Modular Variations of the Human Major Histocompatibility Complex Class III Genes for Serine/Threonine Kinase RP, Complement Component C4, Steroid 21-Hydroxylase CYP21, and Tenascin TNX (the RCCX Module)
A MECHANISM FOR GENE DELETIONS AND DISEASE ASSOCIATIONS*

Zhenyu YangDagger §parallel , Anna R. MendozaDagger , Thomas R. Welch**, William B. ZipfDagger , and C. Yung YuDagger §Dagger Dagger §§

From the Dagger  Children's Hospital Research Foundation, Columbus, Ohio 43205, the § Molecular, Cellular, and Developmental Biology Program, the  Department of Pediatrics, and the Dagger Dagger  Department of Medical Microbiology and Immunology, Ohio State University, Columbus, Ohio 43210, and the ** University of Cincinnati Medical Center, Cincinnati, Ohio 45229

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The frequent variations of human complement component C4 gene size and gene numbers, plus the extensive polymorphism of the proteins, render C4 an excellent marker for major histocompatibility complex disease associations. As shown by definitive RFLPs, the tandemly arranged genes RP, C4, CYP21, and TNX are duplicated together as a discrete genetic unit termed the RCCX module. Duplications of the RCCX modules occurred by the addition of genomic fragments containing a long (L) or a short (S) C4 gene, a CYP21A or a CYP21B gene, and the gene fragments TNXA and RP2. Four major RCCX structures with bimodular L-L, bimodular L-S, monomodular L, and monomodular S are present in the Caucasian population. These modules are readily detectable by TaqI RFLPs. The RCCX modular variations appear to be a root cause for the acquisition of deleterious mutations from pseudogenes or gene segments in the RCCX to their corresponding functional genes. In a patient with congenital adrenal hyperplasia, we discovered a TNXB-TNXA recombinant with the deletion of RP2-C4B-CYP21B. Elucidation of the DNA sequence for the recombination breakpoint region and sequence analyses yielded definitive proof for an unequal crossover between TNXA from a bimodular chromosome and TNXB from a monomodular chromosome.

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Besides the immunoglobulins, complement component C4 is probably the most polymorphic serum protein. There are two isotypes, C4A and C4B, that manifest remarkable differences in chemical reactivities and serological properties (reviewed in Ref. 1). More than 34 allotypes for C4A and C4B have been demonstrated by agarose gel electrophoresis, based on gross differences in electric charge (2). Similar to the protein, the complement C4 genes are unusually complex with frequent variations in gene size and gene number. In addition, the genes surrounding C4A or C4B also exhibit considerable variations. These neighboring genes include RP1 or RP2 at the 5' region, CYP21A, or CYP21B and TNXA or TNXB at the 3' region (Fig. 1). The complex organizations of the C4A and C4B genes, together with the extensive polymorphisms of the C4A and C4B proteins render C4 an excellent marker for MHC1-associated diseases (1, 3). For instance, congenital adrenal hyperplasia (CAH) is mainly caused by mutations or deletions of CYP21B (4), and systemic lupus erythematosus is correlated with C4A deficiencies (5). In addition, insulin-dependent diabetes mellitus (6, 7); sudden infant death syndrome and spontaneous recurrent abortion (8, 9); IgA deficiency and common variable immunodeficiency (10, 11); IgA nephropathy (12); skin vitiligo and pemphigus vulgaris (13, 14); and autism and narcolepsy (15, 16) have all been suggested to be associated with specific alleles or null alleles of C4.

The human C4 genes are either 21 kb (long, L) or 14.6 kb (short, S) in size (17). This dichotomous size variation is due to the presence of an endogenous retrovirus HERV-K(C4) in intron 9 of the long gene (18-20). There may be one, two, or three C4 genes in the MHC class III region of chromosome 6 (21). Most people have two C4 genes in the MHC with one coding for a C4A protein and the other coding for a C4B protein (Fig. 1). C4A has higher affinities to amino group-containing targets; C4B has higher affinities to hydroxyl group-containing targets. These differences are the result of four amino acid changes between positions 1101 and 1106 (22-24). A significant proportion of the population has a single C4 gene in chromosome 6 coding for C4A or for C4B. Deletion or duplication of the C4 genes are always concurrent with their downstream genes, steroid 21-hydroxylase genes, CYP21A or CYP21B (25, 26).


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Fig. 1.   A molecular map of the human MHC complement gene cluster. This map represents the most common organization of the genes in the normal population from complement component C2 gene to extracellular matrix protein TNXB gene. The horizontal arrows represent the direction of gene transcription. Pseudogenes or gene segments are shaded. The negative signs for the intergenic distances between CYP21A and TNXA and between CYP21B and TNXB represent overlaps at the 3'-ends of these genes (data adapted from Ref. 27).

RP1 is one of the four novel genes, RD-SKI2W-DOM3Z-RP1, present in the 30-kb genomic region between complement component genes factor B (Bf) and C4 (27-31). RP2 is a partially duplicated gene segment that contains only 913 bp of the sequence corresponding to the last two and one-half exons of RP1. RP1 transcripts are ubiquitously expressed. Derived amino acid sequence suggested that RP1 codes for a nuclear protein that is probably a serine/threonine kinase (27, 30, 32).

