Comparative Medicine Copyright 2013 by the American Association for Laboratory Animal Science
Vol 63, No 4 August 2013 Pages 348–354
Original Research
Polymorphisms in Canine Platelet Glycoproteins Identify Potential Platelet Antigens Mary Beth Callan,1,* Petra Werner,1,† Nicola J Mason,1 Geralyn M Meny,2,‡ Michael G Raducha,1 and Paula S Henthorn1 Human alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness. More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system; however, there is no previous information on canine platelet-specific alloantigens. Using the HPA system as a model, we evaluated the canine ITGB3, ITGA2B, and GP1BB genes encoding GPIIIa (β3), GPIIb (αIIb), and GPIbβ, respectively, which account for 21 of 27 HPA, to determine whether amino acid polymorphisms are present in the orthologous canine genes. A secondary objective was to perform a pilot study to assess possible association between specific alleles of these proteins and a diagnosis of idiopathic thrombocytopenic purpura (ITP) in dogs. By using genomic DNA from dogs of various breeds with and without ITP, sequencing of PCR products encompassing all coding regions and exon–intron boundaries for these 3 genes revealed 4 single-nucleotide polymorphisms in ITGA2B resulting in amino acid polymorphisms in the canine genome, 3 previously reported and 1 newly identified (Gly[GGG]/Arg[AGG] at amino acid position 576 of ITGA2B. Of 16 possible ITGA2B protein alleles resulting from unique combinations of the 4 polymorphic amino acids, 5 different protein isoforms were present in homozygous dogs and explain all of the genotype combinations in heterozygous dogs. There was no amino acid polymorphism or protein isoform that was specific for a particular breed or for the diagnosis of ITP. Abbreviations: HPA, human platelet antigen; ITP, idiopathic thrombocytopenic purpura; PTR, platelet transfusion refractoriness; SNP, single-nucleotide polymorphism.
Human alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen, through pregnancy or transfusion, include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness (PTR). More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system, with 12 alloantigens grouped into 6 biallelic systems (HPA1 through HPA5 and HPA15) in which alloantibodies against both the common (designated ‘a’) and rare (designated ‘b’) alleles have been identified.5,12 For the remaining 21 platelet alloantigens (HPA6bw through HPA14bw and HPA16bw through HPA27bw), only antibodies against the rare allele (designated ‘bw’) have been detected. Although HPA traditionally were defined by using immune sera, the molecular basis of these antigens has now been characterized.5,12 The HPA reside in platelet membrane glycoproteins, the most common being GPIIIa, which accounts for 14 HPA. In all but one (HPA14bw), the platelet alloantigens are defined by a single amino-acid substitution caused by a single-nucleotide polymorphism (SNP) in the gene encoding the relevant Received: 28 Dec 2012. Revision requested: 15 Feb 2013. Accepted: 27 Feb 2013. 1 Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; 2American Red Cross Blood Services, Penn–Jersey Region, Philadelphia, Pennsylvania. * Corresponding author. Email: [email protected] Current affiliation: †Department of Cardiology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania; ‡University of Texas Health Science Center at San Antonio, San Antonio, Texas.
membrane glycoprotein.5,12 The Platelet Nomenclature Committee, which includes members of both the International Society of Blood Transfusion and the International Society of Thrombosis and Haemostasis, has set guidelines for defining new platelet antigens, which include: 1) determining the genetic basis of the alloantigen by genomic DNA sequence analysis, and 2) demonstrating an association between the genetic mutation and the reactivity of alloantibodies with the allelic forms of the protein. 12 For patients suspected of having alloimmune thrombocytopenia, well-defined platelet genotyping methods and serologic assays for the detection of platelet antibodies are available to guide case management. In contrast to the well-characterized HPA system and wealth of information on alloimmune thrombocytopenic conditions in humans, there is no information on canine platelet-specific alloantigens in the literature. However, early serologic studies using antiHPA1a alloantiserum resulted in direct immunoprecipitation of a 90-kDa protein from canine platelets, suggesting that the antigenic determinant for HPA1a has been conserved between humans and dogs.10 Neonatal alloimmune thrombocytopenia has not yet been documented in dogs, and there is a single case report of suspected posttransfusion purpura in a dog with hemophilia A, in which severe thrombocytopenia was noted 1 to 2 wk after transfusion (cryoprecipitate, fresh whole blood), with an increased amount of platelet surface-associated IgG and rapid resolution (less than 1 wk) of the thrombocytopenia.30 PTR, however, has been well documented in dogs for more than 25 y, because
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Canine platelet glycoprotein polymorphism
this species has been used as an experimental model to evaluate a variety of methods of preventing platelet alloimmunization.21-23 The occurrence of platelet alloimmunization in dogs receiving platelets from dog leukocyte antigen-identical littermates indicates that nondog leukocyte antigen immunizing platelet antigens, perhaps platelet-specific antigens, are responsible for the development of PTR.21 Using the HPA system as a model, our main objective in this study was to evaluate the canine ITGB3, ITGA2B, and GP1BB genes encoding the GPIIIa (β3), GPIIb (αIIb), and GPIbβ, respectively, proteins that account for 21 of 27 HPA, to determine whether there are amino acid polymorphisms that could define canine platelet-specific alloantigens. A secondary objective was to perform a pilot study to assess the possible association between canine platelet antigen protein alleles or single amino acid substitutions and primary immune-mediated thrombocytopenia, also referred to as idiopathic thrombocytopenic purpura (ITP), in dogs. The identification of a canine platelet antigen system would improve our understanding of the molecular basis of alloimmune thrombocytopenic conditions in dogs and help guide effective platelet transfusion support of these critical patients.
Materials and Methods
Dog blood samples. EDTA-anticoagulated blood samples were obtained from healthy blood donor dogs and canine patients with ITP after informed owner consent. The diagnosis of ITP was based on the finding of severe thrombocytopenia (platelet count, less than 30,000/µL), exclusion of all other causes of thrombocytopenia (for example, infectious diseases, neoplasia, drug reaction), and response to corticosteroid therapy. In addition, EDTA-anticoagulated blood samples were obtained from the Clinical Laboratory at the Matthew J Ryan Veterinary Hospital of the University of Pennsylvania. For these samples, medical records were reviewed to determine whether the patient had a history of ITP. Genomic DNA was extracted from blood samples by using a commercial kit (QIAamp DNA blood mini kit, Qiagen, Valencia, CA). Some DNA samples were available from a DNA bank maintained by the investigators (PW, PSH) for other genetic studies. The study was approved by the IACUC of the University of Pennsylvania (POAP no. 221). DNA samples from 43 dogs (Table 1) were used for sequencing ITGB3 and from 23 dogs were used for sequencing ITGA2B (Table 1). In addition, to focus on the regions in the ITGA2B gene where SNP were identified in dogs, DNA samples from an additional 17 dogs were used to sequence these sites of interest (Table 1). DNA samples from 15 dogs were used for sequencing GP1BB. Sequencing of genes encoding canine platelet glycoproteins. To amplify the coding regions and exon–intron boundaries of the ITGB3, ITGA2B, and GP1BB genes, oligonucleotide primer pairs (Table 2) were designed on the basis of the published canine genome sequence (Broad/CanFam2.0 assembly; GenBank assembly no., GCA_000002285.1) by using PrimerSelect, which is part of the Lasergene suite (DNAstar, Madison, WI). Target sequences were amplified by using standard PCR conditions previously described.31 For genomic regions with high GC content (exons 4 through 6, 11, and 12 in ITGA2B and the single exon in GP1BB), KOD Xtreme Hot Start DNA Polymerase (EMD Chemicals, Gibbstown, NJ) was used for amplification in 25-µL reaction mixtures containing approximately 50 ng genomic DNA, 2× Xtreme buffer, 0.2 mM each dNTP, 0.3 µM each primer, and 0.5 U
polymerase under the following conditions: initial denaturation at 94 °C for 2 min, followed by 35 cycles at 98 °C for 1 s, 60 °C for 10 s, and 68 °C for 35 s or 1 min. Sequencing of PCR-amplified products was performed after either electrophoresis and gel extraction (QIAquick gel extraction kit, Qiagen) or PCR spin column purification (QIAquick PCR purification kit, Qiagen). DNA sequences were analyzed for variations by using the computer program SeqMan, a component of the Lasergene suite (DNAstar). Statistical analysis. To determine whether an amino acid polymorphism and the presence or absence of ITP were associated, a 2 × 2 contingency table was created to perform a Fisher exact test and calculate a 2-tailed P value, with values less than 0.05 considered to be statistically significant.
