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início da páginaHLA ANTIGENS AND HAPLOTYPES IN IgA-DEFICIENT BRAZILIAN PEDIATRIC PATIENTS
M. GERBASE-DELIMA, L. C. PINTO, A. GRUMACH*, M. M. S. CARNEIRO-SAMPAIO**
Laboratório de Imunologia Clínica, Departamento de Pediatria, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brasil.
* Instituto da Criança do Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil.
** Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil.
Corresponding author:
Maria Gerbase de Lima, MD
Rua Pirandello, 709
04623-000, São Paulo, SP, Brazil
Phone: 55-11-576-4426
Fax: 55-11-570-1590
E-mail: ger.dped@epm.br
SUMMARY; INTRODUCTION; MATERIALS AND METHODS; RESULTS; DISCUSSION; REFERENCES
In the present study we determined the HLA-A, B and DR antigenic and haplotypic frequencies in unrelated Brazilian Caucasian pediatric patients with IgA-deficiency (IgA-D). Out of 17 IgA-D subjects typed for HLA A and B specificities, 12 (71%) presented B8 and/or B14; of 15 patients also typed for HLA-DR specificities, 14 (93%) were positive for at least one of the HLA markers previously reported to be associated with IgA-D, i.e., B8, B14, DR1, DR3 or DR7. The haplotypes B8,DR3, B14,DR1 and B13,DR7 were present in 43, 21, and 14% of the cases, respectively, versus a frequency in the general population of 2, 2 and 1%, respectively. The concomitant association with of the three IgA-associated haplotypes found in our study probably reflects the admixture of European genetic influences present in the Brazilian Caucasian population of São Paulo.
Running title: HLA antigens and haplotypes in IgA-deficient subjects
Key words: • IgA-deficiency • genetics • HLA haplotypes• HLA
IgA deficiency (IgA-D) is the most common immunodeficiency of infancy. IgA-D subjects may be asymptomatic or may present clinical manifestations such as recurrent infections, allergy and autoimmunity. The IgA-D prevalence in Caucasians has been in general estimated at 1:400 to 1:800 in community-based surveys, while higher figures (1:50 to 1:200) have been observed when the prevalence was assessed in patients with recurrent infections and/or allergy (for reviews see Burks Jr. & Steele, 1986; Cunningham-Rundles, 1990; Hanson et al., 1988; Lawton & Hummell, 1995). African-Americans and Asians present a much lower prevalence of IgA-D (Lawton & Hummell, 1995). In the Brazilian population of São Paulo, the IgA-D prevalence was reported to be 1:965 asymptomatic adults (Carneiro-Sampaio et al., 1989) and 1:50 allergic children (Solé et al., 1985).
In most cases IgA-D represents an isolated immunological disorder, but it may also be associated with other immunoglobulin deficiencies, especially IgG2 and IgG4, probably as the result of a common underlying defect that prevents B lymphocytes from differentiating to plasma cells (Oxelius et al., 1981; Schaffer et al.1989; Cunningham-Rundles et al., 1991; Volanakis et al., 1992; Islam et al., 1994; Lawton & Hummell, 1995).
The participation of genetic factors in IgA-D is well accepted, considering the relatively high familial prevalence of this disorder (Oen et al., 1982) and also the evidence gathered from several studies showing that at least one of the genes involved in the susceptibility to IgA-D (and also to common variable immunodeficiency) is located within the HLA complex.
Reported associations between IgA-D and HLA specificities include HLA A1, A28, B8, B13, B14, B17, DR1, DR3, and DR7 (Ambrus et al., 1977; Seignalet et al., 1978; Oen et al., 1982; Hammarström & Smith, 1983; Hammarström et al., 1984; Haikkilä et al., 1984; Klemola et al., 1988; Cuccia-Belvedere et al., 1989; Cunningham-Rundles et al., 1990; Cobain et al., 1983; Wilton et al., 1985; Fiore et al., 1995). All of these associations may be accommodated into a few extended haplotypes: A1,B8,C4AQ0,C4B1,BfS, DR3,DQ2 (in short: B8,DR3 haplotype); A28,B14,C4A2,C4B1/2(C4B duplication), BfS,DR1,DQ5 (in short: B14,DR1 haplotype); A1,B57(17),C4A6,C4B1,BfS; A29,B44,BfF,C4A3,C4B1,DR7,DQ2 (in short: B44,DR7 haplotype); B13Cw6DR7 (Wilton et al., 1985; Klemola et al., 1988; Cuccia-Belvedere et al., 1989; French & Dawkins, 1990; Volanakis et al., 1992).
