Human Complement Components C4A and C4B Genetic Diversities: Complex Genotypes and Phenotypes

Erwin K. Chung1, Yee Ling Wu1, Yan Yang1, Bi Zhou1, C. Yung Yu1

1 Columbus Children's Research Institute and The Ohio State University, Columbus, Ohio
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 13.8
DOI:  10.1002/0471142735.im1308s68
Online Posting Date:  September, 2005
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Abstract

This unit describes methods that can accurately determine the genotypes and phenotypes of human complement components C4A and C4B. Specifically, they allow investigators to determine how many C4 genes are present in a diploid genome of a human subject and to quantify how many of them encode C4A proteins and how many of them encode C4B proteins. In addition, methods to determine how many long and short C4 genes are present in a diploid genome of a subject are described together with experimental strategies to determine haplotypes and order or configuration of these genes in the MHC. Finally, methods to assess the degree of polymorphism in C4A and C4B proteins and whether low protein levels of plasma C4 may be caused by low C4 gene dosages and/or by mutant C4 genes.

Keywords: C4 polygenic variation; C4 genes; endogenous retrovirus HERV‐K(C4); RP‐C4‐CYP21‐TNX (RCCX) modules; C4A/C4B allotypes; Ch1/Rg1 blood group antigenic determinants; MHC complement gene cluster (MCGC); complement C2 deficiency; labeled‐primer single‐cycle DNA polymerization (LSP); module‐specific PCR

     
 
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Table of Contents

  • Basic Protocol 1: Restriction Fragment Length Polymorphism (RFLP)–Southern Blot for Genomic DNA Analysis of C4 Gene Dosage
  • Basic Protocol 2: Long‐Range Mapping Pulsed‐Field Gel Electrophoresis (PFGE) for Determination of the Modular Structure of RP‐C4‐CYP21‐TNX (RCCX)
  • Support Protocol 1: Probe Labeling and Desalting for Southern Blots
  • Support Protocol 2: Preparation of Genomic DNA Plugs from Unicellular Nucleated Cells
  • Alternate Protocol 1: Module‐Specific PCR for Determination of the Number of RP‐C4‐CYP21‐TNX (RCCX) Modules or Total C4 Gene Dosage
  • Alternate Protocol 2: Labeled‐Primer Single‐Cycle DNA Polymerization (LSP) Reaction Coupled with Restriction Fragment Length Polymorphism (RFLP) for Determination of C4A/C4B Gene Dosages
  • Support Protocol 3: Labeling the 5′ End of Primer E29.3 with T4 Polynucleotide Kinase
  • Basic Protocol 3: Sequence‐Specific Primer (SSP)‐PCR for Determining Known Nonsense Mutations in Complement Components C2 and C4
  • Basic Protocol 4: High‐Voltage Agarose Gel Electrophoresis (HVAGE) for C4 Allotyping
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Restriction Fragment Length Polymorphism (RFLP)–Southern Blot for Genomic DNA Analysis of C4 Gene Dosage

