molecular base of coagulation factor xi deficiency in kerry blue terrier

Bulgarian Journal of Veterinary Medicine (2007), 10, No 4,247−255MOLECULAR BASE OF COAGULATION FACTOR XIDEFICIENCY IN KERRY BLUE TERRIERSummaryE. TCHERNEVA & U. GIGERSchool of Veterinary Medicine, University of Pennsylvania,Philadelphia, PA, USATcherneva, E. & U. Giger, 2007. Molecular base of coagulation factor XI deficiency inKerry Blue Terrier. Bulg. J. Vet. Med., 10, No 4, 247−255.Factor XI (FXI) deficiency has been reported in the Kerry Blue Terrier (KBT) breed and an isolatedfamily of Great Pyrenees and English Springer Spaniel dogs (Knowler et al., 1994). Affected dogshave a mild bleeding tendency, but many remain asymptomatic. The FXI deficiency seems to be inheritedas an autosomal trait with incomplete penetrance. Here we describe the molecular defect responsiblefor FXI deficiency in KBT dogs. Five FXI-deficient KBTs with either no or mild bleedingtendencies, were studied. Their partial thromboplastin times, but not their prothrombin times, weremarkedly prolonged and their plasma FXI activities were

E. Tcherneva & U. GigerDNA screening testThe nature of the mutation – 90 bp insertionmakes the test easier. Our PCR testwas designed for gDNA and it is presentedon Fig. 3.RESULTSSequencing of FXI cDNAWe used a PCR strategy based on conservedamino acid sequences amongknown FXI proteins from variety of speciesto isolate the FXI cDNA from canineblood RNA.The protein sequence predicted fromthe cDNA results is mature protein of 624amino acids and sharing 80 % identitywith hFXI mature protein. A ClustalWalignment of cDNA sequence with thepublished sequences of FXI (NCBI Database;Human Acc.No:NM_000128; MouseAcc.No: NM_028066 and Bovine Acc.No: NM_001008665) is shown on Fig.1*. Using our cDNA sequence we locatedthe FXI gene to the chromosome 16 of thedog genome by BLAST, with the genecomprised of fifteen exons, with exonintronboundaries. Canine chromosome 16shows conservation of synteny with humanchromosome 4 (Lindblad-Toh et al.,2005), where the hFXI gene is located(Asakai et al., 1987).Identification of short interspersed nucleotideelement (SINE) mutation andevaluation of screening testWe analyzed FXI sequences from KBTdogs with a history of bleeding and severaldogs from different breeds with normalcoagulation behavior. Comparison of the* We used Lasergene 7.2 programme(DNASTAR Inc., Madison, WI) to do thisinvestigation.DNA sequences of the entire coding regionand exon-intron junctions from thefirst and second group of dogs revealed aninsertion of 90 bp (SINE) inside of codingexon 7 at +719 bp (the first exon ofFXI gene is not coding; that way the termscoding exon 7 and exon 8 mean one andthe same sequence).The coding structure of the gene andSINE are presented on Fig. 2. Sequenceanalysis of coding exon 7 and boundariesreveled an insertion of the 3’ end-repeatof the gene.We investigated 27 KBT dogs. Mostof them were homozygous carrying normalcopies of the gene, 5 dogs were heterozygous,carrying one normal and onedefective copies of the gene and 2 werehomozygous and affected.In addition to DNA sequence analysis,we developed a rapid detection methodfor the insertion mutation, based on PCR.We designed primers for 168 bp ampliconin normal gene; 258 bp respectively ingene with insertion (Fig. 3). The PCR testdiscriminates successfully between normal,affected and carrier dogs as seenfrom the figure.DISCUSSIONFXI is a unique coagulation enzyme inthat it contains four tandem apple domains(Ap), also known as PAN modules (Fujikawaet al., 1986). These Ap show sequencehomology with each other andother Ap in proteins such as hepatocytegrowth factor and prekallikrein. FXI alsocontains a serine protease (SP) domainthat is homologous to the serin proteasedomains of other coagulation factors.FXI circulates as a dimmer with twoidentical FXI monomers linked by noncovalentinteractions between the Ap4domains and by Cys321-Cys321 disulfideBJVM, 10, No 4 249

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Molecular base of coagulation factor XI deficiency in Kerry Blue Terrier252BJVM, 10, No 4

