The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jaatinen, T.
Right arrow Articles by Lokki, M.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jaatinen, T.
Right arrow Articles by Lokki, M.-L.
The Journal of Immunology, 2002, 168: 5652-5658.
Copyright © 2002 by The American Association of Immunologists

Characterization of a De Novo Conversion in Human Complement C4 Gene Producing a C4B5-Like Protein1

Taina Jaatinen*, Miia Eholuoto*, Tarja Laitinen{dagger} and Marja-Liisa Lokki2,*

* Department of Tissue Typing, Finnish Red Cross Blood Transfusion Service, Helsinki, Finland; and {dagger} Department of Medical Genetics, University of Helsinki, Helsinki, Finland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Complement C4 is a highly polymorphic protein essential for the activation of the classical complement pathway. Most of the allelic variation of C4 resides in the C4d region. Four polymorphic amino acid residues specify the isotype and an additional four specify the Rodgers and Chido determinants of the protein. Rare C4 allotypes have been postulated to originate from recombination between highly homologous C4 genes through gene conversions. Here we describe the development of a de novo C4 hybrid protein with allotypic and antigenic diversity resulting from nonhomologous intra or interchromosomal recombination of the maternal chromosomes. A conversion was observed between maternal C4A3a and C4B1b genes producing a functional hybrid gene in one of the children. The codons determining the isotype, Asp1054, Leu1101, Ser1102, Ile1105 and His1106, were characteristic of C4B gene, whereas the polymorphic sites in exon and intron 28 were indicative of C4A3a sequence. The protein produced by this hybrid gene was electrophoretically similar to C4B5 allotype. It also possesses reversed antigenicity being Rodgers 1, 2, 3 and Chido-1, -2, -3, 4, -5, and -6. Our case describes the development of a rare bimodular C4B-C4B haplotype containing a functional de novo C4 hybrid gene arisen through gene conversion from C4A to C4B. Overall the data supports the hypothesis of gene conversions as an ongoing process increasing allelic diversity in the C4 locus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The complement component C4 has an essential role in the activation of classical and mannan-binding-lectin pathways of complement. Two functionally distinct C4 isotypes, C4A and C4B, are found in plasma. C4A reacts preferentially with amino groups and contributes to immunocomplex clearance in concert with complement receptors on erythrocytes and phagocytes (1, 2). C4B reacts efficiently with hydroxyl groups and has more hemolytic activity than C4A. Isotype specificity is defined by four amino acid differences at positions 1101–1106 within exon 26. C4A has the sequence PCPVLD, whereas LSPVIH is specific for C4B (isotype-specific amino acids indicated with bold) (3, 4).

C4 proteins are among the most polymorphic molecules in serum. C4 is expressed as single-chain precursor, which is cleaved to three-chain structure linked with disulfide bonds. This processing is incomplete, resulting in multiple structural isoforms. In addition to structural variation, >20 allotypes of both C4A and C4B have been found resulting in 24 polymorphic amino acid residues, including the isotypic residues (5, 6). Most of the polymorphic sites are found in the C4d region. Amino acid variations in the C4d region constitute sequential and conformational Rodgers (Rg)3/Chido (Ch) (3) blood group Ags (7). Rg and Ch are not true blood group Ags as they are not located on intrinsic erythrocyte structures, but become bound as covalently attached C4d fragments from plasma. The relevant sequences reside within exons 25, 26, and 28 of the C4 gene. Rg determinants are traditionally associated with C4A and Ch determinants with C4B, but allotypes manifesting reversed antigenicity indicate shuffling of the corresponding sequences between the C4 genes.

The C4 genes are located on 6p21.3 and display substantial variation on genomic level. C4 genes present dichotomous size variation due to a 6.4-kb insertion of human endogenous retrovirus HERV-K(C4) in intron nine. In Caucasians, C4A genes are ordinarily long (20.6 kb) containing the retrovirus. C4B genes can be either long (20.6 kb) or short (14.2 kb) (6). The majority of people have two C4 loci per chromosome encoding C4A and C4B isotypes, but the number of expressed genes can vary from none to four. Bimodular structure with two C4 genes results from ancient duplication of tandemly arranged genes RP, C4, CYP21, and TNX. These four genes form a genetic module, RCCX (8, 9), and usually the deletions and duplications of the C4 genes are accompanied by other genes within RCCX. It is thought that this modular variation causes misalignments and unequal crossovers leading to gene rearrangements. Homology between C4A and C4B genes promotes nonhomologous pairing of these genes, and may result in exchange and homogenization of the polymorphic sequences or hybrid gene formation. Ancient crossover events between C4A and C4B genes are thought to explain infrequent C4A1 and C4B5 allotypes with mixed Rg/Ch Ag patterns (10, 11).

