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The Journal of Immunology, 2003, 171: 2734-2745.
Copyright © 2003 by The American Association of Immunologists

Diversity in Intrinsic Strengths of the Human Complement System: Serum C4 Protein Concentrations Correlate with C4 Gene Size and Polygenic Variations, Hemolytic Activities, and Body Mass Index 1

Yan Yang*,{dagger},{ddagger}, Erwin K. Chung*,{dagger},{ddagger}, Bi Zhou*, Carol A. Blanchong*,{ddagger}, C. Yung Yu2,*,{dagger},{ddagger}, George Füst2,§, Margit Kovács§, Ágnes Vatay§, Csaba Szalai, István Karádi§ and Lilian Varga§

* Center for Molecular and Human Genetics, Columbus Children’s Research Institute, {dagger} Department of Molecular Virology, Immunology, and Medical Genetics, and {ddagger} Department of Pediatrics, Ohio State University, Columbus, OH 43205; § Third Department of Internal Medicine, Semmelweis Medical University, Budapest, Hungary; and Section of Molecular Immunology, Hungarian Academy of Sciences, and Heim Pál Pediatric Hospital, Budapest, Hungary


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Among the genes and proteins of the human immune system, complement component C4 is extraordinary in its frequent germline variation in the size and number of genes. Definitive genotypic and phenotypic analyses were performed on a central European population to determine the C4 polygenic and gene size variations and their relationships with serum C4A and C4B protein concentrations and hemolytic activities. In a study population of 128 healthy subjects, the number of C4 genes present in a diploid genome varied between two to five, and 77.4% of the C4 genes belonged to the long form that contains the endogenous retrovirus HERV-K(C4). Intriguingly, higher C4 serum protein levels and higher C4 hemolytic activities were often detected in subjects with short C4 genes than those with long genes only, suggesting a negative epistatic effect of HERV-K(C4) on the expression of C4 proteins. Also, the body mass index appeared to affect the C4 serum levels, particularly in the individuals with medium or high C4 gene dosages, a phenomenon that was dissimilar in several aspects from the established correlation between body mass index and serum C3. As expected, there were strong, positive correlations between total C4 gene dosage and serum C4 protein concentrations, and between serum C4 protein concentrations and C4 hemolytic activities. There were also good correlations between the number of long genes with serum levels of C4A, and the number of short genes with serum levels of C4B. Thus, the polygenic and gene size variations of C4A and C4B contribute to the quantitative traits of C4 with a wide range of serum protein levels and hemolytic activities, and consequently the power of the innate defense system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Diversities in immune functions are mainly achieved by nucleotide polymorphisms/mutations and somatic recombinations of gene segments (1, 2). Human complement component C4 represents a new paradigm of complex, inborn immune diversity (3). Phenotypically, there are acidic C4A and basic C4B proteins with multiple polymorphic variants in each class (4). The plasma or serum protein levels of total C4 vary between 100 and 1000 mg/L among different individuals, as do the relative quantities of C4A and C4B proteins. Although this phenomenon has been observed over a decade ago (5, 6, 7, 8, 9, 10, 11, 12, 13, 14), the molecular genetic basis for the qualitative and quantitative variations of human complement C4 was often accounted for inaccurately. Until recently, the sophistication of human C4 genetics has not been fully appreciated. A two-locus, C4A-C4B model with null alleles of either locus was used to explain the great variation in the levels of C4A and C4B proteins in a population (15, 16). There are actually a frequent, dichotomous gene size variation and polygenic and modular duplications of C4A and C4B together with their flanking genes RP1 or RP2, CYP21A or CYP21B, and TNXA or TNXB in the MHC (Fig. 1A). Existing at the germline levels and inherent to different individuals, there are monomodular, bimodular, trimodular, or quadrimodular structures of discrete segments in the MHC containing one, two, three, or four RP-C4-CYP21-TNX (RCCX) 3 modules (17, 18, 19). Each C4 gene may code for C4A or C4B. Each C4A or C4B gene may be 21 kb (long) or 14.6 kb (short) in size (20). The long C4 gene contains a 6.36-kb endogenous retrovirus HERV-K(C4) in its intron 9 (21, 22). Recently, we showed that the bimodular RCCX haplotypes constitute slightly more than two-thirds of the Caucasian population in the midwestern United States, while the frequencies of monomodular and trimodular RCCX haplotypes were 0.17 and 0.14, respectively (18). Quadrimodular RCCX haplotypes with four C4 genes in a row are rare in Caucasians, but relatively common in Asians (19, 23). The impacts of the C4 polygenic and gene size variations on C4 protein levels and functional activities have not been accurately studied in a population.



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  		FIGURE 1. The gene size and polygenic variations of human complement C4 and RP-C4-CYP21-TNX (RCCX modules) in the MHC class III region. A, A molecular map of the genes in the MHC complement gene cluster. Horizontal arrows represent gene configurations; filled, vertical arrows represent locations to where the DNA probes hybridized in a TaqI genomic Southern blot analysis to determine the RCCX modular variations; gray, vertical arrows represent locations to where the DNA probes hybridized in a PshAI genomic Southern blot analysis to determine the ratio of C4A and C4B genes. B, Common RCCX length variants in healthy Caucasians. Characteristic TaqI restriction fragments of each RCCX haplotype in a Southern blot analysis are shown on the right.