The cytochrome P450 steroid 21-hydroxylase genes CYP21A or CYP21B are located 3028 bp downstream of C4A or C4B, respectively (17). CYP21 is essential for the biosynthesis of glucocorticoid and mineralocorticoid hormones. The complete absence of CYP21 leads to salt wasting, low activity of CYP21 causes simple virilizing, and below average CYP21 activity causes androgen excess (reviewed in Ref. 4). CYP21A is a pseudogene, because it contains three deleterious mutations: an 8-bp deletion in exon 3 and a T nucleotide insertion in exon 7 that result in frameshift mutations as well as a C to T transition in exon 8 that generates a premature stop codon. In addition, there are many other point mutations in coding and noncoding sequences (33-35). If on both copies of chromosome 6, the deleterious mutations in CYP21A are incorporated into CYP21B or the CYP21B genes are deleted, the subject suffers CAH.

The 3'-ends of CYP21A or CYP21B overlap with the 3'-ends of extracellular matrix protein tenascin TNXA or TNXB by 444 bp, respectively (36). The gene configurations of TNXA and TNXB are opposite to those of RP, C4, and CYP21. The TNXB gene is 68.2 kb in size, consists of 45 exons and encodes a protein of 4289 amino acids (38).2 The derived amino acid sequence of TNXB reveals a heptad, 18.5 epidermal growth factor repeats, 32 fibronectin type III repeats, and a fibrinogen domain. The overall structure of TNXB shows a striking similarity to extracellular matrix proteins tenascin/cytostatin (TN-C) and restrictin (TN-R) (39-41). TN-C is present in the central and peripheral nervous system and in smooth muscle and tendon. It is probably involved in cell adhesion and cell morphology (42). TN-R is expressed in the nervous system and implicated in neural cell attachment (43). The function of TNXB is yet to be determined. Its transcripts are ubiquitously expressed in the fetus (38). TNXA is a partially duplicated gene segment that corresponds to intron 32 to exon 45 of TNXB. In addition, there is a 120-bp deletion at exon 36-intron 36 that results in a frameshift mutation and premature termination of translation (30, 40).

The demonstration of the endogenous retrovirus HERV-K(C4) mediating the size variation of C4 genes (18) and the elucidation of DNA sequences for RP1 and RP2 (30), C4A and C4B (22, 17), CYP21A and CYP21B (33-35), and TNXA and TNXB (30, 38)2 provide important information for resolving the fine structures and the complex organizations of the consecutive genes RP, C4, CYP21, and TNX. The concurrent deletions/duplications of C4 and CYP21 genes (25, 26) prompted us to investigate if RP and TNX also undergo rearrangements in normal individuals and in selected CAH patients. Diagnostic RFLPs for RP1 and RP2, and, for TNXA and TNXB, have been devised. RP-C4-CYP21-TNX genes are organized in variable, modular fashions. The unusually frequent modular variation appears to be the root cause for unequal crossovers and exchange of sequences between the functional and nonfunctional genes of the RCCX.

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Oligonucleotides-- Oligonucleotides were synthesized by an Applied Biosystems model 380B DNA Synthesis machine. The sequences added to facilitate cloning are represented in lowercase type. For amplification and sequencing of TNX genes (38) the following oligonucleotides were used: RDX-5, aga gAA TTC AGT GAA ATC AGG GAG ACC; RDX-3, gag gaa TTC CAG TGC AGC ACG GCG AA; SDX-52, GGA GCC TCA GAG TGT GCA; SDX-32, CAA TCG GAG CCT CCA CCA; XB54H, gtg gaa ttc AAG CGA GCA CCT GAC TCA; and XA31H, gtt gaa ttc TTT TCT TGA CTC CCA CCT G. For amplification of CYP21A probe (35), the following were used: 21A5, TGT GGC CAT TGA GGA GGA A; and 21A3, TGC CAC CGA TCA GGA GGT C.

Isolation of Human Genomic DNA-- Genomic DNAs were isolated following standard protocols from cultured cell lines HepG2 (liver carcinoma) and MOLT4 (T-cell leukemia), peripheral blood of normal individuals, and a congenital adrenal hyperplasia patient (CAH-E1). Appropriate consents from blood donors were obtained according to approved protocols by the Institutional Board of the Columbus Children's Hospital.

Complement C4 Allotyping-- Complement C4A and C4B allotypes from EDTA-blood plasma were determined as described in Refs. 44 and 45. Briefly, 10 µl of plasma was digested with 0.1 unit of neuraminidase (Sigma) at 4 °C overnight and with 0.1 unit of carboxypeptidase B (Sigma) at room temperature for 30 min. Two agarose gels were prepared. Four µl of digested plasma was loaded to each agarose gel and resolved by high voltage gel electrophoresis (46). One of the agarose gels was subjected to immunofixation using goat antiserum against human C4 (Incstar, Stillwater, MN). Plasma proteins in the other agarose gel were subjected to immunoblot analyses using anti-Ch1 or anti-Rg1 monoclonal antibodies (anti-C4B, catalog no. C057-325.2, lot no. 120287; anti-Rg1, RGd1; kindly provided by Dr. Joann M. Moulds, Houston, TX) at a dilution of 1:5000 and 1:1000, respectively. Immune complexes were detected by the chemiluminescence method using the ECL Plus reagents (Amersham Pharmacia Biotech).

HLA Typing-- HLA typing of the E family was kindly performed by The Ohio State University Tissue Typing Laboratory.

PCR of Cosmid and Genomic DNA-- PCR of cosmid and genomic DNA were performed following standard procedures (47). PCR products were purified from 0.8% low gelling temperature agarose and cloned into pBluescript vectors.