The dynamic nature of the C4 gene region might be a positively selected mechanism creating immunological diversity against evolving microbial structures. In contrast, gene rearrangements and deletion haplotypes predisposing to diseases is the cost of instability. C4 null alleles, C4AQ0/C4BQ0, are common and associate with numerous autoimmune and infectious diseases (12, 13, 14).

Here we describe a functional de novo C4 hybrid gene resulting from nonhomologous recombination. The proband expresses a functional C4B protein not inherited from either of the parents. Our results indicate an exchange of genetic information between maternal C4A and C4B genes generating a hybrid gene. Functionally the protein produced by the hybrid gene is similar to C4B5. Our observations reveal a conversion from C4A to C4B increasing diversity in C4 genes and promoting antigenic variability.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects

The proband, his parents, and two siblings were studied. Initially the family was identified by a study on Finnish couples with a history of recurrent spontaneous abortions in early pregnancy (15). Paternity was confirmed by serological HLA typing. The calculations show very high probability values of 99.5% for the proband and his HLA identical sister and 99.6% for the brother, even based on the HLA typing only.

HLA and complement typing

Serological HLA-A, -B, -C, and -DR typing was performed from peripheral blood lymphocytes with two-stage microlymphocytotoxicity test (16). DRB1 genes were analyzed by Taq{alpha}I RFLP (17) and by using LIPA HLA-DRB1 kit (Innogenetics, Zwijndrecht, Belgium). Complement factor B and C4 allotypes were determined from serum samples by electrophoretic segregation (18, 19). The typings have been repeated from independently drawn blood samples.

Detection of hemolytic activity of C4B allotypes

The hemolytic activity of C4B allotypes was studied by applying an overlay of sensitized sheep red blood cells and C4 deficient guinea pig serum on agarose gel after allele segregation by electrophoresis (20). The relative hemolytic activity was detected by densitometric scanning using a 5301 Preference Densitometer (Pharmacia, Uppsala, Sweden).

DNA preparation

Genomic DNA was extracted from peripheral blood samples by salting out method (21).

Southern blot analysis

Genomic DNA was digested with Taq{alpha}I and subjected to Southern blot analysis according to the technique described by the 10th International HLA Workshop (22). Hybridizations were performed using pAT-A and pC21/3c probes for C4 and CYP21 genes, respectively (23, 24).

Screening for the known 2-bp mutation in C4A and C4B genes

To detect a 2-bp insertion in exon 29, 5880–5881insTC, originally described by Barba et al. (25), a mutation-specific primer C4ins29 was used with isotype-specific primers A-down and B-down (Table IGo) (26). PCR was performed under standard reaction conditions with AmpliTaq Gold DNA polymerase (Roche Molecular Systems, Branchburg, NJ) and cycle conditions of 30 s at 94°C, 45 s at 62°C, 1 min at 72°C for 38 cycles on GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA).


View this table:
[in this window]
[in a new window]
  		Table I. PCR and sequencing primers

 
Isotype-specific PCR

Isotype specificity of the C4 genes is based on nucleotide differences in exon 26. With isotype-specific PCR the C4A or C4B gene can be amplified in two fragments. A-down/B-down with C4-e41R amplifies a 6.6-kb fragment downstream from exon 26 (Table IGo). A-up/B-up together with C4-e1F produces a 14-kb PCR fragment for C4A and long C4B genes, and a 7.7-kb fragment for short C4B genes. The PCR amplification was conducted with Expand High Fidelity PCR System (Roche Molecular Biochemicals, Mannheim, Germany). The reaction conditions in a final volume of 50 µl were 0.2 mM dNTPs, 0.3 µM each primer, 100 ng of genomic DNA, and 2.6 U of High Fidelity DNA polymerase. The PCR programs for isotype-specific fragments were performed on GeneAmp PCR System 9700 and are listed in Table IIGo. Amplified DNA was visualized by electrophoresis on 0.8% agarose gel containing ethidium bromide.


View this table:
[in this window]
[in a new window]
  		Table II. Protocols for C4 isotype specific PCR (GeneAmp PCR System 9700; Applied Biosystems)

 
Amplification of the C4d region

The C4d region of the C4 genes of the proband was studied. A 1.5-kb fragment spanning from exon 25 to exon 29 was amplified by PCR using C4-specific primers C4-i24F and C4-i29R (Table IGo). The reaction volume of 100 µl consisted of 500 ng of genomic DNA, PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl2), 0.2 mM dNTPs, 0.3 µM primers, and 2.5 U of AmpliTaq DNA polymerase. Amplification was performed using the following program: 10 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 30 s, and elongation at 72°C for 1 min. The following 20 cycles were 94°C for 30 s, 60°C for 30 s, and elongation at 72°C for 1 min with 5 s time increment in every successive cycle. The size of the amplification product was confirmed with agarose gel electrophoresis using ethidium bromide staining.

