Lack of association between HLA class II antigens polymorphism and leishmaniosis in a peruvian endemic region.

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1 15 Lack of association between HLA class II antigens polymorphism and leishmaniosis in a peruvian endemic region. Piñero, J.*; Martínez, E.*; Pacheco, R.**; Carmelo, E.*; Zurita, A.*; De Armas, F.*; Del Castillo, A. * & Valladares, B. * * Departamento de Parasitología, Ecología y Genética. Facultad de Farmacia. Universidad de La Laguna. Avda. Astrofisico Feo. Sánchez s.n. C.P La Laguna. Tenerife. Islas Canarias. España. ** Departamento de Microbiología. Facultad de Biología. Universidad de San Antonio Abad del Cuzco. Cuzco. Perú. Received: Accepted: Abstract: The possible relationship between the HLA class II antigens polymorphism ofthe human genome and the susceptibility or protection to the cutaneous and mucocutaneous leishmaniosis was studied in this work. The HLA class II antigens in a panel of 110 Peruvians of Quechua ethnic, using PCR amplification with sequence-specific primers (PCR-SSP), was studied. Of the 32 alleles tested, corresponding to the loci DRB 1 (18), DRB3 (2), DRB4 (2), DRB5 (1) and DQB (9), 17 were observed in the examined individuals. The population examinated did not show relationship between the different alleles and haplotypes detected. and susceptibility to cutaneous or mucocutaneous leishmaniosis. Key Words: HLA, Leishmaniosis, Quechua population. Resumen: En este trabajo se estudió la posible relación entre el polimorfismo en los antígenos HLA de clase II y la sensibilidad o protección frente a la leishmaniosis cutánea y mucocutánea. Para ello se estudiaron los antígenos HLA de clase II, en 110 peruanos de la etnia Quechua, utilizando la amplificación por PCR con cebadores específicos. De los 32 alelos estudiados, correspondientes a los locis DRB I (18), DRB3 (2), DRB4 (2), DRB5 (1) Y DQB (9), se encontraron 17 en los individuos examinados. La población estudiada no mostró que existiera relación entre los diferentes alelos y haplotipos detectados y fenómenos de sensibilidad o protección frente a la leishmaniosis cutánea y mucocutánea. Palabras clave: HLA, Leishmaniosis, etnia Quechua. 1. Introduction In recent years evidence has increased for the existence of genetic factors, alleles of the HLA system among them that play an important role in susceptibility to many infectious diseases. Among parasitic diseases, a relationship has been reported between severe malaria and different HLA classes I and II alleles (Hill, 1996; Hill et al., 1991; Bennett et al., 1993), ka1a-azar and HLA classes I and II alle1es (Faghiri et al., 1995; Singh et al., 1997), filariasis and HLA classes I and II alleles (Yazdanbakhsh et al., 1995), etc. Corresponding author: Dr. Basilio Valladares Hernández Departamento de Parasitología. Facultad de Farmacia, Universidad de La Laguna. Avda. Astrofísico Feo. Sánchez s.n. C.P , La Laguna. Tenerife, Islas Canarias, España. Tfno: Fax: bvallada@ull.es This heterogeneous behaviour in the assoclatlon between HLA and disease, according to geographica1 10calization, is re1ated to variations in the antigens of the parasite, differences in the transmission conditions, modifications of the individual immune responses among the different population groups and to other factors currently under study. An apparent link between genetic background (determinants genotype) and infection by Leis hmania has been recently shown. In individua1s affected by American cutaneous leishmaniosis, the ana1ysis of DRb polymorphism in allele frequency reveals a lesser frequency of DR2 in the cases under study with respect to controls (Lara et al., 1991, Cabrera et al., 1995). We report in this work data on the HLA polymorphism of the Quechua population and examine the possib1e association ofhla class II (DR) antigens with 1eishmaniosis, using DNA-based HLA typing methods. Revista Ibérica de Parasitología (2002), 62 (1-2), Sociedad Española de Parasitología (SEP)

