Cairn Terriers Maria Elena Gelain1, Walter Bertazzolo2, Giuseppina Tutino2, Elena Pogliani2, Francesco Cian3, Mary K. Boudreaux4 1
Department of Comparative Biomedicine and Food Science, Faculty of Veterinary Medicine, University of Padua, Padua, Italy; 2 Veterinary Clinic “Tibaldi”, Milan, Italy; 3Department of Veterinary Medicine, University of Cambridge, Cambridge, UK; and 4Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL, USA
Key Words Dog, GTP binding, platelets, single nucleotide polymorphism Correspondence M.E. Gelain, Department of Comparative Biomedicine and Food Science, Faculty of Veterinary Medicine, AGRIPOLIS – Viale dell’Universit a 16, 35020 Legnaro (PD), Italy E-mail: [email protected] DOI:10.1111/vcp.12168
Background: An asymptomatic macrothrombocytopenia, phenotypically similar to asymptomatic inherited macrothrombocytopenia in Cavalier King Charles Spaniels, was described in a group of Norfolk Terriers (NT) from Northern Italy, and isolated cases were also reported in Cairn Terriers (CT). Objectives: The purpose of this work was to evaluate for the presence of a genetic defect in the b1-tubulin gene in macrothrombocytopenic NT and CT. Methods: Samples from 20 healthy dogs (13 NT and 7 CT) were collected at different institutions in Italy (n = 8), United Kingdom (n = 3), and United States (n = 9). Genomic DNA was harvested from EDTA-anticoagulated blood and all coding areas and exon–intron splice sites in the gene encoding b1-tubulin were amplified and sequenced. Results: Twelve dogs (9 NT and 3 CT) showed a single nucleotide polymorphism (SNP) in exon 1 at nucleotide position 5 (G5A) that would result in the change of an arginine to a histidine at amino acid position 2 (R2H). Four dogs (3 NT and one Cairn Terrier) were heterozygous for the SNP, and 4 dogs (one Norfolk Terrier and 3 CT) matched the normal canine genome. Homozygous dogs for the SNP were macrothrombocytopenic with platelet counts ranging from 19,000 to 110,000/lL. Heterozygous and normal dogs had normal platelet counts and morphology. None had the CKCS point mutation. Conclusions: The b1-tubulin N-terminal amino acids form the nucleotidebinding domain and thus this mutation could affect GTP binding enough to influence platelet formation in homozygous but not in heterozygous dogs. The presence of macrothrombocytopenia only in homozygous affected dogs reveals an association between the SNP and the phenotype.
Platelets are the first line of defense against bleeding secondary to vascular injury, and anomaly in their number or function may lead to mucocutaneous bleeding. Similar to that observed in people, inherited thrombopathias and thrombocytopenia have been reported in animals. The severity of bleeding observed with inherited thrombopathias varies as a reflection of their molecular basis and ensuing phenotype.1 However, not all the inherited intrinsic platelet disorders cause hemorrhage. Asymptomatic inherited macrothrombocytopenia is a well-recognized condition in Cavalier King Charles Spaniels (CKCS), due to a point mutation at coding nucleotide 745 (G745A) in the
b1-tubulin gene, resulting in the change of an aspartic acid to an asparagine at amino acid 249 (D249N). This mutation results in impaired microtubule assembly affecting proplatelet formation and platelet production by megakaryocytes.2 Hemostatic function, however, is not altered presumably due to the presence of a normal circulating platelet mass indicated by the measurement of a normal plateletcrit in affected dogs.3,4 The prevalence of asymptomatic macrothrombocytopenia in CKCS is very high with more than 90% of dogs being either carriers or affected.1 Several other breeds as well as mixed-breed dogs have also been identified with this mutation since it was
Vet Clin Pathol 43/3 (2014) 317–321 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
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b1-Tubulin mutation in Norfolk and Cairn Terriers