Cloning

Isotype-specific fragments were ligated into pCR-XL-TOPO vector for cloning according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). C4d-specific PCR products were cloned into pCR2.1 vector using the Original TA Cloning kit (Invitrogen). The isotype of the cloned inserts was determined by A and B specific direct colony PCR with primers A-up/B-up and L3 (Table IGo) (26, 27). Plasmid DNA was purified from 5 ml overnight cultures by QIAprep Spin Miniprep kit (Qiagen, Chatsworth, CA).

Sequencing

Sequencing reactions were performed with BigDye Terminator Cycle Sequencing Ready Reaction kit version 2.0 (Applied Biosystems). Sample electrophoresis was performed on ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Sequencing of the isotype-specific fragments was performed using primers M13 forward (-20), M13 reverse, and C4-specific primers (Table IGo).

Determination of parental C4 allotypes

C4 allotypes of the parents were confirmed by sequencing the polymorphic sites in exons 28 and 29. A 1-kb PCR fragment was amplified with primers A-down/B-down and C4-i29R (Table IGo) using AmpliTaq Gold DNA polymerase in standard PCR conditions with 38 cycles of denaturation at 94°C for 30 s, annealing at 63°C for 45 s, and extension at 72°C for 1 min. The amplicons were used in direct sequencing as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inheritance of MHC haplotypes

The C4 allotypes of the family members are indicated in Fig. 1Go, and HLA haplotypes and complotypes are presented in Fig. 2Go. The father and the mother shared the C4A3 allotype. Allotyping and sequencing of the polymorphic sites in exons 28 and 29 showed that the mother had a C4A3a and a C4B1b gene in both chromosomes. The father carried a C4B2 gene in both chromosomes, and the paternal haplotype a contained the gene for C4A3a. In the paternal haplotype b the C4A gene carried a 2-bp insertion in exon 29, inherited by the brother only (Fig. 3Go). According to segregation of MHC haplotypes (Fig. 2Go), the proband was expected to show C4A3a,B2 inherited from the father, and C4A3a,B1b inherited in the maternal haplotype c. However, in addition to the C4A3a, C4B1b, and C4B2 allotypes, the proband showed an extraordinary C4 protein which was hemolytically intact and had electrophoretic mobility similar to C4B5 control (Fig. 4Go). The HLA identical sister of the proband had only two hemolytically active proteins, C4B1b and C4B2, as expected.



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 1. C4A and C4B allotypes. The samples are in the following order: father (Fa), mother (Mo), C4B5 positive control (Co), proband (Pr), sister (Si), and brother (Br). The extraordinary C4B5-like protein of the proband is indicated with an arrow.

 


View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 2. HLA haplotypes and complotypes. The complotypes represent the phenotypes determined by allotyping. C4A proteins are not produced by the genes on proband’s maternal haplotype c (C4AQ0 in allotyping), whereas a novel C4B5-like protein (indicated with bold), not seen in any other family members, is observed.

 


View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 3. Mutation-specific PCR for the 2-bp insertion in exon 29 of C4 gene. The mutation-specific amplification product of 787 bp was detected in the C4A gene of the father (Fa), brother (Br), and two positive controls (Co), but not in the proband (Pr), mother (Mo), or sister (Si). The mutation accounts for the C4AQ0 in haplotype b.

 


View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 4. Hemolytic assay of the C4B proteins. The hemolytic activity of C4 allotypes is shown on the y-axis. The height of the peak corresponds to the hemolytic activity of the C4B5, C4B2 and C4B1 allotypes. Location of the hemolytic peak for each allotype is shown on the x-axis. The control has the allotypes C4B5 and C4B1. The proband shows three hemolytic peaks corresponding to C4B5, C4B2, and C4B1, whereas the HLA identical sister has only C4B2 and C4B1 proteins.

 
Genomic organization of C4 and CYP21 genes

All family members had the 7.0 kb fragment corresponding to RP1-C4 long gene locus. The father carried only the 5.4-kb fragment specifying the RP2-C4 short gene locus, whereas the mother had the 6.0-kb fragment for the RP2-C4 long gene locus. All three children were heterozygous carrying both short and long RP2-C4 gene loci. RP1 and RP2 genes flank the C4 loci and are partially included in the Taq{alpha}I restriction fragments. The 3.7-kb fragment and the 3.2-kb fragment corresponding to CYP21B and CYP21A genes, respectively, were detected in all family members. The Southern blot analysis established the presence of two genes for C4A, C4B, CYP21A, and CYP21B, and indicates no obvious structural gene rearrangements in the region (Fig. 5Go).



