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DR3 and nonDR3 associated complement component C4A deficiency in systemic lupus erythematosus
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
60,
55-64 (]%I)
DR3 and nonDR3 Associated Complement Component Deficiency in Systemic Lupus Erythematosus ASHOK KUMAR,PRITI
KUMAR,AND
C4A
PETER H. SCHUR'
Department of Rheumatologyllmmunology, 75 Francis Street, Brigham & Women’s Hospital, Harvard Medical School, Boston. Massachusetts 02115 The molecular basis of complement component C4A deficiency in white U.S. and Mexican patients with systemic lupus erythematosus (SLE) was studied. Genomic DNA from SLE patients and non-SLE controls was analyzed for restriction fragments using Hind111 and a 5’ C4 cDNA probe. C4A gene deletion was recognized by the loss of a IS-kb restriction fragment and the appearance of a 8.5-kb fragment. Thirty-two selected U.S. SLE patients, 7 nonSLE controls, and 11 Mexican SLE patients and 9 relatives were studied. The deletion was recognized in all of the 14 HLA-BS;DR3 SLE patients with a C4A protein deficiency. Two SLE patients with DR3 but without B8 also had this gene deletion. None of the 3 U.S. SLE nonDR3, C4A protein deficient patients nor 20 C4A protein deficient Mexican individuals (11 SLE patients and 9 relatives; none had B8 and/or DR3) showed this deletion. Thus the C4A gene deletion failed to account for the C4A protein deficiency in all of the nonDR3 Mexicans and in some U.S. SLE patients. To determine whether gene conversion at the C4A locus would encode a C4B-like protein and be responsible for the C4A protein deficiency tin nonDR3 patients), the C4d region of the gene was amplified by polymerase chain reaction and subjected to Nla IV digestion, and restriction fragment analysis was performed using a C4d region-specific probe. The resulting restriction fragment length polymorphism pattern revealed no changes in the isotype-specific region of the gene as characterized by C4A-specific 276and 191-bp fragments in DR3 or nonDR3 individuals. Thus, homoexpression of C4B at both loci was not responsible for C4A deficiency in nonDR3 SLE patients without C4A gene deletion. 0 1991 Academic press. Inc.
INTRODUCTION
There is a significant association between the deficiency of complement component C4 and systemic lupus erythematosus (SLE) (l-g). C4 genes are located on the short arm of the chromosome 6 within the major histocompatibility complex (MHC) between the HLA-D region and HLA-B locus (9). The two isotypes of C4, C4A, and C4B, are expressed at two loci which are approximately 10 kb apart (9, 10). C4A and C4B proteins are 99% homologous and are highly polymorphic (11) with more than 35 alleles, including null alleles (C4QO) at both loci (12). Null alleles, defined as the absence of C4A or C4B protein in the serum, have been shown to occur in patients with SLE (l-3). An increased frequency of C4A (protein) null alleles (C4AQO) has been demonstrated in patients with SLE of different ethnicity, including whites (l-3), blacks (3, 8, 13) Chinese, and Japanese (5, 6). Heterozygous C4AQO protein deficiency has been reported in 50-W% SLE patients and homozygosity for C4AQO in 10-U% of SLE patients (l-3, 7, 14). A ’ To whom correspondence
should be addressed. 55 0090-1229/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All tights of reproduction in any fomt reserved.
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gene dose effect has been observed with a relative risk for SLE of 3 with the heterozygous and 17 with the homozygous C4A null allele (3). C4AQO null alleles are strongly linked to one extended haplotype HLA-Al ;BS;DR3;SCOl in Caucasians, particularly those of English/Irish ancestry (15). In our studies. C4AQO was found to be increased only in SLE patients of English/Irish ancestry (16). Others have observed C4AQO in SLE in Orientals and Mexicans but not in association with DR3 (3, 5,6,8, 17, 18). In the majority of cases studied to date, C4A protein deficiency, associated with DR3, has been shown to result from an approximately 30-kb gene deletion involving most of the C4A gene and part of the 21 hydroxyiase gene in white SLE patients (19). Similarly, in black SLE patients with HLA-B44; -DR2 and -DR3 alleles, C4A deficiency has been associated with a large C4A, Cyp 21A gene deletion (13). The C4A null allele could also occur due to the expression of identical allotypes at the two C4 loci (20), a phenomenon termed as homoexpression (21). Homoexpression has recently been reported to occur in individuals with C4BQO but not in individuals with C4AQO (22-24). C4A protein differs from C4B by four amino acids due to five nucleotide changes in the C4d region of the gene. The C4A and C4B genes can be distinguished with Nla IV restriction fragment length polymorphism (RFLP) analysis (21). Mutations in this region of the C4A gene could result in a molecule indistinguishable from C4B and an apparent deficiency of C4A protein (21). The occurrence of the tight linkage disequilibrium between B8, DR3, and C4AQO (as part of the extended haplotype HLAAl;BS;DR3;SCOl) makes it difficult to determine the role of each allele, if any, as a primary susceptibility gene. Studies of an ethnic group in which this haplotype occurs normally in low frequency may help in defining the individual alleles in SLE (25). In Mexican mestizos C4AQO occurs frequently but independently of the HLA haplotype B8;DR3 which is rare (~5%) while SLE is particularly frequent (17, 18). The present study defines the role of the C4A gene with particular emphasis on the C4d region of the gene, in C4A protein deficiency in SLE patients from the United States and Mexico. MATERIALS
AND METHODS
Patients. Ail of the patients with SLE included in this study met the American Rheumatism Association classification criteria for the diagnosis of SLE (26). Patients referred to as U.S. SLE were white SLE patients followed at the lupus clinic of this hospital. Mexican SLE patients were followed at Instituto National de la Nutrition Salvador Zubiran, Mexico. SLE patients and non-SLE controls were selected on the basis of presence or absence of a C4A null allele with or without the B8/DR3 phenotype as shown in Table 1. Individuals with a B8, DR3, and C4A null allele on at least one of the chromosomes were grouped as B8;DR3;C4AQO, those with a C4A null allele on both the chromosomes as B8;DR3$4AQOQO. The prefix non was used to signify the absence of the particular allele. HLA typing and C4 allotyping. HLA (A, B, DR) and C4 allotyping was per-
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formed as previously described (16). C4A allotypes including C4AQO were confirmed by family studies. Genomic DNA. Genomic DNA was isolated from EBV-transformed B lymphocytes or from peripheral blood lymphocytes isolated from fresh blood as described before (27). RFLP analysis. RFLP analysis of genomic DNA was performed by Southern blotting (28). Ten micrograms of genomic DNA was digested with Hind111 (New England Biolabs) as per the instructions of the manufacturer. The samples were electrophoresed on an 0.8% agarose gel in TAE buffer (40 mM Tris-acetate, 2 mM EDTA) for 16-20 hr at 40 V. For molecular weight markers, 20 ng of h DNA digested with Hind111 was used. DNA was transferred to the Gene screen plus (DuPont) membrane by the capillary blot method as per the instructions of the manufacturer. The membrane was air dried at room temperature and transferred to a prehybridization solution (50% formamide, 10% dextran sulfate, 2% SDS, 100 t&ml denatured and fragmented salmon sperm DNA, and 1 M sodium chloride) at 42°C for 6-12 hr. A 5’ C4 cDNA probe was used after labeling with 32P-dCTP by the random primer labeling method (29,30). A DNA (250 pg) was also included in the labeling reaction mixture. The membrane was incubated overnight at 42°C in prehybridization solution having the radiolabeled probe. The membrane was washed three times with 0.1 x SSC (15 mM NaCl, 1.5 mM sodium citrate) with 2% SDS at 65°C and authoradiographed at -80°C. Polymerax chain reaction (PCR). For amplification of the C4d region of the C4 gene, 1 p.g of genomic DNA was subjected to PCR. According to the published sequence of the C4 gene (31), oligonucleotide primers A S’-TGCGGATCCAGCAGTTTCGGAAG-3’ and B 5’-ATAGGATCCTAAGGTCCCCTGGGCCT-3’ were used to selectively amplify the 926-bp C4d gene fragment. The optimal PCR conditions were standardized and the reaction was carried out in 10 mM TrisHCl, pH 8.8, 50 mM KCI, 1.25 mM MgCl,, 0.01% gelatin, 2 units of Taq polymerase (Cetus Corp.), 200 mM dNTPs, and 0.2 t~J4 of each primer. Twenty-five thermal cycles, each of denaturation (1 min, 94”C), annealing (2 min, SYC), and extension (2 min. 72”(Z), were carried out using Tempcyler (Coy Corp.). The amplified product was electrophoresed on an 1.5% agarose gel. DNA was recovered from the gel by using the GeneClean kit (Bio 101 Inc.) Probes. A 500-bp cDNA fragment specific for the 5’ region of C4 genes was derived from the BamHIIKpnI digestion of the full-length C4 cDNA in PAT-A (32). The Pb probe derived as a BamHI genomic subclone of C4 for the C4d region of the C4 gene was 927 bp (33). Both probes were kindly provided by Dr. M. C. Carroll, Harvard Medical School, Boston. Another probe specific for the C4d region of the C4 gene was prepared by PCR as described above. The amplified material used as the C4d probe hybridized with the full-length C4 cDNA probe as well as with the Pb probe described above. RFLP analysis of C4d amplified product. Ten nanograms of PCR amplified C4d DNA was subjected to Nla IV (New England Biolabs) digestion as per the manufacturer’s instructions. DNA was electrophoresed on a 12% polyacrylamide gel in TAE buffer along with 20 ng of HaeIII-digested +X 174 DNA as a molecular
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weight marker. DNA was transferred to a Gene screen plus membrane SSC and probed with the C4d probe as described above.