first reported in CKCS. Previous studies found an autosomal recessive manner of inheritance for this condition.5 However, heterozygous dogs often have platelet numbers between 100,000 and 200,000/lL, suggesting an autosomal dominant inheritance.1 Recently, a phenotypically similar macrothrombocytopenia was described in a group of related Norfolk Terriers (NT) from Northern Italy6, and isolated cases of asymptomatic macrothrombocytopenia were also reported in Cairn Terriers (CT).1 The purpose of this work was to evaluate for the presence of a genetic defect in the b1-tubulin gene in macrothrombocytopenic NT and CT in different countries. Peripheral blood samples from 20 healthy NT (n = 13) and CT (n = 7) were collected at different institutions in Italy (n = 8, Department of Veterinary Pathology, Hygiene and Health, University of Milan, from 2008 to 2012), United Kingdom (n = 3, Department of Veterinary Medicine, University of Cambridge, from 2010 to 2012), and United States (n = 9, Department of Pathobiology, College of Veterinary Medicine, Auburn University). There were 11 female (7 intact and 4 neutered) and 9 male (7 intact and 2 neutered) dogs with a mean age of 6 years (min– max 0.8–14). The animals were sampled during the annual visit to a veterinarian for evaluation of their general health status and an informed consent was obtained from all owners. Included in the study were clinically healthy dogs, without alteration in CBC or biochemical profile. Excluded from the analysis were samples from dogs with signs of primary hemostatic dysfunction and/or with thrombocytopenia confirmed by platelet estimation on a peripheral blood smear and after excluding platelet aggregates and/or macroplatelets. CBCs were performed with a Sysmex XT-2000iV (Dasit group S.p.A., Milan, Italy) in Italy and with an ADVIA 120 (Siemens Medical Solutions USA Inc, Malvern, PA, USA) at Auburn University. Blood smears were routinely stained with May–Grunwald–Giemsa or Wright–Giemsa and were examined microscopically to evaluate platelet morphology and for the presence of platelet aggregates.
Gelain et al
Genomic DNA was harvested from EDTA-anticoagulated blood using a commercially available kit (QIAamp DNA Blood Mini Kit; Qiagen Inc, Valencia, CA, USA). Primers were designed to assess all coding areas and exon–intron splice sites in the gene encoding b1-tubulin based on the sequence obtained from the canine genome available on GenBank (Figure 1). The PCR conditions were: annealing at 63°C for 30 s and 40 cycles; the expected product size was 473 bp. PCR products were subjected to electrophoresis on agarose gels and target bands were harvested (QIAquick Gel Extraction Kit; Qiagen Inc) for sequencing (ABI 3100 Genetic Analyzer; Applied Biosystems, Foster City, CA, USA). All sampled dogs were clinically healthy and did not have any history or signs of hemostatic dysfunction. None of the samples had the point mutation reported in CKCS. Twelve dogs (9 NT and 3 CT) showed a single nucleotide polymorphism (SNP) in exon 1 at nucleotide position 5 (G5A). This mutation would result in the change of an arginine to a histidine at amino acid position 2 (R2H). Four dogs (3 NT and one CT) were heterozygous for the SNP and 4 dogs (one NT and 3 CT) had a normal sequence (Figure 2). All dogs with the SNP were macrothrombocytopenic with platelet counts ranging from 19,000 to 110,000/ lL (Table 1). Heterozygous and normal dogs had normal platelet counts and morphology. Eight of the analyzed dogs belong to the same family (Figure 3), where the SNP was passed on from parents to offspring and was present in all the generations. In this study, we describe a novel SNP in the b1-tubulin gene in a selected group of macrothrombocytopenic NT and CT in 3 different countries. Beta1-tubulin is a component of microtubules and is fundamentally important for the maintenance of platelet shape. In addition to maintaining the diskshaped form of circulating platelets, microtubules are the major structural components that drive the orderly fragmentation of platelets from the megakaryocyte cytoplasm. Microtubules are heterodimers composed of alpha and beta monomers. These monomers are
Figure 1. The flanking sequence of the canine b1-tubulin gene. Highlighted in yellow are the forward and reverse primers used for PCR. Exon 1 is underlined and bolded. The location of the single nucleotide polymorphism in Norfolk and Cairn Terriers is highlighted in green.
