BRASS, K. E.

CAETANO, A. R.

SILVA, A. C.

SILVA, C. A. M.

SILVA, L. F.

VINOCUR, M. E.

English

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Equine protease inhibitor system as a marker for the diagnosis of chronicobstructive pulmonary disease (COPD)Myriam E. Vinocur*1, Karin E. Brass1, Alexandre R. Caetano2, Letícia F. Silva1, Aline C. Silva1and Carlos A.M. Silva11Universidade Federal de Santa Maria, Centro de Ciências Rurais, Departamento de Clínicade Grandes Animais, Laboratório de Imunogenética, Santa Maria, RS, Brazil.2Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, Brasília, DF, Brazil.AbstractThe protease inhibitor system (PI) was investigated to ascertain if it can be used as a marker of chronic obstructivepulmonary disease (COPD) in thoroughbred horses. Serum samples were taken from healthy thoroughbreds(n = 13) and those diagnosed as having COPD (n = 24) or inflammatory airway disease (IAD, n = 38) as well as from3,600 undiagnosed thoroughbred horses. PI allelic and genotypic frequencies were estimated using proteinelectrophoresis on starch and polyacrylamide gels. The four groups of horses showed high genotypic similarity andnone of the observed alleles or genotypes of the equine PI system were found to be associated with COPD.Key words: horse, chronic obstructive pulmonary disease, protease inhibitor system.Received: June 4, 2004; Accepted: March 31, 2005.Chronic obstructive pulmonary disease (COPD - alsoknown as chronic pulmonary disease, chronic small airwaydisease and chronic bronchitis/bronchiolitis) is one of themost commonly diagnosed conditions affecting the equinelung (Dixon et al., 1995; Franchini et al., 1998). This syn-drome is considered to be caused by the interaction of envi-ronmental and genetic factors and is characterized byairway obstruction and alveolar neutrophil infiltration(Robinson, 1997; Laurent, 2002). Neutrophils degrade in-haled particles present in the airways by releasing prote-ase-like elastases, peroxidases and nucleases. Theseproteolytic enzymes can destroy airway tissues directly byoxidation or indirectly by activating other proteases and/orinactivating protease inhibitors (PI). Cell damage is nor-mally prevented by the action of protease inhibitors such asalpha-1-antitrypsin (AAT), which control the prote-ase/antiprotease balance by inhibition of neutrophilelastase (Dallegri et al., 1995). In human COPD, low levelsof serum PI caused by autosomal recessive mutations leadto tissue injury at sites of neutrophilic inflammation and ul-timately to the development of pulmonary emphysema(Brantly et al., 1988, Selby et al., 1991, Cavarra et al.,1996). The equine protease inhibitor system is consideredto be equivalent to the human protease inhibitor but prote-ase inhibitor deficiency has never been reported in equineCOPD. The equine PI system is composed of fourclosely-linked serine proteinase inhibitor genes (Spi 1- 4)which have been cytogenetically assigned to chromosomeECA 24q15-16 (Lear et al., 1999). Twenty-two equine pro-tease inhibitor alleles have been characterized (Braend,1970; Juneja et al., 1978; Trommershausen-Smith andSuzuki, 1978) and eight (F, G, I, L, N, S, T and U) are com-monly observed in thoroughbred horses. The high degree ofprotein polymorphism and heterozygosity is the result ofthe co-expression of the four Spi genes (Patterson et al.,1991; Dagleish et al., 1998).In our study we evaluated the potential use of theequine protease inhibitor system as a possible marker fordiagnosing COPD in horses. PI allele and genotype fre-quencies were compared between thoroughbred horseswith COPD, inflammatory airway disease (IAD) andhealthy (control) horses. These horses were examined dur-ing the first semester of 2002 for respiratory diseases at 3different Brazilian racetracks (Tarumã in Curitiba, Paranástate; Cidade Jardim in São Paulo, São Paulo state; andPasso da Areia in Santa Maria, Rio Grande do Sul state) andat different breeding farms located in these areas. The diag-nosis of COPD (n = 24), IAD (n = 38) and identification ofhealthy horses (n = 13) was based on clinical signs, cytol-ogy of bronchoalveolar lavage fluid and intrapleural pres-Genetics and Molecular Biology, 28, 3, 382-385 (2005)Copyright by the Brazilian Society of Genetics. Printed in Brazilwww.sbg.org.brSend correspondence to Karin E. Brass. Universidade Federal deSanta Maria, Centro de Ciências Rurais, Departamento de Clínicade Grandes Animais, Laboratório de Imunogenética, Av. Roraimas/n, Camobi, 97105-900 Santa Maria, Rio Grande do Sul, Brazil.E-mail: kbrass@smail.ufsm.br. *Funding was provided by Conse-lho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Short Communicationsure testing with a Ventigraph (Vinocur et al., 2004;supplier: Boehringer Ingelheim do Brasil, DivisãoVetmédica, São