Mutation Causing von Willebrand's Disease In Scottish Terriers
Patrick J. Venta, Jianping Li, Vilma Yuzbasiyan-Gurkan, George J. Brewer, and William D. Schall
Source: Journal of Veterinary Internal Medicine 2000; 14:10-19. Submitted by Dr. Patrick Venta.
Von Willebrand's Disease (vWD) in the Scottish Terrier is a serious, often fatal, hereditary bleeding disorder. Elimination of the mutated gene by selective breeding is an important goal for the health of this breed. Although the standard protein-based tests are accurate for identification of affected Scottish Terriers, they are not reliable for the identification of carriers of the mutant gene unless multiple replicate assays are performed. A simple, highly accurate test for carriers of the disease is needed so that veterinarians can counsel clients on which animals to use in their breeding programs. The complete coding region of von Willebrand factor (vWF) complementary DNA (cDNA) was sequenced from an affected animal, and a single base deletion in the codon for amino acid 85 of the prepro-vWF cDNA that leads to Scottish Terrier vWD was identified. A highly accurate polymerase chain reaction assay was developed that can distinguish homozygous normal animals from those that are homozygous affected or het erozygous. In a voluntary survey of 87 animals provided by Scottish Terrier owners, 15 were carriers and 4 were affected with vWD, 2 of which had previously been shown to have undetectable vWF. The determination of the complete canine vWF CDNA sequence should facilitate the identification of additional vWD alleles in other breeds and other species.
on Willebrand's disease (vWD) is a bleeding disorder of variable severity that results from a quantitative or qualitative defect in von.Willebrand factor (vWF).1-5 vWD has been observed in many mammalian species, including humans and dogs. This clotting factor has 2 known functions: (1) stabilization of factor VIII (anti-hemophilic factor A) in the blood and (2) aiding the adhesion of platelets to the subendothelium, which allows them to provide hemostasis more effectively. An affected individual may bleed severely if the factor is missing or defective.
The disease is the most common hereditary bleeding disorder in both dogs and humans, and it is genetically and clinically heterogeneous. Three clinical types, 1, 2, and 3 (formerly I, II, and III6, 7), have been described. Type I vWD is thought to be inherited in a dominant, incompletely penetrant fashion in humans. In dogs, Type I vWD appears to be inherited in a recessive fashion in at least some breeds, although it is possibly inherited as dominant with incomplete penetrance in others. Bleeding appears to be due to the reduced amount of vWF rather than a qualitative difference. ___________________________________________
From the Departments of Small Animal Clinical Sciences (Venta, Yuzbasiyan-Gurkan, Schall) and Microbiology (Venta, Yuzbasiyan- Gurkan), College of Veterinary Medicine, Michigan State University, East Lansing, Ml and the Department of Human Genetics University of Michigan, Ann Arbor, MI (Li, Brewer). Preliminary findings re ported at the International Society for Animal Genetics meeting, 1996.
Submitted September 8, 1998; Revived May 6, 1999; Accepted Au gust 12, 1999.
Copyright (D 2000 by the American College of Veterinary Internal Medicine
Although type I is the most common form of vWD found in most mammals and can cause serious bleeding problems, it is generally less severe than the other 2 types.
Type 2 vWD patients have a qualitative defect in vWF, lacking high-molecular-weight multimers. Affected humans and dogs have a severe bleeding tendency.2, 5 Type 2 vWD is inherited in a dominant fashion in humans but is inherited recessively in dogs .4, 8 Type 3 VVID is the severest form of the disease. It is inherited as an autosomal recessive trait, and affected individuals have no detectable vWF in their blood. Serious bleeding episodes require transfusions of blood or cryoprecipitate to supply the missing vWF. Heterozygous carriers have moderately reduced factor concentrations but generally appear to have normal hemostasis.
Scottish Terriers appear to have only type 3 vWD.3, 4 Homozygotes have no detectable vWF and have a severe bleeding disorder. Heterozygotes have reduced amounts of the factor and are clinically normal.9 The prevalence of vVD among Scottish Terriers including both heterozygotes and homozygotes has been variously estimated from 18% to 30%.9-11
Currently, detection of affected and carrier Scottish Terrier dogs is done by vWF antigen testing12, 13 or historically by coagulation assays.14, 15 These procedures yield variable results because the protein-based tests can be influenced by such things as sample handling, estrus, pregnancy, vaccination, age, and perhaps hypothyroidism.16-21 A dog that tests within the normal range on 1 day can test within the carrier range on another day. This variability makes it difficult for breeders to use this information to eliminate the disease-causing allele from their lines. Thus, it is highly desirable that a more definitive test be developed for Scottish Terriers.
Herein we report (1) the complete amino acid sequence as inferred from the canine vWF CDNA, (2) the discovery of the mutation that causes vWD in Scottish Terriers, and (3) the development of a simple direct DNA test for this mutation that distinguishes homozygous normal ("clear"), carrier, and affected animals with complete accuracy. This test gives breeders the potential to dramatically reduce the frequency of the disease-causing allele and the incidence of the disease within the Scottish Terrier breed. In addition, the determination of the complete nucleotide sequence of the canine vWF CDNA should lead to the rapid development of molecular tests for other breeds and for other species.
Materials and Methods
Isolation of RNA
The source of the RNA was a uterus surgically removed because of pyometra from a Scottish Terrier affected with vWD (factor value < 0.1% and a clinical bleeder). Total RNA was extracted from the tissues using Trizol.a The integrity of the RNA was assessed by agarose gel electrophoresis on a 1 x TBE (1 x TBE: 90 mM Tris, pH 8.3, 90 mM borate, 1 mM EDTA), 1% agarose gel and inspection of intact 18S and 28S ribosomal RNA bands. The factor values for the animals reported herein (always less than the sensitivity of the assay for those affected) were determined by the Diagnostic Laboratory at the College of Veterinary Medicine of Cornell University, by the Comparative Laboratory of Hematology at the State of New York Department of Public Health, or by Drs. Read and Brinkhaus and their colleagues at the University of North Carolina and were reported by the owners.
Design of Polymerase Chain Reaction Pyimer Sets
Polymerase chain reaction (PCR) primers were designed for use in 2 regions of the gene where sequences from 2 species were available.22-23 These primers were designed using rules for cross-species amplifications.24 Most of the primers had to be designed for other regions of the gene using the human sequence alone.25 Primers were designed by taking into account number of codons per amino acid, relative amino acid mutabilities, and where applicable, conservation of nucleotide sequence. We used the same methodology to design a series of universal mammalian sequence tagged sites.24 Roughly 66% of the primers designed for the human sequence alone amplified the correct canine target. New primers had to be designed for regions that did not amplify with the original primer sets. After portions of the canine sequence had been determined, new primers were designed for the canine sequence. Primer sets and amplification conditions are available from the authors on request. Appropriate amplification conditions were determined using human and canine genomic DNAs for the initial primer sets. Generally, 94ºC for 1minutes, 57ºC for 2 minutes, and 72ºC for 3 minutes for 35 cycles worked well with the majority of the amplifications. All amplifications used Tris-HCl pH 8.3 (20ºC), 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTPs, and 20 µLM primers in 25- or 100-µl reactions.
Reverse Transcyiplase PCR
Total RNA was reverse transcribed using random primers.16 Reverse transcription was performed using a commercially available kitb under the manufacturer's suggested conditions. Five microliters of reverse transcribed total RNA was included in each 50-µl amplification reaction. The cDNA was amplified using the primer sets shown to work on canine genomic DNA.
DNA Sequence Analysis
Amplification products were isolated from agarose gels by adsorption onto silica gel particles using the manufacturer's method.c Sequences were determined using 32P 5' end-labeled primers (the same ones used for the amplification) and a cycle sequencing kit,c The sequences of the 5' and 3' untranslated regions were determined after amplification using a RACE (random amplification of cDNA ends) kit.e Sequences were aligned using the Eugene software analysis package.f The sequence of canine intron 4 was determined from PCR- amplified genomic DNA. A "long PCR" kit was used to amplify this 5-kb intron.1
Design of a Diagnostic Test
PCR mutagenesis was used to create diagnostic and control BsiE * and Sau96 I restriction enzyme sites for the test. Amplification conditions for the test are 94°C for 1 minute, 61°C for 1 minute, and 72°C for 1 minute for 50 cycles using cheek swab DNA27 (Yuzbasiyan-Gurkan et al, unpublished). Twenty-five microliter reactions were run, and 1 µl of restriction enzyme was added directly to the PCR reaction and incubated for 3 hours at 60°C for BsiE (and 37°C for Sau96 I. Eight microliters of each reaction were loaded onto a 3:1 NuSieve: low EEO agarose 1 x TBE gel and run at 125 V for 1 hour. Gels were stained with ethidium bromide and photographed under 360-nM ultraviolet light.