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Vet Clin Pathol 43/3 (2014) 317–321 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
b1-Tubulin mutation in Norfolk and Cairn Terriers
Gelain et al
A
Table 1. Platelet counts, DNA sequences with and without polymorphism at nucleotide position 5 in exon 1, and corresponding amino acids in 13 Norfolk and 7 Cairn Terriers.
Breed
B
Norfolk Terrier
C
Cairn Terrier Figure 2. DNA sequence of a portion of the gene encoding exon 1 of canine b1-tubulin and the corresponding amino acids. (A) Normal canine sequence with a guanine (G) in position 5 in exon 1. (B) Sequence obtained from a heterozygous Norfolk Terrier. (C) Mutated canine sequence from a Norfolk Terrier with adenine (A) in position 5 in exon 1. The box indicates the position of the nucleotide substitution (G5A).
associated longitudinally head to tail to form protofilaments, which in turn associate laterally to form cylindrical and hollow microtubules. There are many isoforms of alpha and beta monomers encoded by separate genes, but b1-tubulin is the beta isoform primarily expressed in megakaryocytes and platelets.7 Several studies in human and veterinary medicine have supported the importance of b1-tubulin in platelet biology. In people, a double-nucleotide mutation resulting in the substitution of a highly conserved glutamine with a proline (Q43P) has been reported. Heterozygous individuals have normal platelet numbers; however, enlarged and spherocytic platelets are present due to reduced expression of b1-tubulin.8 Homozygous affected individuals have mildly reduced platelet numbers, while impaired platelet function has been documented in both homozygous affected and heterozygous patients.9 Recently, a SNP at nucleotide position 952, resulting in the change of an arginine to a tryptophan at amino acid position 318 (R318W), was described in a boy incidentally found to have macrothrombocytopenia.10
Platelet Count (103/lL); Reference Range: 100–400 9 103/lL Phenotype
DNA Sequence
Amino Acid
19 51 54 62 82 95 95 104 110 308
Affected Affected Affected Affected Affected Affected Affected Affected Affected Carrier
CAT CAT CAT CAT CAT CAT CAT CAT CAT CAT/CGT
324
Carrier
CAT/CGT
361
Carrier
CAT/CGT
368 47 < 100 88 430 441 630
Normal Affected Affected Affected Normal Normal Carrier
CGT CAT CAT CAT CGT CGT CAT/CGT
224
Normal
CGT
Histidine Histidine Histidine Histidine Histidine Histidine Histidine Histidine Histidine Histidine/ arginine Histidine/ arginine Histidine/ arginine Arginine Histidine Histidine Histidine Arginine Arginine Histidine/ arginine Arginine
In cats, in contrast to other mammalian species, platelet size is quite variable, and large platelets, sized comparable to or greater than erythrocytes, are a common finding. Recently, in this species, 2 differences were found in a highly conserved region of gene sequence encoding the M loop of b1-tubulin when compared with sequences from other species. The nucleic acid differences noted are predicted to encode an aspartic acid instead of a glycine (G277D) and an arginine instead of a glutamine (Q279R). The change in charge resulting from these substitutions could result in an alteration in inter-protofilament interaction. Potential consequences are an instability in microtubule assembly and the production of variably sized platelets.11 In dogs, a missense mutation in the b1-tubulin gene resulting in the substitution of an asparagine for an aspartic acid at position 249 (D249N) has been recognized in asymptomatic inherited macrothrombocytopenia in CKCS and more recently in other breeds and mixed-breed dogs.2 This mutation is located at or near the interface between alpha
Vet Clin Pathol 43/3 (2014) 317–321 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
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b1-Tubulin mutation in Norfolk and Cairn Terriers
Figure 3. Pedigree of the group of related Norfolk Terriers in Italy with the observed phenotype and the predicted amino acid. Females are indicated by circles, males by squares. A black symbol indicates an affected dog, a white symbol illustrates an unaffected dog, and a question mark indicates unknown status. Homozygous dogs for histidine at amino acid position 2 in exon 1 (H) are indicated by a pink frame, heterozygous dogs for histidine and arginine at amino acid position 2 in exon 1 (H/R) are indicated by a blue frame.