DNA Probes-- DNA probes used were as follows: for RP, RP1.1, a 1.1-kb insert of RP1 cDNA (30) and RP1 3' probe, a 651-bp NheI-EcoRI fragment of RP1.1; for C4, PA, a 476-bp BamHI-KpnI cDNA fragment isolated from pAT-A (pAT-A contains the almost full-length cDNA insert for the human C4A4 allele (22, 48)) and PB, a C4d-specific 926-bp BamHI DNA fragment subcloned from lambda JM-2a that contains a C4B5 gene (22); for CYP21, a 757-bp fragment of CYP21A, amplified from cos 2 using primers 21A5 and 21A3 (a cosmid isolated from a human genomic library DA (49), cos 2 spans from the 5' region of C4A1 gene to the 3' region of TNXB gene; the genomic DNA of cos 2 derives from the HLA haplotype A3 B47 DR7 that contains a deletion of the CYP21B gene); for TNX, a 600-bp fragment of TNXB, corresponding to exons 35-37 of TNXB, amplified from cos 2 using primers RDX-5 and RDX-3.

Southern Blot Analysis-- Ten µg of genomic DNA was digested to completion with the appropriate restriction enzymes for 16 h, resolved on an agarose gel, blotted onto Hybond-N+ membrane (Amersham Pharmacia Biotech), and hybridized with an appropriate [alpha -32P]dCTP-labeled probe, as described in Ref. 50.

DNA Sequencing and Sequence Analysis-- Sequencing reactions were performed using a Sequenase kit (U.S. Biochemical Corp., Cleveland, OH) and [35S]ATP following the dideoxy sequencing method (51), or by the automated sequencing method using an ABI 377 machine. DNA sequences were compiled using PC/Gene software (Intelligenetics, Mountain View, CA). Comparisons of the sequences were performed by FASTA, BESTFIT, PILEUP, and PRETTY programs in the GCG package through the Pittsburgh Supercomputing Center (52).

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Allotyping of C4-- Allotyping of the plasma C4 proteins from the E family is shown in Fig. 2I. Phenotypically, E1 has C4A1, C4A3, and C4BQ0 (lane 1); E2 has C4A3 and C4BQ0 (lane 2); and E3 has C4A1, C4A3, and C4B1 (lane 3). Lane 4 shows a control plasma with C4A3 and C4B1. Immunoblot analyses revealed positive reactions of C4A1 in E1 (lane 1), C4A1 and C4B1 in E3 (lane 3), and C4B1 in the control plasma (lane 4) with anti-Ch1 monoclonal (panel II). Similarly, positive reactions were observed for the C4A3 allotype in E1, E2, E3, and a control plasma with the anti-Rg1 monoclonal (lanes 1-4, panel III). In separate experiments on family B (data not shown), C4 allotyping showed that B1 has C4AQ0, C4B1; B2 has C4A3, C4B1, C4BQ0; and B3 has C4A3, C4AQ0, C4B1. Immunoblot analyses showed that the C4A3 allotype reacted with anti-Rg1 and the C4B1 reacted with anti-Ch1.


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Fig. 2.   C4 allotypes of the E family. I, human C4A and C4B allotypes detected by immunofixation. Immunoblot analysis is shown of the C4 allotypes using anti-Ch1 monoclonal antibody (II) and anti-Rg1 monoclonal antibody (III). The C4A1 allotype with reverse association of Ch1 epitope is marked by an arrow. Lane 4 is from a control plasma with C4A3 and C4B1.

Modular Variations for RP, C4, CYP21, and TNX-- Whether the flanking genes RP (located 5' to C4) and TNX (located 3' to CYP21) are involved in the C4-CYP21 gene deletion events were investigated. Diagnostic RFLPs were devised to detect and distinguish the presence of the RP1 and RP2 as well as TNXA and TNXB. By using an RP1 3' probe for hybridization, the RP1 gene can be represented by a 9.6-kb BamHI fragment, and the RP2 (and TNXA gene segments) can be represented by a 5.0-kb BamHI fragment. By using a 600-bp TNX probe corresponding to TNXB exons 35-37, the presence of the TNXB gene can be detected by a 9.0-kb ScaI fragment, and the TNXA gene can be detected by a 4.0-kb ScaI fragment (Fig. 3I). Previously, techniques were established for detecting the presence of the C4A and C4B genes by NlaIV RFLP, and their associations with Rg1 or Ch1 antigenic determinants by EcoO 109I RFLP (50). In addition, the presence of CYP21A and CYP21B can be detected by the 3.2- and 3.7-kb TaqI fragments, respectively (34). Fig. 3 (panels II-IV) shows a series of Southern blot analysis of DNA samples isolated from two families, B and E. Members of the B family appear to be normal. For the E family, there is a CAH patient E1.


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Fig. 3.   Modular variations of RP, C4, CYP21, and TNX. DNA samples were isolated family B (B1-B4) and family E (E1-E4), digested by appropriate restriction enzymes, and subjected to Southern blot analyses. Panel I illustrates the structural basis to distinguish RP1 and RP2 by BamHI RFLP, and TNXA and TNXB by ScaI RFLP. Solid bars represent locations of the probes used. Panel II shows detection of RP1 and RP2 genes by BamHI RFLP. The probe used was RP1.1. Panel III-A shows the variation of C4A and C4B isotypes by NlaIV RFLP; panel III-B reveals the association of the Rg1 and Ch1 antigenic determinants in C4 genes by EcoO109I RFLP. The probe used was PB. Panel IV exhibits the presence of CYP21A and CYP21B genes by TaqI RFLP. The probe used was a 757-bp fragment amplified from CYP21A. Panel V shows the detection of TNXA and TNXB and ScaI RFLP. The probe used was a 600-bp fragment corresponding to exons 35-37 of TNXB.