A voluntary survey of Scottish Terriers was conducted to determine an estimate of the frequency of the mutation in the population. Participants were identified and enlisted through the efforts of members of the Scottish Terrier Club of America. All submitted samples was included in the survey. Previously determined factor values were not required, although some participants supplied values for their animals. DNA was collected from 87 Scottish Terriers from 16 pedigrees. DNA was isolated from blood using standard procedures26 or from cheek swab samples27 (Yuzbasiyan-Gurkan et al, unpublished). The genetic status of each animal in the survey was determined using the BsiE I test.
Comparison of the Canine and Human Sequences
The cDNA sequence has been placed in GenBank (accession no. AF099154). Two other groups have independently determined the canine vWF cDNA sequence and have also placed their sequences in GenBank (accession nos. L76227 and U66246). The alignment of the canine and human amino acid sequences is shown in Figure 1. There is 85.1% sequence identity between the prepro-vWF sequences of the canine and human amino acid sequences (Fig 1). The propeptide region is slightly less conserved than the mature protein (81.4% versus 87.5%). There were no other noteworthy sequence identity differences in other regions of the gene or between the known repeats contained within the gene (data not shown).
Fourteen potential N-linked glycosylation sites are present in the canine sequence, all of which correspond to similar sites contained within the human sequence. The 2 integrin-binding sites identified in the human vWF protein sequence 29 also are conserved in the canine sequence (Fig 1). The 3' untranslated region has diverged to a greater extent than the coding region (data not shown), a divergence comparable to that found between the human and bovine sequences derived for the 5' flanking region.30, 31
Two other groups independently determined the sequence for most of exon 28.32, .33 All 3 sequences are in complete agreement, although we have also found 2 silent variants in other breeds (unpublished data). Partial sequences of exons 40 and 41 (cDNA nucleo-tides 6923-7155, from the initiation codon) were also
independently determined by another group as part of the development of a polymorphic simple tandem repeat genetic marker.34 There is a single nucleotide sequence difference between this sequence (T) and ours (C) at nucleotide position 6928.
Scottish Terrier vWD Mutation
A single base deletion was found in exon 4 (Fig 2). This frameshift mutation at codon 88 leads to a new stop codon 103 bases downstream. The resulting severely truncated protein of 119 amino acids does not include any of the mature von Willebrand factor region. The identity of the base in the normal allele was determined from an unaffected dog.
Human MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYL 60
Dog -S-T-LVR ----------K--TK--V ---M-----L-G--I ---E-------D----
Human LAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYL 120
Human ETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEGTL 180
Dog -A -------S-----------N-----------------------------K-------
Human TSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARCHPL 240
Dog ------------------R-K-V ----P--V--D-V-QV ---------A---------
Human VDPEPFVALCEKTLCECAGGLECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGME 300
Human YRQCVSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDZGLCVESTECPCVHSGKRYPPG 360
Human TSLSRDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQD 420
Human HSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDVQLPLLKGDL 480
Human RIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDFLTPSG 540
Human LAEPRVEDFGNAWKLHGDCQDLQKQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVS 600
Human PLPYLRNCRYDVCSCSDGRECLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQ 660
Human CGTPCNLTCRSLSYPDEECNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPED 720
Human IFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADN 780
Human LRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGE 840
Human TVKIGCNTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGS 900
Human NPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGR 960
Human YIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVD 1020
Human FGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPY 1080
Human LDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGY 1140
Human ECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCE 1200
Fig 1. Comparison of the human and canine prepropeptice vWF amino acid sequences. The asterick indicates the location of the Scottish Terrier vWD mutation. Potential N-linked glycosylation sites are shown in bold. The known and postulated integrin-
Human VAGRRFASGKKVTLNPSDPEHCQICRCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVE 1260
Human DISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVE 1320
Human YHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRI 1380
Human ALLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVL 1440
Human SSVDELEQQRDEIVSYLCDLAPEAPPPTLPPHMAQVTVGPGLLGVSTLGPKRNSMVLDVA 1500
Human FVLFGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGD 1560
Human ILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLP 1620
Human GDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTL 1680
Human SPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITT 1740
Human IDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVILV 1800
Human TDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTM 1860
Human VTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLXTHRVNCD 1920
Human RGLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNK 1980
Human EQDLEVILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGNMEV 2040
Human NVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRD 2100
Human GTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKVLAPATFY 2160
Human AICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHC 2220 Dog -M--P----PKK---A--L-------K-------PAN---------------------L-
Human DGNVSSCGDHPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQI 2280
Human CTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHC 2340
Fig 1. (continued) binding sites are boxed. Amino acid numbers based on the prepropeptide vWF sequence are shown on the right side of the figure. The mature protein begins at position 763. The human sequence is derived from GenBank accession no. X04385.46The canine nucleotide sequence has been deposited in GenBank (accession no. AF099154).
Human ERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCCOEYECACNCVN 2400
Human STVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAV 2460
Human MGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKSVGSQ 2520
Human WASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRCERME 2580
Human ACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGEC 2640
Human CGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHK 2700
Human CLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMY 2760
Human SIDINDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK 2813
Fig 1. Continued.
Development of a Diagnostic Test
A PCR primer was designed to produce a BsiE I site in the mutant allele but not in the normal allele (Fig 3). To ensure that the restriction enzyme cut the amplified DNA to completion, an internal control restriction site common to both alleles was designed into the nondiagnostic primer. The test was verified by digestion of the DNA from animals that were affected, obligate carriers, or normal (based on reports of factor values >100% of normal from commonly used testing labs and reported to us by the owners and from breeds in which type 3 vWD has not been observed). The expected results were obtained (e.g., Fig 4). Five vWD-affected animals from a colony of mixed-breed dogs founded from Scottish Terriers35 were homozygous for this mutation. An additional unaffected animal from this same colony was clear.
It would still be possible to misinterpret the results of the test if restriction enzyme digestion were incomplete and if the rates of cleavage of the control and diagnostic sites were vastly different. The rates of cleavage of the 2 BsiE I sites were examined by partially digesting the PCR products and running them on capillary electrophoresis. The rates were nearly equal (the diagnostic site is cut 12% faster than the control site).
The mutagenesis primer was also designed to produce a Sau96 I site in the normal allele but not the mutant allele. This test is the reverse of the BsiE I-dependent test with respect to which allele is cut. Natural internal Sau96 I sites serve as digestion control sites (shown in Fig 3). The test using this enzyme produced genotypic results identical to those using BsiE I for all animals examined (data not shown).
Three pedigrees were examined in which the normal and mutant alletes were segregating. The alleles, as typed by both the BsiE I and Sau96 I tests, showed no inconsistencies with Mendelian inheritance. One of these pedigrees included 2 affected animals, 2 phenotypically normal siblings, and the obligate carrier parents. The 2 parents were found to be heterozygous by the test, the 2 affected animals were found to be homozygous for the mutant allele, and the normal siblings was found to be heterozygotes (Fig 4).
Population Surpey for the Mutation
Cheek swabs or blood samples were collected from 87 animals to determine the prevalence of carriers in the US Scottish Terrier population. Although we attempted to make the sample as random as possible, the results of the tests revealed that these dogs came from 16 pedigrees, several of which were distantly interconnected. This close connection is due to some aseertainment bias based on ownership (as opposed to phenotypic ascertainment bias). In these 87 animals, we found 4 affected and 15 carrier animals.
The determination of the canine vWF CDNA sequence permits the first comparison between 2 complete inferred mammalian vWF protein sequences. The protein has a structure based on 4 kinds of repeat units: the A, B, C, and D repeats.1, 2 There does not appear to be a strict correspondence between these repeat units and the intron-exon structure of the gene. The most notable feature of the gene is the large exon 28, which contains most of the 3 A repeat units. Although most human type 2 mutations occur in this exon, type 3 mutations seem to have a relatively random distribution over the entire length of the gene. Although we have not determined the entire intron-exon structure of the canine gene, the few intron splice sites that we have determined are suggest that the human and canine gene structures are very similar (unpublished results).