and beta subunits, affecting the longitudinal contacts and intra-protofilament interaction. In the NT and CT analyzed in this study, we found a different mutation located in exon 1. The aminoterminal residues (1–206) of b1-tubulin form alternating parallel beta-pleated sheets and helices, and are involved in GTP binding and hydrolysis, which are required for microtubule assembly. This region is highly conserved between different species. So far, an arginine residue was present at position 2 in exon 1 in all species evaluated.11 Interestingly, heterozygous CKCS are not thrombocytopenic, but the number of circulating platelets is lower compared with the number in normal dogs, ranging from 100,000 to 200,000/ lL. In our study, we found that heterozygous NT and CT had normal platelet counts and morphology, which is in contrast to heterozygous CKCS. Thus, it is possible that the amino acid change from arginine to histidine at position 2 (R2H) could affect GTP binding enough to influence platelet formation in homozygous but not in heterozygous dogs. Pedigree analysis revealed that the SNP was present in dogs of all 4 generations analyzed, and the presence of macrothrombocytopenia in homozygous affected dogs may suggest an autosomal recessive mode of inheritance. However, both NT and CT are not very popular breeds, and the relatively low number of dogs analyzed does not allow a definitive conclusion about the prevalence of SNP in these breeds and the mode of inheritance. Similarly, based on limited data available and the lack of information on actual b1-tubulin protein expression, it cannot be
320
Gelain et al
assumed that the identified SNP is the mutation causing the macrothrombocytopenia. In conclusion, in this study, we reported a macrothrombocytopenia similar to the one reported in CKCS, but characterized by a different SNP located in the b1-tubulin gene, a likely candidate gene related to phenotype based on its involvement in platelet maturation and release in people, dogs, and cats.1,7,11 Further investigations on larger study populations are needed to assess the prevalence of macrothrombocytopenia and SNP in NT and CT, and to analyze the actual protein expression to confirm the relationship between the observed mutation and the phenotype and to clarify the role of the SNP described in this study in the pathogenesis of macrothrombocytopenia. Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.
References 1. Boudreaux MK. Inherited platelet disorders. J Vet Emerg Crit Care (San Antonio). 2012;22:30–41. 2. Davis B, Toivio-Kinnucan M, Schuller S, Boudreaux MK. Mutation in beta1-tubulin correlates with macrothrombocytopenia in Cavalier King Charles Spaniels. J Vet Inter Med. 2012;22:540–545. 3. Bertazzolo W, Comazzi S, Sesso L, Scarpa P, Ru G., Paltrinieri S. Comparison of methods for determining platelet numbers and volume in Cavalier King Charles spaniels. J Small Anim Pract. 2007;48:556–561. €k I, Hillstr€ 4. Tvedten H, Lillieh€ oo om A, Haggstrom J. Plateletcrit is superior to platelet count for assessing platelet status in Cavalier King Charles Spaniels. Vet Clin Pathol. 2008;37:266–271. 5. Pedersen HD, H€ aggstrom J, Olsen LH, et al. Idiopathic asymptomatic thrombocytopenia in Cavalier King Charles Spaniels is an autosomal recessive trait. J Vet Inter Med. 2002;16:169–173. 6. Gelain ME, Tutino GF, Pogliani E, Bertazzolo W. Macrothrombocytopenia in a group of related Norfolk terriers. Vet Rec. 2010;167:493–494. 7. Patel SR, Hartwig JH, Italiano JE Jr. The biogenesis of platelets from megakaryocyte proplatelets. J Clin Invest. 2005;115:3348–3354. 8. Freson K, De Vos R, Wittevrongel C, et al. The TUBB1 Q43P functional polymorphism reduces the risk of