E. Tcherneva & U. GigerM N N N C C C A A A300 bp_200 bp_100 bp_Fig. 3. PCR test for discrimination between normal, carriers and affected toward FXI deficiency inKerry Blue Terriers. Non denaturing polyacrylamid gel (8%). Ethidium bromide staining and UVvisualization. M − molecular size DNA marker; N – homozygote normal alleles; C – heterozygote (1normal and 1 affected) alleles; A − homozygote affected alleles, containing SINE.bond, although this bond is not essentialfor dimerization. FXI is activated whenbound to activated platelets. The Ap3domain within FXI binds to the plateletglycoprotein (GP) Ib-IX-V complex in thepresence of high molecular weight kininogen(HK) and zinc ions or prothrombinand calcium ions.Mutations within the FXI gene cancause FXI deficiency leading to a disorderwith a variable clinical phenotype. Homozygotestoward defective FXI gene havelow FXI activity (less than 20%), heterozygoteshave 40 to 60 % FXI. At thisstage we did not investigate the expressionof FXI protein from normal and affecteddogs.The SINE mutation in the canine coagulationFXI gene, which is the object ofour investigation, is the reason for a 30amino acids insertion in the A3 domain ofFXI serine protease (Fig.2D). The 30residues, mostly lysine, are situated in themost important region of the A3 domainwhich is responsible for interactions withheparin and platelets, between 237 and238 residues. Probably the long lysine tailmasks some of the interacting domainswhich causes lack of normal clotting ability.This mutation is in a very conservativeregion − between 273 and 274 residues asit seen from the Fig. 1. If we are inagreement with variants of SINEs impacton gene structure and expression suggestedby Cordaux & Batzer (2006), thatmeans the SINE we occurred is an exonizationof short open reading frame withoutany shift from the normal sequence of thegene. This could be partial explanation ofthe mild clinical signs of FXI disorder inKBT.Statistics of database coverage for FXIin human patients shows a frequency of5% insertions (Saunders et al., 2005).Genome modifications like SINE can occurduring retrotransposition of a donorelement (Kirkness et al., 2003). Whentranscription past its normal termination itBJVM, 10, No 4 253

Molecular base of coagulation factor XI deficiency in Kerry Blue Terriercan lead to the transduction of 3’ sequencesthat flank donor element, as it isin the gene sequenced by us (Fig.2C).Data presented herein suggest that theoligo (dA)-rich tail length determinesphenotype – a disorder in the coagulationway due to one coagulation protease.There are data that variable oligo(dA)-richtail lengths associated with the SINE insertionare presented in different caninebreeds (Clark et al.,2006). But we showthe first coagulation factor coding genewith SINE which is the reason for bleedingdisorders in KBTs. There are twophenotypes of FXI deficiency described(Saunders et al., 2005):Type I is characterized by both lowFXI coagulant activity (FXI:C) and lowantigen (FXI:Ag). This indicates that themutant protein is either present in loweramounts or absent in plasma suggestingthat the mutation has a structural effect onthe mutant protein - ie reducing translation,secretion or stabililty of FXI.Type II is characterized by low FXI:Cwith normal FXI:Ag. This indicates thatthe mutant protein is present in normalamounts in plasma but has reduced orabsent activity, suggesting that the mutationhas a functional effect on the proteine.g. affecting substrate binding.The mutant protein, described in thisinvestigation, is more likely to phenotypeII.Characterization of this mutation anddevelopment of DNA-based screening testwill aid in identification of dogs that arehomozygous or heterozygous for this coagulationdefect, allowing investigators toeffectively eliminate this trait from dogsutilized in pharmacological and toxicologicalresearch studies. Conversely affectedanimals may be useful for thoseinterested in novel approaches to thetreatment of inherited coagulation disorders,supporting the usefulness of thisdisease model in the study of therapeuticstrategies.ACKNOWLEDGEMENTSThis work has been supported in part by NIH02512 and the Kerry Blue Terrier Foundationin the USA.REFERENCESAsakai, R., E. W. Davie & D. W. Chung,1987. Organization of the gene for humanfactor XI. Biochemistry, 26, No 23,7221−7228.Bolton-Maggs, P. H. B., B.Y. Wan-Yin, A. H.McCraw, J. Slack & P. B. A. Kernoff,1988. Inheritance and bleeding in FXI deficiency.British Journal of Haematology,69, 521−528.Brush, P. J., P. H. Anderson & R. F. Gunning,1987. Identification of FXI deficiency inHolstein-Friesian cattle in Britain. TheVeterinary Record, 121, 14−17.Clark, L. A., J. M. Whal, C. A. Rees & K. E.Murphy, 2006. Retrotransposon insertionin SILV is responsible for merle patteringof the domestic dog. Proceedings of the NationalAcademy of Sciences, 103, 1376−1381 (www.pnas.org/cgi/doi/10.1073/ pnas.0506940103).Cordaux, R. & M. A. Batzer, 2006. Teachingan old dog new tricks: SINEs of caninegenomic diversity. Proceedings of the NationalAcademy of Sciences, 103, 1157−1158 (www.pnas.org/cgi/doi/10.1073/pnas.0510 714103).Dodds, W. J. & J. E. Kull, 1971. Canine FXI(plasma thromboplastin antecedent) deficiency.Journal of Laboratory and ClinicalMedicine, 78, 746−752.Fujikawa, K., D. W. Chung, L. E. Hendrixson& E. W. Davie, 1986. Amino acid sequenceof human Factor XI, a blood coagulationfactor with four tandem repeatsthat are highly homologous with plasma254BJVM, 10, No 4