in IO x
RESULTS
Thirty-two selected U.S. SLE patients were included in this study and grouped as shown in Table 1. Two of the patients in this group were homozygous for the C4A null allele (C4AQOQO). Twenty patients in this group were HLA-DR3 and 15 had the HLA-B8 phenotype. Fourteen of the patients had the extended haplotypeB8;DR3;C4AQO. Three patients had a C4A protein deficiency without DR3. On the other hand, all of the 11 Mexican SLE patients studied were nonBSmonDR3 and 6 of them expressed the C4A null allele. Nine individuals who were relatives of the Mexican SLE patients were also studied. Six of the individuals expressed the C4A null allele, including one homozygous for the C4A null allele and others normal for C4A (nonC4AQO). Genomic DNA samples of all these individuals were analyzed for the C4A gene by Southern blot analysis using the Hind111 enzyme and the 5’ C4 cDNA probe (Table 1, Fig. 1). Individuals with only a 8.5-kb restriction fragment were grouped as pattern A, with 26-, 15-, and 8.5kb bands as pattern B and with 26 and 15 kb as pattern C. Analysis of the data revealed that pattern A was observed in those patients who were homozygous for the C4A null allele (C4AQOQO) and were B8;DR3. Individuals who were DR3 and heterozygous for the C4A null allele (C4AQO) showed the pattern B irrespective of the presence or absence of HLA-B8. Patients possessing the C4A null allele and nonDR3;B8 or TABLE 1 RESTRICTION FRAGMENTLENGTH POLYMORPHISM PATTERN OF A C4A GENE IN U.S. AND MEXICAN SLE PATIENTSAND~ONTROLS Group U.S. SLE patients B8;DR3$4AQOQO B8;DR3;C4AQO B8;nonDR3$4AQO nonBS;DR3$4AQO nonB8;nonDR3$4AQO nonB8;DR3;nonC4AQO nonB8;nonDR3;nonC4AQO U.S. non-SLE persons B8;DR3$4AQOQO Mexican SLE patients nonBS;nonDR3$4AQOQO nonBS;nonDR3$4AQO nonBl;nonDR3;nonC4AQO Relatives of Mexican SLE patients nonBl;nonDR3$4AQOQO nonBll;nonDR3$4AQO nonB8;nonDR3;nonC4AQO
NO.
Pattern”
2 12 I 2 2 4 Y
A B C B C C C
7
A
I 5 5
C C C
1 5 3
C C C
a Using the Hind111 enzyme and a 5’ C4 cDNA probe. Patterns A, B, and C describe restriction fragments: A = 8.5 kb; B = 26, IS, 8.5 kb; C = 26, 15 kb.
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IN SLE
M Ae
C
23.1 Kb+ 9.4 Kb-t 6.6 Kb-t FIG. 1. RFLP pattern of genomic DNA digested with the Hind111 enzyme and probed with a 5’ C4 cDNA. DNA from SLE patients with a homozygous C4A null allele (B8;DR3;C4AQOQO) (pattern A), heterozygous C4A null allele (B8;DR3$4AQO) (pattern B), and normal C4A allele (nonBS;nonDR3;nonC4AQO) (pattern C) subjected to Southern blot analysis. HindIII-digested A DNA was used as marker (M).
nonDR3;nonBS showed pattern C as did the individuals who had normal C4A protein (non C4AQO). All of the Mexican patients and their relatives presented the restriction fragment pattern C whether they were homozygous or heterozygous for the C4A null allele or possessed normal C4A protein. In order to further analyze the C4A null alleles, the C4d region of the gene was amplified by PCR using the primers listed under Materials and Methods. The amplification reaction product of 926 bp was seen to hybridize with the C4dspecific probe (data not shown). RFLP analysis of the C4d DNA fragment using the Nla IV enzyme and C4d probe gave restriction fragments of 467,276, and 191 bp for a C4A normal individual (Fig. 2, Table 2). DR3 individuals with a homozygous C4A deficiency (C4AQOQO) showed only a 467-bp fragment while a control C4BQOQO showed 267- and 191-bp fragments. The pattern obtained in the case of heterozygous C4AQO U.S. SLE patients and homozygous or heterozygous C4A null Mexican patients or their relatives was similar to C4A normal individuals (Fig. 2, Table 2).
FIG. 2. RFLP analysis of PCR-amplified DNA from the C4d region of the C4 gene using a Nla IV enzyme and a C4d-specific probe. PCR-amplified C4d DNA from (A) nonB8;nonDR3;nonUQO, (B) nonB8;nonDR3;C4BQOQO (non-SLE control), (C) B8;DR3;C4AQOQO, and (D) nonB8;nonDR3;C4AQOQO individuals were subjected to Southern blot analysis after Ma IV digestion and polyacrylamide gel electrophotesis. +x174 DNA digested with H&II enzyme was used as marker.