Vet Clin Pathol 43/3 (2014) 317–321 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
Gelain et al
cardiovascular disease in men by modulating platelet function and structure. Blood. 2005;106:2356–2362. ~ ez L, Teruel R, Ant 9. Navarro-N un on a I, et al. Rare homozygous status of P43 b1-tubulin polymorphism causes alterations in platelet ultrastructure. Thromb Haemost. 2011;105:855–863. 10. Kunishima S, Kobayashi R, Itoh TJ, Hamaguchi M, Saiot H. Mutation of the beta1-tubulin gene associated
b1-Tubulin mutation in Norfolk and Cairn Terriers
with congenital macrothrombocytopenia affecting microtubule assembly. Blood. 2009;113:458– 461. 11. Boudreaux MK, Osborne CD, Herre AC, Ramos Rivera E, Spanlger EA. Unique structure of the M loop region of b1-tubulin may contribute to size variability of platelets in the family Felidae. Vet Clin Pathol. 2010;39:417– 423.
Vet Clin Pathol 43/3 (2014) 317–321 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
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BRIEF COMMUNICATION
A novel point mutation in the b1-tubulin gene in asymptomatic macrothrombocytopenic Norfolk and Cairn Terriers Maria Elena Gelain1, Walter Bertazzolo2, Giuseppina Tutino2, Elena Pogliani2, Francesco Cian3, Mary K. Boudreaux4 1
Department of Comparative Biomedicine and Food Science, Faculty of Veterinary Medicine, University of Padua, Padua, Italy; 2 Veterinary Clinic “Tibaldi”, Milan, Italy; 3Department of Veterinary Medicine, University of Cambridge, Cambridge, UK; and 4Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL, USA
Key Words Dog, GTP binding, platelets, single nucleotide polymorphism Correspondence M.E. Gelain, Department of Comparative Biomedicine and Food Science, Faculty of Veterinary Medicine, AGRIPOLIS – Viale dell’Universit a 16, 35020 Legnaro (PD), Italy E-mail: [email protected] DOI:10.1111/vcp.12168
Background: An asymptomatic macrothrombocytopenia, phenotypically similar to asymptomatic inherited macrothrombocytopenia in Cavalier King Charles Spaniels, was described in a group of Norfolk Terriers (NT) from Northern Italy, and isolated cases were also reported in Cairn Terriers (CT). Objectives: The purpose of this work was to evaluate for the presence of a genetic defect in the b1-tubulin gene in macrothrombocytopenic NT and CT. Methods: Samples from 20 healthy dogs (13 NT and 7 CT) were collected at different institutions in Italy (n = 8), United Kingdom (n = 3), and United States (n = 9). Genomic DNA was harvested from EDTA-anticoagulated blood and all coding areas and exon–intron splice sites in the gene encoding b1-tubulin were amplified and sequenced. Results: Twelve dogs (9 NT and 3 CT) showed a single nucleotide polymorphism (SNP) in exon 1 at nucleotide position 5 (G5A) that would result in the change of an arginine to a histidine at amino acid position 2 (R2H). Four dogs (3 NT and one Cairn Terrier) were heterozygous for the SNP, and 4 dogs (one Norfolk Terrier and 3 CT) matched the normal canine genome. Homozygous dogs for the SNP were macrothrombocytopenic with platelet counts ranging from 19,000 to 110,000/lL. Heterozygous and normal dogs had normal platelet counts and morphology. None had the CKCS point mutation. Conclusions: The b1-tubulin N-terminal amino acids form the nucleotidebinding domain and thus this mutation could affect GTP binding enough to influence platelet formation in homozygous but not in heterozygous dogs. The presence of macrothrombocytopenia only in homozygous affected dogs reveals an association between the SNP and the phenotype.
