Journal of Heredity Advance Access originally published online on October 26, 2005
Journal of Heredity 2005 96(7):755-758; doi:10.1093/jhered/esi111
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Polymorphism of Slc11a1 (Nramp1) Gene and Canine Leishmaniasis in a Case-Control Study
From the Servei Veterinari de Genètica Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
Address correspondence to Elisenda Sanchez-Robert at the address above, or e-mail: Elisenda.Sanchez{at}uab.es.
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Abstract |
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The prevalence of canine leishmaniasis infection in an endemic area such as the Mediterranean basin (67%) is higher than the prevalence of the disease (10%), suggesting a role of host genetics related to the outcome of the disease. Because Slc11a1 gene affects susceptibility and clinical outcome of autoimmune and infectious diseases, we analyzed five polymorphisms of the Slc11a1 gene in a case-control study with 97 dogs: three new single nucleotide polymorphisms and a G-stretch in the promoter and a microsatellite in intron 1. Haplotype frequency distributions showed significant differences between case and control populations (P = .01), most likely owing to the single nucleotide polymorphisms in the promoter region that were associated to case dogs. The most frequent haplotypes included TAG-8-141, which was present in all the breeds, in both case and control animals; and TAG-9-145, which was overrepresented in the control population and mostly found in boxer dogs. Within the boxer breed, 81% of the healthy dogs were homozygous TAG-9-145, whereas TAG-8-141 was significantly associated to case boxers (P = .02). The special genotype distribution for the Slc11a1 polymorphism associated with the prevalence of the illness in the boxer breed emphasizes the potential importance that breed genetic background has in canine leishmaniasis susceptibility.
Canine leishmaniasis (CL) is a severe systemic infectious canine disease caused by protozoan parasites of the genus Leishmania. In this zoonosis, the dog is considered the main peridomestic reservoir of the parasite. Human leishmaniasis causes visceral disease, particularly in young children and in immunosuppressed individuals, mainly adults infected with human immunodeficiency virus. The disease is usually fatal in people and dogs if left untreated (Alvar et al. 1997; Herwaldt 1999; Molina et al. 1994; Moreno and Alvar 2002; Slappendel 1988; Slappendel and Ferrer 1998).
Canine leishmaniasis is endemic in the Middle East, South America, and the Mediterranean basin, and it has recently been reported in foxhounds in the United States (Enserink 2000; Lindsay et al. 2002; see also, http://www.leishmaniasis.info/linfantum_2.htm). The prevalence of Leishmania infection in an endemic area such as the Mediterranean basin (67%) is higher than the prevalence of the disease (10%; Solano-Gallego et al. 2001), suggesting a role of host genetics related to the outcome of the disease. Candidate genes such as Slc11a1 (solute carrier family 11 member a1) and major histocompatibility complex (MHC) have been analyzed in relation to CL (Altet et al. 2002; Quinnell et al. 2003).
Slc11a1 is a proton/divalent cation antiporter that is better known by its former designation, Nramp1 (natural resistance associated macrophage protein 1), and it is the positionally cloned Ity/Lsh/Bcg gene (Vidal et al. 1993). It was originally described in mice for its roles in regulating resistance and susceptibility to Salmonella, Leishmania, and Mycobacterium. Functional studies with murine Slc11a1 implicate its involvement in macrophage function, including up regulation of chemokine/cytokine genes, such as tumor necrosis factor (TNF) and IL-1b, and induction of nitric oxide synthase (iNOS; Blackwell et al. 2003).
In humans, conclusive evidence has been provided for a functional GT-repeat sequence in the promoter region of Slc11a1 gene that underlies disease susceptibility linked to infectious and autoimmune conditions (Searle and Blackwell 1999). For that reason, this gene has been intensively studied for its role in conferring susceptibility to infectious diseases such as tuberculosis, leprosy, meningococcal meningitis, visceral leishmaniasis, and HIV infection, as well as to autoimmune diseases such as rheumatoid arthritis, diabetes, sarcoidosis, inflammatory bowel disease (reviewed in Blackwell et al. 2001), and (more recently) Kawasaki disease (Ouchi et al. 2003).
In this study we analyzed polymorphisms in the Slc11a1 gene in a case-control study of CL in naturally infected dogs. The polymorphisms analyzed are three new single nucleotide polymorphisms (SNPs) identified in the promoter region and the G-stretch and the intron 1 microsatellite (Altet et al. 2002).