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RESTRICTION FRAGMENT PATTERN OFTHE C4d REGION WITH Nla IV Has2 pair fragment No. United States SLE B8;DR3;C4AQOQO B8;DR3;C4AQO nonB8;DR3$4AQO nonB&nonDR3:C4AQO nonB8;DR3;nonC4AQO nonBS;nonDR3 :nonC4AQO Mexican SLE nonB8;nonDR3$4AQOQO nonBl;nonDR3;C4AQO nonB8;nonDR3;nonC4AQO Controls B8;DR3$4AQOQO nonB8;nonDR3$4BQOQO nonB8;DR3;C4BQOQO
C4A
specific
2 2 3
276.191 276.191 276,191 276.191 276.191
467 467 467 467 467 467
2 6 3
276.191 276,191 276.191
467 467 467
-,-
467 -
2
276,191 276,191
DISCUSSION In our study of white U.S. SLE patients, the loss of 26- and 15kb restriction fragments with the appearance of a 8.5-kb band (pattern A) was the marker of homozygous C4A gene deletion while the presence of all three fragments (pattern B) was found in individuals with the heterozygous C4A gene deletion. The presence of both the 26- and 15-kb fragments (pattern C) was found in individuals with an intact C4A gene (19). Gene deletion, as evident by pattern A or B, was observed whenever the C4A null allele was associated with DR3, presumably as part of the extended haplotype BS;DR3;SCOl (15, 34). In two other DR3 patients who were nonB8, a similar gene deletion pattern was observed. Thus the presence or absence of the B8 allele appeared to demonstrate only a limited association with the C4A gene deletion and susceptibility to SLE. The C4A gene deletion was observed only when the C4A null allele occurred with DR3. Others have interpreted the C4A gene deletion linked to the nonB8 allele to loss of the B8 allele in a recombinational event during the evolution of the haplotype B8:DR3 (19). In the case of the non C4AQO allele, either in the presence or in the absence of DR3 or B8, gene deletion was not observed. However, in the case of the nonBS;nonDR3$4AQO, the C4A gene was intact (Table 1). As only two U.S. SLE patients were available in the nonBS;nonDR3$4AQO category, we studied Mexican patients with the nonB8;nonDR3$4AQO phenotype. Restriction fragment analysis in all these individuals revealed an intact C4A gene (Table l), thus strengthening the finding that the C4A gene deletion is linked to the presence of the DR3 allele. Recently, the lack of a gene deletion causing complement C4A deficiency in Japanese SLE patients has been documented (35) which could also be explained by the rarity of the HLA-DR3 allele/haplotype in the Japanese population (36, 37).
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In the case of nonDR3 individuals, C4AQO appears to be due to a nonfunctional gene which could occur due to the expression of identical allotypes at the two C4 loci (homoexpression) leading to synthesis of a C4B-like protein at locus A. Such a phenomenon has been reported to occur for C4BQO alleles leading to the expression of the C4A protein at locus B in IgA nephropathy in whites (22) and other studies from Germany (23) and Finland (24), presumably from a nondisease population. Partanen and Campbell have speculated that the unidirectional gene conversion observed may be due to the single, ethnically homogeneous population studied (23). In SLE, where the association of the C4A null allele has been rather strong (3,7, 14, 17), the molecular basis of C4A deficiency unaccounted by gene deletion remains unclear. In the nondisease population studied by others in Finland (24) and Germany (23), the C4A gene has been termed as a pseudogene due to possible point mutations or to small deletion or insertions (23,24) which remain unidentified. To study whether the C4A nondeleted gene in SLE patients from the United States (whites) and Mexico undergoes gene conversion to express C4B at Iocus A, the C4d region of the gene was amplified by PCR and subjected to Nla IV restriction fragment analysis for the patients listed in Table 2. The analysis revealed that all the nonDR3 individuals with no major deletions of locus A gave the usual fragments of 276 and 191 bp specific for the C4A isotype in U.S. as well as Mexican SLE patients. Both the U.S. SLE nonDR3 individuals were heterozygous for the C4AQO allele and therefore the pattern with no markable difference in the intensity of restriction fragments (data not shown) (Table 2) was uninformative regarding the status of the C4A gene. However, two of the Mexican nonDR3 individuals included in this study were homozygous for the C4A null allele (C4AQOQO) (Table 2). The data clearly indicated a normal C4d region gene pattern viz. 276- and 191-bp fragments and a 476-bp fragment for both of these people. This implies that in nonDR3 individuals who have an intact C4A gene but express the C4A null allele, a gene conversion event changing C4A to C4B has not occurred, thus showing that the defect leading to the nonexpression of the C4A protein is not due to changes in the isotype-specific region of the gene. In nonDR3 SLE patients the gene defect leading to the absence of C4A protein may be due to a defect at the level of transcription or translation of the C4A gene (38). In guinea pigs, a post-translational defect in the processing of C4 precursor RNA to mature mRNA has been suggested (39). Studies are in progress to identify the possible mutations in the C4A gene leading to protein deficiency as identified in p thalassemia (40, 41), severe combined immunodeficiency (42), acatalasia (43), apolipoprotein CII deficiency (44), hereditary angioedema (45), and osteogenesis imperfecta (46). However, it will be desirable to study more U.S., other Caucasians and Mexican SLE patients, and normal controls with nonDR3 homozygous C4A deficiency to confirm the findings to extrapolate the observation to populations. ACKNOWLEDGMENTS This work was partially supported by grants from USPHS AR35907, AR 07530, and the Lupus Foundation of America. A.K. was the recipient of the Fogarty International Postdoctoral Award. Authors are thankful to Drs. C. A. Alper and 2. Awdeh for providing DNA samples of nonSLE controls. Thanks to Dr. Alarcon-Segovia for providing DNA samples of Mexican SLE patients and
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their relatives. Thanks are due to Dr. M. V. N. Rao for cell culture and Ms. Joanne Davis for her secretarial expertise.
REFERENCES I. Fielder, A. H., Walport, M. J., Batchelor, J. R., Ryes, R. 1.. Black, C. M., Dodi, 1. A., and Hughes, Cl. R., Family study of the major histocompatibility complex in patients with systemic lupus erythematosus: Importance of null alleles of C4A and C4B in determining disease susceptibility. Br. Med. J. 286, 425-428, 1983. 2. Reveille, J. D., Amett, F. C.. Wilson, R. W., Bias, W. B.. and McLean, R. H., Null alleles of the fourth component of complement and HLA haplotypes in familial systemic lupus erythematosus. Immunogenetics 21, 299-31 I, 1985. 3. Howard, P. F., Hochberg, M. C., Bias, W. B., Amett, F. C., and McLean, R. H., Relationship between C4 null genes, HLA-D region antigens and genetic susceptibility to systemic lupus erythematosus in Caucasian and black Americans. Am J. Med. 81, 187-193. 1986. 4. Hauptmann, G., Goetz. J., Uring-Lambert, B., and Grosshans, E., Component deficiencies. 2. The fourth component. Prog. Allergy 39, 232-249, 1986. 5. Hawkins, B. R., Wong, K. L.. Wong, R. W., Chan, K. H.. Dunckley, H., and Sejeantson. S. W., Strong association between the major histocompatibility complex and systemic lupus erythematosus in southern Chinese. J. Rheumatol. 14, 1128-l 131, 1987. 6. Dunckley, H.. Gatenby, P. A., Hawkins, B., Naito, S.. and Sejeantson, S. W., Deficiency of C4A is a genetic determinant of systemic lupus erythematosus in three ethnic groups. J. fmmunogenet. 14, 209-218. 1987. 7. Atkinson, J. P., Complement deficiency: Predisposing factor to auto-immune syndromes. C/in. hp. Rheumafol. 3, 95-101, 1989. 8. Wilson, W. A.. Perez, M. C., and Armatis, P. E., Partial C4A deficiency is associated with susceptibility to systemic lupus erythematosus in black Americans. Arthritis Rheum. 31, 1l711175, 1988. 9. Carroll, M. C., Campbell, R. D., Bentley, D. R., and Porter, R. R., A molecular map of the human major histocompatibility complex class III region linking complement genes C4, C2 and factor B. Nature (London) 307, 237-241, 1984. 10. Carroll, M. C., Belt, K. T., Palsdottir, A., and Porter, R. R., Structure and organization of the C4 genes. Philos. Trans. R. Sot. London B 306, 379-388, 1984. Il. Awdeh, Z. L., and Alper, C. A., Inherited structural polymorphism of the fourth component of human complement. Proc. Natl. Acad. Sci. USA 77, 3576-3580, 1980. 12. Mauff, G., Alper, C. A., Awdeh, Z., Batchelor, J. R., et al.. Statement on the nomenclature of human C4 allotypes. Immunobiology 164, 184-191, 1983. 13. Olsen, M. L., Goldstein, R., Arnett, F. C., Duvic, M., Pollack, M., and Reveille, J. D., C4A gene deletion and HLA associations in black Americans with systemic lupus erythematosus. Immunogenetics 30, 27-33. 1989. 14. Kahl, L. E., and Atkinson, J. P., Autoimmune aspects of complement deficiency. Clin. Aspects Autoimmun. 5, 8-20, 1988. 15. Awdeh, Z. L., Raum, D.. Yunis, E. J., and Alper, C. A., Extended HLA/complement allele haplotypes: Evidence for T/t - like complex in man. Proc. Natl. Acad. Sci. USA 80,25%263, 1983. 16. Schur, P. H., Marcus-Bagley, D., Awdeh, Z. L., Yunis, E. J., and Alper, C. A., The effect of ethnicity on major histocompatibility complex complement allotypes and extended haplotypes in patients with systemic lupus erythematosus. Arthritis Rheum. 33, 985-992, 1990. 17. Granados, J., Olivares, I., Melin, H., Andrade, F., and Alarcon-Segovia, D., Further evidence of the role of complement genotypes in the susceptibility to systemic lupus erythematosus (SLE) obtained from mexican family studies. Arthritis Rheum. JO(Suppl.), S-21, 1987. 18. Alarcon-Segovia, D., and Granados, J., Immunogenetics of SLE in Mexicans. In “Proceedings of the Second International Conference on Systemic Lupus Erythematosus (2630 Nov. 1989, Singapore),” pp. j&51, 1989. 19. Kemp, M. E., Atkinson, J. P., Skanes, V. M., Levine, R. P., and Chaplin, D. D., Deletion of C4A genes in patients with systemic lupus erythematosus. Arthriris Rheum. 30, 1015-1022, 1987.