As shown in Fig. 3II, restriction fragments corresponding to RP1 (9.6-kb BamHI fragment) and RP2 (5.0-kb BamHI fragment) were detected in all individuals except B1 and E1. In these two individuals, only the RP1-specific 9.6-kb fragment is present (lanes 1 and 5).

The presence of the C4A or C4B gene was analyzed by NlaIV restriction patterns shown in in Fig. 3III (A). B1 contains the 467-bp fragment corresponding to C4B genes (lane 1). E1 and E2 have the 276- and 191-bp fragments specific for C4A genes (lanes 5 and 6). Other individuals contain fragments corresponding to both C4A and C4B genes. The associations of C4 genes with the major Chido (Ch1) and Rodgers (Rg1) antigens were revealed in Fig. 3III (B) by the EcoO 109I restriction patterns. The C4B genes in B1 express the Ch1 antigen, since only the 458-bp fragment can be detected (lane 1). The C4A genes in E2 express the Rg1 epitope, since only the 565-bp fragment is present (lane 6). Both Rg1- and Ch1-specific fragments are present in E1 (lane 5). Therefore, one of the C4A genes (C4A1) in E1 expresses Ch1 (that is frequently associated with C4B), and the other C4A gene (C4A3) expresses Rg1. All other individuals contain both C4A and C4B genes. These C4 genes express Rg1 and Ch1 because both 565- and 458-bp fragments were detectable. An additional 344-bp Eco0 109I fragment exists in all individuals, since the 926-bp C4d-specific probe (PB) was used. This fragment is common to C4 genes with Rg1 or Ch1 (50).

The presence of CYP21A and CYP21B was determined by TaqI RFLP (Fig. 3IV). Both CYP21A (3.2-kb fragment) and CYP21B (3.7-kb fragment) were detected in all samples except B1 and E1. B1 only contains the functional CYP21B gene (lane 1), while the CAH patient E1 only contains the pseudogene CYP21A (lane 5).

The presence of TNXA and TNXB genes are determined by ScaI RFLP (Fig. 3V). Restriction fragments for both TNXA (4.0-kb ScaI fragment) and TNXB (9.0-kb ScaI fragment) were detected in all individuals except B1 and E1 (Fig. 3II (B)). These two individuals have the 9.0-kb ScaI restriction fragment corresponding to TNXB but no 4.0-kb ScaI fragment corresponding to TNXA (lanes 1 and 5).

From the above results, it becomes clear that individuals who have a single locus for RP also have single gene loci for C4, CYP21, and TNX. Individuals with both RP1 and RP2 loci also have duplicated loci for C4, CYP21, and TNX. Hence, the four tandemly arranged genes RP, C4, CYP21, and TNX are duplicated or deleted together as a discrete genetic unit. This genetic unit is designated as the RCCX module.

Molecular Basis of the C4 and RP TaqI RFLP and Variations of the RP, C4, CYP21, and TNX (RCCX) Loci in the B and E Families-- The TaqI RFLP is the most widely used technique to illustrate the complexities of polymorphisms in the number and size of C4 genes. Four fragments of 7.0, 6.4, 6.0, and 5.4 kb in size can be detected by Southern blot analysis of TaqI-digested genomic DNA hybridized to PA, a C4 5' probe (26, 50, 53). At the same time, all these TaqI fragments can also be detected by an RP1.1 probe, suggesting that the TaqI RFLP is caused by both RP and C4 gene variations. There is a BamHI site at the 5'-untranslated region of the C4A and C4B genes. To segregate the RP1/RP2 variation from the C4 long/short (L/S) size dichotomy, the BamHI-TaqI double digests of genomic DNA were performed (Fig. 4I). As shown in Fig. 4II, the single 6.4-kb TaqI fragment for B1 (Fig. 4II (A), lane 1) was split into a 3.1-kb TaqI-BamHI fragment corresponding to an RP1 gene (Fig. 4II (B), lane 1) and a 3.2-kb BamHI-TaqI fragment corresponding to a short C4 gene (Fig. 4II (C), lane 1). The 7.0- and 6.0-kb TaqI fragments detected in HepG2 (Fig. 4II (A), lane 2) were split into 3.1- and 2.1-kb TaqI-BamHI fragments corresponding to RP1 and RP2 genes (Fig. 4II (B), lane 2) and a single 4.0-kb BamHI-TaqI fragment corresponding to long C4 genes (Fig. 4II (C), lane 2). The 7.0- and 5.4-kb TaqI fragments detected in MOLT4 (Fig. 4II (A), lane 3) were divided into the RP1-associated 3.1-kb TaqI-BamHI fragment and the RP2-associated 2.1-kb TaqI-BamHI fragment (Fig. 4II (B), lane 3) and the 4.0- and 3.2-kb BamHI-TaqI fragments associated with long C4 genes and short C4 genes, respectively (Fig. 4II (C), lane 3). Therefore, the restriction patterns of the TaqI genomic Southern blot for RP or C4 genes are resulted from a combination of variations of RP1 or RP2, one or two loci of C4 genes, and long or short C4 genes. The basis of the TaqI RFLP is interpreted as follows. The 7.0-kb fragment indicates the presence of an RP1 gene linked to a long C4 gene; the 6.4-kb fragment corresponds to an RP1 gene linked to a short C4 gene; the 6.0-kb fragment represents an RP2 linked to a long C4 gene; and the 5.4-kb fragment stands for an RP2 linked to a short C4 gene. The actual sizes of these TaqI restriction fragments derived from DNA sequences are 0.1 kb greater or smaller than the apparent sizes described above.


