Proc. Nati. Acad. Sci. USA Vol. 86, pp. 8015-8019, October 1989 Immunology Individuals with IgA deficiency and common variable immunodeficiency share polymorphisms of major histocompatibility complex class III genes (B-cell differentiation/complement component C2/complement component C4/steroid 21-hydroxylase/HLA) F. M. SCHAFFER*, J. PALERMOSt, Z. B. ZHUt, B. 0. BARGER*, M. D. COOPER*§, AND J. E. VOLANAKISt¶ *Division of Developmental and Clinical Immunology, Departments of Pediatrics, Medicine, Microbiology, and Pathology, and The Comprehensive Cancer Center, tDivision of Clinical Immunology and Rheumatology, Department of Medicine, *Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294; and the §Howard Hughes Medical Institute, Birmingham, AL 35294 Contributed by M. D. Cooper, July 11, 1989

ABSTRACT IgA deficiency and common variable immu- to B-cell stage in the X chromosome-linked form of congen- nodeficiency are heritable disorders that can occur within the ital agammaglobulinemia (3, 6). same family. Both immunodeficiencies are characterized by The cause ofthe variable B-cell arrests in CVID and IgA-D arrests in B-cell differentiation that vary in the extent of the is unknown. B cells from individuals with CVID are capable immunoglobulin isotypes involved. A high frequency of major of plasma-cell differentiation, as can be demonstrated after histocompatibility complex supratypes associated with a null their infection with Epstein-Barr virus (7). Because T cells allele of the gene encoding the C4A isotype of complement are needed to help B cells respond with antibody production component C4 has been observed in IgA-deficient individuals. to most antigens, a defect of the T cells has long been In search of a genetic linkage between the two immunodefi- suspected in CVID and IgA-D (8-10). Although T cells are ciencies, we examined the major histocompatibility complex usually present in normal numbers, analysis of affected (MHC) class HI genes encoding complement components C2, individuals sometimes suggests either a deficit in helper C4A, and C4B and steroid 21-hydroxylase in addition to the T-cell function or excessive suppressor activity (8-10). Other HLA serotypes in individuals with either common variable studies suggest that T cells from most of these patients are immunodeficiency or IgA deficiency. Twelve of 19 patients with capable of providing all the necessary elements for B-cell common variable immunodeficiency (63%, P < 0.001) and 9 of growth and differentiation (11, 12). 16 patients with IgA deficiency (56%, P < 0.01) had rare C2 While CVID and IgA-D may represent multifactorial dis- alleles and/or C4A and 21-hydroxylase A deletions, whereas eases, susceptibility appears to be heritable, with family these gene features were seen in only 5 of34 healthy individuals studies suggesting an autosomal recessive mode of inheri- (15%) in the control group. Nine of 11 patients with C4A tance (13, 14). CVID and IgA-D have also been observed in deletion had an HLA haplotype consistent with the MHC immediate relatives (13), suggesting a common gene defect. supratype HLA-Al, Cw7, B8, C4AQO, C4B1, BfS, DR3 pre- In search of a shared genetic basis for CVID and IgA-D, we viously found to be associated with IgA deficiency. The data have turned our attention to the major histocompatibility support the hypothesis that common variable immunodefi- complex (MHC) of genes for two main reasons. First, this ciency and IgA deficiency are related disorders, susceptibility region of chromosome 6 encodes a variety of molecules to which is determined by a gene(s) within or near the MHC involved in antigen presentation and T-cell/B-cell interac- class III gene region on chromosome 6. tions (15). Second, studies of individuals with IgA-D have revealed a relatively high incidence of certain HLA haplo- Common variable immunodeficiency (CVID) and selective types (16, 17) and MHC supratypes, the most consistent IgA deficiency (IgA-D) are diagnostic classifications applied feature of which is the presence of null alleles of the gene to groups of immunodeficient individuals exhibiting de- encoding the A isotype of complement component C4 (C4A) creased levels either ofmultiple immunoglobulin classes or of (14). Our present studies reveal that individuals with CVID IgA alone (1). These are not homogeneous entities, but rather and IgA-D have in common a very high incidence of partic- represent opposite ends of a spectrum of immunoglobulin ular restriction fragment length polymorphisms (RFLPs) or deficiencies. Individuals diagnosed as having CVID are fre- deletions of MHC class III genes. quently panhypogammaglobulinemic but may produce sub- stantial levels of IgM. Conversely, individuals with IgA-D may also be deficient in one or more IgG subclasses (2). MATERIALS AND METHODS CVID, typically manifested by recurrent bacterial infections, Participants. All individuals in the is usually recognized in adolescents or young adults and thus Study participating is sometimes called the late-onset form of hypogammaglob- study were Caucasian. Thirty-four healthy individuals (17 ulinemia. In most individuals with CVID, B cells are pro- male and 17 female) randomly selected for study had normal duced in normal numbers but they fail to undergo differen- serum immunoglobulin levels. The 19 CVID patients (12 tiation into antibody-secreting plasma cells (3, 4). Similarly, female and 7 male) and 16 patients with selective IgA-D (6 individuals with IgA-D have normal numbers of IgA-bearing female and 10 male) were selected from a group of immuno- B cells, but these are relatively immature and fail to undergo deficient patients followed in our immunology clinic. In- terminal differentiation into IgA-secreting plasma cells (5). formed consent was obtained from all participants. The arrests in B-cell differentiation seen in CVID and IgA-D are thus similar and contrast with the arrest seen at the pre-B- Abbreviations: MHC, major histocompatibility complex; C2, second component of complement; C4, fourth component of complement; 21-OHase, steroid 21-hydroxylase; IgA-D, IgA deficiency; CVID, The publication costs of this article were defrayed in part by page charge common variable immunodeficiency; RFLP, restriction fragment payment. This article must therefore be hereby marked "advertisement" length polymorphism. in accordance with 18 U.S.C. §1734 solely to indicate this fact. $To whom reprint requests should be addressed.

8015 Downloaded by guest on September 30, 2021 8016 Immunology: Schaffer et al. Proc. Natl. Acad. Sci. USA 86 (1989)

0 5 10 15 20 kb 5' 3'5' C2 }-EU A 6.6* Bam Hi 3.45-32* I 4.4 2.2 1 Sst I 1 2.75-2.5*1 C H 6.6 *----H B 1 2.1 1 4.5 J Taq I

FIG. 1. Schematic representation ofthe C2 gene RFLPs. kb, Kilobases. A partial restriction map ofthe C2 gene (23) is shown with the relative positions and sizes ofpolymorphic fragments indicated by asterisks. Probes A and C are the 5' 2-kbp and 3' 1-kbp EcoRI fragments, respectively, of a full-length cDNA for C2 (22). Probe B is a 300-bp DNA fragment derived from the 5' region of a C2 genomic clone (23). Probe A detects two polymorphisms: (i) A BamHI multiallelic polymorphism with five classes of size variants exhibiting fragments of 3.45, 3.40, 3.35, 3.30, and 3.20 kbp. This polymorphism is also detected by using Sst I-digested DNA and probe B. The Sst I polymorphic fragments have corresponding lengths of 2.75, 2.70, 2.65, 2.60, and 2.50 kbp. (ii) A second BamHI polymorphism characterized by the loss of a 4.4-kbp fragment and the appearance of a 6.6-kbp fragment. Probe C detects a Taq I polymorphism mapping to the 3' end of the C2 gene and characterized by the loss of a 4.5-kbp fragment and the appearance of a 6.6-kbp fragment.