Results
The coding regions and exon–intron boundaries of the ITGB3, ITGA2B, and GP1BB genes were amplified from canine genomic DNA. Sequencing of the PCR product for exon 1 of ITGB3 proved problematic and, because this exon encodes only the signal peptide (which is not present in the mature protein) and a single amino acid of the mature protein, we did not pursue the sequence of this exon. Sequencing of PCR products for the other 14 exons of ITGB3 did not reveal any new amino acid substitution SNP among the 43 dogs tested. There are 2 previously reported silent SNP in the canine ITGB3 gene, rs24604939 in exon 3 and rs24564616 in exon 10 (NCBI dbSNP Short Genetic Variations database, https://www.ncbi.nlm.nih.gov/SNP/). We found 2 dogs (1 Labrador retriever and 1 mixed-breed dog) homozygous and 5 dogs (3 Jack Russell terriers, 1 Boston terrier, and 1 Labrador retriever) heterozygous for the SNP in exon 3, with no candidates carrying the SNP in exon10. PCR products for all 30 exons, encoding the 1036 amino acids of the entire ITGA2B protein, were sequenced for 23 dogs. In addition to 3 known nonsynonymous SNP (that is, those that cause amino acid substitutions), 1 new polymorphism that changed the amino acid sequence was identified (Table 3). The regions containing these 4 missense polymorphisms were sequenced for an additional 17 dogs. The new polymorphism, a G-to-A base substitution resulting in a change from Gly (GGG) to Arg (AGG) at amino acid position 576 of the preprotein, occurred in 26 of the 40 dogs evaluated, whereas 11 dogs were heterozygous for this SNP. Other polymorphisms included a G-to-A substitution, leading to the codon ATG (Met) rather than GTG (Val) at amino acid position 491; 35 of the 40 dogs were homozygous for the G (Val) allele, and 4 dogs were heterozygous for this polymorphism. Only 1 dog, a Labrador retriever, had Met at this position. A T-toC change resulting in Pro (CCC) rather than Ser (TCC) at amino acid position 503 occurred in 7 of the 40 dogs; 16 dogs were heterozygous for the C/T polymorphism. Finally, there was a G-to-A substitution, resulting in a change from Asp (GAC) to Asn (AAC) at amino acid position 201, with 10 dogs being heterozygous (G/A) and 4 dogs homozygous (A/A) for this SNP. There was no difference in allelic frequencies of amino acid polymorphisms between ITP and nonITP dogs (Table 3), although differences in Ser503Pro approached significance (P = 0.0764), with ITP dogs tending toward a lower frequency of Pro and a higher frequency of Ser compared with nonITP dogs. Of 16 possible protein alleles resulting from unique combinations of the polymorphic amino acids, 5 different protein alleles were observed in homozygous dogs (Table 4). Among all 40 dogs
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Table 1. Dogs sequenced for each gene Gene ITGB3 Breed
NonITP
ITGA2B ITP
NonITP
GP1BB ITP
NonITP
ITP
American Cocker spaniel
3
2
2 (3)
3
0
3
Labrador retriever
5
0
1 (2)
0
0
0
Borzoi
5
0
1 (3)
0
0
0
Portuguese water dog
5
0
1
0
0
0
Jack Russell terrier
5
0
0
0
0
0
Cavalier King Charles spaniel
5
0
0
0
0
0
Boston terrier
5
0
1
0
0
0
German Shepherd
5
0
1
0
0
0
Bichon Frise
0
1
(5)
2 (1)
0
3
Golden retriever
1
0
1
0
0
0
Rhodesian ridgeback
0
0
1
0
0
0
West Highland white terrier
0
0
1
1
0
1
English setter
0
0
1
0
0
0
Sharpei
0
0
1
0
0
0
Dachshund
0
0
1
0
1
0
Chihuahua
0
0
1
0
1
0
Collie
0
0
1
0
1
0
Miniature poodle
0
0
0
1
0
1
Yorkshire terrier
0
0
0
1
0
1
Chesapeake Bay retriever
0
0
0
(2)
0
2
Miniature pinscher
0
0
0
(1)
0
1
Mixed-breed dog
1
0
0
0
0
0
Total
40
3
15 (13)
8 (4)
3
12
Numbers in parentheses indicate the number of dogs for which DNA sequencing was performed only for 4 focused areas of interest containing previously reported nonsynonymous amino-acid substitution SNP.
tested, 18 dogs were homozygous for all 4 polymorphic amino acids, whereas 22 dogs were heterozygous for at least 1 amino acid polymorphism. Of the 5 protein alleles observed, proteins c (AspValProArg) and d (AspValSerArg) were the most common, with 26 dogs (4 homozygous and 22 heterozygous) and 23 dogs (7 homozygous and 16 heterozygous), respectively, potentially having these protein isoforms. Protein a (AspMetProGly) occurred with the lowest frequency, with 1 dog being homozygous for all 4 polymorphic amino acids and 3 dogs heterozygous for at least 1 amino acid polymorphisms. Proteins b (AspValProGly) and e (AsnValProArg) occurred with intermediate frequency, with 2 and 4 dogs, respectively, being homozygous for all 4 polymorphic amino acids and 11 dogs being heterozygous for at least 1 amino acid polymorphism each for proteins b and e. No protein allele was present or absent specifically in dogs with ITP. Because only a single dog was tested for most of the breeds, we cannot comment regarding breed-associated differences for the protein alleles. However, protein b (AspValProGly) did not occur among any of the 8 Bichon Frises tested. Sequencing of PCR products for the single exon of the GP1BB gene included 15 dogs, 12 of which were diagnosed with ITP. No amino acid polymorphisms were identified in this region.