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 5. Southern blot analysis. Taq{alpha}I RFLP fragments detected with C4- and CYP21-specific probes indicate the presence of bimodular RCCX in all family members. The 7.0-, 6.0-, and 5.4-kb fragments represent RP1-C4 long, RP2-C4 long, and RP2-C4 short genes, respectively. The 3.7- and 3.2-kb fragments correspond to CYP21B and CYP21A genes, respectively. The samples are in the following order: brother (Br), sister (Si), proband (Pr), mother (Mo), and father (Fa). It should be noted that the 5.4-kb fragment tends to transfer better than the 6.0-kb fragment resulting in slightly stronger hybridization of the fragment in Southern blots.

 
Identification of the C4B genes of the proband

Three different C4B clones were found by sequencing the 6.6-kb C4B downstream fragments. Interestingly, one clone carried C4A3a-specific polymorphic sites in exons 28 and 29 coding for amino acids in positions 1157, 1182, 1188, 1191, and 1267 (Table IIIGo). The rest of the clones showed sequences corresponding with the allotypes C4B1b and C4B2, which share polymorphic codons in exons 28 and 29 (28). The latter C4B clones could be separated based on a cgctcc/ggctc{Delta} ({Delta} deleted base) polymorphism at positions 14 and 19 in intron 28 (29). To detect other polymorphisms or mutations in the three types of C4B isotypic downstream fragments, exons 27 to 41 were sequenced. No mutations were found. In conclusion, the sequencing results of the C4B genes of the proband indicated an exchange of genetic sequences producing a C4B gene that had been converted from a C4A gene (Fig. 6Go). This aberrant gene produced an electrophoretically and hemolytically C4B5-like protein.


View this table:
[in this window]
[in a new window]
  		Table III. Amino acid differences in C4 allotypes of the studied family1

 


View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 6. Schematic representation of proband’s haplotypes. The upper part of the figure indicates the parental chromosomes inherited by the proband. The lower part of the figure displays the sequenced part of the C4d region of the converted gene (C4B5-like), outlined with a gray box within the gene. Single letter codes indicate the polymorphic amino acids and the origin of these polymorphic sites is marked above. Position 1186 presents a silent polymorphism in the codon coding for alanine. The polymorphisms in introns 28 and 29 are indicated with lowercase. The polymorphic site 207 in intron 28 differentiates the parental C4 genes in RP1-C4 long locus. The converted gene of the proband carries t at position 207, seen also in maternal C4A3a genes. The paternal C4A3a gene has c at the corresponding position indicating the maternal origin of the converted gene. Deduced Rg/Ch epitopes are marked below. The 3' recombination break point is between sites His1106 and Asn1157, and the 5' break point region probably lies between Asp1054 and Leu1101. However, the polymorphic site Asp1054 corresponds to both C4A3a and C4B2.

 
The break point region and origin of the converted gene

To characterize the isotype-specific region of the proband’s C4 genes, a C4d fragment containing ten reported polymorphic sites (Table IIIGo) was cloned and sequenced. C4A and C4B clones were separated with rapid colony screening by isotype-specific PCR. All 11 C4A clones were identical and had the allotype C4A3a. The sequence of 10 C4B clones revealed that the proband carried C4B1b and C4B2 genes corresponding to the results from allotyping (Fig. 1Go) and hemolytic studies (Fig. 4Go). Third type of C4B clones had codons Leu1101, Ser1102, Ile1105, and His1106 in exon 26 specific for the C4B isotype. However, in exon 28 the sequence downstream from the polymorphic site Asn1157, including Val1188 and Leu1191, was identical with C4A3a. Position 1186 presents a silent polymorphism of the third base in the codon coding for alanine, which was seen in all C4A3a genes. Thus, the 3' recombination break point is between sites His1106 and Asn1157. The polymorphic site in exon 25 was Asp1054 corresponding to C4A3a and C4B2, hence the 5' break point region probably lies between Asp1054 and Leu1101. The converted gene carried Rg 1, 2, 3 and Ch -1, -2, -3, 4, -5, -6 antigenic determinants (Fig. 6Go). The sequence of the converted gene was verified from three clones. The results confirmed that a functionally active C4B5-like gene had evolved through gene conversion mechanism.