Immunofluorescence Studies. Mononuclear cells were iso- B gene (24). Both were gifts from David Chaplin (Washington lated from heparinized blood samples by centrifugation over University, Saint Louis). The purified DNA probes were Ficoll/Hypaque density gradients. B and T lymphocytes radiolabeled with [32P]dCTP (Amersham) by the random were enumerated by indirect immunofluorescence using primer method (25). monoclonal antibodies and a FACS Scan II flow cytometry HLA Typing. Typing antisera obtained from the National unit (Beckton Dickinson). The anti-B-cell monoclonal anti- Institutes ofHealth, local screening, and commercial sources bodies were specific for cell surface IgM, IgD, IgA, HLA- were utilized for HLA-A, B, DQ, and DR typing by the DR, HLA-DQ, and the HB-2 antigen (18). The T-cell mono- microdroplet lymphocytotoxicity test (26). clonal antibodies were against the CD3, CD4, and CD8 Statistical Analysis. Differences in the frequency of study antigens. Serum immunoglobulin concentrations were deter- parameters between the control and patient populations were mined by rate nephelometry. assessed by the x2 test with correction for continuity, and Southern Blot Analysis. Genomic DNA was extracted from differences between mean values were assessed by the non- the buffy coat fraction of blood samples collected in EDTA paired Student t test. (19). Ten micrograms of genomic DNA was digested with restriction endonucleases (Taq I, BamHI, HindIII, or Sst I) under conditions suggested by the suppliers. DNA restriction RESULTS fragments were separated by electrophoresis through 0.8% Study Population. Immunological parameters of the 19 agarose gels. Phage A DNA digested with HindIII or EcoRI individuals with CVID and 16 individuals with IgA-D are plus HindIII was used as molecular size markers. After summarized in Table 1. None of the patients was lym- electrophoresis, DNA fragments were transferred to nitro- phopenic. All had normal percentages of T cells as indicated cellulose membranes by the method of Southern (20). Pre- by values for the CD3+, CD4+, and CD8+ cells, although the hybridization and hybridization of the membranes was car- CD4/CD8 ratio was lower in the CVID group. In addition, a ried out by standard procedures (21). After hybridization the normal distribution of B lymphocytes was indicated by the membranes were washed under high-stringency conditions percentages of HB2+, IgM+, IgD+, and IgA+ cells. B cells and exposed to x-ray film. expressed the MHC class II antigens DR and DQ at compa- Probes. The 2961-base-pair (bp) insert of a full-length rable levels in the 8 IgA-D, 8 CVID, and 6 normal individuals second component of complement (C2) cDNA clone examined (data not shown). The data in Table 1 thus support (C2HL5-3) isolated from a Agtll human liver library (22) was the conclusion that both CVID and IgA-D are characterized digested with EcoRP to yield a 2-kbp 5' fragment (probe A) by a normal number of B cells with arrested B-cell differen- and a 1-kbp 3' fragment (probe C) (Fig. 1), which were tiation reflected by low serum immunoglobulin concentra- isolated by electrophoresis in low-melting agarose. A 0.3-kbp tions (3, 4). C2 DNA probe (probe B) from the 5' end of the C2 gene (23) C2 Gene RFLPs. Three RFLPs have been described for the was provided by Duncan Campbell (Oxford University). A C2 gene digested with the restriction endonucleases Sst I 0.8-kbp Kpn I/Pst I fragment of the C4A gene was used to (23), BamHI (27), and Taq I (23). The Sst I polymorphism probe the 5' end ofthe C4A and C4B genes, and a 0.5-kbp Sst maps to the 5' region of the C2 gene and is multiallelic, with I/Pst I genomic probe was used to identify the steroid four classes of size variants. We recently reported (28) that 21-hydroxylase (21-OHase) A pseudogene and the 21-OHase this polymorphism can also be detected by probing BamHI- Table 1. Immunologic parameters of immunodeficient individuals T-cell markers, % of MNC positive B-cell markers, % of MNC positive Serum immunoglobulins, mg/dl Diagnosis n Age, yr CD3 CD4 CD8 HB2 sIgM sIgD sIgA IgM IgG IgA CVID 19 45 13 68 12 35 ± 9 26 ± 7 10 ± 3 9 ± 3 9 ± 3 0.8 ± 0.5 21 ± 15 171 ± 58 6 ± 4 IgA-D 16 19 11 67 9 40 ± 8 19 ± 5 15 ± 4 16 ± 4 14 ± 4 1.0 ± 0.7 80 ± 41 1021 ± 285 <6 Normal laboratory range 47-76 23-53 13-23 4-21 6-15 4-13 0.5-1.9 45-226 706-1484 60-375 Mean values ± SD are given for all parameters. MNC, mononuclear cells; s-, surface immunoglobulin. Downloaded by guest on September 30, 2021 Immunology: Schaffer et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8017

A aa bb ab aa aa ae aa ad bd aa B aa bb ab ae ad bd C aa bb ab ae ad bd kb i kb kb " %w 6.90- T -6.60 o W W * 4.50-0 qpI* 4.40- * -3.45 _ _3.40 3.40- to .-. _ _ Ti-3.40 3.35

FIG. 2. Representative polymorphic banding patterns of the C2 gene. Southern blot analyses of genomic DNA from six patients, digested with BamHI (A), Sst I (B), or Taq I (C) and hybridized with probes A, B, and C, respectively. BamHI banding patterns are displayed in pairs with the common a, a pattern, since each pattern is from a different Southern blot. The BamHI a, d pattern contains an artefactual 5-kbp fragment from a previous hybridization of the blot with another probe. C2 alleles exhibit the following BamHI, Sst I, and Taq I restriction fragments: a, 4.4/3.4, 2.7, and 4.5 kbp; b, 6.6/3.2, 2.5, and 6.6 kbp; d, 4.4/3.35, 2.65, and 4.5 kbp; and e, 4.4/3.45, 2.75, and 4.5 kbp.