Discussion
Sequencing of 3 genes, ITGB3, ITGA2B, and GP1BB, encoding the canine platelet glycoproteins GPIIIa (β3), GPIIb (αIIb), and GPIbβ, respectively, identified nonsynonymous SNP only in ITGA2B. Three SNPs, Asp201Asn, Met491Val, and Pro503Ser, were identified provisionally during sequencing of the dog genome (Met491Val in a boxer, Asp201Asn and Pro503Ser in poodles; Table 2; additional details available at www.ncbi.nlm.nih. gov/snp/) but had not been validated prior to the current report. The fourth SNP, Gly576Arg, has not previously been identified. The challenge remains to determine whether these amino acid polymorphisms represent canine platelet-specific alloantigens. Characterization of the HPA system began more than 50 y ago and is ongoing.1,6,7,15,16,18,27-29 The most immunogenic of the platelet alloantigens, HPA1a (originally referred to as Zwa or P1A1), was identified in 1959 by using a platelet agglutination assay and serum from a patient who had developed posttransfusion purpura.28 Posttransfusion purpura is characterized by a sudden and self-limiting thrombocytopenia that typically occurs 5 to 10 d after the transfusion of platelets or RBC and coincides with the development of a platelet alloantibody that destroys not only the allogeneic platelets but also the recipient’s antigen-negative platelets.26 Additional studies with serum from this patient agglutinated platelets from 98% of the normal subjects tested,28 indicating that HPA1a occurs with high frequency in the general population,
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Table 2. Oligonucleotides used for gene exon amplification and sequencing Exon
Forward primer
Reverse primer
2
5′ GTT TGG CCC AGG TTT CAG TCT TGA 3′
5′ ACG GGT GGC ACT CCA GGC AAC AAC 3′
3, 4
5′ GGC TGG GTG AGC TCT AGG CTG TTG 3′
5′ TAG AGG TTT TGG CAG TTA CTA CCC 3′
5, 6
5′ ATT GTG ATG GCT CGG GCG ATG TGA 3′
5′ CCA GCT CGG ACT CCT GCC TTT ACA 3′
7, 8
5′ AGC CCA AAT CAG CTG AGT CCT AAG 3′
5′ GTC TCC AAT CTT GAG GCC GAC ACA 3′
9, 10
5′ GAG GGC GTA GAT GGT TTA GAT TCT 3′
5′ GGC CTT CCT GTG CCT ACC T 3′
11, 12
5′ GGC CCC GCT CTC CTG GCT ATC A 3′
5′ CCT CTG AGG TGG GAA TTA CTC TTC 3′
13
5′ GCT TCA TCA CTG TAG GGC ATA GTA 3′
5′ AGA TGA CAG GCC CTA ACT TAT CAC 3′
14
5′ AAG TGC TAT TCA CAG CCA GTT CAA 3′
5′ GGC CCT ATG TAT GTT CCG TCA GTA 3′
15
5′ CAT CAA AAA GTA ATG GGC TGG ATT 3′
5′ AAG GGT GAC GCA CTT CTA AAC GAA 3′
1
5′ AGC CCT TTC CTC TGC CTA CTG C 3′
5′ TGC CCC TCC CTG ACA CCT TTA C 3′
2, 3
5′ CCT TCC GGG TAG TGC TCT CAA 3′
5′ TCC CTG CCC CCG ACT GCT CT 3′
4, 5, 6
5′ TGG GGG CGT CGG TCG TCA GC 3′
5′ AGG GTG CCC GGG CGG TAA CTC G 3′
7
5′ CCC CCG GCG ATT TTT 3′
5′ TGG CGG TGG CGG GGA TGC 3′
8
5′ CCT GGT GCG GAC GCC TAT TGA C 3′
5′ CTC CCG CCC GCG ATT CTG TAA CC 3′
9
5′ AGC CCC GCA CCG CAC CAA AGT C 3′
5′ GAA CCG GCC TCC ACC TGC TCT C 3′
10, 11
5′ GAG CGG TGA GTG CCC CCT GTC C 3′
5′ CGC GCC CAC CAG CAA GTC GT 3′
12
5′ TGC TCT GGG TCG CCT CCT TTC CTC 3′
5′ CGG CGG TCT TGG TTC TGG CTG TTA 3′
13
5′ GCA GCT GCC TTC TTG ACC 3′
5′ TCT TGG CTA GCT GGA GGC ATT CT 3′
14, 15, 16
5′ CCC CGG GAA AGA TGA GAT GAG GAC 3′
5′ AGG AAG GCG GTG GTG GTG TGG 3′
17,18
5′ AGG GCC GGA GGG TGT TAC TG 3′
5′ TTG GGG GTT TTC ATT AGG GTT TAG 3′
19,20
5′ GCT ACA GCC CGG GAA GGA TGG A 3′
5′ GTT GGG GCT CTC AGG TGG AAA ATG 3′
21
5′ CTC TCG ACC CTA GCC CAC AAG T 3′
5′ CTC CAC GTC CCC ATC ATT ACC T 3′
22
5′ GGT AAT GAT GGG GAC GTG GAG GAT 3′
5′ GGT ATA AGG AGG CGC AGG TCA GG 3′
23, 24, 25
5′ GTG GCA TGC CGT TTG TGG AGT TAG 3′
5′ TGG GGA GGC AAA GAA GCA GTC AGT 3′
26, 27
5′ GCC CCC AGC CCG TCA ACC 3′
5′ TCC TAC CCC CTG GCC TCA TTC TG 3′
28, 29
5′ GGT GGG TGG GGC TTT CTG G 3′
5′ GAC TCC CTC CCG GTG ACT CTT T 3′
30
5′ GAA GGC CTC CGT TTG TGC 3′
5′ GTT GGG ACT CAG CCT CTT TAT TTG 3′
1
5′ CCT GCG GGC CCT GGT GAT AGC 3′
5′ GCG GGC AGC GTG TCC AGC AGT C 3′
1
5′ GCG CCG GGG TGG GGT GGG GAG AT 3′
5′ CTT GGG CGG AGG GCA GTT TC 3′
ITGB3
ITGA2B
GP1BB
a finding later corroborated by several studies.2,4,5,25 Thirty years after its serologic definition, HPA1a (and its allelic form HPA1b) was defined at the molecular level as a T-to-C polymorphism at nucleotide 196 in the ITGB3 gene resulting in the Leu33 (or Pro33 for HP1b) variant of the protein.13 As mentioned previously, early serologic studies using antiHPA1a alloantiserum resulted in direct immunoprecipitation of a 90-kDa protein from canine platelets, suggesting that the antigenic determinant for HPA1a has been conserved between humans and dogs.10 However, among 43 dogs tested, we did not identify a polymorphism corresponding to HPA1; in fact, no nonsynonymous polymorphisms were found in the canine ITGB3 gene. Whereas sera from human patients with neonatal alloimmune thrombocytopenia, posttransfusion purpura, and PTR have been used in the initial serologic characterization of the HPAs, 6,7,14,1820,28,29 sera from dogs with alloimmune thrombocytopenic conditions are unavailable. Therefore, our approach to the investigation of canine platelet-specific antigens was to begin with genomic DNA sequence analysis to determine whether any nonsynonymous SNP that could represent potential platelet alloantigens are
present. Because 14 HPA (HPA1, 4, 6bw, 7bw, 8bw, 10bw, 11bw, 14bw, 16bw, 17bw, 19bw, 21bw, 23bw, and 26bw) are found in platelet membrane GPIIIa (β3), we focused initially on sequencing the canine homolog of its encoding gene, ITGB3, by using genomic DNA from 43 dogs representing 11 breeds. Surprisingly, we did not identify any missense SNP in 14 of 15 exons of ITGB3. As noted previously, exon 1 of the canine ITGB3 gene was not sequenced due to technical difficulties, but none of the HPA SNP are found in exon 1, which is not surprising considering the fact that only one amino acid encoded by this exon is found in the mature, membrane-bound form of the protein (GenBank RefSeq mRNA sequence, NM_000212.2; protein sequence, NP_000203.2). If the frequency of canine platelet-specific antigens is similar to that of HPA in various ethnic groups, the high (for example, HPA1a in China, 0.994) and low (for example, HPA5b in France, 0.13) allelic frequencies5 could explain the lack of missense SNP in a group of 43 dogs. Six HPA (HPA3, 9bw, 20bw, 22bw, 24bw, and 27bw) reside on the platelet membrane GPIIb (αIIb), which is encoded by the ITGA2B gene. In comparing the canine and human GPIIb, there
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Table 3. Amino acid substitution SNP in the canine ITGA2B gene and allele frequencies Amino acid 201
491
503
Base in dog genome (codon / amino acid)
G (GAC /Asp)
A (ATG /Met)
C (CCC /Pro)
G (GGG /Gly)
Alternate base (codon /amino acid)
A (AAC /Asn)
G (GTG /Val)
T (TCC /Ser)
A (AGG /Arg)
rs9158428
rs2455312
rs8870390
ss539225964
Asp 0.78
Met 0.06
Pro 0.63
Gly 0.21
Asn 0.22
Val 0.94
Ser 0.37
Arg 0.79
Asp 0.77
Met 0.05
Pro 0.7
Gly 0.23
Asn 0.23
Val 0.95
Ser 0.3
Arg 0.77
Asp 0.79
Met 0.08
Pro 0.46
Gly 0.17
Asn 0.21
Val 0.92
Ser 0.54
Arg 0.83
Reference sequence number
576
Allelic frequencies All dogs (n = 40)
NonITP dogs (n = 28)
ITP dogs (n = 12)
Table 4. Protein isoforms in dogs homozygous for amino acid substitution SNP in the ITGA2B gene Amino acid Isoform
201
491
503
576
No. of dogs homozygous for isoform
a
G (Asp)
A (Met)
C (Pro)
G (Gly)
1
b
G (Asp)
G (Val)
C (Pro)
G (Gly)
2
c
G (Asp)
G (Val)
C (Pro)
A (Arg)
4
d
G (Asp)
G (Val)
T (Ser)
A (Arg)
7
e
A (Asn)
G (Val)