  Materials
  • High‐molecular‐weight human genomic DNA, prepared from peripheral blood using commercial kit (e.g., Puregene; Gentra Systems)
  • 10 U/µl restriction endonucleases (TaqI or PshAI and, for PshAI‐PvuII double digest only, PvuII) and appropriate buffers (New England Biolabs)
  • Mineral oil (optional)
  • High‐gelling‐temperature agarose, biotechnology grade (Amaresco)
  • 1× TBE (see recipe)
  • 0.5% (v/v) ethidium bromide
  • Glycerol dye (see recipe)
  • Depurination buffer: 0.2 M HCl/0.5 M NaCl, optional
  • Denaturation buffer: 1.5 M NaCl/0.5 M NaOH
  • Neutralization buffer: 1.5 M NaCl/0.5 M Tris·Cl, pH 7.4
  • 10× SSC: 1.5 M NaCl/0.15 M sodium citrate
  • SDS‐SET solution: 0.1% (w/v) SDS in SET solution (see recipe), 42°C
  • 20 mg/ml sheared salmon or fish sperm DNA (ssDNA; Amaresco)
  • Prehybridization solution (see recipe)
  • Appropriate radiolabeled hybridization probes (see protocol 3)
  • 0.1% (w/v) SDS/2× SSC
  • 0.5% (w/v) SDS/0.1× SSC
  • Thermal cycler with heated top or water bath set at 65°C, for TaqI digest only
  • 25° and 37°C water baths for PshAI and PshAI‐PvuII double digest, respectively
  • Horizontal agarose gel apparatus (e.g., model GNA200; Pharmacia) and constant power supply (e.g., model 3000; Pharmacia)
  • 1‐liter conical flask
  • Photodocumentation machine with UV transilluminator
  • Nylon membrane (e.g., Hybond N+; Amersham Biosciences) cut to exact size of gel (e.g., 20 cm × 20 cm)
  • Two pieces Whatman 3MM filter paper cut to exact size of gel (e.g., 20 cm × 20 cm) and additional pieces for supporting membrane
  • Blotting device (e.g., Possiblot machine; Stratagene)
  • UV cross‐linker (e.g., Stratalinker 2400; Stratagene)
  • Hybridization oven including hybridization tubes (e.g., Isotemp; Fisher Scientific), 42°C
  • Boiling water bath
  • Water bath, with shaking (e.g., Techne), 65°C
  • Plastic wrapper (e.g., Clingfilm; Fisher Scientific)
  • X‐ray film and film developer (X‐OMAT; Kodak)
  • Phosphorimager (e.g., Molecular Dynamics, Amersham Biosciences) and accompanying software (e.g., STORM; Amersham Biosciences), optional
  • Additional reagents and equipment for transferring DNA onto a nylon membrane using capillary transfer (unit 10.6), optional

Basic Protocol 2: Long‐Range Mapping Pulsed‐Field Gel Electrophoresis (PFGE) for Determination of the Modular Structure of RP‐C4‐CYP21‐TNX (RCCX)

  Materials
  • Very‐large‐molecular‐weight genomic DNA in low‐gelling‐temperature (LGT)‐agarose plugs (see protocol 4)
  • TE buffer, pH 7.4 ( appendix 2A)
  • 5 U/µl PmeI or PacI restriction enzyme and appropriate buffer
  • Agarose
  • 0.5× TBE (see recipe)
  • λDNA ladder and MidRange Marker II (New England Biolabs) molecular weight markers
  • 0.5% (w/v) InCert LGT agarose (FMC), molten
  • 0.5% (v/v) ethidium bromide
  • Depurination buffer: 0.2 M HCl/0.5 M NaCl, optional
  • Denaturation buffer: 1.5 M NaCl/0.5 M NaOH
  • Neutralization buffer: 1.5 M NaCl/0.5 M Tris·Cl, pH 7.4
  • 10× SSC: 1.5 M NaCl/0.15 M sodium citrate
  • SDS‐SET solution: 0.1% (w/v) SDS in SET solution (see recipe), 42°C
  • 20 mg/ml sheared salmon or fish sperm DNA (ssDNA; Amaresco)
  • Prehybridization solution (see recipe)
  • Radiolabeled hybridization probe E (Table 13.8.1; see protocol 3)
  • 0.1% (w/v) SDS/2× SSC
  • 0.5% (w/v) SDS/0.1× SSC
  • Scalpel, sterile
  • 2‐ml microcentrifuge tubes
  • Pulsed‐field gel electrophoresis system (e.g., CHEF Mapper XA; Bio‐Rad)
  • Glass hooks made from Pasteur pipets, sterile
  • Photodocumentation machine with UV transilluminator
  • Nylon membrane (e.g., Hybond N+; Amersham Biosciences) cut to exact size of gel
  • Two pieces Whatman 3MM filter paper cut to exact size of gel and additional pieces for supporting membrane
  • Blotting device (e.g., Possiblot machine; Stratagene)
  • UV cross‐linker (e.g., Stratalinker 2400; Stratagene)
  • Hybridization oven including hybridization tubes (e.g., Isotemp; Fisher Scientific), 42°C
  • Boiling water bath
  • Water bath, with shaking (e.g., Techne), 65°C
  • Plastic wrapper (e.g., Clingfilm; Fisher Scientific)
  • X‐ray film and film developer (X‐OMAT; Kodak)
  • Additional reagents and equipment for transferring DNA onto a nylon membrane using capillary transfer (unit 10.6), optional