Platelets are the first line of defense against bleeding secondary to vascular injury, and anomaly in their number or function may lead to mucocutaneous bleeding. Similar to that observed in people, inherited thrombopathias and thrombocytopenia have been reported in animals. The severity of bleeding observed with inherited thrombopathias varies as a reflection of their molecular basis and ensuing phenotype.1 However, not all the inherited intrinsic platelet disorders cause hemorrhage. Asymptomatic inherited macrothrombocytopenia is a well-recognized condition in Cavalier King Charles Spaniels (CKCS), due to a point mutation at coding nucleotide 745 (G745A) in the
b1-tubulin gene, resulting in the change of an aspartic acid to an asparagine at amino acid 249 (D249N). This mutation results in impaired microtubule assembly affecting proplatelet formation and platelet production by megakaryocytes.2 Hemostatic function, however, is not altered presumably due to the presence of a normal circulating platelet mass indicated by the measurement of a normal plateletcrit in affected dogs.3,4 The prevalence of asymptomatic macrothrombocytopenia in CKCS is very high with more than 90% of dogs being either carriers or affected.1 Several other breeds as well as mixed-breed dogs have also been identified with this mutation since it was
Vet Clin Pathol 43/3 (2014) 317–321 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
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b1-Tubulin mutation in Norfolk and Cairn Terriers
first reported in CKCS. Previous studies found an autosomal recessive manner of inheritance for this condition.5 However, heterozygous dogs often have platelet numbers between 100,000 and 200,000/lL, suggesting an autosomal dominant inheritance.1 Recently, a phenotypically similar macrothrombocytopenia was described in a group of related Norfolk Terriers (NT) from Northern Italy6, and isolated cases of asymptomatic macrothrombocytopenia were also reported in Cairn Terriers (CT).1 The purpose of this work was to evaluate for the presence of a genetic defect in the b1-tubulin gene in macrothrombocytopenic NT and CT in different countries. Peripheral blood samples from 20 healthy NT (n = 13) and CT (n = 7) were collected at different institutions in Italy (n = 8, Department of Veterinary Pathology, Hygiene and Health, University of Milan, from 2008 to 2012), United Kingdom (n = 3, Department of Veterinary Medicine, University of Cambridge, from 2010 to 2012), and United States (n = 9, Department of Pathobiology, College of Veterinary Medicine, Auburn University). There were 11 female (7 intact and 4 neutered) and 9 male (7 intact and 2 neutered) dogs with a mean age of 6 years (min– max 0.8–14). The animals were sampled during the annual visit to a veterinarian for evaluation of their general health status and an informed consent was obtained from all owners. Included in the study were clinically healthy dogs, without alteration in CBC or biochemical profile. Excluded from the analysis were samples from dogs with signs of primary hemostatic dysfunction and/or with thrombocytopenia confirmed by platelet estimation on a peripheral blood smear and after excluding platelet aggregates and/or macroplatelets. CBCs were performed with a Sysmex XT-2000iV (Dasit group S.p.A., Milan, Italy) in Italy and with an ADVIA 120 (Siemens Medical Solutions USA Inc, Malvern, PA, USA) at Auburn University. Blood smears were routinely stained with May–Grunwald–Giemsa or Wright–Giemsa and were examined microscopically to evaluate platelet morphology and for the presence of platelet aggregates.