C (Pro)
A (Arg)
4
is an 83% identity in the amino acid sequence (both in the open reading frame and in the mature peptide), with the total preprotein length being 1039 amino acids in humans and 1036 amino acids in dogs (with the length difference resulting from an extra amino acid at position 773 in the human mature protein and 2 additional amino acids at the end of the open reading frame). By convention, the amino acid residue numbers start with the first amino acid of the mature protein, and for the specific residues discussed here, are the same in the human and dog sequences. Given that we did not identify any nonsynonymous SNP in the ITGB3 gene among 43 dogs representing 11 breeds, we opted to include a greater number of breeds (n = 19), with fewer dogs of each breed, for the evaluation of ITGA2B, because it is recognized that gene frequencies of HPA vary between races and ethnic groups.2,4,5,8 In dogs, ITGA2B appears to be more polymorphic than is ITGB3, with 4 amino acid substitution SNP noted among 40 dogs with complete (n = 23) or partial (n = 17) sequencing of the ITGA2B gene. Although there were more dog breeds included in sequencing of ITGA2B compared with ITGB3, the lack of identification of nonsynonymous SNP in ITGB3 cannot readily be attributed to the smaller number of breeds tested, given that 3 (1 American Cocker spaniel and 2 Borzois) of 4 dogs homozygous for Asp201Asn and the 1 dog (Labrador retriever) homozygous for Met491Val underwent complete sequencing of ITGB3. Of the 6 SNP defining the HPA on GPIIb, only one, a G-to-A polymorphism resulting in an amino acid change from Val to Met (Val837Met) defining HPA9bw, was reported previously in dogs at amino acid position 491, with the dog genome having Met. Thirty-five of the 40 dogs tested were homozygous for Val, whereas 4 dogs were heterozygotes, and only 1 dog, a Labrador retriever, was homozygous for Met. Although corresponding amino acid
substitution SNP for the Asp/Asn, Pro/Ser, and Gly/Arg polymorphisms observed in dogs of this study have not been identified as HPA, it is not unreasonable to hypothesize that these particular amino acid changes would produce structural changes that could constitute different epitopes on the protein. Clearly, demonstrating an association between an amino acid polymorphism and the reactivity of canine serum alloantibodies with the allelic form of the protein would be required to define Met491 or other protein isoforms as platelet alloantigens. A canine-specific assay based on cloning of the canine ITGA2B gene containing various polymorphisms and their expression in a cell line could be developed for identification of platelet-specific alloantigens. Such recombinant technology has been used for detection of HPA alloantibodies as an alternative to using panels of typed platelets to determine alloantibody reactivity.9,24 In fact, with the use of a combination of DNA sequencing and recombinant antigen expression, the molecular basis of the platelet-specific Vaa antigen, a low-frequency antigen implicated in neonatal alloimmune thrombocytopenia, has been resolved, leading to the designation of this antigen as HPA17bw.24 The 4 amino acid polymorphisms in the ITGA2B gene resulted in 5 observed protein isoforms among the 40 dogs tested. In this small study, we did not note an association of any protein isoform with a specific breed. Once canine platelet alloantigens are characterized, differences in allele frequencies between breeds likely would exist, considering the population bottlenecks and accompanying founder effects that probably occurred in the creation of many dog breeds. In addition to various HPA occurring at different allelic frequencies in various human populations, some HPA polymorphisms, specifically the HPA5b and HPA2a alleles, have been
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associated with the development of acute and chronic refractory ITP, respectively.3,25 There is little information in the veterinary literature regarding the target platelet antigens in dogs with ITP. In a study evaluating canine platelet GPIIb and GPIIIa as target antigens in 17 dogs with ITP through an indirect radioimmunoprecipitation assay using serum from affected dogs, one dog was positive for GPIIb and GPIIIa, whereas 3 dogs were positive for GP IIb only.11 Our inability to identify allelic differences between ITP and nonITP dogs in the current study may have been due to small sample size as well as to our focus on platelet membrane glycoproteins (GPIIb, GPIIIa, and GP1bβ) that may not be the target platelet antigens in the majority of dogs with ITP. Alternatively, there may not be a link between amino acid polymorphisms and canine ITP. A comprehensive study evaluating more dogs with ITP, more healthy dogs for each breed, and additional genes encoding platelet membrane glycoproteins is needed to build on this pilot study to determine whether there is an association between specific alleles and a diagnosis of ITP. Of the alloimmune thrombocytopenic conditions described in human patients, PTR is likely of greatest clinical importance in dogs. Platelet alloimmunization may occur after the administration of packed RBC or platelet concentrate, even though the prevalence of platelet-specific antibodies in multiply transfused human patients is reported to be 3% to 5%.17 With veterinary medicine striving to offer a more sophisticated level of patient care in areas such as oncology and critical care medicine, more canine patients are receiving multiple transfusions, and the development of PTR likely will become more prevalent in the clinical setting. The current study identifies protein polymorphisms in platelet antigens that have the potential to act as alloantigens. An approach using DNA from dogs with PTR and their blood donors to sequence genes encoding for the major platelet membrane glycoproteins may be helpful in the characterization of the canine platelet antigen system.
Acknowledgment
This work was supported by The Barry and Savannah French-Poodle Memorial Fund.
References
1. Bertrand G, Jallu V, Saillant D, Kervran D, Martageix C, Kaplan C. 2009. The new platelet alloantigen Caba: a single point mutation Gln716His on the α2 integrin. Transfusion 49:2076–2083. 2. Bhatti FA, Uddin M, Ahmed A, Bugert P. 2010. Human platelet antigen polymorphisms (HPA1, 2, 3, 4, 5, and 15) in major ethnic groups of Pakistan. Transfus Med 20:78–87. 3. Castro V, Oliveira GB, Origa AF, Annichino-Bizzacchi JM, Arruda VR. 2000. The human platelet alloantigen 5 polymorphism as a risk for the development of acute idiopathic thrombocytopenia purpura. Thromb Haemost 84:360–361. 4. Hadhri S, Gandouz R, Chatti N, Bierling P, Skouri H. 2010. Gene frequencies of the HPA1 to 6 and 15 human platelet antigens in the Tunisian blood donors. Tissue Antigens 76:236–239. 5. Immuno Polymorphism Database (IPD). [Internet]. 2012. HPA allele frequencies. [Cited 16 December 2012]. Available at: www.ebi. ac.uk/ipd/hpa/freqs. 6. Kekomaki R, Raivio P, Kero P. 1992. A new low-frequency platelet alloantigen, Vaa, on glycoprotein IIbIIIa associated with neonatal alloimmune thrombocytopenia. Transfus Med 2:27–33. 7. Kickler TS, Herman JS, Furihata K, Kunicki TJ, Aster RH. 1988. Identification of Bakb, a new platelet-specific antigen associated with posttransfusion purpura. Blood 71:894–898.