 
Complement component C4 is important in immunity, tolerance, and autoimmunity (24, 25, 26, 27). It plays essential roles in linking the recognition pathways of the complement system initiated by Ag-Ab or Ag-mannan-binding lectin complexes to the effectors of the humoral immune response. Complete deficiency of complement C4 (i.e., both C4A and C4B) is one of the most penetrant genetic risk factors in autoimmune disease such as systemic lupus erythematosus (28, 29). Complete or partial deficiencies of C4B are related to vulnerability and severity of microbial infections (30, 31). Complete deficiencies of C4 in humans or guinea pigs have impairments in the secondary immune response and switching of IgM to IgG (32, 33, 34). In an immunization process, it has been shown that low concentrations of C4 in mice (35) or partial deficiencies of C4A in humans are associated with higher frequencies of nonresponders to hepatitis B Ags (36, 37). Therefore, an accurate account of the phenotypic and genotypic diversities of C4A and C4B in a human population both qualitatively and quantitatively is critically important for epidemiological studies of disease associations with C4.

In this study, we report a comprehensive investigation of human C4A and C4B serum protein levels and functional hemolytic activities in a central European population with precisely defined status for C4A and C4B gene dosage, gene size, and RCCX modules. The study reveals higher C4 serum protein levels and hemolytic activities among individuals with short C4 genes than those with long C4 genes only. It also demonstrates that human serum C4 protein levels were determined by the dosage and size of C4 genes and, unexpectedly, the body mass index (BMI) of an individual.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study population

The study was performed in 128 healthy Hungarian subjects (40 males, 88 females). The subjects were 44.7 ± 10 (mean ± SD) years old. Their BMI were 25.5 ± 4.7 kg/m2, and serum total cholesterol and triglyceride concentrations were 5.48 ± 1.02 mM and 1.44 ± 0.90 mM, respectively. These individuals participated in a regular medical survey and gave their informed consent for the use of their serum/plasma and DNA samples for the present study. For ethical reasons, after their computer registration, the data were unlinked from the subjects so that their identities could not be traced. The study was approved by the Ethical Committee of the Semmelweis University (Budapest, Hungary). Peripheral blood samples were obtained by venipuncture and processed immediately. EDTA-plasma and serum samples were aliquoted and stored at -80°C and thawed only immediately before the assays/measurements were performed. Genomic DNA samples were stored at 4°C.

Measurement of serum C4 and C3 concentrations

Concentrations of C4 and C3 were determined immunochemically by single radial immunodiffusion (38), using commercial Abs against C3 and C4 (DiaSorin, Stillwater, MN). Human Serum Protein Calibrator (DAKO A/S, Glostrup, Denmark) was used as standard.

Measurement of the complement C4HA

C4HA were measured by the effective molecule titration method (39). Briefly, different dilutions of the serum samples were incubated with EAC1gp at 30°C for 20 min; purified guinea pig C2 was added and further incubated at 30°C for 15 min. Finally, C-EDTA (guinea pig serum diluted in buffer containing 10 mM EDTA) was added and incubated at 37°C for 60 min. After centrifugation, OD values were read at 412 nm. C4HA in serum was expressed in complement hemolytic units CH63 U/ml, which is the amount of complement component required to lyse 63% of the indicator cells in standard C4 assays. Normal values of the C4HA in the laboratory were between 5,000 and 70,000 CH63 U/ml. Hemolytic titration of C4 was standardized using aliquots stored at -70°C of the same normal human serum pool at each measurement.

Preparation of genomic DNA, Southern blot analysis, and DNA probes

Genomic DNAs were prepared from PBMC (40). Southern blot analyses of TaqI-digested or PshAI-digested genomic DNAs were performed, as described in previous publications (18, 41). The TaqI genomic blots were hybridized with three specific probes corresponding to RP, CYP21, and TNX. The results revealed the presence and dosage of RP1-C4L (7.0 kb), RP1-C4S (6.4 kb), RP2-C4L (6.0 kb), and RP2-C4S (5.4 kb); the presence and relative dosage of CYP21B (3.7 kb) and CYP21A (3.2 kb); and the presence and relative dosage of TNXB (2.5 kb) and TNXA (2.4 kb). The number of RCCX modules was independently confirmed by PshAI-RFLP using a RP 3' probe to determine the relative dosage of RP1 (7.2-kb) and RP2 (8.3-kb) fragments. The relative dosages of C4A and C4B genes were determined by PshAI RFLP using a C4d-specific probe corresponding to C4 gene exons 28–31 (Fig. 1).

Determination of C4 protein polymorphism by immunofixation and immunoblot analysis

EDTA-plasma samples were digested overnight with neuraminidase at 4°C, followed by carboxypeptidase B digest for 30 min at room temperature, and resolved with high voltage agarose gel electrophoresis (HVAGE). C4 proteins were immunofixed with goat anti-human C4 sera. Gels were blotted to remove diffusible proteins, and the immune complexes with C4 proteins were stained with Easy-Stain (Invitrogen, Carlsbad CA), as described previously (41, 42, 43). The relative band intensities of C4A and C4B allotypes in each sample were quantified by densitometry, using an Epson Expression 1600 Scanner, and analyzed by ImageQuant Version 5.0 software. Ambiguous C4A or C4B allotypes were further investigated with immunoblot analysis of HVAGE-resolved gels using anti-Rg1 and anti-Ch1 monoclonals provided by the VIIth Complement Genetics Workshop (4).

Statistical analysis

Parametric tests (two-sample t test, paired t test, Pearson correlation analysis) were used for the normally distributed (C3 and C4 levels, C4HA, BMI) data. All tests were two tailed. Multiple linear regression was used to evaluate potential confounders. Statistical analysis was performed by GraphPad Prism 3.0 (GraphPad Software, San Diego, CA, www.grphpad.com) and SPSS 10.0 (SPSS, Chicago, IL) software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Genotypic variation of human complement components C4A and C4B

The study population consisted of 128 healthy individuals (40 males and 88 females) from Budapest, Hungary. Molecular genetic approaches were applied to determine the genotypic diversities of the constituents of RCCX modules. The total number of C4 genes present in a diploid genome (i.e., gene dosage) was elucidated by TaqI RFLP that also yielded information on the details of the RCCX variants such as the presence and relative dosage of RP1 and RP2 linked to a long or a short C4 gene, and presence and relative dosage of CYP21A and CYP21B, and TNXA and TNXB (Fig. 1B). The TaqI RFLP results were independently confirmed by PshAI RFLP analysis for the relative dosage of RP1 and RP2. The PshAI Southern blots were also used to determine the relative dosage of C4A and C4B using a specific C4d genomic probe, as one of the five nucleotide changes specific for C4A and C4B created a new PshAI cleavage site in C4A (44, 45). The polymorphism of C4A and C4B proteins were elucidated by HVAGE of EDTA-plasma, followed by immunofixations. Selected plasma samples with ambiguous C4A or C4B allotypes were further analyzed by immunoblot analysis using anti-Rg1 and anti-Ch1 monoclonals.

Demonstration of variations in the RCCX constituents. Fig. 2 illustrates the genotypic characterization of the RCCX constituents and phenotypic polymorphism of C4 in 10 individuals. Eight subjects had bimodular RCCX structures from both haplotypes; among them were homozygous LL/LL (L, long) (MK1, 11, and 13), heterozygous LL/LS (S, short) (MK2, 3, and 5), and homozygous LS/LS (MK12). Of these eight individuals who each had a total of four C4 genes in a diploid genome, five had two C4A and two C4B genes (MK2, 3, 8, 12, and 13; Fig. 2C). Two subjects, MK1 and MK5, had three C4A and one C4B. One subject (MK11) had four C4A, but no C4B. The remaining two subjects had heterozygous bimodular and monomodular RCCX structures, i.e., LS/S in MK6 and LS/L in MK9. Both of them had one C4A and two C4B genes.



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  		FIGURE 2. Genotypic variations of RCCX modules, C4A and C4B gene dosages, and protein polymorphisms. A, TaqI RFLP to show RCCX modular variations. Hybridization probes include 3' RP cDNA, CYP21 genomic, and 3' TNX genomic fragments. B, PshAI-RFLP to show number of RCCX modules by determining the relative dosages of RP1 and RP2. A 3' RP cDNA probe was used for hybridization. C, PshAI-RFLP to show relative dosages of C4A and C4B genes. Hybridization probe used was a genomic fragment corresponding to C4 exons 28–31. D, Allotyping of human C4A and C4B proteins. EDTA plasma C4 proteins were resolved by HVAGE, processed by immunofixation using goat polyclonal antisera against human C4. Notice the presence of restriction fragments corresponding to CYP21B and TNXB only (marked by arrows, A), and the presence of a novel 3.5-kb PshAI fragment corresponding to a rearranged TNXA-XB recombinant (B) in subject MK6. Subject MK11 was completely C4B deficient, as there were no C4B gene (marked by an arrow, C) and no C4B protein (marked by an arrow, D).

 
Results of the immunofixation experiment for C4A and C4B protein allotypes are shown in Fig. 2D. In addition to C4A3 that was observed in every individual, C4A2 was present in MK2 and C4A6 was present in MK3. C4B1 was detectable in all except MK5, who had C4B2, and MK11, who was deficient in C4B. The protein band intensities of C4A and C4B were consistent with the C4A and C4B gene dosage results. For examples, MK1 had three C4A and one C4B genes (Fig. 2C), and the protein band intensities of C4A3 allotype were about 3 times greater than that of C4B1 (Fig. 2D); MK11 had complete absence of the C4B gene (Fig. 2C) and that was matched with the absence of C4B protein (Fig. 2D).

The TaqI and PshAI RFLP patterns for the RCCX modules revealed a common pattern for bimodular structures with RP1-C4-CYP21A-TNXA-RP2-C4-CYP21B-TNXB, and a common pattern for monomodular structures with RP1-C4-CYP21B-TNXB (Fig. 1B). A single anomaly was observed in MK6, whose markers for CYP21A and TNXA in the TaqI RFLP appeared to be absent, although he had a bimodular LS and a monomodular S structure. The PshAI RFLP hybridized to RP 3' probe revealed the presence of a 3.5-kb, rearranged fragment that is characteristic of RP2-TNXA + 120. In other words, MK6 has an unusual LS haplotype that is characterized by the presence of two CYP21B genes and a rearranged TNXA with the 120-bp sequence that is normally present in exon 36-intron 36 of the TNXB gene (i.e., RP1-C4-CYP21B-TNXA + 120-RP2-C4-CYP21B-TNXB) (46).

The RCCX and complement C4 polygenic variations in the Budapest population. The frequencies of RCCX length variants, namely, monomodular L and S; bimodular LL and LS; and trimodular LLL, LSS, and LSL, are listed in Table I. The most common RCCX haplotype in the Hungarian population was the bimodular LL with a frequency of 0.500. Another bimodular haplotype LS had a frequency of 0.289. The frequencies for monomodular L and S haplotypes were 0.047 and 0.086, respectively. The frequencies of the trimodular haplotypes LLL, LSS, and LSL were 0.039, 0.027, and 0.004, respectively. Altogether, the frequencies of monomodular, bimodular, and trimodular RCCX haplotypes were 0.133, 0.797, and 0.070, respectively.


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  		Table I. A summary of RCCX length variants and C4 gene dosages in Caucasians

 
In this central European population, the number of C4 genes in a diploid genome varied from two to five. The majority of individuals had four C4 genes per genome, which had a frequency of 0.656. Individuals with C4 gene dosages of three and five had frequencies of 0.211 and 0.117, respectively. Only two individuals had a dosage of two C4 genes (frequency: 0.016). We did not detect any individuals in the study population with six C4 genes.

In the 128 genotyped individuals, there were 496 C4 genes. Among these genes, 77.4% belonged to the long form with HERV-K(C4) integrated into the ninth intron; 22.6% belonged to the short form without the endogenous retrovirus in the C4 gene. The average C4 gene number in the normal Hungarian population (gene index) was 3.875, of which C4A had a gene index of 2.0 and C4B, 1.875. The frequency of individuals with gene dosage of two C4A was 0.570, and with two C4B was 0.700. Overall, one-fifth of the study population had a single C4A gene in a diploid genome; the same frequency was also found in subjects with single C4B genes. The frequency of individuals with a gene dosage of three C4A was 0.190, and that of three C4B was 0.080. Three individuals (frequency: 0.023) had four C4A genes, and one single individual (frequency: 0.008) had four C4B genes in a genome. Two subjects had no C4A gene (frequency: 0.016), and one individual had no C4B gene (frequency: 0.008).

Qualitative variations of C4A and C4B

The polymorphism of C4A and C4B proteins was investigated by immunofixation of EDTA-plasma, resolved by HVAGE. Samples showing ambiguous or unusual C4A and C4B allotypes were further analyzed by immunoblot analysis using anti-Rg1 and anti-Ch1 mAbs. The frequencies of C4A and C4B allotypes detected in this Hungarian study population are listed in Table II.


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  		Table II. Phenotypes of complement components C4A and C4B in a healthy Hungarian population

 
Among the 256 C4A genes in the study population, 249 coded for a detectable protein. The most common C4A allotype was A3, which had a frequency of 85.5%. C4A2, and A6 had frequencies of 7.81 and 1.95%, respectively. Rare C4A allotypes detected in the study population included A92, A12, A4, and A5, which had a combined frequency of 1.95%. In the study population, there were seven suspected C4A mutant genes not producing C4A proteins. Among them, three (1.17%) were confirmed by sequence-specific primer PCR to have nonsense mutations caused by a 2-bp insertion at exon 29 (47, 48).

In the study population, all 240 C4B genes coded for C4B proteins, of which C4B1 had a frequency of 83.8%. C4B2 and B92 had frequencies of 10.8 and 1.25%, respectively. Rare C4B allotypes detected include B94, B12, B15, B3, B4, B5, and B6, which had a combined frequency of 4.17%.

Quantitative diversities of C4A and C4B proteins

The C4 protein level in each individual was measured by single radial immunodiffusion of serum proteins. In the protein-allotyping gels, the band intensities for C4A and C4B were quantified by densitometry and the corresponding C4A and C4B isotype protein levels calculated from the total serum C4 concentrations. The relationship of total C4, C4A, and C4B serum concentrations with respect to the dosages of total C4 (C4A plus C4B), C4A, C4B, long C4 genes, and short C4 genes was analyzed and shown in Fig. 3. For total C4 gene dosage, the population was categorized into three groups: individuals with 4 C4 genes (medium), <4 C4 genes (2 or 3; low), and >4 C4 genes (5; high). With respect to C4A gene dosage, the population was grouped into individuals with <2 C4A genes (0 or 1; low), and >=2 C4A genes (2, 3, or 4; high). The same categorization was applied for C4B genes. With respect to long C4 genes, the population was divided into two groups: 0–2 long C4 genes, and 3–5 long C4 genes. With respect to short C4 genes, the population was divided into two groups: with (i.e., 1–3) and without (i.e., 0) short C4 genes.



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  		FIGURE 3. Analyses of C4 serum concentrations with C4 gene dosage and gene size variations. A–E, Show the relationships of total serum C4 levels with C4 polygenic and gene size variations. F–J, Show the relationships of serum C4A proteins with C4 polygenic and gene size variations. K–O, Show the relationships of serum C4B proteins with C4 polygenic and gene size variations. A, F, and K, The p values for Tukey post hoc test of one-way ANOVA are indicated. For all other panels, p values for unpaired t test are indicated.

 
Total serum C4 protein concentrations and C4 gene dosages. As shown in Fig. 3A, there was a positive correlation of serum C4 concentrations with total C4 gene dosages. The low C4 gene dosage group (gene dosages, 2–3; n = 29) had a mean C4 concentration of 0.33 ± 0.10 g/L, which was significantly lower than that of the medium gene dosage group (gene dosage, 4; n = 84; concentration 0.46 ± 0.15 g/L; p < 0.001) and the high gene dosage group (gene dosage, 5; n = 15; concentration 0.50 ± 0.16 g/L; p < 0.001). The total serum C4 concentration was significantly higher in individuals with 2, 3, or 4 C4A genes (concentration 0.46 ± 0.16 g/L; n = 100; p = 0.0196) than in those with 0 or 1 C4A gene (concentration 0.38 ± 0.13 g/L; n = 28) (Fig. 3B). With respect to the number of the C4B genes, however, there was no relevant correlation with the total C4 serum concentration (Fig. 3C). With respect to the dosage of the long C4 genes, there was also no relevant difference in the C4 serum concentrations between the low dosage and the high dosage groups (Fig. 3D). On the contrary, a higher total C4 serum concentration was observed in the population group with one or more short C4 genes (concentration 0.47 ± 0.17 g/L; n = 80) than the group without any short C4 genes (concentration 0.39 ± 0.11 g/L; n = 48; p = 0.0095; Fig. 3E).

Serum C4A and C4B concentrations and C4 gene dosages. We then analyzed the impact of gene dosages for total C4, C4A, C4B, long genes, and short genes on the serum protein concentrations of C4A and C4B. The mean C4A serum concentrations in the low (i.e., <2), medium (i.e., = 2), and high (i.e., >2) C4 gene dosage groups were 0.13 ± 0.05, 0.23 ± 0.08, and 0.26 ± 0.08 g/L, respectively. There was a close, positive correlation of C4A serum concentrations with the total C4 gene dosage (p < 0.001; Fig. 3F). As expected, the serum C4A concentration was a function of C4A gene dosage (p < 0.0001; Fig. 3G), and was weakly, but inversely related to the C4B gene dosage (Fig. 3H). With respect to C4 gene size, higher serum C4A concentrations were present in individuals with high dosage (concentration 0.23 ± 0.08 g/L) than those with low dosage of long C4 genes (concentration 0.18 ± 0.09 g/L; p = 0.0028; Fig. 3I). The presence of short C4 genes, however, did not appear to have a correlation with the C4A protein concentration (p = 0.9392; Fig. 3J).

The mean C4B serum concentrations were positively correlated with C4B gene dosage (p < 0.0001; Fig. 3M), and a highly significant correlation was noted with the presence of one or more short C4 genes (p < 0.0001; Fig. 3O). The serum C4B level was not related to C4A gene dosage (Fig. 3L). There was a mild, but significant inverse correlation of serum C4B levels with the number of long C4 genes (p = 0.048; Fig. 3N). Unlike the case for C4A, there was no significant correlation of C4B protein concentrations with total C4 gene dosages (Fig. 3K).

Hemolytic activities, C4 gene dosages, C4 gene sizes, and serum C4 concentrations

C4HA of the serum samples were determined by effective molecule titration and expressed in CH63 U/ml. The C4HA from each sample was scatter plotted against the C4 gene dosages (Fig. 4). There was a direct correlation of the mean C4HA with an increase of total C4 gene dosage. This was most obvious when the comparison was made between the high C4 gene dosage group and the low gene dosage group (p < 0.01; Fig. 4A), and also between the groups with one to three short C4 genes and without short C4 genes (p = 0.0063; Fig. 4E). However, there was no clear-cut correlation of C4HA with the gene dosage of C4B (Fig. 4C) or with the dosage of long C4 genes (Fig. 4D).



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  		FIGURE 4. Analyses of C4 hemolytic activities with C4 polygenic and gene size variations (A–E), and with serum C4 concentrations (F–H). Serum hemolytic activity of C4 expressed in CH63 U/ml in the sera of 128 healthy subjects with low (gene dosages = 2–3), medium (gene dosage = 4), or high (gene dosage = 5) dosages of C4 genes (A); low (gene dosage = 0 or 1) or high (gene dosages = 2–4) number of C4A genes (B); or C4B genes (C), low (gene dosages = 0–2) or high (gene dosages = 3 to 5) number of long (L) C4 genes (D), and none or 1–3 short (S) C4 genes (E). A, The p values for Tukey post hoc test of one-way ANOVA are indicated. B–E, The p values for unpaired t test are indicated. F–H, The Pearson correlation coefficients and their significance are indicated.

 
When the relations of serum C4 concentrations and C4HA were examined, it was found that C4HA strongly correlated to the total C4 serum concentrations (Fig. 4F), and to the serum levels of C4A (Fig. 4G) and C4B (Fig. 4H) (p < 0.0001). The correlation coefficients were ~0.5. The strengths of correlation between C4HA and C4A protein levels, and between C4HA and C4B protein levels were similar.

To further illustrate the intricate relations among the size of C4 genes, C4 serum protein concentrations, and hemolytic activities, we focused on the most common gene dosage group, i.e., subjects with four C4 genes (two C4A and two C4B) and organized in one of the following three RCCX configurations: LL/LL, LL/LS, and LS/LS. In comparison with LL/LL homozygotes, individuals with LS/LS structures had higher serum C4 protein concentrations (p < 0.05; Fig. 5A) and higher C4 hemolytic activities (p < 0.01; Fig. 5B). Although not reaching a statistically significant level, heterozygous LL/LS had values between LL/LL and LS/LS for both serum C4 protein concentrations and C4HA. Compared with those having homozygous LL/LL, the C4A and C4B serum protein levels increased by 26.0 and 34.0%, respectively, in LL/LS heterozygotes, and by 34.0 and 40.0% in LS/LS homozygotes. The increase of C4B protein levels was significant in both LL/LS (p < 0.01) and LS/LS (p < 0.01). This phenomenon probably suggested that the presence of short C4 gene(s) at the second locus not only significantly increased the expression of the C4B proteins, but also had a considerable, positive impact on the expression of C4A proteins. It appeared that the presence of HERV-K(C4) reduced the expression of serum C4 and the C4 hemolytic activities in a dose-dependent manner.



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  		FIGURE 5. The impact of long and short C4 genes on the serum C4 protein concentrations and C4HA. Eighty healthy subjects with bimodular/bimodular RCCX haplotypes corresponding to LL/LL, LL/LS, or LS/LS configurations were chosen for analysis. The p values for Tukey post hoc test of one-way ANOVA are indicated.

 
BMI and serum C4 and C3 concentrations

In view of the emerging roles of several complement proteins in lipid metabolism and the strong correlation of serum C3 levels and BMI in healthy people, we investigated whether there were positive relationships between BMI and C4 gene dosage; total C4, C4A, C4B serum protein concentrations; and C4 HA (Fig. 6). BMI was not related to the C4 gene dosage or C4HA (Fig. 6, A and E, respectively). However, there was a modest, but significantly positive correlation between BMI and serum protein concentrations of C4, C4A, and C4B (Fig. 6, B, C, and D, respectively).



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  		FIGURE 6. Analyses of BMI and C4 polygenic variations (A), serum C4 concentrations (B–D), C4HA (E), and serum C3 concentration (F). A, The p values for Tukey post hoc test of one-way ANOVA are indicated. B–F, The Pearson correlation coefficients and their significance are indicated. BMI was expressed in kg/m2.

 
As expected, C3 serum levels were found to be strongly correlated to the BMI (Fig. 6F). Approximately the same coefficients were found in the males and females for each correlation depicted in Fig. 6. The only exception was the correlation between serum C3 concentrations and BMI. In males, we found a very strong correlation (r = 0.675, p = 0.0004), whereas in females the correlation was weaker (r = 0.346, p = 0.0041).

The serum C4 concentration did not correlate to the age of subjects or the serum total cholesterol levels (correlation coefficient r = 0.091, p = 0.316) and triglyceride (r = 0.082, p = 0.362). By contrast, there was a significant positive correlation between C3 levels and the total cholesterol concentration (r = 0.246, p = 0.015), and a very strong correlation between C3 levels and concentrations of triglycerides (r = 0.510, p < 0.001).

Using multiple linear regression analysis, we found the correlation between BMI and serum C4 concentrations to be independent from that between BMI and serum C3 concentrations (Table III). In the whole population, a strong correlation between C3 and BMI was found when no adjustment was applied. However, when serum cholesterol and triglyceride levels were adjusted to the age, gender, cholesterol, and triglyceride levels, the correlation coefficient between C3 concentration and BMI became marginally significant. This difference became more pronounced when the analysis was restricted to the subjects with only bimodular RCCX modules. In this group, no significant correlation (p = 0.356) was noted between C3 levels and BMI after adjustment. Similar disappearance of the correlation between C3 and BMI (p = 0.318) after adjustment was noted in subjects with at least four RCCX modules (i.e., those with monomodular/monomodular and monomodular/bimodular RCCX combinations were excluded). By contrast, adjustments to age, gender, and lipid levels did not negatively affect the strength of the correlation between C4 levels and BMI and in the group of subjects with at least four RCCX modules: the adjusted correlation between C4 concentration and BMI became highly significant instead (p = 0.009) (Table III).


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  		Table III. Linear regression analysis of the relationship between BMI and serum C3 and C4 concentrations in healthy Hungarian subjects

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report three important novel findings. First, we demonstrated that the serum concentrations of C4A and C4B proteins and total amounts of C4 proteins were determined by the number of long and short C4 genes. Second, in individuals with medium or high C4 gene dosages (i.e., >=4 genes), a significant positive correlation was found between BMI and C4 serum levels, which in several aspects was quite different from the correlation between BMI and serum C3 concentrations. Third, by using precise and definitive methods in genotyping and phenotyping experiments, we demonstrated the correlation between C4 gene dosage and C4 protein concentrations. The results provide a foundation for the differences on an important factor of innate immune response. They enable a better understanding of the genetic basis for the qualitative and quantitative diversities of human complement C4, which include variations in immunochemical and functional properties of C4A and C4B, and a wide range of serum protein levels and hemolytic activities.

Factors determining the serum C4 protein levels

Under pathological conditions, low serum or plasma C4 levels may be caused by excessive consumptions due to complement activation as a result of microbial infections or abnormalities such as a deficiency of the complement C1 inhibitor (49, 50), and the presence of nephritic factors or autoantibodies that stabilizes the C1 complex (51, 52). Therefore, the serum concentrations of total C4 and C3 and their split products have been used as indicators for disease activities of autoimmune disease, and inflammatory responses including graft rejections (53, 54, 55). However, there is a more fundamental and sophisticated genetic mechanism leading to a wide range of serum protein levels of total C4, C4A, and C4B in the general population. Besides the polygenic variation from two to five genes among different individuals in our study population, the gene size dichotomy adds an additional dimension to the genetic complexity of human C4. Close to three-quarters (77.4%) of the C4 genes in this study population were long, and about one-quarter (22.6%) of the C4 genes were short. This intrinsic diversity in the C4 gene number and gene size is extraordinary in human genetics, which we suggest to be an adaptation of the innate immune system to provide a wide spectrum of immune efficiencies and strengths to defend against parasites. We have established the sophistication of the C4 genetics in a Caucasian population in midwestern region of the United States (18). This current study does not only support and validate results of the Ohioan study, it also provides an essential link between the C4 genetic diversity with the phenotypic and functional variations. The C4 gene number and gene size and, unexpectedly, the BMI are all relevant in determining the C4 serum protein concentrations, which impact the power of the complement system as reflected by the hemolytic assays. In a separate report, we have delineated in the same Caucasian populations the complex relationship between complement C4 and RCCX modular variations with two common single nucleotide polymorphisms of the TNF gene TNFA, -238 G/A and -308 G/A (56).

The serum C4A protein levels were closely associated with C4A gene dosage, the number of long C4 genes, and the total C4 gene dosages. This phenomenon may reflect the fact that most C4A genes are located in the first RCCX module that frequently contains the endogenous retrovirus HERV-K(C4), and an increase in C4 gene dosage has a higher tendency to increase the number of C4A genes. In contrast, serum C4B protein levels were mainly the functions of gene dosages of C4B and short C4 genes. The latter reflects the fact that more short C4 genes code for C4B proteins than for C4A proteins.

The significance and implication of the long and short C4 genes

It is of interest to note that individuals containing one or more short C4 genes consistently have higher serum total C4 and C4B concentrations than those with long C4 genes only. Higher expression levels of serum C4B proteins than C4A proteins in individuals with equal number of C4A and C4B genes had been observed previously in specific haplotypes such as HLA B18 DR2 C4A4 B2 (57). This B18 DR2 haplotype has a bimodular RCCX structure LS, and it is plausible that the short C4B gene is responsible for the higher serum level of the C4B2 protein than the C4A4 protein. However, the positive impact of short C4 genes seemed more far reaching because it affected not only the C4B (and the total C4 protein levels), but also the levels of C4A proteins, albeit to a lesser extent. This was best illustrated in subjects with bimodular RCCX structures from both copies of chromosome 6 and contained two C4A and two C4B genes. Individuals with homozygous LS/LS had mean serum C4A and C4B protein levels 34.0 and 40.0% higher than those from homozygous LL/LL, respectively. The levels of C4A and C4B proteins from individuals with heterozygous LL/LS were higher than those with LL/LL, but lower than those with LS/LS (Fig. 5). It is likely that the 6.36-kb endogenous retrovirus in the long C4 genes retarded the gene transcription rate because of the larger size of the heteronuclear transcripts, and consequently reduced the corresponding protein products. Consistent with this notion, we found that individuals with LSS/LL have serum C4 protein levels 41.0% higher than those with LLL/LL.

The selection advantage of having an endogenous retrovirus in three-quarters of C4 genes in the Caucasian population is a matter of speculation. It was proposed that the reversely oriented HERV-K(C4) in the intron 9 of the long C4 gene would lead to the production of an antisense transcript of the ancient retrovirus whenever the long C4 gene is transcribed (21). This antisense retroviral RNA transcript with multiple defective mutations could confer the host selection advantage in the defense against retroviral infections. This is because partial hybridizations of the antisense endogenous retroviral RNA transcripts to the proviral RNA genomes would trigger the cellular RNase surveillance and the IFN systems to degrade the double-stranded retroviral hybrids with multiple mismatches. Thus, the host endogenous retroviruses could be a form of innate defense system against retroviral infections (21, 22).

C4HA as a measure of the strength of the complement system

Results of our experiments on the functional activities of C4A and C4B using a conventional hemolytic assay revealed a direct correlation of the hemolytic activities with serum C4 protein concentrations. Higher C4 gene dosage and presence of short C4 gene(s) increased the serum C4 protein levels and hemolytic reactivities. Importantly, this observation reflects the influence of the gene size and polygenic variations of human C4 on the intrinsic strength of the complement system.

In in vitro studies using purified human C4 proteins in most heterologous assays using rabbit Abs and C4-deficient guinea pig complement proteins, activated human C4A proteins have high binding affinities to amino group-containing Ags or immune complexes; activated C4B proteins have an internal catalytic mechanism that favors rapid covalent binding to hydroxyl group-containing Ags through a transesterification mechanism (58, 59). Unexpectedly, the C4 hemolytic activities from 128 samples using conventional hemolytic assays were indifferent to the C4A and C4B isotype composition in the serum. In other words, serum C4A and C4B proteins manifested similar activities in the fluid-phase hemolytic assays, if their serum protein levels were the same. Indeed, differential reactivities between C4A and C4B for bindings to immune complexes, human or sheep erythrocytes, and complement receptor 1, and for prevention of immunoprecipitation and solubilization of immune aggregates could be demonstrated only when the C4A and C4B proteins were purified by an immunochemical method or resolved by gel electrophoresis (16, 60, 61, 62, 63, 64, 65). A differential reactivity between C4A and C4B could not be detected in assay systems when whole human sera were used as a source of C4A or C4B proteins or in reconstituted assays when human C4A- or C4B-deficient sera or human sera with selectively depleted C4 were used (64, 66, 67, 68). One explanation is that the native or activated C4A and/or C4B proteins are regulated or protected by protein(s) in the autologous sera, which mask their differential chemical reactivities in a conventional assay system.

Correlation between BMI and serum concentrations of C4

The observation of a correlation between human serum C4 levels and BMI is novel. When calculated by multiple linear regression analysis adjusted to sex, age, and serum lipid levels, the correlation of C4 and BMI was stronger in the subjects with bimodular RCCX modules only than in the entire study population, and it was the strongest when subjects with monomodular/monomodular and monomodular/bimodular combinations were excluded from the analysis (Table III). The present findings indicate that the mechanism of correlation between C4 and BMI is independent and different from that of complement C3 and BMI. In contrast to C3, there is no correlation between serum lipid levels and serum C4 concentrations. Moreover, at multiple linear regression analysis, C3 and BMI did not significantly correlate after adjustment to serum lipid levels; the same adjustment did not negatively influence the correlation between C4 serum levels and BMI. The correlation between C3 and BMI was stronger in the males than in the females, whereas no gender-related difference could be detected in the relationship of C4 levels and BMI. Why the serum C4 levels are higher in obese than in lean subjects remains to be investigated.

In conclusion, we have shown the C4 polygenic and gene size variations contributing to a wide range of serum levels for C4A and C4B. This phenomenon is relevant. When a patient is diagnosed and treated for a disease, the information on the number of C4A and C4B genes and their serum levels may be helpful. This is because the C4 data would reflect the strength of innate immunity, and also the vulnerability to infections and susceptibility to autoimmune diseases. It is important to note that the serum C4A and C4B are predominantly secreted by the liver and the results presented in this work largely reflected how the C4 gene size and polygenic variations and the BMI influenced the production of C4 by the liver. The influence of C4 polygenic and gene size variations on the regulation mechanisms of C4A and C4B protein biosynthesis in local tissues such as the kidneys, heart, CNS, thyroids, and adrenals is yet to be determined.


   Acknowledgments
 
We sincerely thank the blood donors for participation in this study. We are indebted to Charlene Cameron for excellent secretarial assistance.


   Footnotes
 
1 This work in the United States was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants R01 43969 and 1R01 AR050078, the National Institute of Diabetes and Digestive and Kidney Diseases Grant P01 DK55546, an institutional grant from the Columbus Children’s Research Institute (297401), and Pittsburgh Supercomputing Center through National Institutes of Health Center for Research Resources Cooperative Agreement Grant 1P41 RR06009 (to C.Y.Y.); and in Hungary this work was supported by research grants from the Hungarian Academy of Sciences (2000-107 3,2) and Hungarian National Research Fund (T032661) (to G.F.). Back

2 Address correspondence and reprint requests to Dr. C. Yung Yu, Children’s Research Institute, Department of Pediatrics, The Ohio State University, 700 Children’s Drive, Columbus, OH 43205. E-mail address: cyu{at}chi.osu.edu; or Dr. George Füst, Third Department of Medicine, Semmelweis Medical University, Budapest, Kútvölgyi út 4, H-1125, Hungary. E-mail address: FustGe{at}kut.sote.hu Back

3 Abbreviations used in this paper: RCCX, four-gene module in human MHC with serine/threonine nuclear protein kinase RP, complement C4, steroid 21-hydroxylase CYP21, and extracellular matrix protein tenascin TNX; BMI, body mass index; C4HA, C4 hemolytic activity; HERV-K(C4), human endogenous retrovirus in long C4 gene; HVAGE, high voltage agarose gel electrophoresis; L, RCCX module with long C4 gene; S, RCCX module with short C4 gene. Back

Received for publication February 25, 2003. Accepted for publication July 2, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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Y. L. Wu, S. L. Savelli, Y. Yang, B. Zhou, B. H. Rovin, D. J. Birmingham, H. N. Nagaraja, L. A. Hebert, and C. Y. Yu
Sensitive and Specific Real-Time Polymerase Chain Reaction Assays to Accurately Determine Copy Number Variations (CNVs) of Human Complement C4A, C4B, C4-Long, C4-Short, and RCCX Modules: Elucidation of C4 CNVs in 50 Consanguineous Subjects with Defined HLA Genotypes
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G. Fust, G. J. Arason, J. Kramer, C. Szalai, J. Duba, Y. Yang, E. K. Chung, B. Zhou, C. A. Blanchong, M.-L. Lokki, et al.
Genetic basis of tobacco smoking: strong association of a specific major histocompatibility complex haplotype on chromosome 6 with smoking behavior
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Y. Yang, K. Lhotta, E. K. Chung, P. Eder, F. Neumair, and C. Y. Yu
Complete Complement Components C4A and C4B Deficiencies in Human Kidney Diseases and Systemic Lupus Erythematosus
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