Paulo, Brazil). Serum samples were usedfor protease inhibitor typing which was performed usingstarch and polyacrylamide gel electrophoresis according topreviously published protocols (Juneja et al., 1978; Braend,1970; Trommershausen-Smith and Suzuki, 1978). Proteaseinhibitor typing from 3,600 horses registered at the Brazil-ian Thoroughbred Stud Book (THBI) (132 stallions, 384dams and 3,084 horses born between 1998 and 2001) wereincluded as a 4th group in the genetic analyses, to estimatethe overall PI allele frequency of the Brazilian thorough-bred population. Data from horses in the COPD, IAD andhealthy groups were not included in the THBI group. PIallelic and genotypic frequencies of the 4 horse groupswere estimated using the Genepop program (Raymond andRosset, 1995), version 2001. Allele frequencies were usedto estimate Hardy-Weinberg equilibrium, coefficient of in-breeding (Fis), genetic distance between groups (Fst) and toconstruct a dendrogram using the unweighted pair groupmethod with averages (UPGMA) (Statistic Program, ver-sion 6; StatSoft Inc., 2300 east 14th street, Tulsa, OK74.104, USA). The five-generation pedigree of eachCOPD, AID and healthy horse was used to estimate in-breeding coefficients using the Pedigree Viewer programversion 5.1 (Genetic Information Systems, University ofNew England, Australia). The heterogeneity test was ap-plied to compare genotypic frequencies between COPD,IAD, healthy horse groups and THBI data.We observed seven protease inhibitor alleles (G, I, L,N, S, T and U) in the COPD, IAD and healthy horses andtwo additional alleles in the THBI data (F and L2, Table 1).The inbreeding coefficients estimated using the pedigreedata for each of the COPD, IAD and healthy horses rangedbetween 0.0005 and 0.078 for 18 horses, while a total of 57horses had an estimated inbreeding coefficient of 0.00 (Fig-ure 1C, D). Fisher’s inbreeding coefficient (Fis) estimatedusing the protease inhibitor allele frequency data for eachgroup and the 4 groups pooled was not significant (FisTotal2: 6.12, df: 8, prob: 0.72). Genotypic similarity betweenand among the four groups was assessed by the heterogene-ity test (2HETEROGENEITY = 95.24; df: 44). The observed esti-mates of genetic distances between groups showed that theCOPD group was separated from the IAD group byFst = 0.0097 and both were equidistant from the healthyhorse group by Fst = 0.0116. Genotype distribution and atree diagram using allelic frequencies from the 4 groups areshown in Figures 1A and 1B.It has been shown that human COPD is a result of pro-tease inhibitor dysfunction and it can be inherited and/oracquired (i.e., cigarette-induced) (Gadek, 1988). In horses,the hereditary basis of COPD has only been postulated asone of the predisposing factors (Marti et al., 1991). Asstated above, during the research published in our presentpaper we investigated the possible involvement of theequine protease inhibitor system in the onset of equineCOPD (analogous to the hereditary protease inhibitordysproteinaemia observed in humans) as previously pro-posed by Matthews (1979) who used acid polyacrylamidegel electrophoresis to estimate protease inhibitor allele fre-quencies in a mixed-breed equine population however, thisauthor found no evidence of an association between a spe-cific allele in the equine protease inhibitor system and theonset of COPD. In our study some of the variables used byMatthews (1979) were eliminated because we only usedthoroughbred horses diagnosed with COPD, IAD andhealthy horses (based on physical examination and comple-Vinocur et al. 383Table 1 - Proteinase inhibitor (PI) allele frequencies in thoroughbred horses.Thoroughbreds (present study) Data from other studies (thoroughbreds and non-thoroughbreds)Alleles COPD(n = 24)IAD(n = 38)Healthy(n = 13)THBI(n = 3,600)THBII(n = 14,517)THBIII(n = 63)COPDIII(n = 52)F - - - 0.03 0.030 0.063 0.096G 0.04 0.01 0.08 0.01 0.015 - -I 0.06 0.07 0.04 0.04 0.060 0.040 0.019L2 - - - 0.01 - -L 0.46 0.42 0.50 0.44 0.450 0.539 0.317N 0.25 0.33 0.15 0.29 0.185 0.048 0.048S 0.02 0.03 0.11 0.02 0.130 0.150 0.115T 0.04 0.04 0.04 0.05 - - -U 0.13 0.10 0.08 0.11 0.130 0.151 0.211Others - - - - 0.001 - 0.192Key: chronic obstructive pulmonary disease (COPD); inflammatory airway disease (IAD); healthy control horses (Healthy); a population of 3,600thoroughbreds (132 stallions, 384 dams and 3,084 horses born between 1998 and 2001) from the Brazilian Thoroughbred Stud Book (THBI);,thoroughbreds typed by Bowling & Clark (1985) (THBII); thoroughbreds used as controls (THBIII); and mixed-breed horses with COPD typed byMatthews (1979) (COPDIII).mentary diagnostic methods). In addition, we also usedstarch and alkaline polyacrylamide gel electrophoresis forthe protease inhibitor typing, a methodology that can detectall 22 alleles previously