8. Kim HO, Jin Y, Kickler TS, Blakemore K, Kwon OH, Bray PF. 1995. Gene frequencies of the 5 major human platelet antigens in African American, white, and Korean populations. Transfusion 35:863–867. 9. Kroll H, Yates J, Santoso S. 2005. Immunization against a lowfrequency human platelet alloantigen in fetal alloimune thrombocytopenia is not a single event: characterization by the combined use of reference DNA and novel allele-specific cell lines expressing recombinant antigens. Transfusion 45:353–358. 10. Lane J, Brown M, Bernstein I, Wilcox PK, Slichter SJ, Nowinski RC. 1982. Serological and biochemical analysis of the PIA1 alloantigen of human platelets. Br J Haematol 50:351–359. 11. Lewis DC, Meyers KM. 1996. Studies of platelet-bound and serum platelet-bindable immunoglobulins in dogs with idiopathic thrombocytopenic purpura. Exp Hematol 24:696–701. 12. Metcalfe P, Watkins NA, Ouwehand WH, Kaplan C, Newman P, Kekomaki R, de Haas M, Aster R, Shibata Y, Smith J, Kiefel V, Santoso S. 2003. Nomenclature of human platelet antigens. Vox Sang 85:240–245. 13. Newman PJ, Derbes RS, Aster RH. 1989. The human platelet alloantigens P1A1 and P1A2 are associated with a leucine33/proline33 amino acid polymorphism in membrane glycoprotein IIIa and are distinguishable by DNA typing. J Clin Invest 83:1778–1781. 14. Noris P, Simsek S, de Bruijne-Admiraal LG, Porcelijn L, Huiskes E, van der Vlist GJ, van Leeuwen EF, van der Schoot CE, von dem Borne AEGK. 1995. Maxa, a new low-frequency platelet-specific antigen localized on glycoprotein IIb, is associated with neonatal alloimmune thrombocytopenia. Blood 86:1019–1026. 15. Peterson JA, Gitter ML, Kanack A, Curtis B, McFarland J, Bougie D, Aster R. 2010. New low-frequency platelet glycoprotein polymorphisms associated with neonatal alloimmune thrombocytopenia. Transfusion 50:324–333. 16. Peterson JA, Pechauer SM, Gitter ML, Kanack A, Curtis BR, Reese J, Kamath VM, McFarland JG, Aster RH. 2012. New platelet glycoprotein polymorphisms causing maternal immunization and neonatal alloimmune thrombocytopenia. Transfusion 52:1117–1124. 17. Sanz C, Freire C, Alcorta I, Ordinas A, Pereira A. 2001. Plateletspecific antibodies in HLA-immunized patients receiving chronic platelet support. Transfusion 41:762–765. 18. Shibata Y, Matsuda I, Miyaji T, Ichikawa Y. 1986. Yuka, a new platelet antigen involved in 2 cases of neonatal alloimmune thrombocytopenia. Vox Sang 50:177–180. 19. Shulman NR, Aster RH, Leitner A, Hiller MC. 1961. Immunoreactions involving platelets. V. Posttransfusion purpura due to a complement-fixing antibody against a genetically controlled platelet antigen. A proposed mechanism for thrombocytopenia and its relevance in ‘autoimmunity’. J Clin Invest 40:1597–1620. 20. Simsek S, Vlekke AB, Kuijpers RW, Goldschmeding R, von dem Borne AE. 1994. A new platelet antigen, Groa, localized on glycoprotein IIIa, involved in neonatal alloimmune thrombocytopenia. Vox Sang 67:302–306. 21. Slichter SJ, O’Donnell MR, Weiden PL, Storb R, Schroeder ML. 1986. Canine platelet alloimmunization: the role of donor selection. Br J Haematol 63:713–727. 22. Slichter SJ, Deeg HJ, Kennedy MS. 1987. Prevention of platelet alloimmunization in dogs with systemic cyclosporine and by UV irradiation or cyclosporine-loading of donor platelets. Blood 69:414–418. 23. Slichter SJ, Fish D, Abrams VK, Gaur L, Nelson K, Bolgiano D. 2005. Evaluation of different methods of leukoreduction of donor platelets to prevent alloimmune platelet refractoriness and induce tolerance in a canine transfusion model. Blood 105:847–854. 24. Stafford P, Garner SF, Rankin A, Kekomaki R, Watkins NA, Ouwehand WH. 2008. A single-nucleotide polymorphism in the human ITGB3 gene is associated with the platelet-specific alloantigen Vaa (HPA17bw) involved in fetal maternal alloimmune thrombocytopenia. Transfusion 48:1432–1438.
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25. Thude H, Gatzka E, Anders O, Barz D. 1999. Allele frequencies of human platelet antigen 1, 2, 3, and 5 systems in patients with chronic refractory autoimmune thrombocytopenia and in normal persons. Vox Sang 77:149–153. 26. Tinmouth AT, Semple E, Shehata N, Branch DR. 2006. Platelet immunopathology and therapy: a Canadian blood services research and development symposium. Transfus Med Rev 20:294–314. 27. van der Weerdt CM, Veenhoven-von Riesz LE, van Loghem J. 1963. The Zw blood group system in platelets. Vox Sang 8:513–530. 28. van Loghem JJ Jr, Dorfmeijer H, van der Hart M. 1959. Serological and genetical studies on a platelet antigen (Zw). Vox Sang 4:161–169.
29. von dem Borne AE, von Riesz E, Verheugt FW, Verheugt FWA, ten Cate JW, Koppe JG, Engelfriet CP, Nijenhuis LE. 1980. Baka, a new platelet-specific antigen involved in neonatal alloimmune thrombocytopenia. Vox Sang 39:113–120. 30. Wardrop KJ, Lewis D, Marks S, Buss M. 1997. Posttransfusion purpura in a dog with hemophilia A. J Vet Intern Med 11: 261–263. 31. Werner P, Raducha MG, Prociuk U, Ostrander EA, Spielman RS, Kirkness EF, Patterson DF, Henthorn PS. 2005. The keeshond defect in cardiac conotruncal development is oligogenic. Hum Genet 116:368–377.
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Original Research
Polymorphisms in Canine Platelet Glycoproteins Identify Potential Platelet Antigens Mary Beth Callan,1,* Petra Werner,1,† Nicola J Mason,1 Geralyn M Meny,2,‡ Michael G Raducha,1 and Paula S Henthorn1 Human alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness. More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system; however, there is no previous information on canine platelet-specific alloantigens. Using the HPA system as a model, we evaluated the canine ITGB3, ITGA2B, and GP1BB genes encoding GPIIIa (β3), GPIIb (αIIb), and GPIbβ, respectively, which account for 21 of 27 HPA, to determine whether amino acid polymorphisms are present in the orthologous canine genes. A secondary objective was to perform a pilot study to assess possible association between specific alleles of these proteins and a diagnosis of idiopathic thrombocytopenic purpura (ITP) in dogs. By using genomic DNA from dogs of various breeds with and without ITP, sequencing of PCR products encompassing all coding regions and exon–intron boundaries for these 3 genes revealed 4 single-nucleotide polymorphisms in ITGA2B resulting in amino acid polymorphisms in the canine genome, 3 previously reported and 1 newly identified (Gly[GGG]/Arg[AGG] at amino acid position 576 of ITGA2B. Of 16 possible ITGA2B protein alleles resulting from unique combinations of the 4 polymorphic amino acids, 5 different protein isoforms were present in homozygous dogs and explain all of the genotype combinations in heterozygous dogs. There was no amino acid polymorphism or protein isoform that was specific for a particular breed or for the diagnosis of ITP. Abbreviations: HPA, human platelet antigen; ITP, idiopathic thrombocytopenic purpura; PTR, platelet transfusion refractoriness; SNP, single-nucleotide polymorphism.
Human alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen, through pregnancy or transfusion, include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness (PTR). More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system, with 12 alloantigens grouped into 6 biallelic systems (HPA1 through HPA5 and HPA15) in which alloantibodies against both the common (designated ‘a’) and rare (designated ‘b’) alleles have been identified.5,12 For the remaining 21 platelet alloantigens (HPA6bw through HPA14bw and HPA16bw through HPA27bw), only antibodies against the rare allele (designated ‘bw’) have been detected. Although HPA traditionally were defined by using immune sera, the molecular basis of these antigens has now been characterized.5,12 The HPA reside in platelet membrane glycoproteins, the most common being GPIIIa, which accounts for 14 HPA. In all but one (HPA14bw), the platelet alloantigens are defined by a single amino-acid substitution caused by a single-nucleotide polymorphism (SNP) in the gene encoding the relevant Received: 28 Dec 2012. Revision requested: 15 Feb 2013. Accepted: 27 Feb 2013. 1 Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; 2American Red Cross Blood Services, Penn–Jersey Region, Philadelphia, Pennsylvania. * Corresponding author. Email: [email protected] Current affiliation: †Department of Cardiology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania; ‡University of Texas Health Science Center at San Antonio, San Antonio, Texas.
membrane glycoprotein.5,12 The Platelet Nomenclature Committee, which includes members of both the International Society of Blood Transfusion and the International Society of Thrombosis and Haemostasis, has set guidelines for defining new platelet antigens, which include: 1) determining the genetic basis of the alloantigen by genomic DNA sequence analysis, and 2) demonstrating an association between the genetic mutation and the reactivity of alloantibodies with the allelic forms of the protein. 12 For patients suspected of having alloimmune thrombocytopenia, well-defined platelet genotyping methods and serologic assays for the detection of platelet antibodies are available to guide case management. In contrast to the well-characterized HPA system and wealth of information on alloimmune thrombocytopenic conditions in humans, there is no information on canine platelet-specific alloantigens in the literature. However, early serologic studies using antiHPA1a alloantiserum resulted in direct immunoprecipitation of a 90-kDa protein from canine platelets, suggesting that the antigenic determinant for HPA1a has been conserved between humans and dogs.10 Neonatal alloimmune thrombocytopenia has not yet been documented in dogs, and there is a single case report of suspected posttransfusion purpura in a dog with hemophilia A, in which severe thrombocytopenia was noted 1 to 2 wk after transfusion (cryoprecipitate, fresh whole blood), with an increased amount of platelet surface-associated IgG and rapid resolution (less than 1 wk) of the thrombocytopenia.30 PTR, however, has been well documented in dogs for more than 25 y, because
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this species has been used as an experimental model to evaluate a variety of methods of preventing platelet alloimmunization.21-23 The occurrence of platelet alloimmunization in dogs receiving platelets from dog leukocyte antigen-identical littermates indicates that nondog leukocyte antigen immunizing platelet antigens, perhaps platelet-specific antigens, are responsible for the development of PTR.21 Using the HPA system as a model, our main objective in this study was to evaluate the canine ITGB3, ITGA2B, and GP1BB genes encoding the GPIIIa (β3), GPIIb (αIIb), and GPIbβ, respectively, proteins that account for 21 of 27 HPA, to determine whether there are amino acid polymorphisms that could define canine platelet-specific alloantigens. A secondary objective was to perform a pilot study to assess the possible association between canine platelet antigen protein alleles or single amino acid substitutions and primary immune-mediated thrombocytopenia, also referred to as idiopathic thrombocytopenic purpura (ITP), in dogs. The identification of a canine platelet antigen system would improve our understanding of the molecular basis of alloimmune thrombocytopenic conditions in dogs and help guide effective platelet transfusion support of these critical patients.