The purpose of the present study was to investigate HLA-A,
B and DR associations with IgA-D in the Caucasian population of São Paulo, Brazil. This
population is a heterogeneous mixture of descendants of Europeans, mainly from Portugal,
Spain and Italy, but has also a proportion of other European descendants, particularly
from Central Europe. Data not presented in this paper are able to explain the HLA
frequencies of our "Caucasian" population (Gerbase-DeLima, 1997).
Subjects
Our series comprised 17 unrelated Brazilian Caucasian children from the city of São Paulo (6 males and 11 females aged 1 to 12 years at the time of diagnosis), followed at the outpatient clinic of Instituto da Criança "Pedro de Alcântara", Faculdade de Medicina, Universidade de São Paulo. The diagnostic criterium for IgA-D was serum IgA level below 5 mg/dl. In 15 patients, IgA was undetectable as measured by the single radial immunodiffusion technique. Serum IgG and IgM levels were within the normal range. Recurrent infections and/or allergy were present in 16 patients and one patient (case # 14) presented hyperthyroidism as the sole clinical manifestation. HLA typing of at least one of the parents was performed in order to establish the HLA genotypes of the patients. Two patients had IgA-D siblings who were also typed for HLA antigens.
HLA typing
HLA-A, B, DR and DQ specificities were determined by the microlymphocytotoxicity technique (Terasaki & McClelland, 1964) and commercial antisera (Biotest Diagnostic, Frankfurt, Germany; Pel-Freez Clinical Systems, Brown Deer, WI, USA) were used to define the HLA specificities.
Statistical analysis
The frequencies of HLA specificities and selected HLA haploypes were compared between patients and controls by the Fisher's exact test. The controls consisted of unrelated healthy Brazilian Caucasian individuals from the same city as the patients. In cases of significant positive associations the odds ratio was calculated as an approximation to estimate the relative risk (RR).
The HLA phenotypes and genotypes of the 17 unrelated patients are shown in Table 1. The frequencies of the HLA-B8, B14, DR1 and DR3 specificities were significantly increased in the patients, whereas the frequency of DR5 was diminished (0% x 27,8%, P = 0,013). The frequencies of patients and controls positive for the antigens HLA-B8, B14, DR1 and DR3, and for the haplotypes B8,DR3, B14,DR1 and B13,DR7 are presented in Table 2, along with the P and the relative risks values. Patients number 2 and 13 had IgA-D siblings whose HLA typings were A2,10;B14,18;DR1,7 and A3,31;B8,14;DR1,3, respectively.
The present study has shown one of the highest, albeit heterogenous, associations of HLA markers and IgA-D in comparison to previously reported studies. Out of 17 IgA-D subjects typed for HLA A and B specificities, 12 (71%) presented B8 and/or B14; out of 15 patients also typed for HLA-DR specificities, 14 (93%) were positive to at least one of the HLA markers previously reported to be associated with IgA-D: B8, B14, DR1, DR3, and DR7. Haplotypes B8,DR3, B14,DR1 and B13,DR7 were present in 43, 21, and 14% of cases, respectively, versus a frequency in the general population of 2, 2 and 1%, respectively.
Stronger HLA associations have been reported in cases of total lack in comparison with cases with minute amounts of IgA (0.002-5 mg/dl) of IgA in the serum (Heikkilä et al., 1984). Therefore, the high strength of the association observed in our study could be related to the fact that the majority of the patients (88%) had total lack of IgA.
The concomitant association with B8,DR3, B14,DR1 and B13,DR7 haplotypes in the Brazilian population of São Paulo probably reflects the admixture of European genetic influences present in this population. An association with B8 or with DR3, but not with B14 or DR1, has been observed in IgA-D subjects from Hungary (Ambrus et al, 1977), Canada (Oen et al, 1982), Denmark (Jersild et al., 1983) and Sweden (Hammarström & Smith, 1983; Hammarström et al., 1984), whereas association with B14 or DR1, but not with B8 or DR3, was described in Italians (Cuccia-Belvedere et al., 1989; Fiore et al., 1995). In a study conducted in Spain, both haplotypes were associated with IgA-D (Clerici et al., 1993).
The association of IgA-D with B8,DR3, characteristically observed in Northern European populations, and the association with B14,DR1, found in conjunction with the association with B8,DR3, or as the only association, in Southern European populations, probably reflects the frequency of these haplotypes in these two groups of European populations (Imanishi et al, 1992).
The mechanisms by which the genes of the HLA complex exert their effects are not clear, nor has it been defined which gene (s) is (are) primarily responsible for the associations. To add more complexity to these issues, there are other genetic (Olsson et al., 1992) and nongenetic factors that may play a role in the susceptibility to IgA-D. It has been shown, for instance, that IgA-D prevalence in the offspring of IgA-D females is higher than in the offspring of IgA-D males, possibly due to transplacental passage of IgG anti-IgA antibodies (Petty et al., 1985).
In principle, any gene residing in the IgA-D associated extended haplotypes could be a candidate to mediate the immunologic disorder associated with IgA deficiency. Different suggestions concerning the involved HLA gene have been reported in the literature. Two of them are centered on amino acid sequences of HLA molecules encoded by the DQB1 or DRB1 genes. Olerup et al.(1990) have suggested that the presence of a neutral amino acid (valine or alanine) at position 57 of the DQ b-chain represents a susceptibility factor, whereas the presence of the negatively charged aspartic acid at this same position represents a protective factor. Based on the comparison of amino acids sequences between the molecules encoded by DRB1 alleles, Fiore et al. (1995) suggested that the susceptibility to IgA-D could be related to the presence of specific amino acids between positions 80 and 90. The molecule encoded by the IgA-D associated DRB1*0102 allele presents alanine and valine, whereas the molecule encoded by the non IgA-D associated DRB1*0101 allele presents valine and glycine at the 85 and 86 positions, respectively. We find it difficult to accept that these differences in amino acids could be involved in the mechanism of B cell differentiation arrest responsible for IgA-D.
Investigators from Australia were the first to suggest that the primary HLA association with IgA-D could be with a gene (or genes) located within the HLA class III region (Wilton et al., 1985; French & Dawkins, 1990). This suggestion was based on the observation that a relatively small number of extended haplotypes are shared by the majority of IgA-D individuals, and that the shared haplotypes frequently present alterations in the class III HLA region, such as a deleted C4A gene in haplotype B8,DR3 or a duplicated C4B in the B14,DR1 haplotype. Subsequent studies in other populations have given further support to this hypothesis (Cunninham-Rundles et al., 1991, Volanakis et al., 1992; Ashman et al., 1992). However, the issue of which class III region gene (s) is (are) actually responsible for IgA-D susceptibility is not resolved. In our opinion, the best candidate so far proposed is a still undetermined gene that somehow influences the production of IL-5 (Lio et al., 1995).
A series of immune dysfunctions have been associated with the HLA-B8,DR3 haplotype. HLA-B8,DR3-positive healthy individuals show significantly lower values of serum IgA than HLA-B8,DR3-negative ones (Cryan et al., 1985; Modica et al., 1989). In vitro experiments have shown that lymphocytes from B8,DR3 positive individuals present an impaired ability to produce IL-1, IL-2, the cellular and the soluble form of IL-2 receptor, g -IFN, and IL-5. (Hashimoto et al., 1990; Candore et al. 1994; Lio et al.,1995). Defective expression of CD69, an early activation marker that may be relevant in controlling gene cytokine expression, has also been observed (Candore et al., 1995; Testi et al., 1994).
The impaired production of IL-5 is interesting. It has been demonstrated in the mouse that IL-5 is an efficient stimulating factor for the secretion of IgA (Coffman et al., 1993, Morikawa et al., 1993) and that the level of IL-5 production is regulated by the H-2 complex (the murine counterpart of the human HLA complex) (Dieli et al., 1993; 1995a). It has also been demonstrated that exogenous IL-5 enhances antigen-specific IgA production in MHC genetically determined low IL-5 responder mice both in vitro and in vivo (Dieli et al., 1995b). Furthermore, it has been shown that T cells from IgA-D deficient subjects produce low levels of IL-5 and that the addition of IL-5 to the cell cultures enhances the production of IgA antibodies (Lio et al., 1994).
We believe that it would be interesting to investigate whether the serum and/or secretory IgA levels in the individuals carrying the IgA-D associated haplotypes, besides B8,DR3, are lower than those of the general population, and also to assess whether some of the immunological abnormalities that have been associated with the B8,DR3 haplotype, like the defective production of IL-5 and the lower expression of CD69, are also present in the individuals that carry the other IgA-D associated haplotypes. In addition, a detailed study of the polymorphisms of the many genes of the HLA class III region in all the IgA-D associated haplotypes is of considerable interest. We believe that the search for common immunologic abnormalities and/or genetic polymorphisms among the different IgA-D associated haplotypes could shed some light on the nature of the genes present in the HLA complex that confer susceptibility to IgA deficiency.
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