The expression of ABH and Lewis antigens in Brazilian semi-isolated Black communities

The expression of ABH and Lewis antigens in Brazilian semi-isolated Black communities

Tereza Cristina OLiveira Corvelo, D.C.F. Aguiar and F.E.S. Sagica

Departamento de Genética, Centro de Ciências Biológicas, Universidade Federal do Pará,

Belém, Pará, Brazil.

ABSTRACT

The expression of the ABH and Lewis blood groups was determined in blood and saliva samples from two semi-isolated Black communities of Northern Brazil: Cametá and Alcântara. The distributions of ABO blood group phenotypes and the ABH secretor status frequencies showed no significant differences between these populations. In contrast, there was a difference regarding the frequency of the red blood cell Le(a-b-) phenotypes, associated with erythrocyte/saliva discordance, as confirmed by the observation that individuals with Le(a-b-) red cells have the Lewis antigen in their saliva, resulting in a nongenuine Le(a-b-) phenotype, whose frequency was higher in Alcântara.

Key words: semi-isolated black communities, Lewis blood group, ABH secretor status, dot-blot-ELISA.

Received: June 29, 1999; accepted: July 23, 2002.

INTRODUCTION

The ABH and Lewis blood group systems were initially determined in erythrocytes, followed by other human tissues and secretions. In their systemic relationships, both competition and cooperation exist between the glycolsyltransferases produced by the Secretor, H, Lewis, and ABO loci (as reviewed by Clausen and Hakomori, 1989; Henry et al., 1995; Oriol et al., 1986; Watkins, 1980).

Four phenotype groups are usually observed: Lewis-positive or Lewis-negative, and secretor or nonsecretor of ABH soluble substances. In Lewis-positive and ABH secretor individuals, the main antigen is called Le^b. If allele Se is absent, the type 1 precursor chain is converted by the Le enzyme only into the Le^a determinant. In Lewis-negative individuals, the secretor phenotype has no influence.

The frequency of Le(a-b-) in American Blacks is almost 30%, and some individuals secrete Lewis substances in their saliva (Miller et al., 1954). In African populations, the same phenotype can reach 40% (Mourant et al., 1976). The contribution proportions of the Black component has been evaluated in some interracial populations. In Brazil, the frequency of Le(a-b-) is 25% in the Caucasoid population, and 27% in the Black population (Palatnik et al., 1987).

A number of studies (Hammer et al., 1981; Hirano et al., 1987; Langkilde et al., 1991; Makni et al., 1987; Ærntoft et al., 1991; Stingendal et al., 1984; Yazawa et al., 1988) have shown differences in the phenotypic expression of the Lewis antigens. Individuals with a Le(a-b-) phenotype, presenting no Lewis antigen expression in their erythrocytes, showed Lewis activity in their secretions.

Because of these variations, several studies (Elmgren et al., 1993; Koda et al., 1993; Liu et al., 1996; Mollicone et al., 1994; Nishiara et al., 1993; Pang et al., 1998) were carried out, to determine the most common frequencies of the Lewis phenotypes in different ethnic groups. Thus, the study of semi-isolated Black communities represents a contribution to the investigation of their variability.

The Brazilian Blacks descend from slaves brought from different regions of the African Continent, and are genetically very different from North-American and Caribbean Blacks. Predominantly genes of Bantu origin, but also of Benin, and some very rare ones of Senegambia origin have been observed. (Bortoline et al., 1992; Pante 1995; Vergolino and Figueiredo, 1990).

In the present study, we report on the results of ABH and Lewis blood group system analyses of blood and saliva samples from two Brazilian Black semi-isolated communities.

MATERIAL AND METHODS

Populations sampled

Blood and saliva samples were collected from Black members of semi-isolated communities in the Brazilian cities of Cametá (State of Pará) and Alcântara (State of Maranhão). These communities consist of descendants of former slaves, who fled to isolated areas and formed villages called "quilombos", which still conserve their roots. If we consider all Blacks as one group, the degree of racial mixture may be estimated at about 20% with Caucasoid, and 30% with American Indian genes, respectively.

Voluntary participants were identified, and subsequently specimens were collected. Fifty-eight individuals were tested in Cametá, and 69 in Alcântara.

Samples: blood and saliva

Five-milliliter whole blood samples were drawn with heparin by venipuncture. Samples were stored at 4 °C and tested within 48 h. The remaining red cells were treated with glycerol and frozen at -20 °C. Simultaneously, saliva samples were collected and, within 1 h of collection, boiled during 15 min, centrifuged (at 3500 rpm for 10 min), and supernatants separated and stored at -20 °C for further testing.

Salivary ABH and Lewis Phenotypes

The presence or absence of ABH and Lewis antigens in saliva was determined by Dot-blot-ELISA with monoclonal antibodies (Ortho Diagnostic Systems), including anti-Le^a, anti-Le^b, anti-A, anti-B, and anti-H (Fresenius Diagnostics). The saliva samples were diluted at 1:2; 1:10; 1:50; and 1:100, in TRIS buffer, pH 7.4, and 5 mL of appropriate dilution plotted onto nitrocellulose membranes, as described by Pflug et al. (1989).

Erythrocyte phenotypes

ABO, including the A₁ subgroup, and Lewis were determined in fresh blood samples by standard agglutination methods. Red blood cells were washed and suspended in a 2-3% (v/v) saline solution, 50 mL of this suspension were mixed in tubes with 50 mL of specific antisera, then incubated for 45 min at room temperature, and the results were read by naked eye after centrifugation (at 1000 rpm for 1 min). All assays included appropriate known controls. The antisera used were the following: 1) human polyclonal anti-A and anti-B Ortho Diagnostic Systems; 2) anti-Le^a and Anti-Le^b of goat origin Ortho Diagnostic Systems; 3) anti-H (Ulex europeaus) and anti-A₁ (Dolichus biflorus) lectins were locally prepared, according to methods described by Boyd and Shapleigh (1954).

All red cell samples were also tested by dot-blot-ELISA, with the same monoclonal antibody batches used in the specific assay reported above for the evaluation of salivary antigens. The red cell suspensions were prepared at a concentration of 1:4 (v/v) in 0.09% saline solution, and 5 mL of appropriate dilution were plotted onto the nitrocellulose membrane.

Statistical analysis

The statistical analyses were carried out using the nonparametric Chi-square (c²) and G tests, with a significance level of 5%. In the case of polyallelism, the comparison between the gene frequencies of the ABO system blood groups was made using the graphic method of equivalent circles (Stevens, 1950); by this method, when representative circle images partially overlap, it shows that the population samples are homogeneous: in our case, with no significant difference between the gene frequencies of the ABO system.

RESULTS

The distribution of ABO blood groups in two semi-isolated Black communities of North Brazil (Table I) showed no significant differences between either their phenotype frequencies or their gene frequencies (Figure 1). Both population samples fit the genetic equilibrium estimated for the ABO locus.

Analyses of Lewis antigen expression, both in erythrocytes and in saliva, were used to determine the Lewis blood group phenotypes (Table II). Three groups were identified:

1) Lewis-positive ABH nonsecretor individuals, with red cell phenotype Le(a+b-), also expressing Le^a phenotype in saliva;

2) Lewis-positive, salivary ABH secretor individuals, with red cell phenotype Le(a-b+), producing Le^b antigen in saliva. The majority also produces Le^a antigen, detectable in saliva, but usually not in erythrocytes;

3) Lewis-negative, ABH secretor and nonsecretor individuals, with red cell phenotype Le(a-b-). However, these communities show a high degree of variation in the frequency of red cell Lewis phenotypes. Thus, they can be subdivided into two groups named nongenuine Lewis-negative individuals, with normal Le^a and/or Le^b antigen expression in saliva, and genuine Lewis-negative individuals, not expressing Lewis antigens, neither in erythrocytes nor in saliva.

In this survey, the red cell Lewis phenotype distribution in Cametá differed significantly from that found in the Alcântara population (c² = 12.17; df = 2; p = 0.002). A different range of variation also exists in the red cell Le(a-b-) blood group frequency. Indeed, the Lewis-negative frequency approaches 14% in the Cametá population, whereas, in the Alcântara population, the frequency of the Lewis-negative phenotype exceeds 40%. But, considering all the Lewis-negative salivary phenotypes (Table II), the prevalence of genuine Lewis-negative individuals is about 3.5% in Cametá, and 1.5% in Alcântara. However, the prevalence of nongenuine Lewis-negative individuals is 10.5% in Cametá and 40.5% in Alcântara. In marked contrast to this, secretor and nonsecretor frequencies (Table II) do not significantly differ in the two populations examined (c² = 0.47; df = 1; p = 0.49).

DISCUSSION

The Lewis system is a histo-blood group producing antigens in secretions and plasma, which are subsequently adsorbed onto red cells (Marcus and Cass, 1969; Sneath and Sneath, 1965). However, these antigens can be more accurately detected in saliva secretions, due to secondary acquisition of Lewis antigens by red cells (Makni et al., 1987). The Lewis phenotype is not easily distinguished by the conventional hemoagglutination test, which appears to be partly caused by the labile condition of the Lewis antigen expression and by the small number of Lewis antigens on the erythrocytes, resulting in sometimes very weak hemoagglutination reactions (Wang et al., 1994).

The results presented show that the red cell Lewis antigen reactivity does not appear to be associated with the Lewis phenotype in the saliva, a conclusion supported by the observation that some individuals with Le(a-b-) red cells show Lewis reactivity in their secretions. Such individuals have been classified as nongenuine Lewis-negative, since they present a(1-4) fucosyltransferase activity and Lewis antigens in saliva (Langkilde et al., 1991; Ærntoft et al., 1991).

Analysis of the results shown in Table II raises the question of whether the observed prevalence variability of nongenuine Lewis-negative individuals in the isolated groups studied is biologically true, or rather a result of methodology.

This discrepancy, however, does not seem to conflict with the genetics of the ABH secretor status and Lewis antigen biosynthesis, which is a result of the enzyme products of alleles Se and Le. In saliva, Le^a has been found in Le(a-b-) nonsecretors, while Le^b can be identified in Le(a-b-) secretors.

Other investigators (Makni et al., 1987; Miller et al., 1954; Mollicone et al., 1994; Ærntoft et al., 1991; Palatnik et al., 1987) have reported the existence of paradoxical erythrocyte/saliva discordance, that may have been explained previously by the presence of the weak Lewis allele (Le^w), which originates a fucosyltransferase that is less efficient than that codified by the dominant allele, thus determining a quantitative variation.

Recently published data (Elmgren et al., 1993; Koda et al., 1993; Liu et al., 1996; Mollicone et al., 1994; Nishiara et al., 1993; Pang et al., 1998), however, show the presence of a single-point mutation (T59G) in the transmembrane domain of the Lewis enzyme, which seems to be more globally distributed. This 59 mutation allele, when present in double dose, leads to a reduced enzyme activity, resulting in the nongenuine Le(a-b-) phenotype. This 59 mutation polymorphism was not investigated here, ruling out a comparison with the population samples studied.

Comparing our results with those previously reported for Black populations, we observed a difference in the red cell Lewis-negative phenotype frequency between the Cametá and Alcântara populations. A high frequency of the red cell Le(a-b-) type, already described in Black populations, was observed only in Alcântara (Table II). On the other hand, the results obtained for the ABO system show the expected rise in frequency of the I^B gene in both populations, with no significant differences between the two samples, showing them to be homogeneous with regard to the gene frequencies of ABO blood groups, therefore being considered as originating from the same African parental ethnic stock.

However, in accordance with previous publications (Hammer et al., 1981; Hirano et al., 1987; Langkilde et al., 1991; Makni et al., 1987; Ærntoft et al., 1991; Stingendal et al., 1984; Yazawa et al., 1988), we believe that the high frequency of the Lewis (a-b-) red cell phenotype in the Alcântara population is probably a consequence of the change from Lewis-positive to Lewis-negative, predisposed to the nongenuine Lewis-negative phenotype, due to decreased concentration of circulating Lewis antigens occurring during diseases caused by parasites, infections, or other pathologic conditions.

This change may also have been produced by intrapopulational microdifferentiation of the population structure, caused by stochastic factors, particularly genetic drift, which is frequently observed in small and relatively isolated populations. Further studies focusing on codifier alleles of these fucosyltransferases could determine if this genetic variability adjusts to the differentiated expression of the Lewis antigen.

ACKNOWLEDGEMENTS

We would like to thank Dr. Paula Schneider and her team of collaborators for collecting blood and saliva samples from the Alcântara community, and Mr. Lenor Mandu for technical assistance. This research was supported by Universidade Federal do Pará (UFPa), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Send correspondence toTereza Cristina Oliveira Corvelo. Rua Dom Romualdo Coelho, 169, 66055-190 Belém, Pará, Brazil. E-mail: [e-mail].

• Bortoline MC, Weimer TA, Franco MHLP, Salzano FM, Layrisse Z, Schneider H, Schneider MPC and Harada ML (1992) Genetic studies in three South American Black populations. Gene Geography 6:1-16.
• Boyd WC and Shapleigh E (1954) Diagnosis of subgroups of blood groups A and AB by use of plant agglutinins (lectins). J Lab Clin Med 44:235-237.
• Clausen H and Hakomori S (1989) ABH and related histo-blood group antigens: Immunochemical differences in carrier isotypes and their distribution. Vox Sang 56:1-20.
• Elmgren A, Rydberg L and Larson G (1993) Genotype heterogeneity among Lewis-negative individuals. Biochem Biophys Res Commun 196:515-520.
• Hammer L, Mansson S, Rohr T, Chester T, Ginsburg V, Lundblad A and Zopf D (1981) Lewis phenotype of erythrocytes and Leb active glycolipid in serum of pregnant women. Vox Sang 40:27.
• Henry SM, Oriol R and Samuelsson BE (1995) Lewis histo-blood group system and associated secretory phenotypes. Vox Sang 69:166-182.
• Hirano K, Kawa S, Oguchi H, Kobayashi T, Yonekuni H, Ogata H and Homma T (1987) Loss of Lewis antigen expression on erythrocytes in some cancer patients with high serum CA 19-9 levels. J Nati Cancer Inst 79:1261-1268.
• Koda Y, Kimura H and Mekada E (1993) Analysis of Lewis fucosyltransferase genes from the human gastric mucosa of Lewis-positive and Lewis-negative individuals. Blood 82:2915-2919.
• Langkilde NC, Wolf H, Meldgaard P and Ærntoft TF (1991) Frequency and mechanism of Lewis antigen expression in human urinary bladder and colon carcinoma patients. Br J cancer 63:583-586.
• Liu Y-H, Koda Y, Soejima M, Uchida N and Kimura H (1996) PCR analysis of Lewis-negative gene mutations and the distribution of Lewis alleles in a Japanese population. J Forensic Sci 41:1018-1021.
• Makni S, Dalix AM, Caillard T, Compagnon B, Le Pendu J, Ayed K and Oriol R (1987) Discordance between red cell and saliva Lewis phenotype in patients with hydatic cysts. Exp Clin Immunogenet 4:136.
• Marcus DM and Cass LE (1969) Glycosphingolipids with Lewis blood group activity: Uptake by human erythrocytes. Science 164:553-555.
• Miller EB, Rosenfield RE, Vogel P, Haber G and Gibbel N (1954) The Lewis blood factors in American Negroes. Am J Phys Anthropol 12:427-443.
• Mollicone R, Reguigne I, Kelly RJ, Fletcher A, Watt J, Chatfield S, Aziz A, Cameron HS, Weston BW, Lowe JB and Oriol R (1994) Molecular basis for Lewis a (1,3/1,4) fucosyltransferase gene deficiency (FUT 3). J Biol Chem 296:20987-20994.
• Mourant AE, Kopec AC and Domaniewska-Sobczak K (1976) The distribution of the human blood groups and other polymorphisms. ed 2. Oxford, Oxford University Press. pp 548-576.
• Nishihara S, Yazawa S, Iwasaki H, Nakazato M, Kudo T, Ando T and Narimatsu H (1993) a (1,3/1,4)Fucosyltransferase (FucT-III) gene is inactivated by a single amino acid substitution in Lewis histoblood type negative individuals. Biochem Biophys Res Commum 196:624-631.
• Oriol R, Le Pendu J and Mollicone R (1986) Genetics of ABO, H, Lewis, X and related antigens. Vox Sang 51:161-171.
• Ærntoft TF, Holmes EH, Johnson P, Hakomori S and Clausen H (1991) Differential tissue expression of the Lewis blood group antigens: Enzymatic, immunohistologic, and immunochemical evidence for Lewis a and b antigen expression in Le(a-b-) individuals. Blood 77:1389.
• Palatnik M, Laranjeira NSM, Simões MLMS and Loureiro JB (1987) Genetic heterogeneity of the blood group O, Le(a-b-) phenotype. Rev Brasil Genet 10:761-768.
• Pang H, Liu Y, Koda Y, Soejima M, Jia J, Schlaphoff T D, Du Toit E and Kimura H (1998) Five novel missense mutations of the Lewis gene (FUT3) in African (Xhosa) and Caucasian populations in South Africa. Hum Genet 102:675-680.
• Pante de Souza G (1995) Haplótipos associados ao gene HBB*S na população de Belém (PA) e seu significado antropológico. Dissertação de Mestrado, UFPA, Belém, PA.
• Pflug W, Bässler G and Eberspächer B (1989) ABO and Lewis typing of secretion stains on nitrocellulose membranes using a new dot-blot-ELISA technique. Forensic Science International 43:171-182.
• Sneath JS and Sneath PHA (1955) Transformation of the Lewis groups of human red cells. Nature 176:172.
• Stevens WL (1950) Statistical analysis of the ABO blood groups. Human Biol 22:191-217.
• Stigendal L, Olsson R, Rydberg L and Samuelsson BE (1984) Blood group Lewis phenotype on erythrocytes and saliva in alcoholic pancreatitis and chronic liver disease. J Clin Pathol 37:778.
• Vergolino-Henry A and Figueiredo AN (1990) A presença africana na Amazônia Colonial - Uma notícia histórica. Arquivo público do Pará 1:27-66.
• Wang B-J, Akiyama K and Kimura H (1994) Quantitative analysis of Le^a and Le^b antigens in human saliva. Vox Sang 66:280-286.
• Watkins WM (1980) Biochemistry and genetics of the ABO, Lewis and P blood group systems. Adv Hum Genet 10:131-136.
• Yazawa S, Asao T, Izawa H, Miyamoto Y and Matta KL (1988) The presence of CA 19-9 in serum and saliva from Lewis blood group negative cancer patients. Jap J Cancer Res (Gann) 79:538.

Publication Dates

• Publication in this collection
  06 Dec 2002
• Date of issue
  2002

History

• Accepted
  23 July 2002
• Received
  29 June 1999