Materials and Methods
Dog blood samples. EDTA-anticoagulated blood samples were obtained from healthy blood donor dogs and canine patients with ITP after informed owner consent. The diagnosis of ITP was based on the finding of severe thrombocytopenia (platelet count, less than 30,000/µL), exclusion of all other causes of thrombocytopenia (for example, infectious diseases, neoplasia, drug reaction), and response to corticosteroid therapy. In addition, EDTA-anticoagulated blood samples were obtained from the Clinical Laboratory at the Matthew J Ryan Veterinary Hospital of the University of Pennsylvania. For these samples, medical records were reviewed to determine whether the patient had a history of ITP. Genomic DNA was extracted from blood samples by using a commercial kit (QIAamp DNA blood mini kit, Qiagen, Valencia, CA). Some DNA samples were available from a DNA bank maintained by the investigators (PW, PSH) for other genetic studies. The study was approved by the IACUC of the University of Pennsylvania (POAP no. 221). DNA samples from 43 dogs (Table 1) were used for sequencing ITGB3 and from 23 dogs were used for sequencing ITGA2B (Table 1). In addition, to focus on the regions in the ITGA2B gene where SNP were identified in dogs, DNA samples from an additional 17 dogs were used to sequence these sites of interest (Table 1). DNA samples from 15 dogs were used for sequencing GP1BB. Sequencing of genes encoding canine platelet glycoproteins. To amplify the coding regions and exon–intron boundaries of the ITGB3, ITGA2B, and GP1BB genes, oligonucleotide primer pairs (Table 2) were designed on the basis of the published canine genome sequence (Broad/CanFam2.0 assembly; GenBank assembly no., GCA_000002285.1) by using PrimerSelect, which is part of the Lasergene suite (DNAstar, Madison, WI). Target sequences were amplified by using standard PCR conditions previously described.31 For genomic regions with high GC content (exons 4 through 6, 11, and 12 in ITGA2B and the single exon in GP1BB), KOD Xtreme Hot Start DNA Polymerase (EMD Chemicals, Gibbstown, NJ) was used for amplification in 25-µL reaction mixtures containing approximately 50 ng genomic DNA, 2× Xtreme buffer, 0.2 mM each dNTP, 0.3 µM each primer, and 0.5 U
polymerase under the following conditions: initial denaturation at 94 °C for 2 min, followed by 35 cycles at 98 °C for 1 s, 60 °C for 10 s, and 68 °C for 35 s or 1 min. Sequencing of PCR-amplified products was performed after either electrophoresis and gel extraction (QIAquick gel extraction kit, Qiagen) or PCR spin column purification (QIAquick PCR purification kit, Qiagen). DNA sequences were analyzed for variations by using the computer program SeqMan, a component of the Lasergene suite (DNAstar). Statistical analysis. To determine whether an amino acid polymorphism and the presence or absence of ITP were associated, a 2 × 2 contingency table was created to perform a Fisher exact test and calculate a 2-tailed P value, with values less than 0.05 considered to be statistically significant.
Results
The coding regions and exon–intron boundaries of the ITGB3, ITGA2B, and GP1BB genes were amplified from canine genomic DNA. Sequencing of the PCR product for exon 1 of ITGB3 proved problematic and, because this exon encodes only the signal peptide (which is not present in the mature protein) and a single amino acid of the mature protein, we did not pursue the sequence of this exon. Sequencing of PCR products for the other 14 exons of ITGB3 did not reveal any new amino acid substitution SNP among the 43 dogs tested. There are 2 previously reported silent SNP in the canine ITGB3 gene, rs24604939 in exon 3 and rs24564616 in exon 10 (NCBI dbSNP Short Genetic Variations database, https://www.ncbi.nlm.nih.gov/SNP/). We found 2 dogs (1 Labrador retriever and 1 mixed-breed dog) homozygous and 5 dogs (3 Jack Russell terriers, 1 Boston terrier, and 1 Labrador retriever) heterozygous for the SNP in exon 3, with no candidates carrying the SNP in exon10. PCR products for all 30 exons, encoding the 1036 amino acids of the entire ITGA2B protein, were sequenced for 23 dogs. In addition to 3 known nonsynonymous SNP (that is, those that cause amino acid substitutions), 1 new polymorphism that changed the amino acid sequence was identified (Table 3). The regions containing these 4 missense polymorphisms were sequenced for an additional 17 dogs. The new polymorphism, a G-to-A base substitution resulting in a change from Gly (GGG) to Arg (AGG) at amino acid position 576 of the preprotein, occurred in 26 of the 40 dogs evaluated, whereas 11 dogs were heterozygous for this SNP. Other polymorphisms included a G-to-A substitution, leading to the codon ATG (Met) rather than GTG (Val) at amino acid position 491; 35 of the 40 dogs were homozygous for the G (Val) allele, and 4 dogs were heterozygous for this polymorphism. Only 1 dog, a Labrador retriever, had Met at this position. A T-toC change resulting in Pro (CCC) rather than Ser (TCC) at amino acid position 503 occurred in 7 of the 40 dogs; 16 dogs were heterozygous for the C/T polymorphism. Finally, there was a G-to-A substitution, resulting in a change from Asp (GAC) to Asn (AAC) at amino acid position 201, with 10 dogs being heterozygous (G/A) and 4 dogs homozygous (A/A) for this SNP. There was no difference in allelic frequencies of amino acid polymorphisms between ITP and nonITP dogs (Table 3), although differences in Ser503Pro approached significance (P = 0.0764), with ITP dogs tending toward a lower frequency of Pro and a higher frequency of Ser compared with nonITP dogs. Of 16 possible protein alleles resulting from unique combinations of the polymorphic amino acids, 5 different protein alleles were observed in homozygous dogs (Table 4). Among all 40 dogs
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Table 1. Dogs sequenced for each gene Gene ITGB3 Breed
NonITP
ITGA2B ITP
NonITP
GP1BB ITP
NonITP
ITP
American Cocker spaniel
3
2
2 (3)
3
0
3
Labrador retriever
5
0
1 (2)
0
0
0
Borzoi
5
0
1 (3)
0
0
0
Portuguese water dog
5
0
1
0
0
0
Jack Russell terrier
5
0
0
0
0
0
Cavalier King Charles spaniel
5
0
0
0
0
0
Boston terrier
5
0
1
0
0
0
German Shepherd
5
0
1
0
0
0
Bichon Frise
0
1
(5)
2 (1)
0
3
Golden retriever
1
0
1
0
0
0
Rhodesian ridgeback
0
0
1
0
0
0
West Highland white terrier
0
0
1
1
0
1
English setter
0
0
1
0
0
0
Sharpei
0
0
1
0
0
0
Dachshund
0
0
1
0
1
0
Chihuahua
0
0
1
0
1
0
Collie
0
0
1
0
1
0
Miniature poodle
0
0
0
1
0
1
Yorkshire terrier
0
0
0
1
0
1
Chesapeake Bay retriever
0
0
0
(2)
0
2
Miniature pinscher
0
0
0
(1)
0
1
Mixed-breed dog
1
0
0
0
0
0
Total
40
3
15 (13)
8 (4)
3
12
Numbers in parentheses indicate the number of dogs for which DNA sequencing was performed only for 4 focused areas of interest containing previously reported nonsynonymous amino-acid substitution SNP.