Gelain et al
Genomic DNA was harvested from EDTA-anticoagulated blood using a commercially available kit (QIAamp DNA Blood Mini Kit; Qiagen Inc, Valencia, CA, USA). Primers were designed to assess all coding areas and exon–intron splice sites in the gene encoding b1-tubulin based on the sequence obtained from the canine genome available on GenBank (Figure 1). The PCR conditions were: annealing at 63°C for 30 s and 40 cycles; the expected product size was 473 bp. PCR products were subjected to electrophoresis on agarose gels and target bands were harvested (QIAquick Gel Extraction Kit; Qiagen Inc) for sequencing (ABI 3100 Genetic Analyzer; Applied Biosystems, Foster City, CA, USA). All sampled dogs were clinically healthy and did not have any history or signs of hemostatic dysfunction. None of the samples had the point mutation reported in CKCS. Twelve dogs (9 NT and 3 CT) showed a single nucleotide polymorphism (SNP) in exon 1 at nucleotide position 5 (G5A). This mutation would result in the change of an arginine to a histidine at amino acid position 2 (R2H). Four dogs (3 NT and one CT) were heterozygous for the SNP and 4 dogs (one NT and 3 CT) had a normal sequence (Figure 2). All dogs with the SNP were macrothrombocytopenic with platelet counts ranging from 19,000 to 110,000/ lL (Table 1). Heterozygous and normal dogs had normal platelet counts and morphology. Eight of the analyzed dogs belong to the same family (Figure 3), where the SNP was passed on from parents to offspring and was present in all the generations. In this study, we describe a novel SNP in the b1-tubulin gene in a selected group of macrothrombocytopenic NT and CT in 3 different countries. Beta1-tubulin is a component of microtubules and is fundamentally important for the maintenance of platelet shape. In addition to maintaining the diskshaped form of circulating platelets, microtubules are the major structural components that drive the orderly fragmentation of platelets from the megakaryocyte cytoplasm. Microtubules are heterodimers composed of alpha and beta monomers. These monomers are
Figure 1. The flanking sequence of the canine b1-tubulin gene. Highlighted in yellow are the forward and reverse primers used for PCR. Exon 1 is underlined and bolded. The location of the single nucleotide polymorphism in Norfolk and Cairn Terriers is highlighted in green.
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Vet Clin Pathol 43/3 (2014) 317–321 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
b1-Tubulin mutation in Norfolk and Cairn Terriers
Gelain et al
A
Table 1. Platelet counts, DNA sequences with and without polymorphism at nucleotide position 5 in exon 1, and corresponding amino acids in 13 Norfolk and 7 Cairn Terriers.
Breed
B
Norfolk Terrier
C
Cairn Terrier Figure 2. DNA sequence of a portion of the gene encoding exon 1 of canine b1-tubulin and the corresponding amino acids. (A) Normal canine sequence with a guanine (G) in position 5 in exon 1. (B) Sequence obtained from a heterozygous Norfolk Terrier. (C) Mutated canine sequence from a Norfolk Terrier with adenine (A) in position 5 in exon 1. The box indicates the position of the nucleotide substitution (G5A).
associated longitudinally head to tail to form protofilaments, which in turn associate laterally to form cylindrical and hollow microtubules. There are many isoforms of alpha and beta monomers encoded by separate genes, but b1-tubulin is the beta isoform primarily expressed in megakaryocytes and platelets.7 Several studies in human and veterinary medicine have supported the importance of b1-tubulin in platelet biology. In people, a double-nucleotide mutation resulting in the substitution of a highly conserved glutamine with a proline (Q43P) has been reported. Heterozygous individuals have normal platelet numbers; however, enlarged and spherocytic platelets are present due to reduced expression of b1-tubulin.8 Homozygous affected individuals have mildly reduced platelet numbers, while impaired platelet function has been documented in both homozygous affected and heterozygous patients.9 Recently, a SNP at nucleotide position 952, resulting in the change of an arginine to a tryptophan at amino acid position 318 (R318W), was described in a boy incidentally found to have macrothrombocytopenia.10
Platelet Count (103/lL); Reference Range: 100–400 9 103/lL Phenotype
DNA Sequence
Amino Acid
19 51 54 62 82 95 95 104 110 308
Affected Affected Affected Affected Affected Affected Affected Affected Affected Carrier
CAT CAT CAT CAT CAT CAT CAT CAT CAT CAT/CGT
324
Carrier
CAT/CGT
361
Carrier
CAT/CGT
368 47 < 100 88 430 441 630