Support Protocol 1: Probe Labeling and Desalting for Southern Blots

  Materials
  • Probe DNA (Table 13.8.1): restriction fragments or PCR‐amplified DNA fragments, gel purified (unit 10.5)
  • Multiprime DNA labeling kit (e.g., GibcoBRL Random Primer DNA Labeling System; Invitrogen Life Technologies) containing dNTPs, premix buffer, and Klenow enzyme
  • [α‐32P]dCTP (3000 Ci/mmol; Amersham Biosciences)
  • Microspin G‐50 column (Amersham Biosciences)
  • SDS‐SET solution: 0.1% (w/v) SDS in SET solution (see recipe)
  • Boiling water bath
  • 2‐ml microcentrifuge tubes
  • Tabletop centrifuge

Support Protocol 2: Preparation of Genomic DNA Plugs from Unicellular Nucleated Cells

  Materials
  • Ficoll‐Paque Plus solution (Amersham Biosciences)
  • EDTA or heparinized whole blood ( appendix 3F)
  • PBS (see recipe), 4°C
  • 1% InCert low‐gelling‐temperature (LGT) agarose (FMC): boiled, divided into aliquots in 2‐ml microcentrifuge tubes, and equilibrated at 60°C
  • NDS solution (see recipe)
  • 1.5 mg/ml proteinase K (Fisher)
  • 15‐ml centrifuge tubes
  • Refrigerated tabletop centrifuge (e.g., Eppendorf 5810R), 4°C
  • Plug molds (Bio‐Rad), bottom openings sealed with Scotch tape
  • 2‐ml microcentrifuge tubes
  • Glass hooks made from Pasteur pipets, sterile
  • 50°C water bath

Alternate Protocol 1: Module‐Specific PCR for Determination of the Number of RP‐C4‐CYP21‐TNX (RCCX) Modules or Total C4 Gene Dosage

  Materials
  • Control genomic DNA samples from individuals with three, four, and five C4 genes
  • 100 ng/µl oligonucleotide primers:
    • b: 5′‐GCT CAA GCT GTG AGG AGA ACT‐3′
    • c: 5′‐TAT CAC AGG CTC TGG CCC CA‐3′
    • d: 5′‐TTC GTG GTC CAG TAC AGG GA‐3′
  • Deionized H 2O, autoclaved
  • Platinum Taq PCR x DNA Polymerase (Invitrogen; or equivalent commercial high‐fidelity Taq polymerase kit), including:
    • 10× PCR buffer
    • PCR x enhancer solution
    • 50 mM MgSO 4
    • 5 U/µl Platinum Taq DNA polymerase
  • 2.5 mM dNTP mix: 2.5 mM each dNTP in TE buffer, pH 7.5 ( appendix 2A), store at −20°C
  • Genomic DNA of interest
  • Positive control DNA with bimodular (B)/monomodular (M), B/B, and trimodular (T)/B RCCX modules (Fig. )
  • Thermal cycler and appropriate PCR tubes
  • Photodocumentation machine with UV transilluminator
  • Image quantification software (e.g., ImageQuant TL; Amersham Biosciences)
  • Linear regression software (e.g., Excel; Microsoft)
  • Additional reagents and equipment for DNA agarose gel electrophoresis (unit 10.4)
NOTE: The steps are written for primers b, c, and d (TNXA‐RP2/TNXB). The same method can be used with primers a, b, and c (RP1/TNXA‐RP2).NOTE: The success of this experiment critically relies on the concentrations of the primers because the intensity of each band will need to represent faithfully the actual dosage of the gene segments. For each new batch of primers, this titration needs to be repeated to ensure that the experiment is working.