characterized for the equine prote-ase inhibitor system (Braend, 1970; Juneja et al., 1978;Trommershausen-Smith and Suzuki, 1978). The seven al-leles (G, I, L, N, S, T and U) identified by us in the COPD,IAD and healthy horses, and the two low frequency alleles(F = 0.03; L2 = 0.01) in the THBI population (Table1)showed similar frequencies to those observed in thorough-bred horses typed in the United States of America byBowling and Clark (1985). Matthews (1979) reported thepresence of only six alleles in thoroughbred horses, but thismay have been due to the fact that this author studiedclosely related animals. To avoid genealogical influence inour genetic analysis we estimated inbreeding coefficientsusing the pedigree data of each COPD, IAD and healthyhorse along with the protease inhibitor Fis values of thefour groups of horses (Figure 1 C, D). In spite of this, theheterogeneity test showed genotypic similarity between theCOPD, IAD, healthy groups and the population of 3,600thoroughbred horses and no allele or genotype variants ob-served in this study were found to be associated with the on-set of equine COPD.In humans, the conventional approach for protease in-hibitor allele typing uses a combination of polyacrylamidegel isoelectric focusing of serum at pH 4 to 5 and estimationof seric protease inhibitor levels using Radio Immuno As-says (RIA) and family studies (Wewers et al., 1987). Inhorses, the typing of the protease inhibitor system is morecomplex. Identification of Spi variants using electrophore-sis and the measurements of serum Spi concentration byELISA are not considered ideal procedures (Dagleish et al.,1999, 2000), and the molecular sequence of each Spi hasstill not been fully determined. It therefore appears thatthere is a need to establish more efficient methods to iden-tify the genetic variants derived from each Spi (Godard etal., 1998; Milenkovic et al., 2002) in order to clarify therole of this locus in the onset of equine COPD.ReferencesBraend M (1970) Genetic of horse acidic prealbumins. Genetics65:495-503.Brantly M, Nukiwa Y and Crystal RG (1988) Molecular basis ofalpha1-antitrypsin deficiency. Am J Med 84:13-31.Bowling AT and Clark RS (1985) Blood group and protein poly-morphism gene frequencies for seven breeds of horses in theUnited States. Animal Blood Groups Biochem Genet16:93-108.Cavarra E, Martorana PA, Gambelli F, de Santi M, van Even Pand Lungarella G (1996) Neutrophil recruitment into thelungs is associated with increased lung elastase burden, de-385 Diagnosis of chronic obstructive pulmonary diseaseFigure 1 - (A) PI genotype distribution in thoroughbreds with chronic obstructive pulmonary disease (COPD), inflammatory airway disease (IAD) andhealthy control horses (Healthy). (B) Tree diagram using PI allelic frequencies of horses with COPD, IAD, Healthy and a population of 3,600 thorough-breds (THBI). (C) Year of birth and inbreeding coefficient (Fis) of the 75 thoroughbred horses with COPD, IAD and healthy horses used in this study. (D)Fis of 75 thoroughbred horses with COPD, IAD and healthy horses.creased lung elastin, and emphysema in alpha 1 proteinaseinhibitor-deficient mice. 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Mamm Genome13:524-534.Patterson SD, Bell K and Shaw DC (1991) The equine majorplasma serpin multigene family: Partial characterization in-cluding sequence of the reactive-site regions. BiochemGenet 29:477-499.Raymond M and Rousset F (1995) Population genetics softwarefor exact test and ecumenicism. Genepop (version 1.2). JHeredity 86:248-249.Robinson NE (1997) Pathogenesis and management of airwaydisease. Proc Am Ass Equine Pract 43:106-115.Selby C, Drost E, Lannan S, Wraith PK and MacNee W (1991)Neutrophil retention in the lungs of patients with chronic ob-structive pulmonary disease. Am Rev Respir Dis 143:1359-1364.Trommershausen-Smith A and Suzuki Y (1978) Identity of XKand Pa systems in equine serum. Animal Blood GroupsBiochem Genet 9:127-128.Vinocur ME, Brass KE, Caetano AR, Michelloto PV and SilvaCAM, Bronchoalveolar lavage cytology and ventigraphy asan aid in the diagnosis of pulmonary disease in thoroughbredhorses in Brazil. Brazilian Journal of Veterinary Researchand Animal Science, submitted in 2004.Wewers MD, Casolaro MA, Sellers SE, Swayze SC, McPhaulKM, Wittes JT and Crystal RG (1987) Replacement therapyfor alpha1-antitrypsin deficiency associated withenphysema. N Engl J Med 136:1055-1062.Associate Editor: Pedro Franklin BarbosaVinocur et al. 385

Equine protease inhibitor system as a marker for the diagnosis of chronic obstructive pulmonary disease (COPD).

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Equine protease inhibitor system as a marker for the diagnosis of chronic obstructive pulmonary disease (COPD).

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