Normal Affected Affected Affected Normal Normal Carrier
CGT CAT CAT CAT CGT CGT CAT/CGT
224
Normal
CGT
Histidine Histidine Histidine Histidine Histidine Histidine Histidine Histidine Histidine Histidine/ arginine Histidine/ arginine Histidine/ arginine Arginine Histidine Histidine Histidine Arginine Arginine Histidine/ arginine Arginine
In cats, in contrast to other mammalian species, platelet size is quite variable, and large platelets, sized comparable to or greater than erythrocytes, are a common finding. Recently, in this species, 2 differences were found in a highly conserved region of gene sequence encoding the M loop of b1-tubulin when compared with sequences from other species. The nucleic acid differences noted are predicted to encode an aspartic acid instead of a glycine (G277D) and an arginine instead of a glutamine (Q279R). The change in charge resulting from these substitutions could result in an alteration in inter-protofilament interaction. Potential consequences are an instability in microtubule assembly and the production of variably sized platelets.11 In dogs, a missense mutation in the b1-tubulin gene resulting in the substitution of an asparagine for an aspartic acid at position 249 (D249N) has been recognized in asymptomatic inherited macrothrombocytopenia in CKCS and more recently in other breeds and mixed-breed dogs.2 This mutation is located at or near the interface between alpha
Vet Clin Pathol 43/3 (2014) 317–321 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
319
b1-Tubulin mutation in Norfolk and Cairn Terriers
Figure 3. Pedigree of the group of related Norfolk Terriers in Italy with the observed phenotype and the predicted amino acid. Females are indicated by circles, males by squares. A black symbol indicates an affected dog, a white symbol illustrates an unaffected dog, and a question mark indicates unknown status. Homozygous dogs for histidine at amino acid position 2 in exon 1 (H) are indicated by a pink frame, heterozygous dogs for histidine and arginine at amino acid position 2 in exon 1 (H/R) are indicated by a blue frame.
and beta subunits, affecting the longitudinal contacts and intra-protofilament interaction. In the NT and CT analyzed in this study, we found a different mutation located in exon 1. The aminoterminal residues (1–206) of b1-tubulin form alternating parallel beta-pleated sheets and helices, and are involved in GTP binding and hydrolysis, which are required for microtubule assembly. This region is highly conserved between different species. So far, an arginine residue was present at position 2 in exon 1 in all species evaluated.11 Interestingly, heterozygous CKCS are not thrombocytopenic, but the number of circulating platelets is lower compared with the number in normal dogs, ranging from 100,000 to 200,000/ lL. In our study, we found that heterozygous NT and CT had normal platelet counts and morphology, which is in contrast to heterozygous CKCS. Thus, it is possible that the amino acid change from arginine to histidine at position 2 (R2H) could affect GTP binding enough to influence platelet formation in homozygous but not in heterozygous dogs. Pedigree analysis revealed that the SNP was present in dogs of all 4 generations analyzed, and the presence of macrothrombocytopenia in homozygous affected dogs may suggest an autosomal recessive mode of inheritance. However, both NT and CT are not very popular breeds, and the relatively low number of dogs analyzed does not allow a definitive conclusion about the prevalence of SNP in these breeds and the mode of inheritance. Similarly, based on limited data available and the lack of information on actual b1-tubulin protein expression, it cannot be
320
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assumed that the identified SNP is the mutation causing the macrothrombocytopenia. In conclusion, in this study, we reported a macrothrombocytopenia similar to the one reported in CKCS, but characterized by a different SNP located in the b1-tubulin gene, a likely candidate gene related to phenotype based on its involvement in platelet maturation and release in people, dogs, and cats.1,7,11 Further investigations on larger study populations are needed to assess the prevalence of macrothrombocytopenia and SNP in NT and CT, and to analyze the actual protein expression to confirm the relationship between the observed mutation and the phenotype and to clarify the role of the SNP described in this study in the pathogenesis of macrothrombocytopenia. Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.
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