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Animals
The case-control study included 97 dogs from 14 different breeds: Akita inu, Alaskan malamute, boxer, bulldog, cocker spaniel, Doberman, German shepherd, German shorthaired pointer, Great Dane, pointer, rottweiler, schnauzer, Siberian husky, and Teckel (dachshund). Case animals included 40 dogs who were diagnosed with CL and died (or were euthanized) or were experiencing continuous relapse of the disease. Control animals were 57 healthy dogs from the same breeds of the case animals (to avoid breed effect) who were living in an endemic area of leishmaniasis. They were selected among dogs older than 6 years who were visiting veterinarian hospitals for regular health checks and never developed CL. We assumed that they could have been in contact with the parasite at some time.
Genomic DNA was isolated from peripheral blood leucocytes by standard phenol-chloroform extraction or from bone marrow by direct lyses (Francino et al. 1997).
Genotyping
Analysis in the promoter region for polymorphisms was accomplished by direct sequencing of a 539 bp polymerase chain reaction (PCR) product, amplified with primers sense 5'-CCTCTCAGCTAGTCTGAGCC-3' and antisense 5'-CAGCTGATCTCAGCTGTCCTC-3'. The amplification was performed using a GC-RICH PCR System (Roche) in a final volume of 25 ml. The reaction mixture contained 10 ng to 20 ng of genomic DNA, 0.2 mM of each primer, 1.5 mM MgCl2, 200 mM of each dNTP, 0.5 M GC-rich resolution solution, and 2 U of Taq and Tgo DNA polymerase mix. Thermal cycling profile was 35 cycles of denaturing at 94°C for 45 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, with a final incubation at 72°C for 10 min in a GeneAmp 9700 (Applied-Biosystems, Foster City, CA). PCR products were purified and sequenced with the internal primers 5'-CCTCTCAGCTAGTCTGAGCC-3' and 5'-CAGCTGATCTCAGCTGTCCTC-3' and the Ready Reaction Cycle Sequencing Kit ABI PRISM dGTP Terminator 3.0 on an ABI 3730 DNA Analyzer (Applied-Biosystems, Foster City, CA).
PCR amplification of intron 1 microsatellite was carried out in a final volume of 20 ml containing 10 ng to 20 ng of genomic DNA, 1.5 mM MgCl2, 200mM of each dNTP, 0.2 mM of each primer (5'-FAM-GAGTCTGCTTGAGATTCTCTC-3' and 5'-TATCACCTCCACCCTTCAAAC-3'), and 1 U Taq polymerase (Invitrogen). Thermal cycle profile was as follows: 25 cycles of denaturation at 94°C for 30 s, followed by 55°C for 30 s and 72°C for 30 s, and a final extension at 72°C for 7 min in a GeneAmp 9700 Thermal Cycler (Applied-Biosystems, Foster City, CA). Amplified fragments were analyzed by capillary electrophoresis (ABI 3730 DNA Analyzer, Applied-Biosystems, Foster City, CA), and labeled PCR products were automatically sized relative to the internal standard (CST ROX 70-500; BioVentures, Murfreesboro, TN) with the Genemapper Software 3.5 (Applied-Biosystems, Foster City, CA).
Statistical Analysis
The reconstruction of haplotypes from our population data was performed with the PHASE 2.1 program (Stephens et al. 2001; Stephens et al. 2003). Haplotype frequencies between cases and controls were compared by using chi-square tests. The P value was corrected by the number of alleles observed in our population and was considered statistically significant when P < .05. Haplotype odds ratio (OR) and a 95% confidence interval were estimated by comparison of each haplotype versus all the others together.
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Results and Discussion |
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To assess the possible role of the Slc11a1 gene in determining Leishmania susceptibility, we analyzed the promoter region for polymorphisms and tested a microsatellite located in intron 1 in a case-control study with 97 dogs: 40 dogs diagnosed with canine leishmaniasis and 57 healthy dogs. Direct sequencing analysis revealed three new SNPs and variation in the number of Gs (from seven to nine) in the G-stretch in the promoter region, with G8 as the most frequent allele. Analysis of the intron 1 microsatellite showed five different alleles (133, 137, 139, 141, 145), with alleles 141 and 145 as the most frequent.