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Fig. 4.   Southern blot analysis to segregate the RP1/RP2 variation from the C4 long/short (L/S) size dichotomy. Panel I is a schematic diagram for the molecular basis of the TaqI and TaqI-BamHI RFLP. In panel II (A), genomic DNA isolated from B1 (lane 1), HepG 2 (liver carcinoma, lane 2) and Molt 4 (T-cell leukemia, lane 3) were digested with TaqI, blotted, and hybridized with RP1.1 probe. In panel II (B and C), DNAs were double-digested with TaqI and BamHI and subjected to RP1.1 probe (B) or C4 5' probe PA (C).

The specific combinations of RP1/RP2 genes with C4(L)/C4(S) genes in the B and E family members were examined by TaqI digests and RP1.1 probe as shown in Fig. 5I. The RP1-C4(S) haplotype is present in B1, as represented by the single 6.4-kb TaqI fragment (lane 1). The RP1-C4(L) haplotype is present in E1, as shown by the single 7.0-kb TaqI fragment (lane 5). Therefore, homozygous, single RP1 gene, and single C4 gene are present in B1 and E1. B2 has the homozygous RP1-C4(L)-RP2-C4(L) haplotype as revealed by the presence of the 7.0- and 6.0-kb TaqI fragments (lane 2). B3 and B4 (children of B1 and B2) are heterozygous for the RP1-C4(S) haplotype and the RP1-C4(L)-RP2-C4(L) haplotype, since they both contain the 7.0-, 6.4-, and 6.0-kb TaqI fragments (lanes 3 and 4). E2 and E3 (parents of E1) are heterozygous for the RP1-C4(L)-RP2-C4(L) haplotype and the RP1-C4(L) haplotype, since they both have the 7.0- and 6.0-kb TaqI fragments (lanes 6 and 7).


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Fig. 5.   Taq I polymorphism to detect the TNXA-associated 120-bp deletion in the TNXB gene of CAH patient E1. Genomic DNA isolated from B and E families were digested with TaqI, blotted, and hybridized with appropriate probes to demonstrate different RP-C4 combinations (panel I), the presence of CYP21A and CYP21B genes (panel II), and the variations of TNX genes (panel III). The 2.5-kb Taq I fragment is usually associated with TNXB, while the 2.4-kb Taq I fragment is usually associated with TNXA. An arrow indicates the unusual association of a 2.4-kb fragment with TNXB in E1.

Detection of the TNXA-associated 120-bp Deletion in a TNXB Gene of CAH Patient E1-- Compared with TNXB, TNXA has not only a 63-kb truncation of the 5' region (30, 38)2 but also a 120-bp deletion spanning across the junction of exon 36 and intron 36 (30). This deletion attributes to a TaqI RFLP when a TNX 3' probe is used for Southern blot analysis; TNXA associates with a 2.4-kb fragment (TNX-2.4), and TNXB associates with a 2.5-kb fragment (TNX-2.5). The analysis of this TNX TaqI polymorphism in the B and E families are shown in Fig. 5III. B1, who has a monomodular RCCX (S) structure, exhibited TNX-2.5 (lane 1) that is consistent with the presence of TNXB genes only. B2, who has a bimodular RCCX (L-L) structure, revealed both TNX-2.4 and TNX-2.5 (lane 2), which is concordant with the presence of both TNXA and TNXB. B3 and B4, who are heterozygous for bimodular RCCX (L-L) and monomodular RCCX (S), also revealed both TNX-2.5 and TNX-2.4 fragments.

E3 is heterozygous for monomodular RCCX (L) and bimodular RCCX (L-L), implying that she has two TNXB genes and one TNXA gene. As expected, the relative band intensity of TNX-2.5 to TNX-2.4 is 2:1 (lane 7). E2 is also heterozygous for monomodular RCCX (L) and bimodular RCCX (L-L). He has two TNXB genes and one TNXA gene in a diploid genome. However, the band intensity of TNX-2.5 is only half of that for TNX-2.4 (lane 6), which is opposite from the expected result. The CAH patient E1 is homozygous for monomodular RCCX (L) and has only TNXB but no TNXA. Unexpectedly, he manifested both the TNXB-related 2.5-kb fragment and TNXA-related 2.4-kb fragment. The relative band intensity for TNX-2.5 and TNX-2.4 is 1:1. These results suggest that in both E1 and E2, one of the TNXB genes contains the 120-bp deletion. Hence, the aberrant TNXB gene in E1 originates from the paternal chromosome. Correlation of the HLA typing data for the E family (Table I) and the TaqI RFLP data suggests that the aberrant TNXB gene is present in HLA haplotype A3 B35 DR1.

                              
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Table I
RCCX modular structures of the B and E families

Based on the phenotypes of the C4A and C4B proteins and haplotypes of the RCCX modules, the organizations of RCCX genotypes for the B and E families were deduced and are shown in Table I. The haplotype c of the B family and the haplotype a of the E family both have bimodular RCCX structures coding for C4A3 protein from the two C4 loci.