C4A
DEFICIENCY
IN SLE
63
20. Campbell, R. D., Dunham, I., Kendall, E., and Sargent, C. A., Polymorphism of the human complement component C4. Exp. C/in. Immunogener. 7, 69-84, 1990. 21. Yu, C. Y., and Campbell, R. D., Definitive RFLPs to distinguish between the human complement C4A/C4B isotypes and the major Rogers/Chide determinants: Application to the study of C4 null alleles. Immunogenetics 25, 383-390, 1987. 22. Welch. T. R., Beischel, L. S., and Choi. E. M., Molecular genetics of C4B deliciency in IgA nephropathy. Hum, Immunol. 26, 353-363, 1989. 23. Partanen. J., and Campbell, R. D., Restriction fragment analysis of nondeleted complement C4 null genes suggests point mutations in C4A null allele, but gene conversions in C4B null alleles. Immunogenetics
30, 520-523,
1989.
24. Braun, L., Schneider, P. M., Giles, C. M., Bertrams, J., and Rittner, C.. Null alleles of human complement C4: Evidence for pseudogenes at the C4A locus and for gene conversion at the C4B locus. J. Exp. Med. 171, 129-140, 1990. 25. Woodrow, J. C., immunogenetics of systemic lupus erythematosus. J. Rheumarol. 15, 197-199. 1988. 26. Tan, E. M.. Cohen. A. S., Fries, J. F., Masi, A. T., McShane. D. J.. Rothtield, N. F., Schaller, J. G., Talal, N., and Winchester, R. J., The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 25, 1271-1277, 1982. 27. Tebib, J. G., Alcocer-Varela, J., Alarcon-Segovia, D., and Schur, P. H., Association between a T cell receptor restriction fragment length polymorphism and systemic lupus erythematosus. J. Clin. Invest. 86, 1961-1967, 1990. 28. Southern, E. M., Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. MO/. Biol. 98, 503-517, 1975. 29. Feinberg, A. P., and Vogelstein. B.. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13, 1983. 30. Feinberg, A. P.. and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137, 266-267, 1984. 31. Belt, K. T.. Yu, C. Y., Carroll, M. C.. and Porter, R. R.. Polymorphism of human complement component C4. fmmunogenetics 21, 173-180. 1985. 32. Belt, K. T.. Carroll, M. C., and Porter, R. R., The structural basis ofthe multiple forms of human complement component C4. Ce// 36, 907-914, 1984. 33. Yu, C. Y., Belt, K. T., Giles, C. M., Campbell, R. D., and Porter, R. R., Structural basis of the polymorphism of human complement components C4A and C4B: Gene size, reactivity, and antigenicity. EMBO 1. 5, 2873-2881, 1986. 34. Schneider, P. M., Carroll, M. C., Alper, C. A., Rittner, C.. Whitehead, A. S., Yunis, E. J., and Colten, H. R., Polymorphism of the human complement C4 and steroid 21-hydroxylase genes: Restriction fragment length polymorphisms revealing structural deletions. homoduplications and size variants. J. C&n. Invest. 78, 650-657. 1986. 35. Yamanda, M., Watanabe, A., Mimori. A., et a/., Lack of gene deletion for complement C4A deficiency in Japanese patients with systemic lupus erytbematosus. J. Rheumatol. 17, 1054-1057, 1990.
36. Hashimoto. M., Tsuda, H., Matsumoto, T., er al., HLA antigens associated with systemic lupus erythematosus in Japan. J. Rheumafol. 12, 919-923, 1985. 37. Tokunaga, K., Omoto, K., Akaza, T., et al., Haplotype study on C4 polymorphism in Japanese: Associations with MHC alleles, complotypes, and HLA-complement haplotypes. Immunogenetits 22, 359-365,
1985.
38. Goldstein, R., Amett, F. C., McLean, R. H., Bias, W. B., and Duvic, M., Molecular heterogeneity of complement component C4-null and 2l-hydroxylase genes in systemic lupus erythematosus. Arthritis
Rheum.
31, 736-744,
1988.
39. Whitehead, A. S., Goldberger, G., Woods, D. E., Markham, A. F., and Cohen, H. R., Use of a cDNA clone for the fourth component of human complement (C4) for analysis of a genetic deticiency of C4 in guinea pig. Proc. Natl. Acad. Sci. USA 80, 5387-5391, 1983. 40. Humphries, R. K., Ley, T. J., Anagnou, N. P., Baur, A. W.. and Nienhuis, A. W., B-39 Thalassemia gene: A premature termination codon causes beta mRNA deficiency without affecting cytoplasmic beta mRNA stability. Blood 64, 23-32, 1984.