The three new SNPs gave rise to three new haplotypes (TAA, TGA, CGA), which were identified in four case dogs from different breeds: German shorthaired pointer, Doberman, rottweiler, and German shepherd. The SNPs are located upstream of the G-stretch, and one of them is adjacent to the one described by Altet et al. (2002) for a Rottweiler dog. As shown in Figure 1, the three SNPs affect different genomic binding sites of eukaryotic transcription factors, suggesting a possible role of differential gene expression. In humans, a Z-DNA-forming repeat and a –237 C/T polymorphism have been associated with a different ability to drive gene expression and have been correlated with infectious versus autoimmune disease susceptibility (Searle and Blackwell 1999; Zaahl et al. 2004).
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The polymorphisms of the five loci analyzed together generated 14 different haplotypes in our case-control study. Haplotype frequencies distribution between case and control populations showed significant differences (P = .01), most likely due to the SNPs of the promoter region, given that no significant differences were found either for the variation in the G-stretch or for the intron 1 microsatellite when analyzed separately. However, if we take into account the SNPs of the promoter together, significant differences in haplotype distribution are found between case and control population (P = .003) because three mutations are present only in case animals (data not shown).
The most frequent haplotypes are TAG-8-141 and TAG-9-145 (frequencies of 0.35 and 0.32, respectively). Haplotype TAG-8-141 is present in all breeds, but TAG-9-145 is found mostly in boxer dogs, which suggests that the haplotype distribution is breed specific. The existence of a different haplotype distribution in this breed leads us to perform the statistical analysis in the case-control animals via two groups: boxer dogs (see Table 1) and dogs other than boxers.
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The haplotype frequencies in boxer dogs vary between controls and cases. Within the boxer breed, haplotype TAG-9-145 is the most frequent, and it is preferentially observed in the homozygous state in the control animals (P = .03; 81% of the control boxers versus 37% of the case boxers). On the other hand, haplotype TAG-8-141 is significantly associated to case animals (P = .02), and 50% of the boxers with CL carried this haplotype, all heterozygous TAG-8-141/TAG-9-145 (P = .01). Boxer dogs carrying the TAG-8-141 allele are more likely to be susceptible to the disease (odds ratio = 10.33), whereas allele TAG-9-145 would be protective in homozygous state for the boxer breed. However, in dogs other than boxer, haplotype TAG-8-141 is the most frequent and is evenly distributed between case and control animals (frequencies of 0.36 and 0.47, respectively), indicating a nonstraightforward genotype-phenotype relationship.
Previous works suggests a role of host genetics in the predisposition or progression of CL, which could be different among breeds. For instance, visceral leishmaniasis was diagnosed in 41% of foxhounds from the index kennel in New York but in none of the beagles and basset hounds from the same kennel (Gaskin et al. 2002), suggesting a special predisposition of foxhounds to the infection. On the other hand Ibizan hounds, dogs living in an endemic area (Balearic Islands, Mediterranean basin), have been reported to be more resistant to Leishmania infection than other breeds living in the same area, owing to a predominant cellular immune response against to the infection (Solano-Gallego et al. 2000). Moreover, a recent epidemiological study has demonstrated a bimodal distribution of CL, with the first peak including dogs diagnosed at 2 years to 4 years of age and the second peak including dogs about 7 years old (and, in these latter cases, being mostly concomitant with other infections or neoplastic diseases). Dogs genetically predisposed to CL were expected to be in the first age peak, but the boxer breed was overrepresented in it (68% of the sick boxers are diagnosed for CL before 4 years of age; Miranda et al. 2005). It is important to remark here that, even though case animals were not breed selected for our study, the boxer breed represents 20% of our case group.
In conclusion, we found a significant association between Slc11a1 and susceptibility to CL, either for the SNPs in the promoter region in the case-control study or for the association of TAG-8-141 to case animals in the boxer breed. These results, taken together with previous work that has demonstrated an association between MHC class II and susceptibility to visceral leishmaniasis in dogs (Quinnell et al. 2003), point to genetic factors involved in the disease. Expression analysis of the different Slc11a1 haplotypes and characterization of other candidate genes are necessary to further advance our knowledge and understanding of the immunological factors that underlie canine leishmaniasis.
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Acknowledgments |
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This work was supported by the Veterinary Molecular Genetic Service from the Universitat Autònoma de Barcelona.
We are thankful to the Veterinary Teaching Hospital from the Universitat Autònoma de Barcelona and all the clinicical veterinarians for providing the samples used in this study. We are also thankful to the anonymous referees for their critical review. This article was presented at the 2nd International Conference on the "Advances in Canine and Feline Genomics: Comparative Genome Anatomy and Genetic Disease," Universiteit Utrecht, Utrecht, The Netherlands, October 14–16, 2004.
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Footnotes |
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Corresponding Editor: Bernard van Oost
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