C2 DEFICIENCY IN BLOOD DONORS AND LUPUS PATIENTS: PREVALENCE, CLINICAL CHARACTERISTICS AND HLA-ASSOCIATIONS IN THE BRAZILIAN POPULATION
Maria Neile Torres Araújo3, Neusa Pereira da Silva1, Luis Eduardo Coelho Andrade1, Emília Inoue Sato1, Maria Gerbase-DeLima2, and Paulo Guilherme Leser1
Division of Rheumatology1, Department of Medicine; Division of Allergy, Clinical Immunology and Rheumatology2, Department of Pediatrics, Universidade Federal de São Paulo, São Paulo, SP; Department of Patology3, Universidade Federal do Ceará, CE. Brazil.
SHORT RUNNING TITLE: C2 deficiency in the Brazilian population
Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
SUMMARY; INTRODUCTION; MATERIAL AND METHODS; RESULTS; DISCUSSION; REFERENCES
The objective of the present study was to investigate the prevalence, clinical characteristics, and HLA associations of C2 deficiency in the Brazilian population. The frequency of C2 deficiency profile (C2Q° profile) was 2.2% among 1503 blood donors and 6.6% among 166 patients with systemic lupus erythematosus (SLE). A higher incidence of clinical manifestations possibly related to immune complex disease was observed among blood donors with C2Q° profile and their relatives with C2Q° profile when compared to the normal C2 relatives. The comparison of clinical and laboratory features between SLE patients with C2Q° profile and those with normal C2 revealed earlier disease onset, higher frequency of oral ulcerations and lower frequency of anti-native DNA antibodies in the first group. The HLA study conducted on 18 individuals with C2Q° profile (11 blood donors and 7 SLE patients) confirmed the previously reported association with the antigens HLA-A25, B18 and DR2, supporting the concept that probably most C2 deficiency cases, throughout the world, are due to a single mutation in the C2 gene in linkage disequilibrium with the A25B18DR2 haplotype.
Key Indexing Terms: C2 DEFICIENCY, SYSTEMIC LUPUS ERYTHEMATOSUS, HLA, GENETICS
C2 deficiency is one of the most common inherited complement component deficiency (1,2). It may occur in asymptomatic individuals but it is frequently associated with autoimmune manifestations, particularly with discoid or systemic lupus erythematosus (SLE) (3,4).
Studies of C2 deficiency in samples of Caucasian populations including North American blood bank donors showed a prevalence of heterozygous ranging from 1 to 2% (5-8). The prevalence of homozygous individuals, C2Qo, has been calculated in the range of 1 in 40,000 (7) to 1 in 10,000 individuals (5). In a survey of 145,640 blood donors in Japan, no case of C2 deficiency was found (9).
Family studies in Caucasian populations have established that C2Qo allele, located within the HLA (human leukocyte antigens) complex, is in strong linkage disequilibrium with the HLA genes encoding for A25(10), B18, DR2, BfS, C4A4 and C4B2 (6,7,10-15). The purpose of the present study was to evaluate the prevalence of C2 deficiency, to determine possible clinical manifestations, and its association with HLA-A, B and DR alleles, among Brazilian blood donors and SLE patients.
Blood donors and patients. C2 deficiency was investigated among blood bank donors and SLE patients from the city of São Paulo, Brazil. The population of São Paulo is racially mixed; Whites are estimated to comprise 70% of it and the remainder to be represented by Orientals, Blacks, and Amerindians with varied degrees of admixture with Whites.
The blood donor sample comprised 1503 individuals. Those with a C2 deficiency profile, designated C2Q° profile, (see below) were contacted for clinical evaluation by a rheumatologist and blood sampling for autoantibody screening, profile validation, and HLA typing.
The SLE sample was composed of 166 patients meeting at least four criteria of the American College of Rheumatology (16), and randomly selected from the Rheumatology Outpatient Clinic at Universidade Federal de São Paulo. All patients were subjected to clinical examination and serum samples were obtained for complement and autoantibody screening. Patients with a C2 Q° profile were selected for HLA typing.
Relatives of individuals presenting C2Q° profile were contacted for clinical evaluation by a rheumatologist and blood sampling for autoantibody screening and C2 profile determination. HLA typing was performed in families presenting at least one affected relative.
C2 deficiency profile (C2Q° profile) definition. The term C2Q° profile was applied to groups of individuals with either homozygous or heterozygous profile. A homozygous C2Q° profile was defined as undetectable levels of C2 and CH50 associated with normal levels of C1q, C3 and C4. A heterozygous C2Q° profile was defined as low C2 level, low to normal CH50 level and normal C1q, C3 and C4 levels.
C2 deficiency classification [adapted from Glass et al. (7)]. Homozygous C2 deficiency, C2Qo-hm, was determined by the characteristic homozygous C2Q° profile. Heterozygous C2 deficiency, C2Q° -ht, was ascertained by the presence of heterozygous C2Q° profile in at least two independent occasions and validated by family studies (at least one relative with C2Q° profile). Possible C2 deficiency, possible C2Q° , refers to those individuals that presented heterozygous C2Q° profile in one or more determinations but could not be validated by family study (either because there was no available relative or because the available ones had normal C2 levels).
CH50 and complement components assays. Sera were separated immediately after clotting and either processed in the same day or stored at -70°C. CH50 and C2 levels were determined by radial immunohemolysis (17,18). C1q, C3 and C4 levels were determined by radial immunodiffusion (19) with commercial plates from Behring, Germany, following manufacturer instructions.
Autoantibody screening. Antinuclear antibodies (ANA) and anti-native DNA antibodies were detected by indirect immunofluorescence (IIF). Anti-ENA antibodies (aSm, aU1-RNP, aSS-A/Ro and aSS-B/La) were detected by double immunodiffusion.
HLA typing. HLA antigens were determined by the standard complement-dependent microlymphocytotoxicity method using a battery of 140 anti-HLA class I sera (Biotest, Pel-Freez and other sera) capable of recognizing 13 HLA-A (HLA A1-3, 9, 25 (10), 26(10), 11, 28-33) and 17 HLA-B (HLA B5, 7, 8, 12-18, 21, 22, 27, 35, 37, 40, 41) specificities. HLA-DR specificities (DR1-DR7) were determined with a battery composed of 70 antisera (Biotest and Pel-Freez sera). Identical by descent HLA haplotypes were assigned by comparing the HLA phenotypes among family members.
Statistical analysis. The Mann-Whitney test was used to compare the age of disease onset between SLE patients with C2Q° profile and normal C2 SLE patients. The Fisher's exact test was used for the other comparisons. When significant differences were detected (p<0.05), the degree of association was assessed using the odds ratio as an approximation to estimate the relative risk (RR).
The prevalence of C2Q° profile among blood donors was found to be 2.2% (33/1503), corresponding to a gene frequency of 1.1% and an expected C2Qo-hm prevalence of 1.2/10,000 individuals. The prevalence of profile among SLE patients was 6.6% (11/166).
Considering the C2 profile and, when available, the family study, among the 33 blood donors with C2Q° profile, eight were C2Q° -ht cases, one C2Q° -hm, and 24 were possible C2Q° cases. The prevalence of C2Q° (definite cases) among blood donors was 0,6% (9/1503). Among 11 SLE patients with C2Q° profile, four cases were C2Q° -ht patients and 7 were possible C2Q° . The prevalence of C2Q° (definite cases) among SLE patients was 2,4% (4/166). Antinuclear antibodies were not detected among the 33 blood donors with C2Q° profile.
Studies were possible on nuclear families from 21 propositi, comprising 75 relatives (49 relatives of 12 blood donors and 26 relatives of 9 SLE patients). C2Q° profile was found in 22 relatives of 9 blood donors and in 3 relatives of 3 SLE patients.
Among all individuals that underwent clinical evaluation, relevant findings were observed in 33.3% (7/21) blood donors with C2Q° profile, in 31.8% (7/22) of their relatives with C2Q° profile and in 14.8% (4/27) of their normal C2 relatives (Table I). Taking into account only those manifestations that might be related to immune complex disease, we found a 25.6% frequency of clinical manifestations among 43 individuals with C2Q° profile (21 blood donors and 22 relatives) and a 3.7% frequency among 27 normal C2 relatives of the C2Q° profile blood donors. This difference was statistically significant (p=0.022).
Regarding the relatives of C2Q° profile SLE patients, relevant clinical findings (SLE, Hashimoto's disease, and allergy) were found in 4 out of 23 normal C2 relatives and in none of the 3 relatives with a C2Q° profile.
The comparison of clinical and laboratory features between 11 C2Q° profile SLE patients and 155 normal C2 SLE patients showed that those with a C2Q° profile presented earlier disease onset (mean of 21 years vs 27 years; p=0.028), higher frequency of oral ulcerations (45.5% vs 13.5%; p=0.015; RR=5.3) and lower frequency of anti-native DNA antibodies (18.2% vs 52.9%; p=0.031; RR=0.19). Anti-Sm antibodies were found in 21.9% of the normal C2 SLE patients but in none of those with C2Q° profile. This difference, however, did not reach statistical significance (p=0.12).
The HLA-A, B, and DR phenotypes of 11 blood donors and 7 SLE patients with C2Q° profile are listed in Table II. C2Q° -hm and C2Q° -ht cases from both groups were pooled for comparison of HLA antigenic frequencies with the control population. As shown in Table III, they presented significantly higher frequencies of HLA-A25 (44.4% vs 3.8% in the normal population; p=0.0007; RR= 20.4), B18 (55.6% vs 6.5%; p=0.003; RR=9.2) and DR2 (66.6% vs 19.0%; p=0.003; RR=8.5). Possible C2Q° individuals were analysed as a separate group and presented also higher frequencies of those HLA antigens as compared to controls (Table III).
HLA studies were performed on 5 families with more than one affected individual (5 parents, 13 C2Q° profile siblings and 6 normal C2 siblings were HLA typed). In 4 of the 5 families, the HLA haplotype A25B18DR2 was present in all C2Q° profile relatives and in none of the normal C2 ones. In the family where the C2Q° -hm relative was found, the HLA haplotypes involved were A25B18DR2 and A25B18DR7. The HLA haplotype A2B21DRblank was associated with C2Q° profile in the only family that did not present the A25B18DR2 haplotype.
The 2.2% (33/1503) incidence of C2Q° profile observed in the present study in a sample of 1503 Brazilian blood donors is similar to those reported for blood donors of predominantly Caucasian populations (5-8). The present finding of 6.6% individuals with C2Q° profile among 166 patients with SLE is also very close to the figure of 5.9% reported by Glass et al.(7).
The frequency of manifestations that could be related to immune complex disease was higher among blood donors and relatives with C2Q° profile than among normal C2 relatives (p=0.022). This frequency (25.6%) is higher than that of 13.4% reported by Agnello (3) but lower than that of 55.8% reported by Lachmann for homozygous C2 deficiency (21). It is noteworthy that the search for antinuclear antibodies in the blood of all the 33 C2Q° profile blood donors was negative.
The earlier disease onset observed in C2Q° profile SLE patients when compared to normal C2 SLE patients (mean of 21 years vs 27 years, p=0.028) was in agreement with the finding of Glass et al.(7). In addition, C2Q° profile SLE patients disclosed a lower frequency of anti-native DNA antibodies than normal C2 SLE patients (18.2% vs 52.9%; p=0.031) as reported earlier (3), and a higher frequency of oral ulcerations, a finding not previously described. No significant differences were observed concerning other autoantibodies.
Considering that complement level determination is a widely employed and useful ancillary method for estimating SLE disease activity and that C2 deficiency can affect CH50 determination, we emphasize the importance of C2 deficiency diagnosis in SLE patients for a correct clinical interpretation of serum complement assays.
HLA-A, B and DR typing revealed a strong association of heterozygous and homozygous C2 deficiency with antigens HLA-A25 (44.4% vs 3.8% in the normal population), B18 (55.6% vs 6.5%), and DR2 (66.6% vs 19.0%), and particularly with the HLA haplotype A25B18DR2. These findings confirm that the Brazilian population presents the same associations between C2 deficiency and HLA that have been reported for other populations (6,7,10-13,22). It should be emphasized that the adopted classification for heterozygous C2 deficiency, based in persistent low C2 serum level in the presence of normal C1q, C3, and C4 levels associated to familial clustering, although not including a formal demonstration of C2Q° allele (due to local technical limitations), was further substantiated by the expected haplotype association observed in this study. As can be observed from Table III, possible C2Q° cases presented HLA A25, B18 and DR2 frequencies comparable to those observed among C2Q° -hm and C2Q0-ht cases, and higher than in the normal population. These results suggest that the possible C2Q° cases could be C2 deficient individuals.
The mechanism underlying the association of C2 deficiency and SLE has not been clearly established. One explanation is a linkage disequilibrium between the C2Q° allele and some SLE susceptibility gene of the HLA complex. Alternatively, or additionally, considering that deficiencies of other early complement components, namely C1q, C1r/1s and C4, are also associated with immune complex disease and particularly with SLE (21,23), and that clinical manifestations compatible with immune complex deposition were observed in C2Q° profile individuals without an overt autoimmune disease, it might be hypothesized that C2 deficiency could play a direct role in the pathogenesis of immune complex diseases via abnormalities in the clearance of immune complexes. This interpretation is consistent with the concept that the failure to properly handle immune complexes is a leading phenomenon in the pathophysiology of immune complex-mediated diseases (21,23).
ACKNOWLEDGMENTS. The authors wish to thank Prof. Walter Leser and Prof. Paulo A. Otto for fruitful discussions and helpful suggestions.
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