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41. Takeshita. K., Forget, B. G.. Scarpa, A., and Benz, E. J.. Intranuclear defect in beta globin mRNA accumulation due to a premature translation termination codon. B/o& 64, 13-22. 1984. 42. Reith, W., Satola, S.. Sauchez. C. H., et ul.. Congenital Immunodeficiency with regulatory defect in MHC class 11 gene expression lacks a specific HLA-DR promoter binding protein RF-X. C&l 53, 897-906, 1988. 43. Crawford, D. R.. Mirault, M., Remy. M.. Zbinden. I.. and Cerutti, P. A.. Molecular defect in human Acatalasia libroblasts. Biochem. Biophys. Res. Common. 153, 59-66. 1988. 44. Davison, P. J.. Stalenhoef, A. F. H.. and Humphries. R. K., Apolipoprotein C II (ApoCII) gene expression defect in an individual with familial APOCII deficiency. Biochem. Biophys. Res. Commun. 148, 320-328, 1987. 45. Stoppa-Lyonnet, D.. Tosi, M.. Laurent, J., Sobel, A.. Lagrue, G.. and Meo. T., Altered Cl inhibitor genes in type 1 hereditary angioedema. N. Engl. J. Med. 317, 1-6. 1987. 46. Prockop, D. J.. and Kivirikko. K. I., Heritable diseases of collagen. N. Ens/. /. Med. 311, 376386, 1984. Received October 29, 1990; accepted with revision March 7. 1991
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
60,
55-64 (]%I)
DR3 and nonDR3 Associated Complement Component Deficiency in Systemic Lupus Erythematosus ASHOK KUMAR,PRITI
KUMAR,AND
C4A
PETER H. SCHUR'
Department of Rheumatologyllmmunology, 75 Francis Street, Brigham & Women’s Hospital, Harvard Medical School, Boston. Massachusetts 02115 The molecular basis of complement component C4A deficiency in white U.S. and Mexican patients with systemic lupus erythematosus (SLE) was studied. Genomic DNA from SLE patients and non-SLE controls was analyzed for restriction fragments using Hind111 and a 5’ C4 cDNA probe. C4A gene deletion was recognized by the loss of a IS-kb restriction fragment and the appearance of a 8.5-kb fragment. Thirty-two selected U.S. SLE patients, 7 nonSLE controls, and 11 Mexican SLE patients and 9 relatives were studied. The deletion was recognized in all of the 14 HLA-BS;DR3 SLE patients with a C4A protein deficiency. Two SLE patients with DR3 but without B8 also had this gene deletion. None of the 3 U.S. SLE nonDR3, C4A protein deficient patients nor 20 C4A protein deficient Mexican individuals (11 SLE patients and 9 relatives; none had B8 and/or DR3) showed this deletion. Thus the C4A gene deletion failed to account for the C4A protein deficiency in all of the nonDR3 Mexicans and in some U.S. SLE patients. To determine whether gene conversion at the C4A locus would encode a C4B-like protein and be responsible for the C4A protein deficiency tin nonDR3 patients), the C4d region of the gene was amplified by polymerase chain reaction and subjected to Nla IV digestion, and restriction fragment analysis was performed using a C4d region-specific probe. The resulting restriction fragment length polymorphism pattern revealed no changes in the isotype-specific region of the gene as characterized by C4A-specific 276and 191-bp fragments in DR3 or nonDR3 individuals. Thus, homoexpression of C4B at both loci was not responsible for C4A deficiency in nonDR3 SLE patients without C4A gene deletion. 0 1991 Academic press. Inc.
INTRODUCTION
There is a significant association between the deficiency of complement component C4 and systemic lupus erythematosus (SLE) (l-g). C4 genes are located on the short arm of the chromosome 6 within the major histocompatibility complex (MHC) between the HLA-D region and HLA-B locus (9). The two isotypes of C4, C4A, and C4B, are expressed at two loci which are approximately 10 kb apart (9, 10). C4A and C4B proteins are 99% homologous and are highly polymorphic (11) with more than 35 alleles, including null alleles (C4QO) at both loci (12). Null alleles, defined as the absence of C4A or C4B protein in the serum, have been shown to occur in patients with SLE (l-3). An increased frequency of C4A (protein) null alleles (C4AQO) has been demonstrated in patients with SLE of different ethnicity, including whites (l-3), blacks (3, 8, 13) Chinese, and Japanese (5, 6). Heterozygous C4AQO protein deficiency has been reported in 50-W% SLE patients and homozygosity for C4AQO in 10-U% of SLE patients (l-3, 7, 14). A ’ To whom correspondence
should be addressed. 55 0090-1229/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All tights of reproduction in any fomt reserved.
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gene dose effect has been observed with a relative risk for SLE of 3 with the heterozygous and 17 with the homozygous C4A null allele (3). C4AQO null alleles are strongly linked to one extended haplotype HLA-Al ;BS;DR3;SCOl in Caucasians, particularly those of English/Irish ancestry (15). In our studies. C4AQO was found to be increased only in SLE patients of English/Irish ancestry (16). Others have observed C4AQO in SLE in Orientals and Mexicans but not in association with DR3 (3, 5,6,8, 17, 18). In the majority of cases studied to date, C4A protein deficiency, associated with DR3, has been shown to result from an approximately 30-kb gene deletion involving most of the C4A gene and part of the 21 hydroxyiase gene in white SLE patients (19). Similarly, in black SLE patients with HLA-B44; -DR2 and -DR3 alleles, C4A deficiency has been associated with a large C4A, Cyp 21A gene deletion (13). The C4A null allele could also occur due to the expression of identical allotypes at the two C4 loci (20), a phenomenon termed as homoexpression (21). Homoexpression has recently been reported to occur in individuals with C4BQO but not in individuals with C4AQO (22-24). C4A protein differs from C4B by four amino acids due to five nucleotide changes in the C4d region of the gene. The C4A and C4B genes can be distinguished with Nla IV restriction fragment length polymorphism (RFLP) analysis (21). Mutations in this region of the C4A gene could result in a molecule indistinguishable from C4B and an apparent deficiency of C4A protein (21). The occurrence of the tight linkage disequilibrium between B8, DR3, and C4AQO (as part of the extended haplotype HLAAl;BS;DR3;SCOl) makes it difficult to determine the role of each allele, if any, as a primary susceptibility gene. Studies of an ethnic group in which this haplotype occurs normally in low frequency may help in defining the individual alleles in SLE (25). In Mexican mestizos C4AQO occurs frequently but independently of the HLA haplotype B8;DR3 which is rare (~5%) while SLE is particularly frequent (17, 18). The present study defines the role of the C4A gene with particular emphasis on the C4d region of the gene, in C4A protein deficiency in SLE patients from the United States and Mexico. MATERIALS
AND METHODS
Patients. Ail of the patients with SLE included in this study met the American Rheumatism Association classification criteria for the diagnosis of SLE (26). Patients referred to as U.S. SLE were white SLE patients followed at the lupus clinic of this hospital. Mexican SLE patients were followed at Instituto National de la Nutrition Salvador Zubiran, Mexico. SLE patients and non-SLE controls were selected on the basis of presence or absence of a C4A null allele with or without the B8/DR3 phenotype as shown in Table 1. Individuals with a B8, DR3, and C4A null allele on at least one of the chromosomes were grouped as B8;DR3;C4AQO, those with a C4A null allele on both the chromosomes as B8;DR3$4AQOQO. The prefix non was used to signify the absence of the particular allele. HLA typing and C4 allotyping. HLA (A, B, DR) and C4 allotyping was per-
C4A
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formed as previously described (16). C4A allotypes including C4AQO were confirmed by family studies. Genomic DNA. Genomic DNA was isolated from EBV-transformed B lymphocytes or from peripheral blood lymphocytes isolated from fresh blood as described before (27). RFLP analysis. RFLP analysis of genomic DNA was performed by Southern blotting (28). Ten micrograms of genomic DNA was digested with Hind111 (New England Biolabs) as per the instructions of the manufacturer. The samples were electrophoresed on an 0.8% agarose gel in TAE buffer (40 mM Tris-acetate, 2 mM EDTA) for 16-20 hr at 40 V. For molecular weight markers, 20 ng of h DNA digested with Hind111 was used. DNA was transferred to the Gene screen plus (DuPont) membrane by the capillary blot method as per the instructions of the manufacturer. The membrane was air dried at room temperature and transferred to a prehybridization solution (50% formamide, 10% dextran sulfate, 2% SDS, 100 t&ml denatured and fragmented salmon sperm DNA, and 1 M sodium chloride) at 42°C for 6-12 hr. A 5’ C4 cDNA probe was used after labeling with 32P-dCTP by the random primer labeling method (29,30). A DNA (250 pg) was also included in the labeling reaction mixture. The membrane was incubated overnight at 42°C in prehybridization solution having the radiolabeled probe. The membrane was washed three times with 0.1 x SSC (15 mM NaCl, 1.5 mM sodium citrate) with 2% SDS at 65°C and authoradiographed at -80°C. Polymerax chain reaction (PCR). For amplification of the C4d region of the C4 gene, 1 p.g of genomic DNA was subjected to PCR. According to the published sequence of the C4 gene (31), oligonucleotide primers A S’-TGCGGATCCAGCAGTTTCGGAAG-3’ and B 5’-ATAGGATCCTAAGGTCCCCTGGGCCT-3’ were used to selectively amplify the 926-bp C4d gene fragment. The optimal PCR conditions were standardized and the reaction was carried out in 10 mM TrisHCl, pH 8.8, 50 mM KCI, 1.25 mM MgCl,, 0.01% gelatin, 2 units of Taq polymerase (Cetus Corp.), 200 mM dNTPs, and 0.2 t~J4 of each primer. Twenty-five thermal cycles, each of denaturation (1 min, 94”C), annealing (2 min, SYC), and extension (2 min. 72”(Z), were carried out using Tempcyler (Coy Corp.). The amplified product was electrophoresed on an 1.5% agarose gel. DNA was recovered from the gel by using the GeneClean kit (Bio 101 Inc.) Probes. A 500-bp cDNA fragment specific for the 5’ region of C4 genes was derived from the BamHIIKpnI digestion of the full-length C4 cDNA in PAT-A (32). The Pb probe derived as a BamHI genomic subclone of C4 for the C4d region of the C4 gene was 927 bp (33). Both probes were kindly provided by Dr. M. C. Carroll, Harvard Medical School, Boston. Another probe specific for the C4d region of the C4 gene was prepared by PCR as described above. The amplified material used as the C4d probe hybridized with the full-length C4 cDNA probe as well as with the Pb probe described above. RFLP analysis of C4d amplified product. Ten nanograms of PCR amplified C4d DNA was subjected to Nla IV (New England Biolabs) digestion as per the manufacturer’s instructions. DNA was electrophoresed on a 12% polyacrylamide gel in TAE buffer along with 20 ng of HaeIII-digested +X 174 DNA as a molecular
58
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weight marker. DNA was transferred to a Gene screen plus membrane SSC and probed with the C4d probe as described above.