To conclude the origin of the conversion, the isotype-specific sites from exon 26 to intron 29 were sequenced in both parents. In intron 28 of C4A gene we found a polymorphic site at the position 207, where the father had c and the mother had t. The C4B genes of both parents showed c at the corresponding position. In the proband, converted C4B5-like gene carried t confirming the maternal origin of C4A3a. The proband had c in his other C4A and C4B genes. The converted gene carried the motif cgctcc in intron 28. This motif was also present in maternal and paternal C4A3a allotypes.

Interestingly, the motif was detected in the maternal C4B1b allotype as well. In C4B isotype, this polymorphism has previously been found in C4B3 and C4B5 allotypes only (11), suggesting that the motif is not isotype nor allotype specific. At the position 25 in intron 29, the C4B5-like gene of the proband shared g with maternal C4A genes, whereas the father was heterozygous having either c or g (Fig. 6Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Duplications and gene conversions have been shown to account for allelic diversity in the HLA Ags (30). However, gene conversions and unequal crossover events can also promote homogeneity by transferring sequence motifs between homologous loci. Both mechanisms are active in the MHC class III region (31, 32). Homoexpression of two C4B genes on a bimodular RCCX is extremely rare (0.67%) compared with that of C4A-C4A haplotypes (13.3%) in the Caucasian population (33). The converted genes in these homoexpression haplotypes produce hybrid proteins possessing reversed antigenicity. Structural variation in RCCX causes misalignments and unequal exchange of genetic information resulting in deletion and duplication haplotypes of the C4 and CYP21 loci. Erroneous alignment can create hybrid genes, which have been reported between human TNXA and TNXB, or CYP21A and CYP21B (34, 35).

Genetic instability through deletions and loss-of-function mutations has been revealed in MHC class III-associated disease conditions. Unequal pairing of maternal monomodular and bimodular haplotypes have been shown to cause the deletion of CYP21B and C4B genes in a congenital adrenal hyperplasia patient (36). Point mutations in the CYP21B gene have been found to result from microconversion between the pseudogene and its functional counterpart (37). Among congenital adrenal hyperplasia patients, the reported frequency of de novo conversions is high in the intron 2 of the CYP21B gene (38). Also, a recombination event has been shown to produce a double null haplotype (C4AQ0,C4BQ0) leading to increased susceptibility to infections (39).

Successful recombinations often escape detection if phenotype is not altered. In the present study, the recombination between maternal alleles produced a novel, hemolytically active C4B5-like hybrid gene in the proband. Southern blots indicated that the parents and the proband had the C4 and CYP21 genes organized in a bimodular fashion. The hybrid gene contained the polymorphic sites in the exon 28, intron 28, and intron 29 identical with the maternal C4A3a allotype, while the isotype determining sequence was specific for C4B (exon 26). Due to the homology of maternal C4B alleles (Fig. 2Go), we were not able to distinguish between intrachromosomal or interchromosomal gene conversions. However, maternal chromosomes have revealed intrachromosomal recombinations, whereas interchromosomal recombinations have been shown to take place preferably in spermatogenesis (40). In addition, maternal sexual preference in conversion events has been reported in mouse H-2 genes (41). Our results suggest that the 3' recombination breakpoint resides between codons 1106 in the exon 26 and 1157 in the exon 28, contrary to previously reported codons 1157 and 1186 in the exon 28 (11). Because the Asp (1054) in the exon 25 was not informative in this family, the 5' breakpoint could not be located precisely.

The hybrid protein displayed electrophoretic and functional characteristics similar to C4B5 allotype having partially reversed antigenicity relative to Rg and Ch epitopes (42). The function of Rg/Ch determinants is still obscure. The increased number of Rg/Ch Ags on old erythrocytes suggests that they may play a role in removal of aged erythrocytes (43). They may also mediate the clearance of immune complexes through the attachment to complement receptors on erythrocytes (2). C4B5 is much more common among the Japanese population (8.8%) (44) than among the Caucasians, in which C4B5 is most often found in the haplotype HLA-B55, C4A4 (45). A C4B5 with Rg 1, 2, 3 and Ch -1, -2, -3, 4, -5, -6, identical to our case, has been reported but not confirmed from sequence data (46), and would be called C4B*0508 (47). The C4B5Rg+ subtype associates also with HLA-B60 (40). C4 deletions and mutations have been found in this haplotype (25, 48). In the studied family, the father expressed recurrent spontaneous abortion risk haplotypes (15). Certain HLA haplotypes may be prone to rearrangements leading to unfavorable rearrangements. The de novo mutation of maternal origin described here serves as an example for successful rearrangement. Even though the mechanism for the genesis of this de novo mutation cannot be fully elucidated, it provides evidence for high incidence of instability in the C4 gene region resulting in variable C4 genes and in the generation of rare C4 allotypes.