tested, 18 dogs were homozygous for all 4 polymorphic amino acids, whereas 22 dogs were heterozygous for at least 1 amino acid polymorphism. Of the 5 protein alleles observed, proteins c (AspValProArg) and d (AspValSerArg) were the most common, with 26 dogs (4 homozygous and 22 heterozygous) and 23 dogs (7 homozygous and 16 heterozygous), respectively, potentially having these protein isoforms. Protein a (AspMetProGly) occurred with the lowest frequency, with 1 dog being homozygous for all 4 polymorphic amino acids and 3 dogs heterozygous for at least 1 amino acid polymorphisms. Proteins b (AspValProGly) and e (AsnValProArg) occurred with intermediate frequency, with 2 and 4 dogs, respectively, being homozygous for all 4 polymorphic amino acids and 11 dogs being heterozygous for at least 1 amino acid polymorphism each for proteins b and e. No protein allele was present or absent specifically in dogs with ITP. Because only a single dog was tested for most of the breeds, we cannot comment regarding breed-associated differences for the protein alleles. However, protein b (AspValProGly) did not occur among any of the 8 Bichon Frises tested. Sequencing of PCR products for the single exon of the GP1BB gene included 15 dogs, 12 of which were diagnosed with ITP. No amino acid polymorphisms were identified in this region.
Discussion
Sequencing of 3 genes, ITGB3, ITGA2B, and GP1BB, encoding the canine platelet glycoproteins GPIIIa (β3), GPIIb (αIIb), and GPIbβ, respectively, identified nonsynonymous SNP only in ITGA2B. Three SNPs, Asp201Asn, Met491Val, and Pro503Ser, were identified provisionally during sequencing of the dog genome (Met491Val in a boxer, Asp201Asn and Pro503Ser in poodles; Table 2; additional details available at www.ncbi.nlm.nih. gov/snp/) but had not been validated prior to the current report. The fourth SNP, Gly576Arg, has not previously been identified. The challenge remains to determine whether these amino acid polymorphisms represent canine platelet-specific alloantigens. Characterization of the HPA system began more than 50 y ago and is ongoing.1,6,7,15,16,18,27-29 The most immunogenic of the platelet alloantigens, HPA1a (originally referred to as Zwa or P1A1), was identified in 1959 by using a platelet agglutination assay and serum from a patient who had developed posttransfusion purpura.28 Posttransfusion purpura is characterized by a sudden and self-limiting thrombocytopenia that typically occurs 5 to 10 d after the transfusion of platelets or RBC and coincides with the development of a platelet alloantibody that destroys not only the allogeneic platelets but also the recipient’s antigen-negative platelets.26 Additional studies with serum from this patient agglutinated platelets from 98% of the normal subjects tested,28 indicating that HPA1a occurs with high frequency in the general population,
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Table 2. Oligonucleotides used for gene exon amplification and sequencing Exon
Forward primer
Reverse primer
2
5′ GTT TGG CCC AGG TTT CAG TCT TGA 3′
5′ ACG GGT GGC ACT CCA GGC AAC AAC 3′
3, 4
5′ GGC TGG GTG AGC TCT AGG CTG TTG 3′
5′ TAG AGG TTT TGG CAG TTA CTA CCC 3′
5, 6
5′ ATT GTG ATG GCT CGG GCG ATG TGA 3′
5′ CCA GCT CGG ACT CCT GCC TTT ACA 3′
7, 8
5′ AGC CCA AAT CAG CTG AGT CCT AAG 3′
5′ GTC TCC AAT CTT GAG GCC GAC ACA 3′
9, 10
5′ GAG GGC GTA GAT GGT TTA GAT TCT 3′
5′ GGC CTT CCT GTG CCT ACC T 3′
11, 12
5′ GGC CCC GCT CTC CTG GCT ATC A 3′
5′ CCT CTG AGG TGG GAA TTA CTC TTC 3′
13
5′ GCT TCA TCA CTG TAG GGC ATA GTA 3′
5′ AGA TGA CAG GCC CTA ACT TAT CAC 3′
14
5′ AAG TGC TAT TCA CAG CCA GTT CAA 3′
5′ GGC CCT ATG TAT GTT CCG TCA GTA 3′
15
5′ CAT CAA AAA GTA ATG GGC TGG ATT 3′
5′ AAG GGT GAC GCA CTT CTA AAC GAA 3′
1
5′ AGC CCT TTC CTC TGC CTA CTG C 3′
5′ TGC CCC TCC CTG ACA CCT TTA C 3′
2, 3
5′ CCT TCC GGG TAG TGC TCT CAA 3′
5′ TCC CTG CCC CCG ACT GCT CT 3′
4, 5, 6
5′ TGG GGG CGT CGG TCG TCA GC 3′
5′ AGG GTG CCC GGG CGG TAA CTC G 3′
7
5′ CCC CCG GCG ATT TTT 3′
5′ TGG CGG TGG CGG GGA TGC 3′
8
5′ CCT GGT GCG GAC GCC TAT TGA C 3′
5′ CTC CCG CCC GCG ATT CTG TAA CC 3′
9
5′ AGC CCC GCA CCG CAC CAA AGT C 3′
5′ GAA CCG GCC TCC ACC TGC TCT C 3′
10, 11
5′ GAG CGG TGA GTG CCC CCT GTC C 3′
5′ CGC GCC CAC CAG CAA GTC GT 3′
12
5′ TGC TCT GGG TCG CCT CCT TTC CTC 3′
5′ CGG CGG TCT TGG TTC TGG CTG TTA 3′
13
5′ GCA GCT GCC TTC TTG ACC 3′
5′ TCT TGG CTA GCT GGA GGC ATT CT 3′
14, 15, 16
5′ CCC CGG GAA AGA TGA GAT GAG GAC 3′
5′ AGG AAG GCG GTG GTG GTG TGG 3′
17,18
5′ AGG GCC GGA GGG TGT TAC TG 3′
5′ TTG GGG GTT TTC ATT AGG GTT TAG 3′
19,20
5′ GCT ACA GCC CGG GAA GGA TGG A 3′
5′ GTT GGG GCT CTC AGG TGG AAA ATG 3′
21
5′ CTC TCG ACC CTA GCC CAC AAG T 3′
5′ CTC CAC GTC CCC ATC ATT ACC T 3′
22
5′ GGT AAT GAT GGG GAC GTG GAG GAT 3′
5′ GGT ATA AGG AGG CGC AGG TCA GG 3′
23, 24, 25
5′ GTG GCA TGC CGT TTG TGG AGT TAG 3′
5′ TGG GGA GGC AAA GAA GCA GTC AGT 3′
26, 27
5′ GCC CCC AGC CCG TCA ACC 3′
5′ TCC TAC CCC CTG GCC TCA TTC TG 3′
28, 29
5′ GGT GGG TGG GGC TTT CTG G 3′
5′ GAC TCC CTC CCG GTG ACT CTT T 3′
30
5′ GAA GGC CTC CGT TTG TGC 3′
5′ GTT GGG ACT CAG CCT CTT TAT TTG 3′
1
5′ CCT GCG GGC CCT GGT GAT AGC 3′
5′ GCG GGC AGC GTG TCC AGC AGT C 3′
1
5′ GCG CCG GGG TGG GGT GGG GAG AT 3′
5′ CTT GGG CGG AGG GCA GTT TC 3′
ITGB3
ITGA2B
GP1BB
a finding later corroborated by several studies.2,4,5,25 Thirty years after its serologic definition, HPA1a (and its allelic form HPA1b) was defined at the molecular level as a T-to-C polymorphism at nucleotide 196 in the ITGB3 gene resulting in the Leu33 (or Pro33 for HP1b) variant of the protein.13 As mentioned previously, early serologic studies using antiHPA1a alloantiserum resulted in direct immunoprecipitation of a 90-kDa protein from canine platelets, suggesting that the antigenic determinant for HPA1a has been conserved between humans and dogs.10 However, among 43 dogs tested, we did not identify a polymorphism corresponding to HPA1; in fact, no nonsynonymous polymorphisms were found in the canine ITGB3 gene. Whereas sera from human patients with neonatal alloimmune thrombocytopenia, posttransfusion purpura, and PTR have been used in the initial serologic characterization of the HPAs, 6,7,14,1820,28,29 sera from dogs with alloimmune thrombocytopenic conditions are unavailable. Therefore, our approach to the investigation of canine platelet-specific antigens was to begin with genomic DNA sequence analysis to determine whether any nonsynonymous SNP that could represent potential platelet alloantigens are
present. Because 14 HPA (HPA1, 4, 6bw, 7bw, 8bw, 10bw, 11bw, 14bw, 16bw, 17bw, 19bw, 21bw, 23bw, and 26bw) are found in platelet membrane GPIIIa (β3), we focused initially on sequencing the canine homolog of its encoding gene, ITGB3, by using genomic DNA from 43 dogs representing 11 breeds. Surprisingly, we did not identify any missense SNP in 14 of 15 exons of ITGB3. As noted previously, exon 1 of the canine ITGB3 gene was not sequenced due to technical difficulties, but none of the HPA SNP are found in exon 1, which is not surprising considering the fact that only one amino acid encoded by this exon is found in the mature, membrane-bound form of the protein (GenBank RefSeq mRNA sequence, NM_000212.2; protein sequence, NP_000203.2). If the frequency of canine platelet-specific antigens is similar to that of HPA in various ethnic groups, the high (for example, HPA1a in China, 0.994) and low (for example, HPA5b in France, 0.13) allelic frequencies5 could explain the lack of missense SNP in a group of 43 dogs. Six HPA (HPA3, 9bw, 20bw, 22bw, 24bw, and 27bw) reside on the platelet membrane GPIIb (αIIb), which is encoded by the ITGA2B gene. In comparing the canine and human GPIIb, there