in IO x
RESULTS
Thirty-two selected U.S. SLE patients were included in this study and grouped as shown in Table 1. Two of the patients in this group were homozygous for the C4A null allele (C4AQOQO). Twenty patients in this group were HLA-DR3 and 15 had the HLA-B8 phenotype. Fourteen of the patients had the extended haplotypeB8;DR3;C4AQO. Three patients had a C4A protein deficiency without DR3. On the other hand, all of the 11 Mexican SLE patients studied were nonBSmonDR3 and 6 of them expressed the C4A null allele. Nine individuals who were relatives of the Mexican SLE patients were also studied. Six of the individuals expressed the C4A null allele, including one homozygous for the C4A null allele and others normal for C4A (nonC4AQO). Genomic DNA samples of all these individuals were analyzed for the C4A gene by Southern blot analysis using the Hind111 enzyme and the 5’ C4 cDNA probe (Table 1, Fig. 1). Individuals with only a 8.5-kb restriction fragment were grouped as pattern A, with 26-, 15-, and 8.5kb bands as pattern B and with 26 and 15 kb as pattern C. Analysis of the data revealed that pattern A was observed in those patients who were homozygous for the C4A null allele (C4AQOQO) and were B8;DR3. Individuals who were DR3 and heterozygous for the C4A null allele (C4AQO) showed the pattern B irrespective of the presence or absence of HLA-B8. Patients possessing the C4A null allele and nonDR3;B8 or TABLE 1 RESTRICTION FRAGMENTLENGTH POLYMORPHISM PATTERN OF A C4A GENE IN U.S. AND MEXICAN SLE PATIENTSAND~ONTROLS Group U.S. SLE patients B8;DR3$4AQOQO B8;DR3;C4AQO B8;nonDR3$4AQO nonBS;DR3$4AQO nonB8;nonDR3$4AQO nonB8;DR3;nonC4AQO nonB8;nonDR3;nonC4AQO U.S. non-SLE persons B8;DR3$4AQOQO Mexican SLE patients nonBS;nonDR3$4AQOQO nonBS;nonDR3$4AQO nonBl;nonDR3;nonC4AQO Relatives of Mexican SLE patients nonBl;nonDR3$4AQOQO nonBll;nonDR3$4AQO nonB8;nonDR3;nonC4AQO
NO.
Pattern”
2 12 I 2 2 4 Y
A B C B C C C
7
A
I 5 5
C C C
1 5 3
C C C
a Using the Hind111 enzyme and a 5’ C4 cDNA probe. Patterns A, B, and C describe restriction fragments: A = 8.5 kb; B = 26, IS, 8.5 kb; C = 26, 15 kb.
C4A DEFICIENCY
59
IN SLE
M Ae
C
23.1 Kb+ 9.4 Kb-t 6.6 Kb-t FIG. 1. RFLP pattern of genomic DNA digested with the Hind111 enzyme and probed with a 5’ C4 cDNA. DNA from SLE patients with a homozygous C4A null allele (B8;DR3;C4AQOQO) (pattern A), heterozygous C4A null allele (B8;DR3$4AQO) (pattern B), and normal C4A allele (nonBS;nonDR3;nonC4AQO) (pattern C) subjected to Southern blot analysis. HindIII-digested A DNA was used as marker (M).
nonDR3;nonBS showed pattern C as did the individuals who had normal C4A protein (non C4AQO). All of the Mexican patients and their relatives presented the restriction fragment pattern C whether they were homozygous or heterozygous for the C4A null allele or possessed normal C4A protein. In order to further analyze the C4A null alleles, the C4d region of the gene was amplified by PCR using the primers listed under Materials and Methods. The amplification reaction product of 926 bp was seen to hybridize with the C4dspecific probe (data not shown). RFLP analysis of the C4d DNA fragment using the Nla IV enzyme and C4d probe gave restriction fragments of 467,276, and 191 bp for a C4A normal individual (Fig. 2, Table 2). DR3 individuals with a homozygous C4A deficiency (C4AQOQO) showed only a 467-bp fragment while a control C4BQOQO showed 267- and 191-bp fragments. The pattern obtained in the case of heterozygous C4AQO U.S. SLE patients and homozygous or heterozygous C4A null Mexican patients or their relatives was similar to C4A normal individuals (Fig. 2, Table 2).
FIG. 2. RFLP analysis of PCR-amplified DNA from the C4d region of the C4 gene using a Nla IV enzyme and a C4d-specific probe. PCR-amplified C4d DNA from (A) nonB8;nonDR3;nonUQO, (B) nonB8;nonDR3;C4BQOQO (non-SLE control), (C) B8;DR3;C4AQOQO, and (D) nonB8;nonDR3;C4AQOQO individuals were subjected to Southern blot analysis after Ma IV digestion and polyacrylamide gel electrophotesis. +x174 DNA digested with H&II enzyme was used as marker.
60
KUMAR.