Alternate Protocol 2: Labeled‐Primer Single‐Cycle DNA Polymerization (LSP) Reaction Coupled with Restriction Fragment Length Polymorphism (RFLP) for Determination of C4A/C4B Gene Dosages

  Materials
  • Commercial PCR amplification kit (e.g., FailSafe PCR System; Epicentre Technologies)
  • Genomic DNA of interest
  • Forward primers (choose one):
    • Y24IN: 5′‐CAG AAG GGT GAG TGT CAC CTG AG‐3′
    • E26.5: 5′‐GCT CAC AGC CTT TGT GTT GAA‐3′
  • Reverse primer:
    • E29.3: 5′‐TTG GGT ACT GCG GAA TCC CC‐3′
  • Commercial PCR purification kit (e.g., Amicon Microncon‐PCR Centrifugal Filter Devices; Fisher)
  • [γ‐32P]E29.3 primer (see protocol 7)
  • Deionized water, autoclaved
  • 10 U/µl PshAI or 5 U/µl XcmI restriction enzyme and appropriate 10× buffer
  • Glycerol dye (see recipe)
  • High‐gelling‐temperature agarose, biotechnology grade (Amaresco)
  • Thermal cycler
  • Scalpel
  • Plastic wrapper
  • Phosphorimager, cassette, and screen (e.g., STORM; Molecular Dynamics)
  • Image analysis software (e.g., ImageQuant; Amersham Biosciences)
  • Additional reagents and equipment for DNA agarose gel electrophoresis (unit 10.4) and Southern blotting (see protocol 1)
NOTE: The difference between the two forward primers is that Y24IN yields a PCR product of 1.3 kb in size, whereas E26.5 yields a 1.1‐kb fragment. It takes less time to separate the PshAI‐digested PCR product (∼900 bp) from the undigested 1.3‐kb C4B PCR product if the Y24IN primer is used.

Support Protocol 3: Labeling the 5′ End of Primer E29.3 with T4 Polynucleotide Kinase

  Materials
  • 500 ng/µl primer E29.3 (5′‐TTG GGT ACT GCG GAA TCC CC‐3′)
  • 10 U/µl T4 polynucleotide kinase and 10× T4 polynucleotide kinase reaction buffer (New England Biolabs)
  • 10 mM spermidine
  • 250 µCi [γ‐32P]ATP (6000 Ci/mmol; Amersham Biosciences)
  • Deionized H 2O, autoclaved
  • Sephadex G‐25 buffered in SET solution (see recipe)
  • SDS‐SET solution: 0.1% (w/v) SDS in SET solution
  • Color dye: 0.01 g of 0.1% (w/v) xylene cyanol and 0.01 g bromphenol blue in 10 ml TE, pH 7.4 ( appendix 2A)
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 95% (v/v) ethanol, 4°C
  • 70% (v/v) ethanol, room temperature
  • Pasteur pipets and small piece of glass wool
  • Clamp stand
  • 2‐ml microcentrifuge tubes
  • Microcentrifuge, 4°C
  • Glass vacuum chamber

Basic Protocol 3: Sequence‐Specific Primer (SSP)‐PCR for Determining Known Nonsense Mutations in Complement Components C2 and C4

  Materials
  • Mutation‐specific forward and reverse primers (Table 13.8.4)
  • >100 ng/µl genomic DNA of interest
  • DNA PCR amplifying kit (e.g., FailSafe PCR System; Epicentre Technologies)
  • 10 U/µl MboI restriction enzyme and appropriate 10× buffer (e.g., New England Biolabs), for C4 Mutation 4 only
  • Molecular weight markers
  • Thermal cycler
  • Additional reagents and equipment for DNA agarose gel electrophoresis (unit 10.4)
    Table 3.8.4   MaterialsProbe Design for Sequence‐Specific Primer PCR for Determining C2 and C4 Nonsense Mutations