   Acknowledgments
 
We thank the proband and his family for their interest and willingness to take part into this study. We thank also the staff of the Department of Tissue Typing and especially Marjaana Mustonen for her technical assistance. The collaboration with Dr. Maija Tulppala (Infertility Clinic of Helsinki, Helsinki, Finland) is gratefully acknowledged. We thank Dr. Yung Yu for providing C4 primers to our laboratory.


   Footnotes
 
1 This study was supported by the Medical Research Fund of Finnish Red Cross Blood Transfusion Service. Back

2 Address correspondence and reprint requests to Dr. Marja-Liisa Lokki, Department of Tissue Typing, Finnish Red Cross Blood Transfusion Service, Kivihaantie 7, 00310 Helsinki, Finland. E-mail address: maisa.lokki{at}bts.redcross.fi Back

3 Abbreviations used in this paper: Rg, Rodgers blood group Ag; Ch, Chido blood group Ag. Back

Received for publication November 5, 2001. Accepted for publication March 27, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schifferli, J. A., G. Steiger, J. P. Paccaud, A. G. Sjöholm, G. Hauptmann. 1986. Difference in the biological properties of the two forms of the fourth component of human complement (C4). Clin. Exp. Immunol. 63:473.[Medline]
  2. Birmingham, D. J., L. A. Herbert. 2001. CR1 and CR1-like: the primate immune adherence receptors. Immunol. Rev. 180:100.[Medline]
  3. Belt, K. T., M. C. Carroll, R. R. Porter. 1984. The structural basis of the multiple forms of human complement component. Cell 36:907.[Medline]
  4. Yu, C. Y., K. T. Belt, C. M. Giles, R. D. Campbell, R. R. Porter. 1986. Structural basis of the polymorphism of human complement components C4A and C4B: gene size, reactivity and antigenicity. EMBO J. 5:2873.[Medline]
  5. Mauff, G., B. Luther, P. M. Schneider, C. Rittner, B. Stradmann-Bellinghausen, R. Dawkins, J. M. Moulds. 1998. Reference typing report for complement component C4. Exp. Clin. Immunogenet. 15:249.[Medline]
  6. Blanchong, C. A., E. K. Chung, K. L. Rupert, Y. Yang, Z. Yang, B. Zhou, J. M. Moulds, C. Y. Yu. 2001. Genetic, structural and functional diversities of human complement components C4A and C4B and their mouse homologues, Slp and C4. Int. Immunopharmacol. 1:365.[Medline]
  7. Yu, C. Y., R. D. Campbell, R. R. Porter. 1988. A structural model for the location of the Rodgers and the Chido antigenic determinants and their correlation with the human complement component C4A/C4B isotypes. Immunogenetics 27:399.[Medline]
  8. Kawaguchi, H., C. O’Huigin, J. Klein. 1993. Evolution of primate C4 and CYP21 genes. J. Klein, and D. Klein, eds. Molecular Evolution of the Major Histocompatibility Complex 357. Springer-Verlag, Berlin Heidelberg.
  9. Shen, L., L. C. Wu, S. Sanlioglu, R. Chen, A. R. Mendoza, A. W. Dangel, M. C. Carroll, W. B. Zipf, C. Y. Yu. 1994. Structure and genetics of the partially duplicated gene RP located immediately upstream of the complement C4A and the C4B genes in the HLA class III region: molecular cloning, exon-intron structure, composite retroposon, and breakpoint of gene duplication. J. Biol. Chem. 269:8466.[Abstract/Free Full Text]
  10. Giles, C. M., J. R. Batchelor, I. A. Dodi, A. H. Fielder, C. Rittner, G. Mauff, K. Bender, C. Levene, G. M. Schreuder, L. J. Wells. 1984. C4 and HLA haplotypes associated with partial inhibition of anti-Rg and anti-Ch. J. Immunogenet. 11:305.[Medline]
  11. Martinez-Quiles, N., E. Paz-Artal, M. A. Moreno-Pelayo, J. Longas, S. Ferre-Lopez, M. Rosal, A. Arnaiz-Villena. 1998. C4d DNA sequences of two infrequent human allotypes (C4A13 and C4B12) and the presence of signal sequences enhancing recombination. J. Immunol. 161:3438.[Abstract/Free Full Text]
  12. Lokki, M. L., H. R. Colten. 1995. Genetic deficiencies of complement. Ann. Med. 27:451.[Medline]