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AND
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2
RESTRICTION FRAGMENT PATTERN OFTHE C4d REGION WITH Nla IV Has2 pair fragment No. United States SLE B8;DR3;C4AQOQO B8;DR3;C4AQO nonB8;DR3$4AQO nonB&nonDR3:C4AQO nonB8;DR3;nonC4AQO nonBS;nonDR3 :nonC4AQO Mexican SLE nonB8;nonDR3$4AQOQO nonBl;nonDR3;C4AQO nonB8;nonDR3;nonC4AQO Controls B8;DR3$4AQOQO nonB8;nonDR3$4BQOQO nonB8;DR3;C4BQOQO
C4A
specific
2 2 3
276.191 276.191 276,191 276.191 276.191
467 467 467 467 467 467
2 6 3
276.191 276,191 276.191
467 467 467
-,-
467 -
2
276,191 276,191
DISCUSSION In our study of white U.S. SLE patients, the loss of 26- and 15kb restriction fragments with the appearance of a 8.5-kb band (pattern A) was the marker of homozygous C4A gene deletion while the presence of all three fragments (pattern B) was found in individuals with the heterozygous C4A gene deletion. The presence of both the 26- and 15-kb fragments (pattern C) was found in individuals with an intact C4A gene (19). Gene deletion, as evident by pattern A or B, was observed whenever the C4A null allele was associated with DR3, presumably as part of the extended haplotype BS;DR3;SCOl (15, 34). In two other DR3 patients who were nonB8, a similar gene deletion pattern was observed. Thus the presence or absence of the B8 allele appeared to demonstrate only a limited association with the C4A gene deletion and susceptibility to SLE. The C4A gene deletion was observed only when the C4A null allele occurred with DR3. Others have interpreted the C4A gene deletion linked to the nonB8 allele to loss of the B8 allele in a recombinational event during the evolution of the haplotype B8:DR3 (19). In the case of the non C4AQO allele, either in the presence or in the absence of DR3 or B8, gene deletion was not observed. However, in the case of the nonBS;nonDR3$4AQO, the C4A gene was intact (Table 1). As only two U.S. SLE patients were available in the nonBS;nonDR3$4AQO category, we studied Mexican patients with the nonB8;nonDR3$4AQO phenotype. Restriction fragment analysis in all these individuals revealed an intact C4A gene (Table l), thus strengthening the finding that the C4A gene deletion is linked to the presence of the DR3 allele. Recently, the lack of a gene deletion causing complement C4A deficiency in Japanese SLE patients has been documented (35) which could also be explained by the rarity of the HLA-DR3 allele/haplotype in the Japanese population (36, 37).
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61
In the case of nonDR3 individuals, C4AQO appears to be due to a nonfunctional gene which could occur due to the expression of identical allotypes at the two C4 loci (homoexpression) leading to synthesis of a C4B-like protein at locus A. Such a phenomenon has been reported to occur for C4BQO alleles leading to the expression of the C4A protein at locus B in IgA nephropathy in whites (22) and other studies from Germany (23) and Finland (24), presumably from a nondisease population. Partanen and Campbell have speculated that the unidirectional gene conversion observed may be due to the single, ethnically homogeneous population studied (23). In SLE, where the association of the C4A null allele has been rather strong (3,7, 14, 17), the molecular basis of C4A deficiency unaccounted by gene deletion remains unclear. In the nondisease population studied by others in Finland (24) and Germany (23), the C4A gene has been termed as a pseudogene due to possible point mutations or to small deletion or insertions (23,24) which remain unidentified. To study whether the C4A nondeleted gene in SLE patients from the United States (whites) and Mexico undergoes gene conversion to express C4B at Iocus A, the C4d region of the gene was amplified by PCR and subjected to Nla IV restriction fragment analysis for the patients listed in Table 2. The analysis revealed that all the nonDR3 individuals with no major deletions of locus A gave the usual fragments of 276 and 191 bp specific for the C4A isotype in U.S. as well as Mexican SLE patients. Both the U.S. SLE nonDR3 individuals were heterozygous for the C4AQO allele and therefore the pattern with no markable difference in the intensity of restriction fragments (data not shown) (Table 2) was uninformative regarding the status of the C4A gene. However, two of the Mexican nonDR3 individuals included in this study were homozygous for the C4A null allele (C4AQOQO) (Table 2). The data clearly indicated a normal C4d region gene pattern viz. 276- and 191-bp fragments and a 476-bp fragment for both of these people. This implies that in nonDR3 individuals who have an intact C4A gene but express the C4A null allele, a gene conversion event changing C4A to C4B has not occurred, thus showing that the defect leading to the nonexpression of the C4A protein is not due to changes in the isotype-specific region of the gene. In nonDR3 SLE patients the gene defect leading to the absence of C4A protein may be due to a defect at the level of transcription or translation of the C4A gene (38). In guinea pigs, a post-translational defect in the processing of C4 precursor RNA to mature mRNA has been suggested (39). Studies are in progress to identify the possible mutations in the C4A gene leading to protein deficiency as identified in p thalassemia (40, 41), severe combined immunodeficiency (42), acatalasia (43), apolipoprotein CII deficiency (44), hereditary angioedema (45), and osteogenesis imperfecta (46). However, it will be desirable to study more U.S., other Caucasians and Mexican SLE patients, and normal controls with nonDR3 homozygous C4A deficiency to confirm the findings to extrapolate the observation to populations. ACKNOWLEDGMENTS This work was partially supported by grants from USPHS AR35907, AR 07530, and the Lupus Foundation of America. A.K. was the recipient of the Fogarty International Postdoctoral Award. Authors are thankful to Drs. C. A. Alper and 2. Awdeh for providing DNA samples of nonSLE controls. Thanks to Dr. Alarcon-Segovia for providing DNA samples of Mexican SLE patients and
62
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AND SCHUR
their relatives. Thanks are due to Dr. M. V. N. Rao for cell culture and Ms. Joanne Davis for her secretarial expertise.
REFERENCES I. Fielder, A. H., Walport, M. J., Batchelor, J. R., Ryes, R. 1.. Black, C. M., Dodi, 1. A., and Hughes, Cl. R., Family study of the major histocompatibility complex in patients with systemic lupus erythematosus: Importance of null alleles of C4A and C4B in determining disease susceptibility. Br. Med. J. 286, 425-428, 1983. 2. Reveille, J. D., Amett, F. C.. Wilson, R. W., Bias, W. B.. and McLean, R. H., Null alleles of the fourth component of complement and HLA haplotypes in familial systemic lupus erythematosus. Immunogenetics 21, 299-31 I, 1985. 3. Howard, P. F., Hochberg, M. C., Bias, W. B., Amett, F. C., and McLean, R. H., Relationship between C4 null genes, HLA-D region antigens and genetic susceptibility to systemic lupus erythematosus in Caucasian and black Americans. Am J. Med. 81, 187-193. 1986. 4. Hauptmann, G., Goetz. J., Uring-Lambert, B., and Grosshans, E., Component deficiencies. 2. The fourth component. Prog. Allergy 39, 232-249, 1986. 5. Hawkins, B. R., Wong, K. L.. Wong, R. W., Chan, K. H.. Dunckley, H., and Sejeantson. S. W., Strong association between the major histocompatibility complex and systemic lupus erythematosus in southern Chinese. J. Rheumatol. 14, 1128-l 131, 1987. 6. Dunckley, H.. Gatenby, P. A., Hawkins, B., Naito, S.. and Sejeantson, S. W., Deficiency of C4A is a genetic determinant of systemic lupus erythematosus in three ethnic groups. J. fmmunogenet. 14, 209-218. 1987. 7. Atkinson, J. P., Complement deficiency: Predisposing factor to auto-immune syndromes. C/in. hp. Rheumafol. 3, 95-101, 1989. 8. Wilson, W. A.. Perez, M. C., and Armatis, P. E., Partial C4A deficiency is associated with susceptibility to systemic lupus erythematosus in black Americans. Arthritis Rheum. 31, 1l711175, 1988. 9. Carroll, M. C., Campbell, R. D., Bentley, D. R., and Porter, R. R., A molecular map of the human major histocompatibility complex class III region linking complement genes C4, C2 and factor B. Nature (London) 307, 237-241, 1984. 10. Carroll, M. C., Belt, K. T., Palsdottir, A., and Porter, R. R., Structure and organization of the C4 genes. Philos. Trans. R. Sot. London B 306, 379-388, 1984. Il. Awdeh, Z. L., and Alper, C. A., Inherited structural polymorphism of the fourth component of human complement. Proc. Natl. Acad. Sci. USA 77, 3576-3580, 1980. 12. Mauff, G., Alper, C. A., Awdeh, Z., Batchelor, J. R., et al.. Statement on the nomenclature of human C4 allotypes. Immunobiology 164, 184-191, 1983. 13. Olsen, M. L., Goldstein, R., Arnett, F. C., Duvic, M., Pollack, M., and Reveille, J. D., C4A gene deletion and HLA associations in black Americans with systemic lupus erythematosus. Immunogenetics 30, 27-33. 1989. 14. Kahl, L. E., and Atkinson, J. P., Autoimmune aspects of complement deficiency. Clin. Aspects Autoimmun. 5, 8-20, 1988. 15. Awdeh, Z. L., Raum, D.. Yunis, E. J., and Alper, C. A., Extended HLA/complement allele haplotypes: Evidence for T/t - like complex in man. Proc. Natl. Acad. Sci. USA 80,25%263, 1983. 16. Schur, P. H., Marcus-Bagley, D., Awdeh, Z. L., Yunis, E. J., and Alper, C. A., The effect of ethnicity on major histocompatibility complex complement allotypes and extended haplotypes in patients with systemic lupus erythematosus. Arthritis Rheum. 33, 985-992, 1990. 17. Granados, J., Olivares, I., Melin, H., Andrade, F., and Alarcon-Segovia, D., Further evidence of the role of complement genotypes in the susceptibility to systemic lupus erythematosus (SLE) obtained from mexican family studies. Arthritis Rheum. JO(Suppl.), S-21, 1987. 18. Alarcon-Segovia, D., and Granados, J., Immunogenetics of SLE in Mexicans. In “Proceedings of the Second International Conference on Systemic Lupus Erythematosus (2630 Nov. 1989, Singapore),” pp. j&51, 1989. 19. Kemp, M. E., Atkinson, J. P., Skanes, V. M., Levine, R. P., and Chaplin, D. D., Deletion of C4A genes in patients with systemic lupus erythematosus. Arthriris Rheum. 30, 1015-1022, 1987.