    Mutation Definition Forward primer Reverse primer
    Mutation 1 2‐bp deletion in C4 exon 13 C4e13D5: 5′‐ATC CCG AGG GCA GAT CGT TC‐3′ C4e14.3: 5′‐CTT GCC CAT GTT GAG GGG CT‐3′
    Mutation 2 1‐bp deletion in C4 exon 13 13DELB: 5′‐CAT CAC CTG GCA CCC TCC TTT A‐3′ C4e14.3: 5′‐CTT GCC CAT GTT GAG GGG CT‐3′
    Mutation 3 1‐bp deletion in C4 exon 20 20DELF: 5′‐AGT CCA GCT CCG GGT GTT CG‐3′ C4e23.3: 5′‐GTA ACC CTG ACG TAG CTG TT‐3′
    Mutation 4 G→A mutation in C4 intron 28 I27F: 5′‐CAA GAC CCT CCT CCC GTT TTC‐3′ MBO‐28R: 5′‐GCC AGA GCC CCT CAC CCC TGA‐3′
    Mutation 5 2‐bp insertion in C4 exon 29 C4e26.5: 5′‐GCT CAC AGC CTT TGT GTT GAA‐3′ 29InsR: 5′‐GAG AAC CAG TGA CTG AGA GC‐3′
    Mutation C2 28‐bp deletion in C2 exon 6‐intron 6 5C2: 5′‐GCC TGG GCC GTA AAA TCC AAA TCC A‐3′ 3C2: 5′‐GCA CAG GAA GGC CTC TGC TGC AGG C‐3′

Basic Protocol 4: High‐Voltage Agarose Gel Electrophoresis (HVAGE) for C4 Allotyping

  Materials
  • EDTA‐plasma sample of interest
  • PBS (see recipe)
  • 100 U/3 ml neuraminidase (Sigma) in PBS, store in 100‐µl aliquots at −80°C
  • Control EDTA‐plasma samples with C4A3 and C4B1 allotypes, or with any other known C4 allotypes of interest
  • 10 µg/µl carboxypeptidase B (Sigma) in 0.1 M NaCl
  • 0.5× and 1× allotype running buffer (see recipe)
  • Medium electroendosmosis agarose (e.g., Agarose‐ultrapure; Invitrogen Life Technologies)
  • Hemoglobin marker: 1 g lyophilized human hemoglobin (Sigma) resuspended in 600 µl PBS
  • Goat anti–human C4 (DiaSorin)
  • Simplyblue Safestain (Invitrogen)
  • Horizontal gel apparatus and cooler (e.g., Multiphor II and MultiTemp III system; Amersham Biosciences)
  • Gelbond film (Amersham Biosciences)
  • Humidified container (plastic box with lid, lined with wet paper towel)
  • 11 × 12–cm pieces of Whatman 3MM filter paper
  • Hair dryer
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Figures