  13. Jaatinen, T., O. Ruuskanen, L. Truedsson, M. L. Lokki. 1999. Homozygous deletion of the CYP21A-TNXA-RP2–C4B gene region conferring C4B deficiency associated with recurrent respiratory infections. Hum. Immunol. 60:707.[Medline]
  14. Seppänen, M., M. L. Lokki, T. Timonen, M. Lappalainen, H. Jarva, A. Järvinen, S. Sarna, V. Valtonen, S. Meri. 2001. Complement c4 deficiency and hla homozygosity in patients with frequent intraoral herpes simplex virus type 1 infections. Clin. Infect. Dis. 33:1604.[Medline]
  15. Laitinen, T.. 1993. A set of MHC haplotypes found among Finnish couples suffering from recurrent spontaneous abortions. Am. J. Reprod. Immunol. 29:148.
  16. Amos, D., H. Badir, W. Boyle, M. McQueen, A. Tiilikainen. 1969. A simple microcytotoxicity test. Transplantation 7:220.[Medline]
  17. Bidwell, J. L., E. A. Bidwell, B. A. Bradley. 1990. HLA class II genes: typing by DNA analysis. Baillieres Clin. Haematol. 3:355.[Medline]
  18. Marcus, D., C. A. Alper. 1986. Methods for allotyping complement proteins. N. R. Rose, and H. Friedman, and J. L. Fahey, eds. Manual of Clinical Laboratory Immunology 3rd Ed.185. American Society for Microbiology, Washington, DC.
  19. Sim, E., S. J. Cross. 1986. Phenotyping of human complement component C4, a class-III HLA antigen. Biochem. J. 239:763.[Medline]
  20. Awdeh, Z. L., C. A. Alper. 1980. Inherited structural polymorphism of the fourth component of human complement. Proc. Natl. Acad. Sci. USA 77:3576.[Abstract/Free Full Text]
  21. Miller, S. A., D. D. Dykes, H. F. Polesky. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16:1215.[Free Full Text]
  22. Marcadet, A., P. O’Connell, D. Cohen. 1989. Standardized Southern blot workshop technique. B. Dupont, ed. Immunobiology of HLA 553. Springer-Verlag, New York.
  23. Belt, K. T., M. C. Carroll, R. R. Porter. 1984. The structural basis of the multiple forms of human complement component C4. Cell 36:907.
  24. White, P. C., M. I. New, B. Dupont. 1986. Structure of human steroid 21-hydroxylase genes. Proc. Natl. Acad. Sci. USA 83:5111.[Abstract/Free Full Text]
  25. Barba, G., C. Rittner, P. M. Schneider. 1993. Genetic basis of human complement C4A deficiency: detection of a point mutation leading to nonexpression. J. Clin. Invest. 91:1681.
  26. Barba, G. M., L. Braun-Heimer, C. Rittner, P. M. Schneider. 1994. A new PCR-based typing of the Rodgers and Chido antigenic determinants of the fourth component of human complement. Eur. J. Immunogenet. 21:325.[Medline]
  27. Braun, L., P. M. Schneider, C. M. Giles, J. Bertrams, C. Rittner. 1990. 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.[Abstract/Free Full Text]
  28. Campbell, R. D., I. Dunham, E. Kendall, C. A. Sargent. 1990. Polymorphism of the human complement component C4. Exp. Clin. Immunogenet. 7:69.[Medline]
  29. Belt, K. T., C. Y. Yu, M. C. Carroll, R. R. Porter. 1985. Polymorphism of human complement component C4. Immunogenetics 21:173.[Medline]
  30. Little, A. M., P. Parham. 1999. Polymorphism and evolution of HLA class I and II genes and molecules. Rev. Immunogenet. 1:105.[Medline]
  31. Martinsohn, J. T., A. B. Sousa, L. A. Guethlein, J. C. Howard. 1999. The gene conversion hypothesis of MHC evolution: a review. Immunogenetics 50:168.[Medline]
  32. Donohoue, P. A., C. van Dop, R. H. McLean, P. C. White, N. Jospe, C. J. Migeon. 1986. Gene conversion in salt-losing congenital adrenal hyperplasia with absent complement C4B protein. J. Clin. Endocrinol. Metab. 62:995.[Abstract/Free Full Text]
  33. Blanchong, C. A., B. Zhou, K. L. Rupert, E. K. Chung, K. N. Jones, J. F. Sotos, W. B. Zipf, R. M. Rennebohm, C. Y. Yu. 2000. Deficiencies of human complement component C4A and C4B and heterozygosity in length variants of RP-C4-CYP21-TNX (RCCX) modules in caucasians: the load of RCCX genetic diversity on major histocompatibility complex-associated disease. J. Exp. Med. 191:2183.[Abstract/Free Full Text]