E. Tcherneva & U. Gigerprekallikrein. Biochemistry, 25, No 9,2417−2424.Kirkness, E. F., V. Bafna, A. L. Halpern, S.Levy, K. Remington, D. B. Rusch, A. L.Delcher, M. Pop, W. Wang, C. M. Fraser& J. C. Venter, 2003. The dog genome:Survey sequencing and comparative analysis.Science, 301, 1898−1903.Knowler, C., U. Giger, W. J. Dodds & M.Brooks, 1994. FXI deficiency in KerryBlue Terrier. Journal of the American VeterinaryMedical Association, 205, 1557−1561.Kociba, G. J., O. D. Ratnoff, W. F. Loeb, R. L.Wall & L. E. Heider, 1969. Bovine plasmatromboplastin antecedent (factor XI) deficiency.Journal of Laboratory and ClinicalMedicine, 74, 37−41.Lindblad-Toh, K., C. M. Wade, T. S. Mikkelsen,E. K. Karlsson, D. B. Jaffe, M. Kamal,M. Clamp, J. L. Chang, E. J. KulbokasIII, M. C. Zody, E. Mauceli, X. Xie,M. Breen, R. K. Wayne, E. A. Ostrander,C. P. Ponting, F. Galibert, D. R. Smith, P.J. deJong, E. Kirkness, P. Alvarez, T. Biagi,W. Brockman, J. Butler, C.-W. Chin,A. Cook, J. Cuff, M. J. Daly, D. DeCaprio,S. Gnerre, M. Grabherr, M. Kellis, M.Kleber, C. Bardeleben, L. Goodstadt, A.Heger, C. Hitte, L. Kim, K.-P. Koepfli, H.G. Parker, J. Pollinger, S. M. J. Searle, N.B. Sutter, R. Thomas, C. Webber, BroadSequencing Platform Members* & E. S.Lander, 2005. Genome sequence, comparativeanalysis and haplotype structureof the domestic dog. Nature, 438,803−819.Saunders, R. E., N. M. O’Conell, A. C. Lee,D. J. Perry & S. J. Perkins, 2005. FactorXI deficiency database: An interactive webdatabase of mutations, phenotypes, andstructural analysis tools. Human Mutation,26, No 3, 192−198.Seligsohn, U., 1993. FXI deficiency. Thrombosisand Haemostasis, 70, 68−71.Sun, M.-F., M. Zhao & D. Gailani,1999. Identificationof amino acids in the FXI appledomain 3 required for activation of FXI.Journal of Biological Chemistry, 274,36373−36378.Troxel, M. T., M. B. Brooks & M. L. Esterline,2002. Congenital FXI deficiency in adomestic shorthair cat. Journal of theAmerican Animal Hospital Association,38, 549−553.* All Broad Sequencing Platform Memberscould be found at http://www.nature.com/ nature/journal/v438/n7069/full/nature04338.html#Broad%20Sequencing%20Platform%20members.Paper received 02.05.2007; accepted forpublication 12.11.2007Correspondence:Dr. Eva TchernevaINVITROGEN Co.Environmental Diagnostic7335 Executive WayFrederick, MD-21704e-mail: evatcherneva@hotmail.comBJVM, 10, No 4 255