An Unequal Crossover between Monomodular and Bimodular RCCX Chromosomes Leading to Gene Deletion and Gene Duplication-- The presence of a monomodular RCCX structure in E1 with RP1-C4(L), CYP21A, and a TNXB gene with TNXA-associated 120-bp deletion leads us to hypothesize that there was an unequal crossover between TNXA and TNXB from the homologous chromosomes. This unequal crossover resulted in the formation of an XB-XA recombinant and the deletion of the RP2-C4B-CYP21B genes (Fig. 6I). To test this hypothesis, the genomic region spanning the puta-tive 120-bp deletion was amplified by PCR using the CAH-E1 genomic DNA. The strategies for PCR are depicted in Fig. 6II. When primers RDX5 and RDX3 were used, two PCR products of 493 and 613 bp were obtained. These products were cloned and sequenced. Both sequences correspond to exons 35-37 of TNX. The sequence of the 613 bp is identical to that of the regular TNXB, while the 493-bp product appears to contain a 120-bp deletion similar to that observed in TNXA. To further prove that this is an aberrant TNXB gene with a TNXA-associated 120-bp deletion, a 2.6-kb genomic DNA fragment was amplified using RYM25 and XA31H. RYM25 is a TNXB-specific primer because it is located in exon 32 and is 220 bp upstream of the gene duplication breakpoint for TNXA and TNXB. The XA31H primer spans across the 120-bp deletion and therefore is TNXA-specific. The 2.6-kb PCR product was cloned and sequenced to completion. The sequences of the 2.6- and 493-bp fragments overlap and so were compiled. The resulting sequence E1XB-A was aligned with two normal TNXB sequences (TNXB-H and TNX-M) (38)2 and two normal TNXA sequences (TNXA-M and TNXA-Y) (30, 54) obtained from three different laboratories. In addition, we included a recombinant TNXA sequence in a pauciarticular juvenile rheumatoid arthritis (JRA) patient, L1XA-B, that acquired the described 120-bp genomic DNA sequence (64). The alignment starts at the breakpoint of gene duplications for TNXA-TNXB. The alignment reveals a picture for the original DNA recombination leading to the reciprocal recombinant sequences in E1XB-A and L1XA-B (Fig. 7I). There are nine single nucleotide changes or informative sites (marked by asterisks) which can differentiate TNXA from TNXB. A Chi sequence related to DNA recombination in bacteriophage lambda  is located at nucleotides 450-457. Five of the TNXA/TNXB informative sites (nucleotides 14, 60, 63, 73, and 278) are 5' to the Chi site. The other four sites (nucleotides 2234, 2268, 2273, and 2423) are 3' to the Chi site and they are flanking the 120-bp deletion. For E1XB-A, the sequence is TNXB-specific for the first five informative sites but is TNXA-specific for the last four sites. It also acquires the TNXA-specific 120-bp deletion (Fig. 7II). This suggests that E1XB-A is the result of a recombination between TNXB and TNXA, occurring between nucleotides 278 and 2234. 


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Fig. 6.   I, a model for an unequal crossover between a bimodular RCCX chromosome and a monomodular chromosome to generate a TNXB/XA recombinant and a TNXA/XB recombinant; II, PCR strategy to amplify the breakpoint region of gene recombination and its corresponding exon-intron structure.


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Fig. 7.   I, an alignment of the normal and recombinant TNXA and TNXB genomic DNA sequences. Only the sequences around the informative sites are shown. The nucleotide numbering starts at the breakpoint region (boldface and underlined) of the TNXB and TNXA gene duplication. The normal TNXB sequences, TNXB-H (GenBankTM accession no. U89337) and TNXB-M (GenBankTM accession no. X71937), are taken from Footnote 2 and Ref. 38, respectively. The normal TNXA sequences, TNXA-Y (GenBankTM accession no. L20263) and TNXA-M (GenBankTM accession no. S38953) are taken from Ref. 30 and Ref. 38, respectively. E1XB-XA was generated by this work. L1XA-XB (GenBankTM accession no. AF077974) is from Ref. 72. TNXA- and TNXB-specific sequences or informative sites are marked by asterisks. II, a schematic diagram showing the reciprocal locations of TNXA and TNXB informative sites in the TNX recombinants of CAH-E1 and JRA-L1. Informative sites are shown as vertical strokes; an open box represents the 120-bp deletion. The continuous DNA sequence for the breakpoint region of the TNXB/TNXA recombinant (E1XB-A) is available from GenBankTM under the accession number AF086641.

For L1XA-B, it has sequences characteristic of TNXA at the first five informative sites but has sequences characteristic of TNXB at the last four sites and without the 120-bp deletion. This aberrant TNXA sequence in L1XA-B appears to be the reciprocal product of E1XB-A resulting from DNA recombination between TNXA and TNXB.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we show definitively that in human MHC class III region, four tandemly arranged genes serine/threonine kinase RP, complement C4, steroid 21-hydroxylase CYP21, and tenascin TNX are organized as a genetic unit designated as an RCCX module. In a monomodular RCCX haplotype, the "full-length" genes RP1 and TNXB are always present, implying relevant cellular functions for RP1 and for TNXB. In an RCCX bimodular haplotype, duplication of the RCCX module occurs by the addition of a C4 gene and a CYP21 gene together with the TNXA and RP2 gene segments. This additional, modular genomic fragment is either 32.5 or 26.2 kb in size, depending on whether the C4 gene contains the endogenous retrovirus HERV-K(C4). The three pseudogenes/gene segments, CYP21A, TNXA, and RP2, present between the two C4 loci, probably do not encode for functional protein products. The concurrent deletions of a C4A or C4B gene with a CYP21A or CYP21B gene is a well established phenomenon (25, 26). This report provides the detailed documentation for the modular deletions or duplications of RP and TNX genes together with C4 and CYP21.

Although this multiple-gene modular variation observed in RCCX is uncommon in mammalian genetics, a similar phenomenon with concurrent variations of at least three tandem genes has been observed in a genomic region at chromosome 5q12-13 (reviewed in Ref. 55). The three genes are BTFp44 (a p44 subunit of transcriptional factor TFIIH), NAIP (neuronal apoptosis inhibitory protein), and SMN (survival motor neuron). One, two, or three modules of these three genes (each module spans about 500 kb in size) are present in the population (56). The divergence of sequences in C4A and C4B is analogous to that in SMNT and SMNC; the presence of the pseudogene CYP21A and the functional CYP21B is analogous to the pseudogene NAIPDelta (with deletion of exon 5) and the intact NAIP. The absence of the SMNT gene is associated with most spinal muscular atrophy, and deletion of the NAIP gene in most severe forms of spinal muscular atrophy (57).

The frequency of the RCCX modular variations has been studied in a population of 150 normal Caucasian females. It has been discovered that 75.4% of the C4 genes are long, and 24.6% are short. Bimodular and monomodular RCCX organizations are present in about 71.6 and 16.2% of the chromosome 6, respectively. Trimodular RCCX haplotypes have a frequency about 12.2% (58).3 Excluding the trimodular haplotypes (2, 59), there are four major RCCX modular structures in the Caucasian population: bimodular long-long (L-L), bimodular long-short (L-S), monomodular long (L), and monomodular short (S) (Fig. 8). These four RCCX structures can be detected conveniently by TaqI RFLPs. The widely applied TaqI RFLP analysis of C4 genes yields information on the combination of RP1 or RP2 with C4(L) or C4(S). It does not yield definitive information, however, on whether the C4 gene codes for C4A or C4B proteins. The information for the presence of C4A and C4B genes may be obtained by NlaIV RFLP analysis or by direct DNA sequencing. From these four RCCX organizations, 12 haplotypes for RP1/RP2, C4A/C4B, CYP21A/CYP21B, TNXA/TNXB are observable in the normal and in disease populations. Six of these haplotypes are more common in the normal population and they are highlighted (haplotypes 1, 2, 5, 9, 10, and 12). RCCX bimodular haplotypes with two C4B genes (haplotypes 4 and 8) are yet to be shown definitively. Bimodular haplotypes with two CYP21A genes (haplotypes 3 and 6) and a monomodular haplotype with a CYP21A gene (haplotype 11) are present in CAH patients. In two Brazilian tribes, a fifth RCCX organization with two short C4 genes is found (60). This bimodular C4(S)-C4(S) combination (haplotype 13) is extremely rare in other ethnic groups.


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Fig. 8.   RCCX modular variations in the human population. Monomodular and bimodular RCCX structures with 13 different haplotypes of RP, C4, CYP21, and TNX gene combinations are shown. The common haplotypes are in boldface type. The bimodular and monomodular haplotypes are present in 71.6 and 16.2% of the normal Caucasian population (C.A. Blanchong and C. Y. Yu, manuscript in preparation). Disease haplotypes associated with CAH are marked with asterisks. 21A, CYP21A; XA, TNXA. The presence of haplotypes 4 and 8 (indicated by question marks) has not been definitively demonstrated.

The diversities in the number and size of the RCCX modules probably contribute to the genetic variability and instability of the HLA class III region. A recombination between a bimodular RCCX chromosome and a monomodular RCCX chromosome may lead to the exchange or homogenization of polymorphic or mutant sequences between complement C4A and C4B loci. This has been demonstrated by the presence of the C4A-associated amino acid residues in many C4B allotypes, and vice versa. The typical examples are the reverse associations of Ch1 antigenic determinant with C4A1 and C4A13 and of Rg1 antigenic determinant with C4B5 (22, 61, 62). It was also shown that in a systemic lupus erythematosus patient, there is an acquisition of a 2-bp insertion in exon 29 from C4AQ0 to C4BQ0 (63, 64). The same type of recombination may also lead to the acquisition of mutations from pseudogene CYP21A or gene segment TNXA to their corresponding functional genes (65, 66). This is manifested in many disease-associated haplotypes such as the presence of two CYP21A pseudogenes in RCCX bimodular haplotypes (65) and a CYP21A/CYP21B hybrid gene in the HLA haplotype A3 B47 DR7 with RCCX monomodular structure of CAH patients (67, 68). Another example comes from the presence of a TNXB/TNXA hybrid gene with the 120-bp deletion together with the deletion of the RP2-C4B-CYP21B gene in CAH patient(s), as demonstrated in this paper.

In CAH patient E1, the 120-bp deletion in the TNXB-XA recombinant will cause a premature termination and therefore truncation of the carboxyl-terminal sequences. The truncation includes three fibronectin type III repeats (with four N-linked glycosylation sites) and the entire fibrinogen domain. This mutation may diminish or knockout the function of TNX. The haplotype for the recombinant chromosome with monomodular RCCX characterized the presence of a CYP21A pseudogene linked to the TNXB/XA hybrid is HLA A3 B35 DR1, RP1 C4A3 CYP21A TNXB-XA.

Three independent observations on the deletions of the CYP21B genes, which could have arisen by a mechanism similar to that for haplotype b of CAH-E1, were reported. In the first study, a salt-losing CAH patient was found to have haplotype HLA B35 DR1, the presence of a single C4A3 gene and a CYP21A gene, and a deletion of C4B with CYP21B and (69). This is similar to haplotype b of CAH-E1. Whether this patient has a recombinant TNXB-XA in the monomodular RCCX was not determined.

In the second study, a de novo deletion of C4B together with CYP21B was suggested to derive from a meiotic unequal crossover between the maternal homologous chromosomes (70). From our current knowledge, this de novo deletion probably resulted from an unequal crossover between TNXA from a bimodular L-S chromosome (HLA A30 B13 DR7) and TNXB from a monomodular S chromosome (HLA A1 B8 DR3). The recombinant has a monomodular L chromosome (HLA A30 B13 DR3) with CYP21A and a 2.4-kb TaqI fragment at its 3'-end, which is indicative of a 120-bp deletion in the TNXB gene.

In the third study, one of the chromosomes 6 in a CAH patient appears to have a monomodular RCCX with the 120-bp deletion in TNXB. The other chromosome 6 has a bimodular RCCX with two CYP21A genes and no CYP21B gene, as revealed by Southern blot analyses using TaqI-, BglII-, and BssHII-digested genomic DNA. Immunoblot analysis showed that this patient did not produce any TNXB protein. Therefore, both TNXB genes from the homologous chromosomes were presumed to be nonfunctional. Since the patient suffers Ehlers Danlos syndrome in addition to CAH, it is proposed that malfunction of TNXB is associated with the connective tissue disease (71).

Another important piece of evidence for the described unequal crossover comes from studies on the molecular genetics of a pauciarticular JRA patient in our laboratory. This JRA patient has an RCCX bimodular haplotype with two CYP21B genes and a 5'-TNXA/XB-3' hybrid with the TNXA-specific truncation of exons 1-32 at the 5' region, and the presence of the TNXB-specific 120-bp sequence at the 3' region (72). The TNXA-XB hybrid appears to be the reciprocal product in CAH-E1 5'-TNXB/XA-3', attributable to the genetic recombination between TNXA and TNXB. This notion is substantiated by the reciprocal associations of the informative sites for TNXB and for TNXA at the two ends of the hybrid sequences (Fig. 7).

In the bimodular (or trimodular) RCCX haplotypes, one of the duplicated genes or gene segments could undergo sequence mutations without the immediate deleterious effect of knocking out the gene function. The RCCX modular variations in the population allowed sequence variations and enhanced the incorporation of diversified or mutant sequences among the paralogous genes, pseudogenes, or gene segments. The selection advantage is probably the emergence of various polymorphic forms of complement C4A and C4B to tackle different microbial antigens (73). The burdens are the accompanying genetic or autoimmune diseases such as CAH, systemic lupus erythematosus, and possibly EDS, caused by unequal crossovers and incorporations of deleterious mutations in the constituents of the RCCX.

In the HLA class II region between DRB1 and DRA genes, there may be 1-3 DRB pseudogenes. In addition, DRB3, DRB4, or DRB5 can be present (74). The DR locus is about 350 kb centromeric to the RCCX modules. Between heterozygous chromosomes with different DR gene number and RCCX modules, misalignments and unequal crossovers would occur during meiosis. This may result in deletion or duplication of structural genes between these two variable regions. More than 10 structural genes with important functions have been discovered between DR and RCCX (37).2 Recombinant chromosomes with the essential structural genes deleted would be lethal. Therefore, the apparent productive recombination frequencies between certain MHC haplotypes would be less than expected. This would have contributed to the linkage disequilibrium of the MHC genes, since many of the "ancestral" MHC haplotypes remain largely conserved in the population.

    ACKNOWLEDGEMENTS

We are indebted to Dr. Joann Moulds for kind instruction in C4 allotyping techniques, the Ohio State University Tissue Typing Laboratory for HLA typing, Bi Zhou for excellent technical assistance, and Dr. Arthur Burghes for helpful discussion.

    FOOTNOTES

* This work was supported by NIAMSD, National Institutes of Health (NIH), Grant R01 AR43969; March of Dimes Birth Defects Foundation Grant FY95-1087 (Basil O'Conner Starter Scholar Research Award); Children's Hospital Research Foundation (Columbus, OH) Grant 210698; and the Pittsburgh Supercomputing Center through the NIH Center for Research Resources Cooperative Agreement (Grant 1P41 RR06009) (to C. Y. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel Supported by an Ohio State University Presidential Fellowship.

§§ To whom all correspondence should be addressed: Room W208, Children's Hospital Research Foundation, 700 Children's Dr., Columbus, OH 43205. Tel.: 614-722-2821; Fax: 614-722-2774; E-mail: cyu{at}chi.osu.edu.

2 L. Rowen, C. Dankers, D. Baskin, J. Faust, C. Loretz, M. E. Ahearn, A. Banta, S. Schwartzell, T. M. Smith, T. Spies, and L. Hood, GenBankTM accession no. U89337.

3 C. A. Blanchong and C. Y. Yu, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: MHC, major histocompatibility complex; RCCX, RP, complement C4, steroid 21-hydroxylase CYP21, and tenascin TNX; CAH, congenital adrenal hyperplasia; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction; JRA, juvenile rheumatoid arthritis.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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