Videos

Literature Cited

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   Giles, C.M. and Robson, T. 1991. Immunoblotting human C4 bound to human erythrocytes in vivo and in vitro. Clin. Exp. Immunol. 84:263‐269.
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   Lokki, M.‐L., Circolo, A., Ahokas, P., Rupert, K.L., Yu, C.Y., and Colten, H.R. 1998. Deficiency of complement protein C4 due to identical frameshift mutations in the C4A and C4B genes. J. Immunol. 162:3687‐3693.
   Lorenz, M., Regele, H., Schillinger, M., Exner, M., Rasoul‐Rockenschaub, S., Wahrmann, M., Kletzmayr, J., Silberhumer, G., Horl, W.H., and Bohmig, G.A. 2004. Risk factors for capillary C4d deposition in kidney allografts: evaluation of a large study cohort. Transplantation 78:447‐452.
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   Rupert, K.L., Moulds, J.M., Yang, Y., Arnett, F.C., Warren, R.W., Reveille, J.D., Myones, B.L., Blanchong, C.A., and Yu, C.Y. 2002. The molecular basis of complete C4A and C4B deficiencies in a systemic lupus erythematosus (SLE) patient with homozygous C4A and C4B mutant genes. J. Immunol. 169:1570‐1578.
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   Sim, E. and Cross, S. 1986. Phenotyping of human complement component C4, a class III HLA antigen. Biochem. J. 239:763‐767.
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   van den Elsen, J.M.H., Martin, A., Wong, V., Clemenza, L., Rose, D.R., and Isenman, D.E. 2002. X‐ray crystal structure of the C4d fragment of human complement component C4. J. Mol. Biol. 322:1103‐1115.
   Yang, Y., Chung, E.K., Zhou, B., Lhotta, K., Hebert, L.A., Birmingham, D.J., Rovin, B.H., and Yu, C.Y. 2004a. The intricate role of complement C4 in human SLE. Curr. Direct. Autoimmun. 7:98‐132.
   Yang, Y., Lhotta, K., Chung, E.K., Eder, P., Neumair, F., and Yu, C.Y. 2004b. Complete complement components C4A and C4B deficiencies in human kidney diseases and systemic lupus erythematosus. J. Immunol. 173:2803‐2814.
   Yang, Z., Mendoza, A.R., Welch, T.R., Zipf, W.B., and Yu, C.Y. 1999. Modular variations of HLA class III genes for serine/threonine kinase RP, complement C4, steroid 21‐hydroxylase CYP21 and tenascin TNX (RCCX): A mechanism for gene deletions and disease associations. J. Biol. Chem. 274:12147‐12156.
   Yu, C.Y. 1991. The complete exon‐intron structure of a human complement component C4A gene: DNA sequences, polymorphism, and linkage to the 21‐hydroxylase gene. J. Immunol. 146:1057‐1066.
   Yu, C.Y. and Whitacre, C.C. 2004. Sex, MHC and complement C4 in autoimmune diseases. Trends Immunol. 25:694‐699.
   Yu, C.Y., Belt, K.T., Giles, C.M., Campbell, R.D., and Porter, R.R. 1986. Structural basis of the polymorphism of human complement component C4A and C4B: Gene size, reactivity and antigenicity. EMBO J. 5:2873‐2881.
   Yu, C.Y., Campbell, R.D., and Porter, R.R. 1988. A structural model for the location of the Rodgers and the Chido antigenic determinants and their correlation with the human complement C4A/C4B isotypes. Immunogenetics 27:399‐405.
   Yu, C.Y., Blanchong, C.A., Chung, E.K., Rupert, K.L., Yang, Y., Yang, Z., Zhou, B., and Moulds, J.M. 2002. Molecular genetic analyses of human complement components C4A and C4B. In Manuals of Clinical Laboratory Immunology (N.R. Rose, R.G. Hamilton, B. Detrick, eds.) pp. 117‐131. American Society for Microbiology Press, Washington, D.C.
   Yu, C.Y., Chung, E.K., Yang, Y., Blanchong, C.A., Jacobsen, N., Saxena, K., Yang, Z., Miller, W., Varga, L., and Fust, G. 2003. Dancing with complement C4 and the RP‐C4‐CYP21‐TNX (RCCX) modules of the major histocompatibility complex. Prog. Nucl. Acid Res. Mol. Biol. 75:217‐292.
Key References
   Chung et al., 2002b. See above.
  A basic reference for current C4 genotyping techniques.
   Mauff et al., 1998. See above.
  An important reference for C4A and C4B phenotypes.
   Sim and Cross, 1986. See above.
  A seminal paper for C4 allotypes.
   Yu et al., 2003. See above.
  A comprehensive review on the genetics of human and mouse complement C4.
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