2 16 Piñero, J. el al., Non association HLA antigens - Leishmaniosis. 2. Materials and Methods The samples used in this study were obtained from individuals from the Province of La Convención in Cuzco, Pem. A total of 110 unrelated Quechua ethnic individuals were examined for DNA typing. Thirty eight samples were from patients presenting clinical symptoms of cutaneous or mucocutaneous leishmaniosis, metastatic lesions which involve the mucose. Diagnosis in patients with leishmaniosis was corroborated by usual methods, microscopy and culture, and by PCR-ELISA (Piñero et al., 1999). Individuals included were not affected by other pathologies. Gn the other hand, seventy two samples were from individuals who had no previus history of leishmaniosis. Genomic DNA was isolated from 200 mi of blood wich was lysed with sarkosyl 1 %, EDTA O.lM and 0.25 mg/ml proteinase K at 56C for 1 hour. Total nucleic acid was isolated by phenol extraction and ethanol precipitation. The extract was stored at _20 0 C until used. All samples were typed by PCR-SSP (sequence specific primers) according with the conditions previously described by Bunce et al. (1995). Calculation of phenotype frequencies was performed by direct counting while allele frequencies were estimated by the maximum likelihood method (Ceppelini et al., 1955; Yasuda and Kimura, 1968; Petzl-Erler et al., 1993). Calculation of allelic associations (linkage disequilibrium: DAB) was estimated by equation described by Mattiuz et al. (1970). The significance associations levels detected was calculated by c2 corrected by Yates. The degree of association of the studied alleles to disease was calculated by measure of the relative risk (RR) and the ethiological fraction (d) (Bengtsson and Thomson, 1981; Thomson et al., 1983). 3. Results and Discussion Cutaneous or mucocutaneous leishmaniosis caused by Leishmania braziliensis is endemic in the Province of La Convención in Cuzco, Pem. This paper reports HLA class II polymorphism in Quechua population detected by DNA typing using PCR sequence-specific primers (PCR SSP) and explores the possibility that those HLA antigens are associated with leishmaniosis. The control group used consisted of healthy members of Quechua population, without relationship with patients group. As with other ethnic groups, sorne significant differences in the polymorphism ofhla-drbl, -DRB3, -DRB4, -DRB5 and DQB 1 were found. Of the group of DRBl alleles, DRB1 *0101,0102,0104 (DRl not 103) is present with a percentage of 1.8% while allele DRBl *0103 (DR103) was not detected (Table 1). This low percentage of DR1 alleles coincides with the data obtained in Colombian Amerindian populations (Trachtenberg et al. 1996) where these alleles were rare or absent. Compared with the data published about other American populations such as the Mexican, Brazilian and Bolivian (Gojorobi and Inoko, 1991; Sasazuki and Kimura, 1991) it is observed that the percentages are greater in the first two, while none of these alleles is present in the Bolivian population. The predominant alleles in the DR2 group were DRBl * (DRI5) and, on the other side, DRB1 * (DRI6) were not found (Table 1). The DR2 alleles are the most frequent in Amerindian populations in Colombia, and although also present, they present 10wer values in other American populations (Gojorobi and Inoko, 1991; Sasazuki and Kimura, 1991). The most predominant among the DR3 alleles were DRBl *0301, 0304 (DRI7), while alleles DRB1 *03011, 03012,0302,0303,0304,0305,1107 (DR17, 18, 1107) were not found in the population studied and the alleles DRBl *0302,0305, 1302, 1305, 1109, 1120, 1402, 1403, 1409, 1413, 1419 (DRI8 + sorne DR13's & 14's) were found in a low percentage (Table 1). Other studies detected the absence of DR3 alleles in Colombian Amerindian populations (Trachtenberg et al., 1996) or very low percentages in Bolivians (Gojorobi and Inoko, 1991; Sasazuki and Kimura, 1991). With regard to DR4 alleles, it was found that 20% of the population studied presented the alleles DRB1 * , 1410,1122 (DR , 1122) (Table 1). This percentage is lower than that found in the Bolivian population (Gojorobi and Inoko, 1991; Sasazuki and Kimura, 1991) and in Brazilian Amerindian populations (Petzl-Erler et al., 1993) but similar to that of the Amerindian populations of Colombia (Trachtenberg et al., 1996) and greater than that of the Mexican population (Gojorobi and Inoko, 1991; Sasazuki and Kimura, 1991). The allele DRBl *0901 (DR9) appears in a low percentage in the American populations for which data are available, but in the population under study the percentage observed is high (Table 1). Neither the alleles of group DR10 or those of group DR12 were detected in the population under study (Table 1). With regard to DRB3 alleles, it should be pointed out that alleles DRB3*020l, 0301 (DR52 (3*0201, 0301 subset)) appear in a lower percentage than allele DRB3*0101 (DR52 (3*0101 subset)) in the population studied (Table 1).

3 Piñero, J. el al., Non association HLA antigens - Leishmaniosis. 17 Table 1.- Phenotype and genotype frequeneies for HLA elass II loei (HLA-DR) in Peruvian population. Allele Phenotype Frequency (%) Genotype or Allele Frequency T. L. w.l. T. L. w.l. (N= 11O) (n=38) (n=72) (N=11O) (n=38) (n=72) DRBl *0101,0102,0104 1,82 2,78 O 0,009 0,014 O DRBI-0103 O O O O O O DRBl* ,73 33,33 31,58 0,18 0,18 0,1 7 DRBl* ,82 2,78 O 0,009 0,014 O DRBl *0301, ,18 66,67 42,1 0,35 0,42 0,24 DRB10302, 0305, 1302, 1305,1109, 1120, 1,82 2,78 O 0,009 0,014 O 1402, 1403,1409,1413,1419 DRB1* , 1410, ,44 21,05 0,1 0,1 0,11 DRBl *0701 5,45 5,55 5,26 0,028 0,028 0,037 DRB1*080l-0811, ,64 22,22 26,31 0,13 0,12 0, 14 DRB1* ,45 33,33 10,53 0,14 0,18 0,054 DRB11001 O O O O O O DRB1* ,1411 1,82 O 5,26 0,009 O 0,026 DRB11201, 1202, 1203 O O O O O O DRB1*1301-2, 1304, 1308, , , 9,1 8,33 10,53 0,046 0,043 0, , 1416, , 1111,1114,1116, DRBl*1301, 1302, 1308, 1309, 13 16, 1320, 9,1 11,11 5,26 0,046 0,057 0, , 1120, 1416 DRBl*1401, 1407, 1416 O O O O O DRBl *1402-3, 1406, 1409, ,1417, O O O O O O ( , 1421),1318 DRB5*0101, 0102, 0201, ,54 13,89 15,79 0,075 0,07 0,082 DRB3* ,1 33,33 21,05 0,16 0,18 0,11 DRB3*0201, ,73 13,89 10,53 0,066 0,07 0,054 DRB4* , 0102, , ,68 0,49 0,5 0,49 DRB4* N O O O O O O DQB1 *0305 O O O O O O DQBl*02 O O O O O O DQB l*04 O O O O O O DQBl*05 5,454 2,78 10,53 0,03 0,014 0,054 DQB 1 *060 1/2/3 O O O O O O DQB 1 * O O O O O DQBl *0301,0304 O O O O O O DQBl*0302 O O O O O O DQB l*03032 O O O O O T: Total population studied. L. Population studied with leishmaniosis. W.L. Population studied without leishmaniosis. It is also noteworthy that the frequency observed of DRB4*0101l02N (DR53 not expressed) was not detected. alleles DRB4* , 0102, 0103 (DR53 expressed) in On the other hand, group DRB5 alleles were found the individuals studied was very high, while allele in 14.5% ofthe population investigated (Table 1).

4 18 Piñero, J. el al., Non association HLA antigens - Leishmaniosis. It should be noted that the phenotype and genotype frequencies of the population with and without leishmaniosis do not show statistical1y significant variations. It has already been stated that in the determination of the alleles of locus DQB1, amplification was only obtained for the allele DQBl *05 (DBB5) (Table 1). Regarding the linkage disequilibrium of the 36 haplotypes studied, in total of population studied, only three of these associations presented statistical significance (Table 2). Taking into account only the population group that did not present symptomatology of leishmaniosis, it was found that these haplotypes did not present any appreciable statistical significance. To conclude this work, an attempt was made to verify the existence of some type of association between the different alleles and haplotypes found in the population studied and mucocutaneous leishmaniosis. Leishmaniosis has been related to HLA class 11 al1eles (Blackwel1, 1996). Recent studies associated susceptibility to mucocutaneous leishmaniosis with the frequency of HLA-DR2 al1eles, which was very low in patients, and that of HLA -DQw3 al1eles, which was lower in controls than in patients (Petzl-Erler el al., 1991). On the other hand, it was also observed a lesser frequency of HLA-DR2 alleles in patients diagnosed with cutaneous leishmaniosis than in controls (Lara el al., 1991; Cabrera el al., 1995). In the case of the population studied in this work, no relationship was observed between the different alleles and haplotypes detected and the absence or presence of leishmaniosis. The ethiological fraction does not seem to be useful in negative associations and varies between O (absence of linkage) and 1 (maximum linkage) (Bengtsson and Thomson, 1981; Thomson el al., 1983). In no case values of relative risk and etiological fraction observed indicate a possible statistical association with protective or exacerbating effects upon the presence of the disease since the probability in every case was greater than Likewise, no statistically significant association could be established between the alleles under study and the type of leishmaniosis that affects the population suffering from this disease. 4. References Bengtsson, B. and Thomson, G Measuring the strength of associations between HLA antigens and diseases. Tissue Antigens, 18, Bennett, S. ; Allen, S.J.; 0lerup, P.; Jackson, D.J.; Wheeler, J.G.; Rowe, P.A.; Riley, E.M. and Greenwood, B.M Human leucocyte antigen (HLA) and malaria morbidity in a Gambian community. Trans Royal Soc n "Op Med Hyg, 87, Blackwell, lm Genetic susceptibility to leishmanial infections: studies in mice and mano Parasitology, 112, S Bunce, M.; O'Neill, C.M.; Barnardo, M.C.N.M.; Krausa, P.; Browning, M.J.; Morris, P.J. and Welsh, K.I Phototyping: comprehensive DNA typing for HLA-A, B, C DRB1, DRB3, DRB4, DRB5 & DQBl by PCR with 144 primer mixes utilizing sequence-specific primers (PCR SSP). Tissue Antigens, 46, Cabrera, M.; Shaw, M.A.; Sharples, C.; Williams, H.; Castes, M.; Convit, l and Blackwell, J.M Polymorphism in TNF genes associated with mucocutaneous leishmaniosis. J Exp Med, 182, Ceppelini, R. ; Siniscalco, M. and 8mith, C.A.B The estimation of gene frequencies in a random-mating population. Ann Hum Genet, 20, 97. Faghiri, Z.; Tabei, S.Z. and Taheri, F Study of the association of HLA class 1 antigens with kala-azar. Hum Hered, 45, 5, Gojorobi, T. and Inoko, H The data book. Vol. 1. HLA XIth International Histocompatibility Workshop, Yokohama, November, Hill, A.V.S.; Allsopp, C.E.M.; Kwiatkowski, D.; Anstey, N.M.; Twumasi, P.; Rowe, P.A.; Bennett, S. ; Brewster, D.; McMichael, A.l and Greenwood, B.M Comrnon west African HLA antigens are associated with protection from severe malaria. Nature, 352, Table 2. - HLA-DRBI and - DRB3 haplotypes with high positive disequilibrium in total population studied. DRBI DRB3 r~ a HF b p value 0301, , ,1121, 0201, , , ,1302,1308,1309,1316, 1320, 1116, 1120, 1416 a: Relative Delta value x 1000; b: Haplotype frequency x 100

5 Piñero, J. el al., Non association HLA antigens - Leishmaniosis. 19 Hill, A.V.S Genetic susceptibility to malaria and other infectious diseases: from the MHC to the whole genome. Parasitology, 112, S75-S84. Lara, M.L.; Layrisse, Z.; Scorza, J.v.; Garcia, E.; Stoikow, Z.; Granados, J. and Bias, W Immunogenetics ofhuman American cutaneous leishmaniosis. Study of HLA haplotypes in 24 families from Venezuela. Hum Immunol, 30, Mattiuz, P.L.; Ihde, D.; Piazza, A.; Ceppelini, R. and Bodmer, w.f New approaches to the population genetics and segregation analysis of the HLA system. Histocompatibility Testing, Munskaard, Copenhagen, Petzl-Erler, M.L.; Belich, M.P. and Queiroz-Telles, F Association of mucosal leishmaniosis with HLA. Hum Immunol, 32, 4, Petzl-Erler, M.L.; Luz, R. and Santos Sotomaior, V The HLA polymorphism of two distinctive South-American Indian tribes: The Kaingang and the Guarani. Tissue Antigens, 41, Piñero, l; Martínez, E.; Pacheco, R.; Aragón, Z.; de Armas, F.; del Castillo, A. and Valladares, B PCR-ELISA for diagnosis of mucocutaneous leishmaniosis. Acta Trap, 73, Sasazuki, T. andkimura,a DNAcomponent. HLA XIth International Histocompatibility Workshop, Yokohama, November, Singh, N.; Sundar, S.; Williams, F.; Curran, M.; Rastogi, A.; Agrawal, S. and Middleton, D Molecular typing of HLA c1ass I and c1ass 11 antigens in Indian kala-azar patients. Trop Med Int Health, 2, 5, Thomson, G.; Motro, V. and Selvin, S Statistical aspects of measuring the strength of association between HLA antigens and diseases. Tissue Antigens, 21, Trachtenberg, E.A.; Keyeux, G.; Bernal, le.; Rhodas, M.C. and Erlich, H.A Results of Expedition Humana 1. Analysis of HLA c1ass 11 (DRBI-DQAI-DQBI-DPB1) alleles and DR-DQ haplotypes in nine Amerindian populations from Colombia. Tissue Antigens, 48, 3, Yasuda, N. and Kimura, M A gene-counting method of maximum likelihood for estimating gene frequencies in ABO and ABO-like system. Anns Hum Genet, 31,409. Yazdanbakhsh, M.; Sartono, E.; Kruize, Y.C.; Kurniawan, A.; Partono, F.; Maizels, R.M.; Schreuder, G.M.; Schipper, R. and de Vries, R.R HLA and elephantiasis in lymphatic filariasis. Hum Immunol, 44, 1,58-61.