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Table 3. Amino acid substitution SNP in the canine ITGA2B gene and allele frequencies Amino acid 201
491
503
Base in dog genome (codon / amino acid)
G (GAC /Asp)
A (ATG /Met)
C (CCC /Pro)
G (GGG /Gly)
Alternate base (codon /amino acid)
A (AAC /Asn)
G (GTG /Val)
T (TCC /Ser)
A (AGG /Arg)
rs9158428
rs2455312
rs8870390
ss539225964
Asp 0.78
Met 0.06
Pro 0.63
Gly 0.21
Asn 0.22
Val 0.94
Ser 0.37
Arg 0.79
Asp 0.77
Met 0.05
Pro 0.7
Gly 0.23
Asn 0.23
Val 0.95
Ser 0.3
Arg 0.77
Asp 0.79
Met 0.08
Pro 0.46
Gly 0.17
Asn 0.21
Val 0.92
Ser 0.54
Arg 0.83
Reference sequence number
576
Allelic frequencies All dogs (n = 40)
NonITP dogs (n = 28)
ITP dogs (n = 12)
Table 4. Protein isoforms in dogs homozygous for amino acid substitution SNP in the ITGA2B gene Amino acid Isoform
201
491
503
576
No. of dogs homozygous for isoform
a
G (Asp)
A (Met)
C (Pro)
G (Gly)
1
b
G (Asp)
G (Val)
C (Pro)
G (Gly)
2
c
G (Asp)
G (Val)
C (Pro)
A (Arg)
4
d
G (Asp)
G (Val)
T (Ser)
A (Arg)
7
e
A (Asn)
G (Val)
C (Pro)
A (Arg)
4
is an 83% identity in the amino acid sequence (both in the open reading frame and in the mature peptide), with the total preprotein length being 1039 amino acids in humans and 1036 amino acids in dogs (with the length difference resulting from an extra amino acid at position 773 in the human mature protein and 2 additional amino acids at the end of the open reading frame). By convention, the amino acid residue numbers start with the first amino acid of the mature protein, and for the specific residues discussed here, are the same in the human and dog sequences. Given that we did not identify any nonsynonymous SNP in the ITGB3 gene among 43 dogs representing 11 breeds, we opted to include a greater number of breeds (n = 19), with fewer dogs of each breed, for the evaluation of ITGA2B, because it is recognized that gene frequencies of HPA vary between races and ethnic groups.2,4,5,8 In dogs, ITGA2B appears to be more polymorphic than is ITGB3, with 4 amino acid substitution SNP noted among 40 dogs with complete (n = 23) or partial (n = 17) sequencing of the ITGA2B gene. Although there were more dog breeds included in sequencing of ITGA2B compared with ITGB3, the lack of identification of nonsynonymous SNP in ITGB3 cannot readily be attributed to the smaller number of breeds tested, given that 3 (1 American Cocker spaniel and 2 Borzois) of 4 dogs homozygous for Asp201Asn and the 1 dog (Labrador retriever) homozygous for Met491Val underwent complete sequencing of ITGB3. Of the 6 SNP defining the HPA on GPIIb, only one, a G-to-A polymorphism resulting in an amino acid change from Val to Met (Val837Met) defining HPA9bw, was reported previously in dogs at amino acid position 491, with the dog genome having Met. Thirty-five of the 40 dogs tested were homozygous for Val, whereas 4 dogs were heterozygotes, and only 1 dog, a Labrador retriever, was homozygous for Met. Although corresponding amino acid
substitution SNP for the Asp/Asn, Pro/Ser, and Gly/Arg polymorphisms observed in dogs of this study have not been identified as HPA, it is not unreasonable to hypothesize that these particular amino acid changes would produce structural changes that could constitute different epitopes on the protein. Clearly, demonstrating an association between an amino acid polymorphism and the reactivity of canine serum alloantibodies with the allelic form of the protein would be required to define Met491 or other protein isoforms as platelet alloantigens. A canine-specific assay based on cloning of the canine ITGA2B gene containing various polymorphisms and their expression in a cell line could be developed for identification of platelet-specific alloantigens. Such recombinant technology has been used for detection of HPA alloantibodies as an alternative to using panels of typed platelets to determine alloantibody reactivity.9,24 In fact, with the use of a combination of DNA sequencing and recombinant antigen expression, the molecular basis of the platelet-specific Vaa antigen, a low-frequency antigen implicated in neonatal alloimmune thrombocytopenia, has been resolved, leading to the designation of this antigen as HPA17bw.24 The 4 amino acid polymorphisms in the ITGA2B gene resulted in 5 observed protein isoforms among the 40 dogs tested. In this small study, we did not note an association of any protein isoform with a specific breed. Once canine platelet alloantigens are characterized, differences in allele frequencies between breeds likely would exist, considering the population bottlenecks and accompanying founder effects that probably occurred in the creation of many dog breeds. In addition to various HPA occurring at different allelic frequencies in various human populations, some HPA polymorphisms, specifically the HPA5b and HPA2a alleles, have been
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Canine platelet glycoprotein polymorphism
associated with the development of acute and chronic refractory ITP, respectively.3,25 There is little information in the veterinary literature regarding the target platelet antigens in dogs with ITP. In a study evaluating canine platelet GPIIb and GPIIIa as target antigens in 17 dogs with ITP through an indirect radioimmunoprecipitation assay using serum from affected dogs, one dog was positive for GPIIb and GPIIIa, whereas 3 dogs were positive for GP IIb only.11 Our inability to identify allelic differences between ITP and nonITP dogs in the current study may have been due to small sample size as well as to our focus on platelet membrane glycoproteins (GPIIb, GPIIIa, and GP1bβ) that may not be the target platelet antigens in the majority of dogs with ITP. Alternatively, there may not be a link between amino acid polymorphisms and canine ITP. A comprehensive study evaluating more dogs with ITP, more healthy dogs for each breed, and additional genes encoding platelet membrane glycoproteins is needed to build on this pilot study to determine whether there is an association between specific alleles and a diagnosis of ITP. Of the alloimmune thrombocytopenic conditions described in human patients, PTR is likely of greatest clinical importance in dogs. Platelet alloimmunization may occur after the administration of packed RBC or platelet concentrate, even though the prevalence of platelet-specific antibodies in multiply transfused human patients is reported to be 3% to 5%.17 With veterinary medicine striving to offer a more sophisticated level of patient care in areas such as oncology and critical care medicine, more canine patients are receiving multiple transfusions, and the development of PTR likely will become more prevalent in the clinical setting. The current study identifies protein polymorphisms in platelet antigens that have the potential to act as alloantigens. An approach using DNA from dogs with PTR and their blood donors to sequence genes encoding for the major platelet membrane glycoproteins may be helpful in the characterization of the canine platelet antigen system.
Acknowledgment
This work was supported by The Barry and Savannah French-Poodle Memorial Fund.
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