  34. Rupert, K. L., R. M. Rennebohm, C. Y. Yu. 1999. An unequal crossover between the RCCX modules of the human MHC leading to the presence of a CYP21B gene and a tenascin TNXB/TNXA-RP2 recombinant between C4A and C4B genes in a patient with juvenile rheumatoid arthritis. Exp. Clin. Immunogenet. 16:81.[Medline]
  35. Chu, X., L. Braun-Heimer, C. Rittner, P. M. Schneider. 1992. Identification of the recombination site within the steroid 21-hydroxylase gene (CYP21) of the HLA-B47,DR7 haplotype. Exp. Clin. Immunogenet. 9:80.[Medline]
  36. Sinnott, P., S. Collier, C. Costigan, P. A. Dyer, R. Harris, T. Strachan. 1990. Genesis by meiotic unequal crossover of a de novo deletion that contributes to steroid 21-hydroxylase deficiency. Proc. Natl. Acad. Sci. USA 87:2107.[Abstract/Free Full Text]
  37. Collier, S., M. Tassabehji, P. Sinnott, T. Strachan. 1993. A de novo pathological point mutation at the 21-hydroxylase locus: implications for gene conversion in the human genome. Nat. Genet. 3:260.[Medline]
  38. Tusie-Luna, M. T., P. C. White. 1995. Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms. Proc. Natl. Acad. Sci. USA 92:10796.[Abstract/Free Full Text]
  39. Fasano, M. B., J. A. Winkelstein, T. LaRosa, W. B. Bias, R. H. McLean. 1992. A unique recombination event resulting in a C4A*Q0,C4B*Q0 double null haplotype. J. Clin. Invest. 90:1180.
  40. Lopes, J., S. Tardieu, K. Silander, I. Blair, A. Vandenberghe, F. Palau, M. Ruberg, A. Brice, E. LeGuern. 1999. Homologous DNA exchanges in humans can be explained by the yeast double-strand break repair model: a study of 17p11.2 rearrangements associated with CMT1A and HNPP. Hum. Mol. Genet. 8:2285.[Abstract/Free Full Text]
  41. Loh, D. Y., D. Baltimore. 1984. Sexual preference of apparent gene conversion events in MHC genes of mice. Nature 309:639.[Medline]
  42. Roos, M. H., C. M. Giles, P. Demant, E. Mollenhauer, C. Rittner. 1984. Rodgers (Rg) and Chido (Ch) determinants on human C4: characterization of two C4 B5 subtypes, one of which contains Rg and Ch determinants. J. Immunol. 133:2634.[Abstract]
  43. Atkins, C. J.. 1985. Chido and Rodgers: a serological study of their variations on the red cell and in plasma Brunel University, Uxbridge, Middlesex, U.K.
  44. Tokunaga, K., K. Omoto, T. Akaza, N. Akiyama, H. Amemiya, S. Naito, T. Sasazuki, H. Satoh, T. Juji. 1985. Haplotype study on C4 polymorphism in Japanese: associations with MHC alleles, complotypes, and HLA-complement haplotypes. Immunogenetics 22:359.[Medline]
  45. Hing, S. N., C. M. Giles, A. H. Fielder, J. R. Batchelor. 1986. HLA haplotypes with C4B5; evidence for further allelic heterogeneity. Immunogenetics 23:151.[Medline]
  46. Giles, C. M.. 1987. Three Chido determinants detected on the B5Rg+ allotype of human C4: their expression in Ch-typed donors and families. Hum. Immunol. 18:111.[Medline]
  47. Schneider, P. M., B. Stradmann-Bellinghausen, C. Rittner. 1996. Genetic polymorphism of the fourth component of human complement: population study and proposal for a revised nomenclature based on genomic PCR typing of Rodgers and Chido determinants. Eur. J. Immunogenet. 23:335.[Medline]
  48. Lokki, M. L., A. Circolo, P. Ahokas, K. L. Rupert, C. Y. Yu, H. R. Colten. 1999. Deficiency of human complement protein C4 due to identical frameshift mutations in the C4A and C4B genes. J. Immunol. 162:3687.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
C. K. Doyle, B. K. Davis, R. G. Cook, R. R. Rich, and J. R. Rodgers
Hyperconservation of the N-Formyl Peptide Binding Site of M3: Evidence that M3 Is an Old Eutherian Molecule with Conserved Recognition of a Pathogen-Associated Molecular Pattern
J. Immunol., July 15, 2003; 171(2): 836 - 844.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jaatinen, T.
Right arrow Articles by Lokki, M.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jaatinen, T.
Right arrow Articles by Lokki, M.-L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS