Real-time PCR quantification of human complement C4A and C4B genes
© Szilagyi et al; licensee BioMed Central Ltd. 2006
Received: 29 July 2005
Accepted: 10 January 2006
Published: 10 January 2006
The fourth component of human complement (C4), an essential factor of the innate immunity, is represented as two isoforms (C4A and C4B) in the genome. Although these genes differ only in 5 nucleotides, the encoded C4A and C4B proteins are functionally different. Based on phenotypic determination, unbalanced production of C4A and C4B is associated with several diseases, such as systemic lupus erythematosus, type 1 diabetes, several autoimmune diseases, moreover with higher morbidity and mortality of myocardial infarction and increased susceptibility for bacterial infections. Despite of this major clinical relevance, only low throughput, time and labor intensive methods have been used so far for the quantification of C4A and C4B genes.
A novel quantitative real-time PCR (qPCR) technique was developed for rapid and accurate quantification of the C4A and C4B genes applying a duplex, TaqMan based methodology. The reliable, single-step analysis provides the determination of the copy number of the C4A and C4B genes applying a wide range of DNA template concentration (0.3–300 ng genomic DNA). The developed qPCR was applied to determine C4A and C4B gene dosages in a healthy Hungarian population (N = 118). The obtained data were compared to the results of an earlier study of the same population. Moreover a set of 33 samples were analyzed by two independent methods. No significant difference was observed between the gene dosages determined by the employed techniques demonstrating the reliability of the novel qPCR methodology. A Microsoft Excel worksheet and a DOS executable are also provided for simple and automated evaluation of the measured data.
This report describes a novel real-time PCR method for single-step quantification of C4A and C4B genes. The developed technique could facilitate studies investigating disease association of different C4 isotypes.
In addition to length variations, C4 genes have two main isotypes, C4A and C4B encoding functionally different proteins, as C4A is more reactive with targets containing free amino groups while C4B has a higher affinity to hydroxyl groups [4, 5]. Most individuals have the same number of the two different C4 genes, while about 30% of the population has a lower level of either C4A or C4B proteins. The unbalanced production of C4A and C4B proteins has been associated to several diseases. Complete deficiency of the C4A or C4B gene in a haplotype module is referred to as C4A*Q0 and C4B*Q0, respectively. C4A*Q0, which is an essential constituent of the 8.1. ancestral haplotype, was found to be associated with systemic lupus erythematosus [6, 7], insulin-dependent diabetes mellitus [8, 9], myasthenia gravis , other autoimmune diseases and abnormalities of the immune system (reviewed in ). On the other hand, carriers of the C4B*Q0 have a highly increased risk for myocardial infarction , stroke  and an increased vulnerability for microbial infections . Interestingly autism  and narcolepsy  have also been described to be associated with C4B deficiency although no responsible haplotype was identified.
For several decades the number of the C4A and C4B genes has been evaluated by phenotyping, i.e. by measuring the relative amount of the C4A and C4B proteins employing immunofixation electrophoresis. Direct quantification of C4A and C4B is more difficult as these genes are highly homologous with only five isotypic nucleotide differences [16, 17]. This sequence variation can be detected by restriction fragment length polymorphism (RFLP) combined with Southern blot analysis . Determination of the RCCX module number is possible with Taq I RFLP, while Psh A I RFLP was earlier used to define the C4A/C4B ratio . Beside these techniques, there are several methods to demonstrate of the complete absence of C4A and C4B isoforms. C4 null alleles with non-expressed or absent C4A/C4B genes can be detected by high voltage agarose gel electrophoresis of carboxypeptidase and neuraminidase treated serum or plasma samples , as well as by RFLP analysis . A rapid screening method was developed to determine the main form of the C4A null allele (C4A deletion) by long PCR . Man and co-workers described a polymerase chain reaction (PCR) procedure with sequence specific primers (PCR-SSP) to determine the frequency of C4A and C4B null alleles in SLE patients .
Real-time PCR is one of the most applicable and up-to-date methods for DNA quantification, which allows to track the accumulation of the PCR product during the reaction. It provides the possibility to report the results as a threshold cycle (CT) value, which is the cycle number where the measured fluorescence reaches a given threshold. This threshold is adjusted to the initial section of the exponential phase of the amplification, thus the CT value is highly proportional to the copy number of the template DNA. Double-stranded DNA binding non-specific dyes, such as SYBR Green or sequence-specific probes (double-dye oligonucleotides) can be used to detect the amplified PCR products during the reaction. Employment of TaqMan probes is one of the most general applications of the latter approach, where fluorescence is generated based on the 5' nuclease activity of the Taq polymerase. The 3' quencher dye of the intact probe absorbs the light emitted by the reporter dye at the 5' end of the oligonucleotide and emits at much longer wavelengths that is not detected by the real-time PCR machine. On the other hand, the DNA polymerase cleaves the TaqMan probe during the extension step of the PCR, thus the emitted light of the reported dye is not quenched any more . One of the most commonly used quencher dyes is TAMRA, an emerging alternative is however the employment of the MGB (minor groove binder) probes instead, which possesses several advantages. Although TAMRA emits at 582 nm, it is not completely dark at those wavelengths that are detected by the real-time PCR instruments. MGB is a non-fluorescent "dark" quencher, moreover it stabilizes the probe-template DNA duplex providing enhanced mismatch discrimination and higher precision at quantitative assays [25, 26].
Here, we report a novel and rapid qPCR method for the gene dosage determination of the complement C4A and C4B genes. Our system employs real time PCR, and affiliates the two major applications of TaqMan probes: quantitative assay and SNP detection.
Determination of the number of C4A and C4B genes
A novel robust and high throughput method was developed for C4 gene dosage determination by quantitative real time polymerase chain reaction (qPCR). Sequence specific TaqMan® probes with minor groove binding (MGB) non-fluorescent quencher were applied to determine the number of the two isotypes, the quantitative assay of the RNase P gene was used as a reference. The copy number of C4A and C4B genes was determined in two separate tubes. Reaction mixture I contained the VIC-labeled C4A-specific probe and the FAM-labeled RNase P system, while the FAM-labeled C4B-specific probe and the VIC-labeled RNase P reference were applied in reaction mixture II (for sequences see Methods). To obtain the most consistent CT values, automatic baseline and manually adjusted threshold to the lowest possible level (approximately to 0.04 fluorescence (ΔRn) value) were applied. The number of C4A and C4B genes (nC4A, nC4B) was calculated according to equations (1) and (2),
The real number of C4A and C4B genes is the rounded value of nC4A and nC4B respectively, CT(RF), CT(C4A),CT(RV)and CT(C4B)are the determined threshold cycle values of the FAM labeled reference (RNase P), the C4A, the VIC labeled reference and the C4B reactions. qC4A:RF and qC4B:RV are the efficiency quotients for VIC labeled C4A and FAM labeled reference in reaction I, and for FAM labeled C4B and VIC labeled reference in reaction II, respectively.
Calculation of efficiency quotients
"Individual" (rows 1–7) and "overall" (last row) efficiency quotients (q values) and the corresponding error of C4A and C4B gene qunatification
0.38 ± 0.03
0.64 ± 0.04
0.40 ± 0.02
0.71 ± 0.03
0.36 ± 0.04
0.67 ± 0.07
0.39 ± 0.03
0.70 ± 0.03
0.36 ± 0.02
0.66 ± 0.03
0.41 ± 0.03
0.67 ± 0.05
0.42 ± 0.02
0.65 ± 0.05
0.39 ± 0.04
0.67 ± 0.05
Reliability of C4A and C4B gene quantification
To analyze the reliability and reproducibility of the developed qPCR system, we investigated the error of the C4A and C4B gene quantification by calculating either with the overall q of our 7 pilot experiments or with the values defined by each individual experiment ("individual q") respectively. Error of the gene number quantification was calculated as the absolute value of the difference of the exact (equations (1) and (2)) and rounded n values according to equation (3).
e = |nC4 - int(nC4 + 0.5)| (3)
Reliability of gene number determination in a large DNA concentration range
Application of the developed qPCR system for C4 gene dosage analysis in a Hungarian population
C4A and C4B gene dosage in a healthy cohort determined by the presented qPCR method. Comparison to earlier data obtained by Southern-blot analysis 
p = 0.7023
p = 0.3632
Software provided for calculations
An Excel sheet (MHC.XLS) and a stand-alone software (MHC.EXE) were designed to improve the processing of the measured C T values. The software reads the "csv" file created by the "Export Ct values..." function of the Sequence Detection Software (SDS) of the Real Time PCR Instrument of Applied Biosystems. It optimizes the q values in a default range of 0.19–1.59 for both qC4A:RF and qC4B:RV, however these ranges can be modified by the user. The measure of stringency ("error level") has to be entered, and the software creates a data file containing the C4A and C4B gene numbers and the reliability of the results. "+++" means that e (see equation 4) is smaller than the error level, "++" shows that e is higher than this limit, but lower than its double. One or zero "+" shows even higher distance of the calculated number from an integer, in this case the gene dosages are not determined. The software can export sample names and C T values in the format required by the Excel sheet.
The Excel file, MHC.XLS calculates the same results in a more user-friendly Windows based environment. In this case the user has to type the q values, however the calculated optimal ratios are shown. Moreover red background of these cells warns if the entered numbers are too far away from the optimal values. In this file green, yellow and red dots show the reliability of the calculations of the number of the C4A and C4B genes, based on the entered "error limit" and the same assumptions described above. Further details about the software and the Excel sheet can be read in their manual that can be downloaded together with the files as "additional files of the paper".
Real-time PCR is a useful tool for quantitative measurements, thus it is readily applicable for gene expression analyses as well as for the investigation of gene dosage (i.e. for the determination of the copy number of different genes in the genome or in transgenic organisms). Melo et al. developed a real-time PCR based system for the quantification of glucocorticoid receptor alpha isoform. Similarly to our observation they demonstrated that the method is reliable in a very wide template concentration range of higher than 3 orders of magnitude . Bubner and Baldwin reviewed the use of real-time PCR for determining copy number and zygosity in transgenic plants. It was shown that carefully optimized reaction conditions and the application of MGB probes in combination with the comparative method () provided the possibility to detect as low as two-fold differences which is a key issue in gene dosage analyses .
The quantification of C4A and C4B genes is of great clinical as well as theoretical importance, because either the deficiency or the exceeding amount of any of the two C4 variants may adversely influence immune processes. Although the sequence of the two isoforms differs in less than 1%, this variance alter their hemolytic and serological reactivity as well as their affinity to antigens and immune complexes . The disease association of the C4A*Q0 or C4B*Q0 phenotypes have been widely investigated. C4A*Q0, an essential constituent of the 8.1 ancestral haplotype  was found to be related to systemic lupus erythematosus [31–34], Graves' disease  and systemic sclerosis . In contrast the association of the C4B*Q0 and shorter life-expectancy , increased susceptibility for myocardial infarction , stroke , autism , Henoch-Schonlein purpura glomerulonephritis , bacterial meningitis, angio-oedema and "lupus-like" disease , and bacteremia with encapsulated organisms  and meningococcal disease  was also described.
This wide range of diseases underlines the relevance of a simple and high throughput method for C4 gene dosage analysis to improve our knowledge about the role of functionally different isotypes in physological as well as in the above mentioned pathological immune procedures. RFLP and gel electrophoresis based methods developed earlier [18, 20] are of low throughput, moreover they are technically difficult and labor intensive. RFLP analysis in combination with Southern-blot is the only method at present which is suitable for characterization of the whole RCCX module, but it requires a large amount of DNA and the employment of radioisotopes.
Our report presents a rapid qPCR technique for the determination of the number of C4A and C4B genes. The developed method is a single step procedure, where no subsequent post-PCR analyses are required. Computational applications are also provided for automated allele-calling procedure, which makes the evaluation of the results fast, simple and reliable. The determined C4 gene dosages were compared to the data of an earlier study that investigated the same group of healthy Hungarian subjects applying Southern blot for C4A and C4B gene quantification. Moreover 33 samples were analyzed by two independent methods. There was no significant difference between the results of these studies underlining the accuracy of our novel method. Although the demonstrated technique has some limitations, for example it is not suitable for the identification of the non-functional C4 genes caused by 1- or 2-bp-deletions , this comprehensive system could facilitate the investigation of complement C4A and C4B genes in the future.
Genomic DNA was isolated from peripheral blood using the Flexigene DNA isolation kit (Qiagen). Primers and fluorogenic probes for the 5' nuclease assay were designed by the Primer Express software. Fig (4) shows a short segment of the sequence of complement C4B gene [GenBank:U24578] demonstrating the position of the primers (arrows) and the TaqMan probe. The black boxes indicate those nucleotides that are different in the two isoforms, this sequence variation was used to design the C4A and C4B specific probes. For quantifying the C4A and C4B genes two separate reactions were used, three parallels were carried out for each measurement. Both reaction mixtures contained 6 μM forward (5' GCA GGA GAC ATC TAA CTG GCT TCT 3') and 6 μM reverse (5' CCG CAC CTG CAT GCT CCT 3') primer (see Fig (4)), 1× TaqMan Universal PCR Master Mix (AmpliTaq Gold® DNA Polymerase, dNTPs with dUTP, Passive Reference, No AmpErase UNG®) and genomic DNA template. Reaction mixture I contained furthermore the C4A specific TaqMan probe (5' VIC-ACC C CT G TC CAG TGT TAG-MGB 3'; MGB: minor groove binding non-fluorescent quencher) and the FAM-labeled 1× RNase P Detection Mix (ABI Cat. No. 4316831), while the FAM-labeled C4B specific TaqMan probe (5' FAM-ACC T CT C TC CAG TGA TAC-MGB 3') and the VIC-labeled 1× RNase P Detection Mix (ABI Cat. No. 4316844) was added to reaction mixture II in a total volume of 25 μl. (The bold and underlined letters show the sequence differences of the two probes corresponding to the nucleic acid variations that distinguish the C4A and C4B genes.) DNA amplification was carried out in an ABI 7500 Real Time PCR System. Thermocycle was initiated by incubating the mixtures at 95°C for 10 minutes to denature genomic DNA and to activate AmpliTaq Gold® DNA Polymerase. This was followed by 40 cycles of two steps of 95°C for 15 sec and 60°C for 1 minute, the fluorescence intensity was measured during the step of 60°C.
DNA samples of 173 healthy Hungarian individuals were used for the present study. These individuals participated in a regular medical survey and gave their informed consent for the use of their sample for the study. For ethical reasons after their computer registration the data were unlinked from the subjects so their identities could not be traced. The study was approved by the Ethical Committee of the Semmelweis University (Budapest, Hungary)
This work was supported by Hungarian grants GVOP AKF 311 2004 05 0324_3.0, OTKA F42730 and by National Office for Research and Technology (NKTH).
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