The order and length of these repeats has remained conserved between the 2 species. There are no inserted or deleted amino acids, and thus the lengths of the human and canine prepropeptide sequences are identical at 2,813 aniino acids. The integrin receptor sites of the human factor are both conserved in the canine molecule (Fig 1). All of the 234 cysteine residues are conserved between the 2 species, suggesting that these residues are of importance for the maintenance of the structure of the molecule. The determination of the canine sequence will also allow cross-species PCR amplifications to be conducted more easily, which should lead to a greater understanding of the structure, function, and evolution of this important coagulation protein.
The results reported here also establish that the single base deletion found in exon 4 of the vWF gene causes vWD in the Scottish Terrier breed. The putative protein produced from the mutant allele is extremely short and does not include any of the mature vWF protein. Four Scottish Terriers known to be affected with the disease were homozygous for the mutation. Five other mixed-breed dogs descended from Scottish Terriers and affected with vWD were also homozygous for the mutation. No phenotypically normal animals were homozygous for the mutation. Unaffected obligate carriers are always heterozygous for the mutation.
The gene frequency in the population surveyed appears to be around 0.13, resulting in a heterozygote frequency of about 23% and expected frequency of affected animals of about 2%. It is extremely difficult to obtain truly random samples from domestic animal populations, and thus caution is needed in extrapolating the results from the survey conducted in this report to the whole Scottish Terrier population. Although the sample size is relatively small and probably biased in unknown ways, these data are in general agreement with the results of protein-based surveys5, 10 and demonstrate that the mutant allele is not uncommon.
The data collected so far indicate that this mutation could account for most and possibly all of the VVVD found in Scottish Terriers. This result is consistent with the allelic homogenity found for other genetic diseases of comestic animals that have been defined at the molecular level.36-38 This homogenity is most likely due to the pronounced founder effect (usually popular sire effect) that occurs in domestic animals, in contrast to the situation in most human and wild animal populations.
Other vWD alleles may exist or may occasionally arise in the Scottish Terrier breed. However, seveal facts suggest that the mutation described in the current report accounts for most of the disease in the breed: (1) the number of carriers for this mutation is of the same magnitude as that found in previous protein-based surveys, 4, 9, 10 which would be consistent with the characterization of this mutation as the major mutation; (2) all of the results obtained in this study are explained by the 1 mutation; (3) only type 3 vWD has ever been reported in Scottish Terriers, which supports the probability of allelic homogeneity; and (4) the founder effect is known to be an important element in domestic animal genetic diseases. Although there is still a possibility that other vWD alleles exist at low frequency in the Scottish Terriers, it seems likely that the elimination of this 1 mutation would cause vWD to become a rare disease in the breed.
Published data using the protein-based factor assays have shown that, at least in several instances, obligate carriers have had factor values that would lead to a diagnosis of "clear" of the disease allele. For example, in 1 study an obligate carrier had a factor value of 78%.39 In another study, at least some of the obligate carriers had factor values of >65%.35 In both of these studies, however, researchers used older method-ologies. (Laurell electrophoresis and venom coagula-tion), although even enzyme-linked immunosorbent assays can produce results with considerable variation.40 In addition, the number of animals
Clear Carrier Affected
G A T C G A T C G A T C
Fig 2. Nucleotide sequencing ladders for the vWD mutation region for clear, carrier, and affected Scottish Terriers. The sequences were obtained directly from PCR products derived from genonmic DNA in exon 4. The arrowheads show the location of the C nucleotide that is deleted in the disease-causing allele. The carrier ladder of each base above the point of the mutation has a doublet appearance, as predicted for deletion mutations. The factor values reported by the owners of these Scottish Terriers were 54% clear, 34% carrier, and <0. 1% affected.
Fig 3. Molecular test to detect the Scottish Terrier vWD mutation. An asterick indicates the position of the deleted nucleotide. BsiE I restriction sites were created by PCR primer mutagenesis. The altered nucleotides in each primer are underlined. The normal and mutant allele can also be distinguished using Sau96 I. The naturally occurring Sau96 I sites are shown by double underlines. The highly conserved donor and acceptor dinucleotide splice sequences are shown in bold.
Fig 4. Scottish Terrier pedigree segregation of the mutant and normal vWF alleles. Exon 4 of the vWF gene was simplified from genomic DNA using PCR techniques. The PCR products were examined for the presence of the normal and mutant vWF alleles by agarose gel electrophoresis after digestion with BsiE I (see Fig 3). The affected animals are homozygous for the mutant allele (229 bp; lanes 3, 5). The other animals in this pedigree are heterozygotes (251 bp and 229 bp; lanes 1, 2, 4 , 6), including the obligate carrier parents. One hundred base-pair ladders were run in the marker lanes (M).
that fall into an equivocal range can be substantial. In 1 study, 19% of Scottish Terriers fell in this range (50-65% of the normal vWF antigen value).10 Thus, although the protein-based tests have been useful, the certainty of the DNA-based test should relieve the necessity of repeated testing and the variability associated with the protein-based assays. In a recent report, the authors provided evidence that daily biological variability is more important than the variability found in the test, and they stated that "multiple tests may be necessary to obtain a reliable estimate of vWF concentration in dogs."40
A direct test for the mutation should also be more useful than a linkage-based test using a vWF-specific polymorphic site, such as the known microsatellite marker contained within intron 40 of the canine vWF gene,34 because the mutation will probably be present on only a proportion of chromosomes that have a particular allele of the microsatellite. Although linkage disequilibrium between the mutation and 1 of the alleles may exist, there will still be many dogs with the same repeat number that do not have the mutation. In addition, a rare recombination between the mutation and the marker or a mutation in the microsatellite repeat could affect the accuracy of the diagnosis.
Although it should also be possible to deduce the genotypic status of the animal using family information, this method is less reliable than a direct test because it requires obtaining accurate information about relatives, which is often not possible. Genetic counseling using a linkage-based test requires considerably more effort, both on the part of the veterinarian or breeder and the service provider, than does analysis of blood or cheek swabs. Also, linkage-based tests do not consistently allow the determination of the genotype at the disease locus, often because the marker s not informative.41
The Scottish Terrier mutation is present in the propeptide portion of the vWF molecule. The propeptide is removed prior to delivery of the mature protein into the plasma. The propeptide is also important for the assembly of the multimeric mature vWF protein.42, 43 With the Scottish Terrier frameshift vWD mutation, neither the propeptide nor any of the mature factor would ever be produced. To our knowledge, no factor has ever been detected in the blood of affected Scottish Terriers, as predicted for the frameshift mutation that was identified.
The determination of the complete canine vWF cDNA sequence will have an impact upon the development of carrier tests for other breeds and other species. Currently, Shetland Sheepdogs and Dutch Kooikers are known to have a significant amout of type 3 vWD.9, 44 Type 3 vWD also has occassionally been seen in other breeds.39 All type 3 vWD mutations described in humans to date have been found within the vWF gene itself. The availability of the canine sequence will make it easier to find the mutations in other breeds. The mutation for the Dutch Kooiker breed has been recently reported.44 In addition, at leaset some type 1 mutations have been found within the human vWF gene, and thus type 1 mutations may be found within the canine vWF gene for breeds affected with that form of the disease. The availability of 2 divergent mallalian vWF cDNA sequences will also make it much easier to sequence the gene from other mammalian species using cross-species PCR methods.24, 45
The test that we have developed for the detection of the mutation in Scottish Terriers can be performed using only small amounts of DNA from any tissue. The tissues that can be obtained using the least invasive methods are blood and buccal cells. A cheek swab can be easily obtained by the veterinarian or breeder.
Care should be taken not to produce genetic bottlenecks when using results from this test. The production of affected animals can be avoided by using at least 1 clear animal in a mating. Genes conferring desirable characteristics in carrier animals can be maintained in a line until the disease-causing allele is lost after a few generations. Using this approach, genetic variability will be maintained at otherloci, and the potential detrimental effects of stringent selection against a deleterious gene will be avoided.
We thank Murat Gurkan, Paul Ferguson, Dr Yueying Cao, and Chris Willett for conducting some of the experiments tor this work and Dr Mary A. McLoughlin, Dr Stephen R DiBartola, Ms Helen Harbulak, Dr Majorie S. Read, Dr Kenneth M. Brinkhous, and Mrs Barbara DeSaye. This work was supported by the American Kennel Club, the Ortho-pedic Foundation for Animals, the Morris Animal Foun- dation, and the Companion Animal Fund at Michigan State University and by contributions from Scottish Terrier clubs and owners.
Some of the authors (GJB, PJV, VYG) have equity in the technology transfer company that they founded to pro vide the vV*9D test and other molecular genetic services to veterinarians and dog breeders.
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Original Doc: vwdgene.doc
von Willebrand's Disease in Dogs
The Doctor's Forum
Von Willebrand's disease is the most common, mild, inherited bleeding disorder of animals, and affects many breeds of dogs.
By W. JEAN DODDS, DVM
Division of Laboratories and Research, New York State Dept of Health, Empire State Plaza, Albany, NY 12201.
Adapted from the Veterinary Reference Laboratory Newsletter, Vol 7, No 3, 1983, courtesy of Veterinary Reference Laboratory, Inc.
The following material was submitted by a smooth fox terrier breeder who discovered VWD in her dogs. The article has been edited for readability and in intended for informational use only. For more in-depth information, use the References listed at the end of this article.
Source: The Fox Terrier Forum, April/May 1995, pp 4-7.
Von Willebrand's disease, the most common, mild, inherited bleeding disorder of animals, is a disease which generally has many physical characteristics but a low death rate. It affects many breeds of dogs. Clinical signs include hematuria, epistaxis, gingival or genital mucosal bleeding, lameness, and prolonged bleeding from cut nails or wounds. Hypothyroidism in a dog with von Willebrands will greatly increase the severity of the disease. Affected dogs and carriers should not be bred and dogs should be tested for von Willebrand's factor before breeding. Treatment of von Willebrand's involves intravenous infusion of fresh whole blood or plasma, at 3-5 ml/lb, with topical use of blood clotting compounds, and avoidance of drugs that interfere with blood clotting.
Von Willebrand's disease (VWD) is the most common, mild, inherited bleeding disorder of people and animals. It is a physical disease with two forms of clinical and genetic expression. It can be a disease that is physically recessive. In that event clinically affected individuals are homozygous for the VWD gene and have two asymptomatic, heterozygous (carrier) parents; or it can be a disease with an autosomal, incompletely dominant expression (variable penetrance), in which both homozygotes and heterozygotes can have a bleeding tendency. Homozygosity is often lethal in this form of VWD.
The recessive form of VWD has been recognized in Poland-China swine, Scottish Terriers and Chesapeake Bay Retrievers. The incompletely dominant for is much more common and has been recognized in 28 breeds of dogs, though virtually any breed may be affected. Several breeds have a high prevalence of the disease (15-60% gene frequency).
Many physical characteristics (but a low death rate) are generally associated with VWD. Typically, there is mild to severe bleeding, usually from mucosal surfaces. Bleeding is increased by physical, emotional and physiologic stress, hormonal imbalances, (especially hypothyroidism), and by other existing diseases in the effected dog such as parasitic, viral and bacterial infections. Typical clinical signs of VWD include: recurrent hematuria; epistaxis; gingival, vaginal or penile bleeding; lameness; stillbirths or neonatal deaths (“fading pups”), with evidence of bleeding at autopsy; prolonged estrual or postpartum bleeding; hematoma formation on the surface of the body, limbs or head; excessive umbilical cord bleeding at birth; and excessive bleeding from toe nails cut too short, or after tail docking, ear cropping or dewclaw removal. Severely affected dogs may bleed to death from surgical prodedures. A parvoviral infection often aggravates the bleeding tendency, which probably explains the high prevalence of severe parvoviral disease in Doberman Pinschers. About 58% of the Dobermans tested have the VWD trait.
RELATIONSHIP OF HYPOTHYROIDISM TO VWD
Several breeds of dogs have a high prevalence of hypothyroidism; the common occurrence of both VWD and hypothyroidism in some of these breeds (Dobermans, Golden Retrievers, Scotties, Corgis, Manchester Terriers) suggests a causal relationship between the synthesis and/or metabolic regulation of thyroid hormones and von Willebrand's factor (VWF) protein, which is deficient or abnormal in VWD. Hypothyroidism in people has been associated with a bleeding tendency caused by abnormal platelet function and low levels of factor VIII activity and VWF. Reduction in these abnormalities has been reported after oral thyroid supplementation. In our studies of Doberman Pinschers, there was an increased frequency and severity of bleeding episodes in animals with the VWD gene that were also hypothyroid. Clinical signs of bleeding in these dogs were lessened or controlled within 48 hours of initiating daily thyroid supplementation; VWF levels also increased. Thus, animals with thyroid dysfunction can have fluctuating levels of VWF. When these dogs are given thyroid supplementation, VWF levels can increase to within normal limits, which could prevent accurate diagnosis of their genetic status for VWD. For example, carriers of VWD might test as normal when on thyroid medication. Because of the apparent increase of the bleeding tendency in dogs with VWD and hypothyroidism, breeders and veterinarians must be aware of the increased risk and be more cautious about breeding or performing surgery on dogs with both problems.
GENETICS AND RECOMMENDATIONS TO BREEDERS
One form of VWD is inherited as an autosomal dominant trait which can reveal itself with varying physical signs. Or, in other words, the dog can have a differing levels of the VWD gene. This type of inheritance is termed 'incompletely dominant.' Both sexes can be equally affected in VWD, unlike in hemophilia, which is an X-linked recessive trait that classically affects only males. While the dominant expression of the VWD gene produces reduced levels of VWF in homozygotes with a double dose of the gene, it also does so in heterozygotes with a single-gene dosage. Clinical expression of VWD as a bleeding tendency, however, is inherited in the two forms mentioned at the beginning of this article. The common form is an autosomal incompletely dominant condition in which homozygosity is usually lethal, and heterozygotes can be either carriers with no physical symptoms, or affected to a varying degree. The more rare form of VWD is an autosomal recessive disorder in which clinically affected individuals are homozygous for the trait and express a double dose of the gene, one from each heterozygous, asymptomatic parent.
The following possibilities occur when animals with the VWD gene are mated:
If a normal dog, free of the VWD gene, is mated to one affected with or carrying the gene, about half of the litter will be affected or will be carriers, like the VWD parent, and the other half will be normal, free of VWD.
If two affected animals or two carriers are mated, three-quarters of the puppies will have the VWD gene. One quarter of the litter will be severely affected (perhaps fatally at birth), one-half will be affected or carriers like the parents, and the remaining one-quarter will be normal.
The more severely affected the parent, the more likely that it will produce severely affected bleeder puppies. Thus, a VWD carrier, which is clinically normal but transmits the VWD gene (trait) to some of its progeny, is the least likely to produce a clinically affected bleeder pup, provided the mate was VWD free. Mating two carriers of VWD, however, doubles the VWD gene, and puppies that get a double dose (one gene from each parent) are more severely affected than either parent. This is obviously an undesirable situation.
The following recommendations are offered to reduce the prevalence of VWD in a population: Blood tests for VWD should be taken in animals that are related to those bloodlines known to have the problem, as well as other top-producing or winning foundation stock. Pedigrees can be sent to the New York State Department of Health for evaluation of whether the animals or bloodlines are closely, distantly or not related to those of known affected families. Ideally, affected animals and carriers of VWD should not be bred. However, it may not be feasible for a breeder to eliminate these dogs as breeders, especially if they are desirable for other reasons. Therefore, if breeding affected animals is necessary to preserve important bloodlines or type, the following should be considered:
Breed asymptomatic carriers to normal dogs and blood test the pups. Half of the litter should be normal and the other half should be carriers. This is the best alternative to preserve bloodlines.
Mate two asymptomatic carriers only with caution and blood test the pups. Remember that only one-fourth of the litter will be normal and another one-fourth will be more severely affected than either parent.
Breed mildly or moderately affected animals only if essential to the breed and then breed the affected dog only to a normal animal. Be sure to blood test the pups, since half of the litter should be normal, and the other half will be affected like the parent. We do not advise this type of mating.
Do not mate two affected animals.
Never breed a severely, clinically affected animal.
Diagnostic tests for canine VWD are similar to those used for human VWD. Specialized VWF assays are required because VWD is indicated in the blood by a deficiency or abnormality or one or more proteins of the factor VIII complex. Screening coagulation tests (APTT, PT and TCT) are nondiagnostic. A brief explanation of the factor VIII complex is given in the sidebar on the following page to help make the tests used to diagnose VWD easier to understand. Dogs clinically affected with VWD also have prolonged bleeding times, abnormal platelet retention in vitro, and variable factor VIII coagulant activity levels(normal to moderately reduced). Definitive diagnosis is by finding reduced levels or no levels of FVIIIR:Ag and/or the platelet-related assays of VWF (ristocetin cofactor). A practical way to distinguish between asymptomatic heterzygotes and heterozygotes clinically affected with VWD (both have reduced levels of FVIIIR:Ag) is to determine the cuticle bleeding time. This test is performed by laying the dog on his side and cutting one or more toe nails too short with a standard, guillotine-type (Resco) nail clipper. With the foot undisturbed, the bleeding should cease within five minutes in normal dogs. Dogs with hemostatic defects, such as VWD or platelet dysfunction, have prolonged initial bleeding times (some never stop bleeding and must be cauterized) or begin bleeding again after initial clotting. A convenient way to perform this test is as a presurgical screen once the animal is anesthetized and the surgical site is being prepared. Clinical experience with this technic as a presurgical screen has been useful in identifying dogs that may bleed excessively at surgery.
GENETIC SCREENING FOR VWD
Screening for genetic defects has been used successfully in people for many years and more recently has been applied to animals. In the mid-1960's, veterinarians began screening dog populations for inherited eye diseases and hip dysplasia. Eventually, organizations were developed to register animals free of the major hereditary conditions known to affect purebred dogs. In New Zealand and Australia, mass screening of cattle for mannosidosis has been used effectively since the mid-1970's to control this devastating disease. The common practice of line breeding and inbreeding purebred dogs facilitates both the transmission and recognition of all types of genetic defects. Also, because companion animals live in close daily contact with their owners, illnesses are more likely to be noticed and treated.
The screening program developed in our laboratory for inherited bleeding disorders of purebred dogs evolved from an ongoing consultation and referral practice in veterinary hematology. The number of dogs screened for VWD by blood testing has increased steadily from the inception of our program in 1976 through 1980. Since 1980, the number has stabilized; this reflects implementation of planned matings between normal and carrier stock to eliminate the gene once heterozygotes have been identified.
In developing an accurate test to detect heterozygotes, we found, as in bovine mannosidosis, a skewed distribution between normal and abnormal populations, with a small but defined area of overlap. Despite this overlap, we have developed an effective program to identify heterozygotes and to gradually eliminate them from the breeding population. Data in support of this conclusion have shown a significant decrease in the overall prevalence of VWD in Scottish Terriers, Golden Retrievers and Miniature Schnauzers. The prevalence of VWD has been reduced in Scotties from 35% to 11%, in Goldens from 15% to 6%, and in Schnauzers from 25% to 18%. The prevalence in the other commonly affected breeds is essentially unchanged. This probably reflects a combination of insufficient numbers tested to implement planned matings for control of the gene and/or the need to increase breeder and veterinary awareness and acceptance of the importance of VWD.
The accuracy of the testing program in detecting the VWD genotype has been evaluated by retrospective analysis of the results to date for all three mating types (normal x normal, normal x carrier, carrier x carrier). The rate of misclassification of genetic status by this test is only 2-3%, which means that in 97% or more cases, the test is a reliable predictor for genotype. Cumulative data from these studies are currently being entered into a newly developed computer program to facilitate subsequent analyses.
FACTOR VIII COMPLEX
Factor VIII circulated in plasma as a complex of two proteins: the factor VIII-coagulant activity protein, which is severely deficient in hemophilia A and often is reduced in severe forms of VWD; and the VWF protein (also called after factor VIII-related protein), which has several important biologic properties that control bleeding and is deficient or abnormal in VWD but normal in hemophilia. Thus, one can readily distinguish between VWD and hemophilia even without a family history to establish the inheritance pattern, because animals with VWD have abnormal or low levels of VWF protein and hemophiliacs do not.
One of the properties of the VWF protein is its ability to cross-react in immunologic tests with antibodies formed against it in other species. This immunologic property of VWF protein is called factor VIII-related antigen (FVIIIR:Ag) and is measured routinely in plasma by the Laurell electroimmunoassay. Our screening program is based on this technic, using antibodies formed against VWF protein purified from normal canine plasma. (Such antibodies are not commercially available for animals, and cross-reactivity with readily available human antibodies varies among species, has less avidity, or is absent.)
The sensitivity, or lower limit of detection of this technic is about 7%. Thus, levels of 7% of normal or less of this plasma protein are undetectable (zero) in the assay. However, by using a more sensitive research technic that detects as little as 0.1% of the antigen, dogs affected with VWD can be classified into two groups. Scottish Terriers and Chesapeake Bay Retrievers that are homozygous for the autosomal recessive form of VWD have no detectable antigen and are truly zero level animals, whereas their heterozygous (carrier) parents and those with the other form of VWD (incompletely dominant expression) have reduced but measurable amounts of antigen.
To establish the normal range for canine FVIIIR:Ag, we measured levels in plasma from over 125 healthy purebred dogs of breeds in which VWD has yet to be recognized. The antigen levels in these animals ranged from 60-72% of normal, with a mean value of 93 ± 33%.
MANAGEMENT AND TREATMENT: GENERAL CONSIDERATIONS
Patients with bleeding disorders cannot be properly treated without an appropriate physiologic and physical environment for hemostasis, tissue repair and prevention of recurrence. An extremely important aspect of medical management is avoiding use of drugs known to interfere with hemostasis. For example, aspirin, phenylbutazone, phenothiazine tranquilizers, estrogens, plasma expanders (Dextran, HES), nitrofurans, sulfonamides, antiinflammatory drugs, penicillins, and local anesthetics. These are contraindicated for patients with moderate or severe hemostatic defects, since they impair platelet function and further compromise the stability of the hemostatic plug.
Any live-virus vaccine or viral infection can impair platelet and/or endothelial cell production and turn over. The effect occurs during the viremic phase after vaccination or exposure (usually at 5-10 days) and results in relative thrombocytopenia or endothelial injury, which may prolong bleeding time and predispose the animal to hemorrhagic problems. Platelet reductions of 100,000/yl can occur. During this period, animals with hemostatic defects are at risk and should be evaluated carefully for signs of bleeding. Elective surgical procedures, such as ear cropping, ovariohysterectomy, castration and dental surgery, should be performed within 48 hours after vaccination or should be postponed for 10-14 days. In most cases, animals admitted for elective surgery are vaccinated immediately or within 24 hours of surgery, which accounts for the relatively few vaccine related bleeding problems.
As mentioned earlier, hypothyroidism produces a bleeding tendency and exacerbates the clinical expression of concomitant VWD. Thus, asymptomatic heterozygotes for VWD can exhibit a bleeding tendency if they also become hypothyroid. This is a common situation in the Doberman Pinscher; many of these dogs have very low levels of T4 and/or T3, with abnormal TSH response tests, yet show none of the typical overt signs of thyroid disease. Hypothyroid animals with concomitant VWD usually show slightly to moderately reduced platelet counts (70,000-100,000/yl) and very long cuticle bleeding times, in addition to their low VWF activity.
Recent clinical experience with Dobermans admitted for bleeding episodes, such as nosebleeds or severe hematuria, has shown that thyroid supplementation alone reduces and then controls the bleeding within 24-48 hours. This finding supports our unpublished experimental studies that showed that when treated with T4, seven Dobermans with VWD and mild hypothyroidism had a two- or three-fold increase in their baseline levels of VWF activities within 24 hours. This peaked at three days and was sustained for another three to four days. The standard therapeutic dosage, based on body weight as recommended by the manufacturer, should be used and the animal should have a pretreatment serum specimen collected for resting T4 and T3 determinations. Follow-up of Dobermans treated with thyroid medication to control bleeding has shown that most were severely hypothyroid and the remainder were mildly abnormal or borderline normal. Therefore, our recommendation for all Dobermans with serious bleeding is to collect a serum specimen for thyroid and VWD measurements and then treat with thyroid replacement hormones pending results of blood tests.
Topical Treatment: Microcrystalline collagen (Avitene: Alcon), a topical hemostat, is superior hemostatically to pressure alone and/or oxidized cellulose cloth (Oxycel: Parke-Davis) or thrombin-soaked gelatin sponge (Gelfoam: Upjohn). Thrombin itself also has been used as a topical agent to control bleeding.
Blood or Blood-Component Replacement: Details for the type and volume of blood or blood products recommended to treat moderate or severe bleeding disorders are discussed elsewhere. The preferred anticoagulants for collection of whole blood for transfusion are acid-citrate-dextrose or citrate phosphate dextrose. Use of heparin is not recommended because it activates platelets, causing them to clump. Blood plasma products used to control and treat bleeding should be as fresh as possible or fresh-frozen, because coagulation factors and platelets are labile. As mentioned above, animals with bleeding disorders are likely to require repeated transfusions during their lifetimes and thus are at risk for transfusion incompatibilities. Use of unmatched whole blood, therefore, is contraindicated except in life-threatening emergencies. Appropriate therapy involved IV infusion of fresh universal donor or cross-matched blood at 3-5 ml per pound of body weight. The treatment of choice for VWD, when RBC's are not required, is canine plasma factor VIII concentrates (cryoprecipitates), but these are not commercially nor readily available for animals. An alternative is to give fresh-frozen, homologous plasma at 3-5 ml per pound of body weight once or twice daily.
Because platelets and coagulation factors have relatively short in vivo lives, control of bleeding episodes requires that the daily amount be divided and given as two regularly spaced transfusions. This regimen also reduces the risk of circulatory overload. For elective or other surgery, clinically affected VWD patients should receive fresh, compatible whole blood, at 3-5 ml/lb, two to four hours beforehand. The ability of the transfused
VWF to reduce the bleeding time lasts for up to four hours, whereas the other properties of the factor VIII complex remain active for 10-24 hours. For surgery on a VWD heterozygote with an unknown or no bleeding history, the cuticle bleeding time should be performed as a presurgical screen. If the time is within five to six minutes (normal), this does not preclude the existence of a mild underlying hemostatic defect but indicates that the surgeon probably will not encounter significant bleeding.
Instructions for Preparation
of von Willebrand's Disease Samples
Fill a blue-top Vacu-Tainer to capacity, mix well and centrifuge at 2500-3000 rpm for 15 minutes. Aspirate the supernatant plasma with a plastic pipette, siliconized pipette or a small plastic syringe, such as a tuberculin syringe. Place the plasma into a small plastic tube and freeze. Ship within 1-2 weeks after drawing.
All samples should be frozen initially and sent on cold pack to the laboratory. The sample may thaw in transit but samples in shipment longer than three days may have slightly reduced values. Dry ice is not necessary, but samples should arrive in a cool condition. Shipping by regular mail is quite satisfactory if the above criteria are met (cool, in three days or less).
Test Restrictions For Genetic Screening of Healthy Dogs
Do not test unhealthy animals or animals receiving any type of medication for recent illness or vaccination within the previous 14 days. If the animal is being given long-term medication or heartworm preventive, please indicate type and duration (e.g., Caricide or thyroid medication). A fasting blood sample is not necessary.
Tests with borderline findings between "normal" and "suspect" carriers will be repeated. Therefore, in these cases, additional time will be required to repeat the test. Those with <60% FVIIIR:Ag activity are normal, those with 40-60% are carriers, and those with less than 40% are considered FVIIIR:Ag deficient.
* These instructions are for specimens to be submitted to the veterinary Reference Laboratory for analysis. Readers should consult with their local diagnostic laboratory for instructions if the veterinary Reference Laboratory is not used.
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Original Doc: vwd02.doc
Update On Von Willebrand’s Disease In Scottish Terriers With Emphasis On The Acquired Disorder
W. Jean Dodds, DVM
Wadsworth Center for Laboratories & Research
New York State Department of Health
P.O. Box 509
Albany, NY 12201-0509*
Scottish terrier fanciers have contacted us recently requesting information about the influence of autoimmune diseases and thyroid disease in particular upon the level of von Willebrand factor (vWF) and the expression of von Willebrand’s disease (vWD) in the breed. Questions and concerns have been raised about some recent vWD test results because owners were expecting to have normal testing offspring from parents that have tested normal or came from normal testing ancestry. Some puppies from these litters were coming up with results at the lower end of normal range (60-69% vWF antigen, vWF:Ag), borderline (50-59%), or even an occasional one testing below 50% vWF:Ag. How could this occur?
In order to help explain the various factors that could pertain here, we thought an article about the variables affecting vWF production and the influence of autoimmune disease on its regulation and clinical expression would be helpful.
Influence of Sample Collection, Processing and Transport
Errors or difficulties encountered when collecting and processing blood samples for vWD screening still remain the most common reason for inconsistent results. We realize that its not always easy to obtain proper samples with clean venipuncture and good blood flow rate, especially with puppies or breeds that like to express their displeasure at having to endure this indignity! Samples containing significant hemolysis (red blood cell breakdown) or clots, even small ones, indicate that the animal was stressed and/or the sample was not obtained cleanly. Results for vWF:Ag on these samples may be artificially raised, lowered, or invalid. We routinely examine the quality of each sample, note any unusual findings on the written report, and advise you to retest the animal if indicated. We realize that this is more easily said than done. However, in reviewing records of earlier tests when future generations appear to be testing lower than expected, we quite often see that original results of questionable validity were never confirmed by a retest. So one way to assure more consistent interpretation of vWD genotype for a breeding program is to retest all foundation stock if there is or was any question about the sample quality on an original test. Having more than one test result gives you more confidence about the true reading of your stud dog or brood bitch. As expected, animals with normal levels are rarely retested whereas those with low levels are rechecked one or more times. Unfortunately, the higher readings on rechecks are often the ones that breeders remember or elect to proceed with -- which is only human nature, but may not reflect the animal’s true status. If there was no apparent reason to obviate one or more of the test results, then a prudent approach would be to consider averaging all values or at least keeping in mind that some other factor(s) might be influencing vWF levels in this animal.
When transporting samples to us for testing they should stay cold or frozen in transit; an
*Home: 938 Stanford Street, Santa Monica, CA 90403 [(213) 828-4804].
overnight mail or package express system is preferred over regular mail. Samples should be placed in an insulated container with a frozen cold pack even in wintertime, because during transport the box could be kept indoors near a heating unit. Sending the samples directly from your location to us is preferable to having a commercial laboratory pick up the specimen at your veterinarian’s clinic and ship it to one location where it is then reshipped to us or another place for testing. The fewer times a sample has to be reshipped prior to analysis, the less chance for transit artifacts affecting results. In fact if samples become heated or otherwise altered by shipment, levels of vWF:Ag often go up. The owners may be pleased to obtain high testing results but the actual level could be lower. (as an example, we invalidate all results above 200% vWF:Ag.)
Disparity Between Duplicate or Repeat Test Results from Different Laboratories
On several occasions, fanciers have indicated that they split samples or sent two samples within a few days to our laboratory with different animal identifiers, or have sent them to us and another university or commercial laboratory at the same time. The purpose has been to check the reliability and validity of the testing process. We have no problem with anyone wishing to do so because it is the cornerstone of proper quality control. If a problem exists in our testing procedure we want to hear about it in order to investigate the situation. If you do this, however, please let us know afterwards so we can correct our records when duplicate samples were submitted under different names.
The assay we run routinely is basically the same as its been for the past 10 or more years. We have recently developed a newer, more sensitive and equally reliable test that we’ve been running in parallel with the traditional assay. So far results of the two tests have been remarkably consistent. The same supervisory laboratory staff has coordinated and reviewed our vWD testing results for this entire period! We have very strictly standardized quality control procedures and the reproducibility of the assay is + 3% with a coefficient of variation of less than 9%. While any system can experience an occasional human error, including our own, the error factor is minimized by our quality control/review procedure.
A group of commercial veterinary testing laboratories offer vWD testing now. Most of these laboratories send their samples directly to us but a few do their own testing in-house. If you use this system through your veterinarian, find out how or where the laboratory does the test. A few human clinical laboratories also offer veterinary testing. Be cautious here because if the routinely used human assay has not been adapted for use in the dog, the results may be erroneous. Secondly, if a veterinary laboratory performs the test in-house using our reagents their staff may be less aware of the need to check sample type and quality and note any unusual findings on the report. We know that this situation occurs because of healthy animals that received test reports of < 7% vWF:Ag when the specimen submitted turned out to be serum (already clotted and so no vWF:Ag was left) rather than plasma.
The most frequent problem encountered with divergent results between so-called “duplicates” is that the specimens are not truly duplicates. For example, the ideal way to collect true duplicates is to preload an empty syringe with the correct amount of trisodium citrate anticoagulant to achieve a final concentration of 1 part citrate to 9 parts blood (1 in 10 final dilution). After collecting a free-flowing sample from a clean venipuncture, the sample is mixed and transferred to a test tube for centrifugation. After centrifugation, the plasma at the top of the tube is removed, placed into another empty test-tube, mixed thoroughly and then half of it is transferred to a third empty test tube. Only in this manner are the two specimens equivalent. The mistakes most commonly made in trying to send duplicates are taking two separate samples from different veins one after the other (the animal is more stressed for the second one), or the plasma is incorrectly divided after spinning the blood down because the top part is removed and put in one tube and the bottom part is placed in the second tube. As plasma proteins centrifuge according to weight and mass, the heavier proteins are at the bottom of the plasma and lighter ones at the top; this does not produce true duplicate samples.
Finally, one other common problem occurs with the use of VacutainersR to collect the sample, if they’re not used as originally intended. In our experience many veterinary clinics collect blood first into a dry, empty syringe and then add it to the VacutainerR containing the citrate anticoagulant, instead of drawing the blood directly through the needle by vacuum into the tube. This modification allows for clotting to begin before the anticoagulation occurs and so samples are partially clotted and will most likely have lower readings for vWF:Ag.
Inherited and Acquired vWD
vWD in humans and dogs is now recognized to be congenital (present at birth) and inherited and/or acquired secondary to familial autoimmune thyroid disease. Presently, 44 of 53 dog breeds known to have vWD also transmit familial hypothyroidism. The Scottish terrier is one of the breeds having both disorders. While the prevalence of thyroid disease has increased rapidly over the last decade within Scottish terrier stock, vWD has declined until recently through the collective efforts of breeders to test and screen out carriers from their breeding programs.
1. Inherited (Congenital) vWD
In Scottish terriers, vWD is an autosomal recessive disorder classified as Type III vWD. Clinically affected animals are homozygous for the vWD gene and have two asymptomatic, heterozygous (carrier) parents. The summary and recommendations about vWD’s clinical and gene expression as described in the Scottish Terrier Association’s Handbook are still correct.
To review, vWD in Scottish terriers expresses a mild to severe bleeding diathesis that usually involves mucosal surfaces and is exacerbated by physical, emotional and physiological stresses as well as by other concurrent diseases. Typical clinical signs include: recurrent gastrointestinal hemorrhage with or without diarrhea; recurrent hematuria; nosebleeds; gingival, vaginal, and penile bleeding; lameness that mimics eosinophilic panosteitis; stillbirths or neonatal deaths (“fading pups”) with evidence of bleeding at necropsy; prolonged estrual or postpartum bleeding; hematoma formation on the surface of the body, limbs, or head; excessive umbilical cord bleeding at birth, and excessive bleeding from toe nails cut too short or after dewclaw removal. Affected dogs may bleed to death from surgical procedures. Diagnostic tests require specialized vWF assays. Screening coagulation tests (APTT, PT and TCT) are nondiagnostic. Animals affected with the recessive Type III disease are homozgotes, have long bleeding times and zero vWF:Ag, whereas their heterozygous parents have reduced levels (15-60% of normal) and have normal bleeding times unless some other hemostatic problem coexists.
2. Acquired VWD
In dogs, vWD is exacerbated by concurrent hypothyroidism, so that asymptomatic carriers of vWD may exhibit a bleeding tendency if they subsequently become hypothyroid, a common situation found in many breeds including Scottish terriers. The concomitant prevalence of vWD and hypothyroidism confirms the link between the synthesis and/or metabolic regulation of thyroid hormones and vWF. Furthermore, hypothyroid dogs may exhibit low platelet counts (thrombocytopenia) and associated mucosal surface bleeding. In humans, a bleeding tendency may be an early manifestation of hypothyroidism. This includes easy bruising and thrombocytopenia, abnormal platelet function which returns to normal after treatment with thyroid hormone, and acquired vWD similar to the common Type I form of this disorder (low VWF:Ag). It is generally impossible to distinguish between the inherited and acquired types of vWD in an individual patient with currently available techniques.
Circulating antithyroid antibodies are often present in humans and dogs with autoimmune thyroid disease (called thyroiditis or Hashimoto’s thyroiditis) several years before the thyroid disease becomes clinically apparent. Thus animals with thyroid dysfunction can have fluctuating levels of vWF and, when placed on thyroid supplementation, levels can increase to within normal limits, which could preclude the accurate diagnosis of their genetic status for vWD (i.e., carriers of vWD might test as normal when on thyroid medication). Because of this apparent exacerbation of bleeding tendencies in dogs with vWD and hypothyroidism, breeders and veterinarians need to be aware of the increase risk and be more cautious about breeding or performing surgery on dogs with both problems.
In humans, antithyroid antibodies can be transferred from mothers with Hashimoto’s thyroiditis to their infants in breast milk. Neonatal hypothyroidism can occur and is picked up on the routine newborn screening programs run by state and local public health agencies. Whether a parallel transfer of maternal antithyroid antibody occurs via the colostral milk to newborn puppies is unknown but is likely. What effect, if any, the transfer of these antibodies could have on the development and health of puppies is also unknown . Both humoral and cell-mediated immunity could be affected, especially when growth hormone and thyroxine modulate the thymus and thereby immune function. Perhaps the presence of maternal circulating antithyroid antibodies during the first several months of life could alter immune recognition or development sufficiently to render these puppies more susceptible to other immune-mediated stressors (viruses, vaccines, drugs, toxins, etc.). These antibodies could play a role in stimulating the active immunological process that leads to the development of immune thyroiditis.
3. Magement and Treatment
The severely limited availability of blood products for clinical use in veterinary medicine has necessitated the development of alternate strategies for the treatment and management of bleeding disorders. In those breeds like Scottish terriers in which both vWD and hypothyroidism occur relatively often, the administration of oral thyroid hormone has been effective in controlling bleeding episodes exacerbated by these two disease. Clinical signs of bleeding are lessened or controlled within 48 hours after therapy is initiated. In man, L-thyroxine therapy has been shown to reduce levels of circulating antibodies responsible for thyroiditis and hypothyroidism. Presumably this plays a role in the reversal of the bleeding tendency associated with thyroid disease. In dogs, the toenail bleeding time is dramatically shortened and in some cases may even be corrected to within normal limits (<5 mins) from pre-treatment values beyond 20 minutes. Thus, thyroid supplementation alone may suffice to control bleeding in mild to moderate vWD, a situation analogous to the use of desmopressin (DDAVP) or danazol to control bleeding in man. DDAVP treatment has recently been shown to improve vWF levels in normal dogs and dogs with vWD, although relatively high doses are required and the response is transient (1-3 hours); refractoriness to this treatment can develop rapidly.
The standard dosage of L-thyroxine for treatment of vWD [enhances platelet stickiness (adhesion) and improves vWF activity] is 0.1 mg/10 lb. body weight given twice daily. The therapeutic response should be monitored with serial toenail or other bleeding times and periodically by thyroid function tests; dosages of medication should be adjusted accordingly to maintain thyroid levels (total T4, free T4, total T3) within the upper half to third of the adult normal ranges.
Production of vWF and Causes of Autoimmune Thyroid Disease
The endothelial cells lining blood vessels are the primary and essentially sole source of vWF production in the dog. Thyroid dysfunction from familial autoimmune thyroiditis is by far the most commonly recognized cause of acquired vWD in dogs. Production and/or secretion of vWF from endothelial cells is clearly altered by thyroiditis. The most likely cause of lower than expected vWF:Ag scores on dogs with parents known to be free of the vWD gene is therefore this thyroid disorder, whether or not it is expressed to date, because the pathologic process associated with autoimmune antithyroid antibody production can take several years to induce the end stage of clinical hypothyroidism. If you breed them, they will transmit susceptibility for the thyroid problems to some of their offspring.
Our cumulative data on many breeds affected by familial autoimmune thyroid disease, including Scottish terriers, indicate that reduction in vWF:Ag production associated with thyroiditis is becoming quite common. To repeat, the clinical and laboratory features of this acquired form of vWD are indistinguishable from the congenital inherited form of vWD. Thus while the inheritance and carrier detection program for vWD have not changed, the situation over the past decade and especially in the last 5 years is complicated by the rapid rise in the incidence of thyroid and other autoimmune disease in your breed.
This is illustrated by a summary of our latest statistics on the prevalence of vWD over the past 8 years.
The dramatic increase since 1987 in the number of dogs having abnormal levels of vWF:Ag is real because the number of dogs tested each year continues to be large enough for statistical validation. There are three possible explanations for this increase, which by the way has also occurred in the five other breeds that we test most often (Doberman pinchers, golden retrievers, standard poodles, Shetland sheepdogs, Pembroke Welsh corgis). The first envokes a shift in the measurement of vWF:Ag; this possibility is ruled out because our assay has been standardized internally both vertically and horizontally over time and so varies less that + 5% of the actual value. Secondly, the prevalence of inherited vWD is increasing because tested or untested carriers continue to be bred. Lastly, the increasing prevalence of thyroid disease has produced a parallel increase in the acquired vWD. The most likely explanation is a combination of the last two possibilities.
There are 4 primary causes of autoimmune disease and autoimmune endocrine (e.g., thyroid ) disease in particular: a) genetic predisposition; b) viral infection or exposure; c) hormonal influences especially of sex hormones; and d) stress. In humans with a parallel form of thyroid disease (Hashimoto’s thyroiditis or lymphocytic thyroiditis), they have identified a specific profile of the major histocompatability complex, HLA-DR, which conveys genetic susceptibility to the disease. Furthermore, in May 1989 a research team in England discovered a novel retrovirus, distinct from but related to the HIV virus that transmits AIDS, that is associated with human thyroiditis. The bottom line is that viruses capable of inducing immune dysregulation in genetically susceptible stock can initiate autoimmune thyroid disease under the appropriate environmental conditions (e.g., hormonal imbalance, stress, drug or toxin exposure, dietary factors, etc.). There is every reason to believe that the canine disease has similar causation. In the past several months, three veterinary groups have demonstrated reverse transcriptase activity ( a result of retrovirus exposure) in canine lymphoma/leukemia cell lines. Dogs and especially closely related linebred/inbred purebreds have the genetic makeup that conveys susceptibility, the viral elements are there, and reproductive hormonal and stress influences are prevalent when one breeds for performance, etc. The occurrence of low levels of vWF:Ag and the increased risk of bleeding it conveys from acquired vWD is one of several major undesirable consequences of this common autoimmune endocrine disease.
We believe it is very important for Scottish terrier fanciers to understand the influence autoimmune thyroid disease has on vWF levels and the expression of acquired vWD. This is a more complex and important problem which impacts the survival and vigor of the Scottish terrier breed than is the Type III autosomal recessive form of vWD which an be controlled relatively easily with an open, honest testing and planned breeding program.
About 90% of thyroid abnormalities in the dog have an inherited autoimmune basis. Linked to these disorders are other immune-mediated problems such as autoimmune hemolytic anemia and thrombocytopenia; bone marrow failure; leukemia and lymphoma; systemic lupus erythematosus; seizure disorders; chronic infections; immunosuppressive viral infections like distemper, parvovirus and retrovirus diseases; chronic active hepatitis (liver disease); immune kidney and adrenal (Addison’s) diseases; chronic allergic and immune skin and muscle disorders.
Thyroiditis is the immune-mediated process characterized by the presence of antithyroid antibodies in the blood or tissues. This condition usually progresses eventually to thyroid disease. Antithyroid antibodies can be present in euthyroid (normal), hypothyroid, or hyperthyroid humans or animals, i.e., can be present while a dog is still testing normal for thyroid function, and can be doing things to other tissues such as bone marrow, fetuses, and other fast-growing cells. Part of the management of autoimmune disorders is to test for the presence of thyroid dysfunction and antithyroid antibodies and to treat with thyroid hormones to inhibit the process. Therefore, we need a way to diagnose the presence of antibodies before the disease develops.
A typical thyroid test measures the total presence of the T4 hormone in the body. But an important measurement is the small fraction of the total T4 which is not bound in the body and is therefore available to the tissues. This “free” T4 is converted in the tissues to T3 which is important for intracellular function. In the newer thyroid profiles available at specialized veterinary laboratories in the US and Canada, they test for total T4, total T3, free T4, cholesterol, and free T3. Cholesterol should be measured from the same blood sample as the thyroid tests. In dogs with thyroid dysfunction the higher the cholesterol level, the lower the level of free T4. Owners can obtain a printout listing all the test results for their dog, as well as the “K” value, which is a linear constant between Free T4 and cholesterol.
With respect to K values, those which are above +1 and preferable above +5 are considered to be normal. Less than -4 indicates an animal with primary thyroid disease, even if there are no overt symptoms. Values between -4 and +1 are either early thyroid disease and/or a nonthyroidal illness (another illness which depresses thyroid levels). Thyroid deficiency even at early subclinical stages can cause irreversable damage in the body, and the value of this more sensitive test is that problems can be detected before damage has become so severe that clear visible symptoms are present.
For a dog displaying symptoms of autoimmune disease, the K value can be helpful to indicate whether the dog has low, normal or high thyroid function, and if the autoimmune problems are likely to be responsive to thyroid hormone supplementation. If test results are normal, and the dog is nevertheless ill, proceed to do other diagnostic tests such as that for antithyroid antibodies [antithyroglobulin, anti T4 and anti T3 autoantibodies].
Michigan State University has recently introduced antithyroid hormone antibody (anti T4 and anti T3) testing as part of their complete thyroid panel. This is an important step towards more effective thyroid testing. Contact the Endocrinology Section, Animal Health Diagnostic Laboratory, P.O. Box 30076, Lansing, MI 48909 (517-353-0621). Our laboratory and the Veterinary College at University of Florida, Gainesville, offer antithyrogobulin antibody testing.
These thyroid panels and antibody tests can also be used for genetic screening of apparently healthy animals to evaluate their fitness for breeding. A bitch with antithyroid antibodies in her blood may pass these along to her puppies in her colostral milk. Also any dog having circulating antithyroid antibodies can eventually develop clinical symptoms of thyroid or other autoimmune diseases. Therefore thyroid screening can be very important for potential breeding stock.
Thyroid testing for genetic screening purposes is less likely to be meaningful before puberty. Healthy young dogs (less than 15-18 months of age) should have thyroid baseline levels for all parameters in the upper third of the adult normal ranges. In fact, for optimum thyroid function in screening breeding stock, levels should be at least at the mid-point of the so-called “normal” ranges because lower levels may well be indicative of the early stages of thyroiditis among relatives of dog families known to have thyroid disease.
When treating with thyroid hormones: T4 is very forgiving, T3 can be easily toxic. With T4, dosage must be twice per day, as about half of it is metabolized and excreted from the body within 12 hours. For follow-up testing 4 to 6 weeks after initiating treatment, test at 4 to 6 hours after the morning dosage. For optimum levels, dogs should test in the upper third of the normal ranges after 4-6 weeks of therapy.
Dogs on long-term supplementation with thyroid hormones, should be monitored with complete panels on a regular basis (every 6 to 12 months), and dosages should be adjusted accordingly.
For Clubs Holding Joint Blood Testing for vWD or Other Bleeding Diseases and ThyroidDysfunction
Blood samples should be drawn for the coagulation testing (vWD or other tests) first because anticoagulated specimens are needed from unstressed animals to optimize valid results.
A separate sample of clotted blood to obtain serum is required for thyroid function profiling.
We would appreciate your assistance in interpreting the test results from your dog(s). Please indicate when submitting samples from your animals whether they have any type of health problem or family history of liver and/or thyroid disease. If your dog(s) is completely healthy at the time of testing, please also indicate this fact so that we can avoid assumptions that may be incorrect.
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