digested genomic DNA with the 2-kbp 5' fragment of a HindIII Southern blots can demonstrate fragments of 26, 15, full-length cDNA. These data suggest the presence of vari- and 8.5 kbp associated with the C4B gene, the C4A gene, and able number of tandem repeats (29) at the 5' end of the C2 a C4A gene deletion, respectively (30). Taq I Southern blots gene. In the present study we identified a fifth size variant can yield fragments of7.0, 6.0, 5.4, and/or 6.4 kbp associated exhibiting a 2.75-kbp Sst I fragment and a corresponding with a long C4A gene, a long C4B gene, a short C4B gene, and 3.45-kbp BamHI fragment. The location of the polymorphic a deletion of the C4A gene with fusion to a short C4B gene, sites of the C2 gene and the probes used to detect them are respectively (31). However, not all C4A deletions are asso- shown diagrammatically in Fig. 1. By considering the results ciated with a 6.4-kbp Taq I fragment. BamHI Southern blots obtained from Southern blot analyses together, eight banding can result in a 4.8-kbp fragment associated with long C4 genes patterns were observed, six in IgA-D and CVID patients and and a 3.5-kbp fragment associated with short C4 genes (30). two additional ones in healthy control individuals. The pat- Densitometric assessment of fragments from Southern blots terns observed in the patient population are shown in Fig. 2. generated by the three restriction enzymes allows for eval- On the basis of family studies these banding patterns are uation of C4B gene duplications and deletions. Fig. 3 A, B, attributed to five alleles, termed a, b, c, d, and e, in the order and C illustrates the RFLP patterns of 3 representative of their observed frequency (28). The a allele defined by patients. Assessment of our patient (Table 2) and control BamHI/Sst I 3.4/2.7-kbp, BamHI 4.4-kbp, and Taq I 4.5-kbp populations demonstrated that 5 of the 16 IgA-D patients fragments was observed in 63 of 68 (92.6%) chromosomes of (31%) and 6 of the 19 CVID patients (32%) had heterozygous healthy controls. All other alleles were relatively rare. An C4A deletions, whereas only 1 of the 34 healthy controls did increased frequency of rare alleles was observed in both (3%, Table 3). There were no differences in the relative CVID and IgA-D patients (Tables 2 and 3) although the frequencies of long or short C4B genes or of C4B gene differences from the controls were not statistically signifi- deletions or duplications between the patient and healthy cant. control populations. These results indicate that a significant C4 and 21-OHase Gene RFLPs. To determine potential number of CVID and IgA-D patients have heterozygous C4A RFLPs in the C4 loci, we hybridized genomic DNA digested deletions in comparison to healthy individuals. with Taq I, HindIII, orBamHI with a genomic C4A probe (24) The 21-OHase genes were assessed by hybridizing Taq which identifies the 5' end ofboth the C4A and the C4B genes. I-digested genomic DNA with a genomic probe (24) that Table 2. HLA serotypes and MHC class III genotypes of immunodeficient individuals CVID IgA-D MHC class II MHC class III* MHC class I MHC class II MHC class HII* MHC class I Patient HLA- HLA- 21-OHase HLA- HLA- Patient HLA- HLA- 21-OHase HLA- HLA- no. DQ DR C4B A:B ratio C4A C2 B A no. DQ DR C4B A:B ratio C4A C2 B A 1 w2, w3 3, 9 L, Sf 1:2 L, 0 a, b 8, 44 1,26 1 w2 3 L, Sf 1:2 L, 0 a,a 8 1,3 2 wl, w2 2, 3 L, Sf 1:2 L, 0 a,a 8 1 2 wl, w2 3, 6 L, Sf 1:2 L, 0 a,a 7, 8 2,31 3 w2, w3 3, 4 L, Sf 1:2 L, 0 a,a 8 1 3 w2, w3 3, 4 L, Sf 1:2 L, 0 a,a 8 1 4 w2, w3 3, 4 L, Sf 1:2 L, 0 a, a 7, 8 1,3 4 w2 3, 7 S, Sf 1:2 L, 0 a, a 8,14 1 5 wl, w3 1,4 S, Sf 1:2 L, 0 a,a 35, 60 24, 26 5 wl, w2 3,11 L, Sf 1:2 L, 0 a,a 8, 35 1,25 6 wl, w3 4,10 S, S, Sf 1:1 L, 0 a,a 8,14 11, 32 6 w2 7 S, S 1:1 L, L a, b 44 28, 29 7 wl, w3 4, 6 S. 0 1:2 L, L a,e 18, 35 3, 25 7 w2 7 L, S 1:1 L, L a, b 39, 44 2 8 wl, w3 9,10 L, S 1:1 L, L a,d 60 2, 24 8 w2 7 S, S 1:1 L, L b, b 44 29 9 w1 2, 6 L, L 1:1 L, L a, b 35, 44 2, 29 9 w2, w3 4, 7 L, S 1:1 L, L b, d 8, 60 1, 3 10 w2 7 L, S 1:1 L, L a, e 13, 39 2, 30 10 w2 7 L, S 1:1 L, L a, a 35, 38 2,11 11 w2 7 S, S 1:1 L, L a, b 14, 44 1,29 11 w2, w3 7, 9 L, S 1:1 L, L a,a 27, 57 2, 28 12 wl, w2 2, 3 L, S 1:1 L, L a, b 44, 61 29, 32 12 w3 4, 8 L, L 2:3 L, L a,a 16 29 13 wl, w2 1, 7 L, S 1:1 L, L a,a 44, 45 1, 2 13 w3 4 L, L 1:1 L, L a,a 40 24 14 w2, w3 3, 4 L, S 1:1 L, L a,a 39 1 14 w1 10,14 L, L 1:1 L, L a,a 8, 47 1, 28 15 w2, w3 4, 7 L, S 1:1 L, L a,a 44, 52 26, 28 15 wl, w2 6, 7 L, S 1:1 L, L a,a 16, 44 2 16 w1 1, 2 L, L 1:1 L, L a,a 49, 70 1, 3 16 w2, w3 4,11 L, L 1:1 L, L a,a 27, 60 1, 24 17 w3 4, 6 L, L 1:1 L, L a,a 18, 60 2, 26 18 w2, w3 4, 7 L, L 1:1 L, L a,a 35, 52 2, 32 19 w3 4,11 L, L 1:1 L, L a,a 38, 51 1, 2 *C4B and C4A genes: L, long gene; S, short gene; Sf, short fused C4B gene; 0, deleted gene. The 21-OHase A:B gene ratio was determined by densitometric scanning of Taq I Southern blots. Downloaded by guest on September 30, 2021 8018 Immunology: Schaffer et al. Proc. Natl. Acad. Sci. USA 86 (1989) Table 3. Summary of MHC class III gene polymorphisms DISCUSSION No. of individuals (%) The results of the present study, demonstrating a high No. in C4A Rare incidence of C4A and 21-OHase A gene deletions and of C2 Diagnosis group deletion C2 alleles Total RFLPs in CVID and IgA-D, support the hypothesis of a immu- Healthy 34 1 (3) 4 (12) 5 (15) common genetic basis for the two disorders. Neither IgA-D 16 5 (31)* 4 (25) 9 (56)t nodeficiency is a homogeneous disease, and consequently CVID 19 6 (32)* 7 (37) 12§ (63)t complete concordance can probably never be expected with any single gene defect. However, taken together with previ- *P < 0.02. tp < 0.01. tP < 0.001. All comparisons to healthy controls ous data on IgA-D, our results strongly implicate involve- by j test with correction for continuity. within the in the §One of the CVID patients (CVID patient no. 1, Table 2) has both a ment of a gene(s) MHC region pathogenesis C4A deletion and a rare C2 allele. of the arrests in B-cell differentiation that characterize the spectrum of immunoglobulin deficiencies, which range from selective IgA-D to the panhypogammaglobulinemia of a identifies both genes. Taq I RFLPs demonstrate 3.7- and typical CVID patient. 2.8-kbp fragments associated with the 21-OHase B gene and Wilton et al. (14) have examined the expression of MHC 3.2- and 2.6-kbp fragments associated with the 21-OHase A genes in IgA-D patients. Their studies revealed a high inci- pseudogene. Fig. 3D illustrates the 21-OHase Taq I RFLP dence of specific MHC supratypes containing C4A null patterns for the 3 patients. Densitometry studies (Table 2) alleles. We have extended these findings by performing a revealed that 7 ofthe 19 CVID patients and 5 ofthe 16 IgA-D genotypic evaluation of the genes encoding C2, C4A, C4B, patients have 21-OHase A heterozygous deletions, usually in and 21-OHase in individuals with IgA-D and CVID. Southern association with a C4A gene deletion, whereas only 1 of the blot analysis revealed that approximately one-third of these 34 control individuals has a heterozygous deletion ofthe C4A individuals have heterozygous C4A gene deletion, usually in and 21-OHase A genes. association with 21-OHase A gene deletion. The incidence of MHC Supratypes. Previous studies of IgA-D individuals C4A deletion that we observed is significantly lower than the have documented the frequent occurrence of the MHC reported 70% incidence of C4A null alleles in IgA-D (14). A supratype HLA-A1, Cw7, B8, C4AQO, C4B1, BfS, DR3 (14). similar difference exists between the incidence of C4A gene Nine of our 11 patients with heterozygous C4A deletions deletion in our healthy control population and that reported (CVID patients 1-4, IgA-D patients 1-5, Table 2) have at least for null alleles in larger Caucasian populations (32). These one haplotype consistent with HLA-A1B8DR3. Family stud- differences are not unexpected, since lack of expression of ies, although presently incomplete, indicate that the C4A C4A genes may also be due to transcriptional or translational deletion occurs on the same chromosome as that expressing defects. Differences in the study populations could also this haplotype. We also found that 67% (6/9) ofthe CVID and influence these frequency estimates. IgA-D patients expressing HLA-B44 have the rare C2 b allele C4A null alleles and C4A and 21-OHase A gene deletions there is an association with DR7 have also been associated with other diseases, notably sys- and in 4 of these 6 patients temic lupus erythematosus (33). Since C4A plays an essential and C4BS (CVID patient 11, IgA-D patients 6, 7, and 8, Table role in the normal processing and elimination of circulating 2). Our results therefore suggest that the MHC supratype of immune complexes (34), and immune complexes are impli- DR7-C4BS-C4A-C2b-B44 is also associated with CVID and cated in the pathogenesis ofthis disease, it has been postulated IgA-D. that C4A null alleles may represent the disease susceptibility genes (33). The fact that individuals with CVID and IgA-D A D often develop autoimmune disorders, such as systemic lupus 1 2 3 kb 1 2 3 kb 1 2 3 1 2 3 kb kb erythematosus or rheumatoid arthritis, or have close relatives 26.0_ 3.7- .*wqUNo _ 4.8- with these disorders (35) is compatible with this proposal. 15.0- w 'we vo 6.4- II 3.2- However, it seems unlikely that the arrests in B-cell differen- 8.5- -a 5.4- 44o3.5- tiation underlying these immunodeficiencies are causally re- 2.8- lated to C4A gene deletion or lack of expression. A more likely 2.6- interpretation of the results is that C4A deletion may be in FIG. 3. Representative polymorphic banding patterns of the C4 linkage disequilibrium with another MHC region gene which and 21-OHase genes. Southern blot analyses of genomic DNA from affects plasma cell maturation and antibody secretion. three patients, digested with HindIII (A), Taq I (B and D), or BamHI Analysis of 29 independent HLA supratypes from 17 IgA-D (C) and hybridized with the C4 (A, B, and C) or the 21-OHase probe individuals by Wilton et al. (14) revealed that 22 of these (D). Patient 1 (IgA-D patient 8, Table 2) has a pattern consistent with haplotypes included deletions or duplications of C4 and 21- equal numbers of C4A and short C4B genes and of 21-OHase A and OHase genes or contained the C4A6 allele that gives unique B genes. In patient 2 (CVID patient no. 5, Table 2) the presence of RFLPs. They suggested that an IgA-D gene is located within the 8.5-kbp HindIII (A) and the 6.4-kbp Taq I (B) bands indicates the MHC class III gene region, perhaps between the C4A and deletion of a C4A gene and fusion to a short C4B gene. The 1:2 ratio data neither nor refute this of the intensities of the 4.8- and 3.5-kbp BamHI bands (C) is C4B genes. Our present support consistent with this interpretation. The ratio ofthe 3.2- to the 3.7-kbp hypothesis, but family studies on our CVID and IgA-D pa- fragments and that of the 2.6- to the 2.8-kbp fragments of the tients may prove informative in this regard. The observed 21-OHase genes (D) is 1:2, consistent with heterozygous deletion of relatively high incidence of C2 rare alleles in both CVID and a 21-OHase A gene. In patient 3 (CVID patient no. 6, Table 2), the IgA-D will be particularly helpful in ascertaining a possible 8.5-kbp HindII1 (A) and the 6.4-kbp Taq I (B) bands indicate deletion linkage between the putative gene defect and MHC class III of a C4A gene with fusion to a short C4B gene. The intensity of the genes. 5.4-kbp Taq I band is twice that of the 7.0- and 6.4-kbp Taq I bands, The Sst I/BamHI multiallelic RFLP of the C2 gene is indicating duplication of a short C4B gene. This interpretation is probably due to the presence of variable number of tandem corroborated by the BamHI blot (C), where the intensity ratio of the within an intron near the 5' end of the C2 gene 4.8- to the 3.5-kbp band is 1:3. The ratio of the 3.2- to the 3.7-kbp repeats (29) fragments and that of the 2.6- to the 2.8-kbp fragments of the (23). This RFLP has no discernible effects on the structure, 21-OHase genes (D) is 1:1, indicating deletion of a 21-OHase gene in function, or expression ofC2, but it may be a valuable marker association with the C4A gene deletion and duplication of a 21-OHase for analysis of disease associations with gene(s) within the A gene in association with the duplication of the short C4B gene. MHC class III region. This region ofchromosome 6 is located Downloaded by guest on September 30, 2021 Immunology: Schaffer et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8019 between the MHC class I and class II gene clusters and 12. Cassidy, J. T., Oldham, G. & Platts-Mills, T. A. E. (1979) Clin. contains the genes encoding complement proteins C2, factor Exp. Immunol. 35, 296-305. C4A, and C4B, as well as the microsomal cytochrome 13. Wollheim, F. A., Belfrage, S., Coster, C. & Lindholm, H. B, (1964) Acta Med. Scand. 176, 1-16. P-450 21-OHase (36). Other genes that have been mapped in 14. Wilton, A. N., Cobain, T. J. & Dawkins, R. L. (1985) Immu- this region include genes encoding the tumor necrosis factors nogenetics 21, 333-342. a and 8 (36) and a protein with an unusual periodic structure, 15. Korman, A. J., Boss, J. M., Spies, T., Sorrentino, R., Okada, termed RD (37), the human homolog ofthe mouse gene B144 K. & Strominger, J. L. (1985) Immunol. Rev. 85, 45-83. (38), a duplicated gene encoding the major heat shock protein 16. Ambrus, M., Hernadi, E. & Bajtai, G. (1977) Clin. Immunol. HSP70 (39), and five genes encoding "HLA-B-associated Immunopathol. 7, 311-314. 17. Hammarstrom, L. & Smith, C. I. (1983) Tissue Antigens 21, transcripts" (38). An additional six genes have recently been 75-79. identified in the region between the genes encoding C4 and 18. Landay, A., Gartland, G. L., Abo, T. & Cooper, M. D. (1983) tumor necrosis factor ,8 (40). The function of the products of J. Immunol. Methods 58, 337-347. these recently identified genes is presently unknown, al- 19. Sykes, B. C. (1983) Lancet Ui, 787-788. though several of them are actively transcribed in B cells (38, 20. Southern, E. (1975) J. Mol. Biol. 98, 503-517. 40). It seems possible that the C4A and/or C2 RFLPs 21. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) in Molecular observed in this study are in linkage disequilibrium with Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 387-389. defective alleles of one or more of the newly identified, or 22. Horiuchi, T., Macon, K. J., Kidd, V. J. & Volanakis, J. E. other as yet unidentified, MHC class III genes. It is tempting (1989) J. Immunol. 142, 2105-2111. to speculate further that the product of this gene is involved 23. Bentley, D. R., Campbell, R. D. & Cross, S. J. (1985) Immu- in B-cell differentiation and that its putative defect predis- nogenetics 22, 377-390. poses to the development of IgA-D and CVID. The existence 24. White, P. C., Grossberger, D., Jonufer, B. J., Chaplin, D. D., of both genetic and nongenetic components in the pathogen- New, M. I., Dupont, B. & Strominger, J. L. (1985) Proc. Natl. esis of these diseases is suggested by family studies, by the Acad. Sci. USA 82, 1089-1093. 25. Feinberg, A. P. & Vogelstein, B. (1983) Anal. 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