C4A
DEFICIENCY
IN SLE
63
20. Campbell, R. D., Dunham, I., Kendall, E., and Sargent, C. A., Polymorphism of the human complement component C4. Exp. C/in. Immunogener. 7, 69-84, 1990. 21. Yu, C. Y., and Campbell, R. D., Definitive RFLPs to distinguish between the human complement C4A/C4B isotypes and the major Rogers/Chide determinants: Application to the study of C4 null alleles. Immunogenetics 25, 383-390, 1987. 22. Welch. T. R., Beischel, L. S., and Choi. E. M., Molecular genetics of C4B deliciency in IgA nephropathy. Hum, Immunol. 26, 353-363, 1989. 23. Partanen. J., and Campbell, R. D., Restriction fragment analysis of nondeleted complement C4 null genes suggests point mutations in C4A null allele, but gene conversions in C4B null alleles. Immunogenetics
30, 520-523,
1989.
24. Braun, L., Schneider, P. M., Giles, C. M., Bertrams, J., and Rittner, C.. Null alleles of human complement C4: Evidence for pseudogenes at the C4A locus and for gene conversion at the C4B locus. J. Exp. Med. 171, 129-140, 1990. 25. Woodrow, J. C., immunogenetics of systemic lupus erythematosus. J. Rheumarol. 15, 197-199. 1988. 26. Tan, E. M.. Cohen. A. S., Fries, J. F., Masi, A. T., McShane. D. J.. Rothtield, N. F., Schaller, J. G., Talal, N., and Winchester, R. J., The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 25, 1271-1277, 1982. 27. Tebib, J. G., Alcocer-Varela, J., Alarcon-Segovia, D., and Schur, P. H., Association between a T cell receptor restriction fragment length polymorphism and systemic lupus erythematosus. J. Clin. Invest. 86, 1961-1967, 1990. 28. Southern, E. M., Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. MO/. Biol. 98, 503-517, 1975. 29. Feinberg, A. P., and Vogelstein. B.. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13, 1983. 30. Feinberg, A. P.. and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137, 266-267, 1984. 31. Belt, K. T.. Yu, C. Y., Carroll, M. C.. and Porter, R. R.. Polymorphism of human complement component C4. fmmunogenetics 21, 173-180. 1985. 32. Belt, K. T.. Carroll, M. C., and Porter, R. R., The structural basis ofthe multiple forms of human complement component C4. Ce// 36, 907-914, 1984. 33. Yu, C. Y., Belt, K. T., Giles, C. M., Campbell, R. D., and Porter, R. R., Structural basis of the polymorphism of human complement components C4A and C4B: Gene size, reactivity, and antigenicity. EMBO 1. 5, 2873-2881, 1986. 34. Schneider, P. M., Carroll, M. C., Alper, C. A., Rittner, C.. Whitehead, A. S., Yunis, E. J., and Colten, H. R., Polymorphism of the human complement C4 and steroid 21-hydroxylase genes: Restriction fragment length polymorphisms revealing structural deletions. homoduplications and size variants. J. C&n. Invest. 78, 650-657. 1986. 35. Yamanda, M., Watanabe, A., Mimori. A., et a/., Lack of gene deletion for complement C4A deficiency in Japanese patients with systemic lupus erytbematosus. J. Rheumatol. 17, 1054-1057, 1990.
36. Hashimoto. M., Tsuda, H., Matsumoto, T., er al., HLA antigens associated with systemic lupus erythematosus in Japan. J. Rheumafol. 12, 919-923, 1985. 37. Tokunaga, K., Omoto, K., Akaza, T., et al., Haplotype study on C4 polymorphism in Japanese: Associations with MHC alleles, complotypes, and HLA-complement haplotypes. Immunogenetits 22, 359-365,
1985.
38. Goldstein, R., Amett, F. C., McLean, R. H., Bias, W. B., and Duvic, M., Molecular heterogeneity of complement component C4-null and 2l-hydroxylase genes in systemic lupus erythematosus. Arthritis
Rheum.
31, 736-744,
1988.
39. Whitehead, A. S., Goldberger, G., Woods, D. E., Markham, A. F., and Cohen, H. R., Use of a cDNA clone for the fourth component of human complement (C4) for analysis of a genetic deticiency of C4 in guinea pig. Proc. Natl. Acad. Sci. USA 80, 5387-5391, 1983. 40. Humphries, R. K., Ley, T. J., Anagnou, N. P., Baur, A. W.. and Nienhuis, A. W., B-39 Thalassemia gene: A premature termination codon causes beta mRNA deficiency without affecting cytoplasmic beta mRNA stability. Blood 64, 23-32, 1984.
64
KUMAR,
KUMAR,
AND
SCHUR
41. Takeshita. K., Forget, B. G.. Scarpa, A., and Benz, E. J.. Intranuclear defect in beta globin mRNA accumulation due to a premature translation termination codon. B/o& 64, 13-22. 1984. 42. Reith, W., Satola, S.. Sauchez. C. H., et ul.. Congenital Immunodeficiency with regulatory defect in MHC class 11 gene expression lacks a specific HLA-DR promoter binding protein RF-X. C&l 53, 897-906, 1988. 43. Crawford, D. R.. Mirault, M., Remy. M.. Zbinden. I.. and Cerutti, P. A.. Molecular defect in human Acatalasia libroblasts. Biochem. Biophys. Res. Common. 153, 59-66. 1988. 44. Davison, P. J.. Stalenhoef, A. F. H.. and Humphries. R. K., Apolipoprotein C II (ApoCII) gene expression defect in an individual with familial APOCII deficiency. Biochem. Biophys. Res. Commun. 148, 320-328, 1987. 45. Stoppa-Lyonnet, D.. Tosi, M.. Laurent, J., Sobel, A.. Lagrue, G.. and Meo. T., Altered Cl inhibitor genes in type 1 hereditary angioedema. N. Engl. J. Med. 317, 1-6. 1987. 46. Prockop, D. J.. and Kivirikko. K. I., Heritable diseases of collagen. N. Ens/. /. Med. 311, 376386, 1984. Received October 29, 1990; accepted with revision March 7. 1991