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

互联网2013-12-31

3224

Abstract
Table of Contents
Materials
Figures
Literature Cited

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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

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 (Taq I or Psh AI and, for Psh AI‐Pvu II double digest only, Pvu II) 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 Taq I digest only
25° and 37°C water baths for Psh AI and Psh AI‐Pvu II 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 Pme I or Pac I 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‐³² P]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 H₂ O, 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 MgSO₄
- 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‐³² P]E29.3 primer (see protocol 7 )
Deionized water, autoclaved
10 U/µl Psh AI or 5 U/µl Xcm I 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 Psh AI‐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‐³² P]ATP (6000 Ci/mmol; Amersham Biosciences)
Deionized H₂ O, 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 Mbo I 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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 13.8.1 Polygenes and gene size dichotomy of human complement C4 and RP‐C4‐CYP21‐TNX (RCCX) modular variations. (A ) A molecular map of gene organization of the MHC‐complement gene cluster with a bimodular RCCX structure. Horizontal arrows represent transcriptional orientations. Lettered vertical arrows indicate locations and names of DNA probes used for genomic Southern blot analyses (Table ). C2 is a serine proteinase subunit for the C3 and C5 convertases in the classical and lectin complement activation pathways. Bf is a serine proteinase subunit for the C3 and C5 convertases in the alternative complement activation pathway. RD is an RNA binding protein that contains 24 copies of the Arg‐Asp (RD) motif. RD is a negative transcription elongation factor. SKI2W is a nucleolar and ribosomal protein with an RNA helicase domain. DOM3Z is a cytoplasmic protein that might be involved in RNA turnover. (B ) Structures of eleven RCCX length variants. The fragment sizes of Taq I restriction fragments for RP‐C4 , CYP21 , and TNX are shown on the right. The Pme I restriction fragment sizes representing the entire RCCX resolved by pulsed‐field gel electrophoresis (PFGE) are also listed (modified from Yang et al., ).

View Image

Figure 13.8.2 Molecular basis of different Taq I restriction fragments linking RP and C4 in different RCCX modules. Solid, black boxes represent exons of complement C4 (Yu, ). Solid gray boxes represent exons of the neighboring genes RP , CYP21 , or TNX . Numbers above the C4 gene indicate exon numbers. Notice that in the first RCCX module, RP1 is located 5′ to a C4 gene, but CYP21‐TNXA‐RP2 is in this position in the second, third, and fourth RCCX modules (Bristow et al., ; Yang et al., ). HERV‐K(C4) is an endogenous retrovirus located in intron 9 of long C4 genes (Dangel et al., ); included in the diagram are its long terminal repeats (LTRs) and viral genes or genetic elements env , AT , pol , and gag . SVA is a composite retroelement in the RP1 gene (Shen et al., ). The small square box above exon 24 indicates the sequences encoding the thioester bond; the asterisk above exon 26 indicates the location of the C4A or C4B isotypic sequences; the small inverted triangle above exon 28 indicates the location of the sequences encoding the Rg1 or Ch1 antigenic determinant.

View Image

Figure 13.8.3 Genotyping of RCCX length variants and complement C4 gene dosages. Genomic DNA samples from five different subjects with two, three, four, five, and six C4 genes are used to depict the variations. (A ) Taq I genomic restriction fragment length polymorphism (RFLP) to elucidate constituents of RCCX modules: the presence and relative dosages of RP1 or RP2 linked to a long (L) or a short (S) C4 gene, steroid CYP21B (21B) functional gene versus CYP21A (21A) pseudogene, and tenascin TNXB (XB) versus TNXA (XA) gene fragment. Probes A, E, and F (Table ) were used for hybridization. The two haplotype organizations of RCCX gene constituents in each subject are shown on the right panel. (B ) Psh AI genomic RFLP using probe A (RP 3′) to illustrate the relative band intensities of RP1 and RP2 . There are two copies of RP1 genes in a diploid genome. Each duplication of the RCCX module generates one copy of the RP2 gene fragment. Therefore, the total number of RCCX modules can be deduced by the presence and relative band intensities of the Psh AI restriction fragments for RP1 (8.8 kb) and RP2 (7.3 kb). (C ) Long‐range mapping of RCCX modular variants by pulsed‐field gel electrophoresis (PFGE) of Pme I‐digested genomic DNA samples in agarose plugs. Probe E was used for hybridization. The RCCX length variants in each subject are interpreted on the right panel. S, monomodular structure with a short C4 gene; LS, bimodular structure with a long C4 gene in the first module and a short C4 gene in the second module; LL, bimodular structure with two long C4 genes; LSL, trimodular structure with a long C4 gene in the first module, a short C4 gene in the second module, and a long C4 gene in the third module; LLL, trimodular structure with three long C4 genes (modified from Chung et al., ).

View Image

Figure 13.8.4 Module‐specific PCR to elucidate the number of RCCX modules and C4 gene dosages. (A ) Schematic presentation for module‐specific PCR with primer set a, b, and c, or with primer set b, c, and d. Primer a is RP1 specific. Primer b hybridizes to the 3′ ends of RP1 and RP2 . Primer c hybridizes to the 3′ ends of TNXA and TNXB . Primer d is TNXB specific. Primers with filled and open arrows are used to produce results shown in the left and right panels, respectively, in B. (B ) Module‐specific PCR of genomic DNA samples from five human subjects with two, three, four, five, and six C4 gene copies. Left panel, primer set a, b, and c was used. Right panel, primer set b, c, and d was used. The lower panels depict the ratios of band intensities of the PCR products (modified from Chung et al., ).

View Image

Figure 13.8.5 Labeled‐primer single‐cycle DNA polymerization (LSP) and Psh AI restriction fragment length polymorphism (RFLP) analyses to determine the relative dosages of C4A and C4B . (A ) I and II: heteroduplex formation among DNA strands from allelic variants during the denaturation and annealing processes. ³² P‐labeled primer (shown with a star) was used for one cycle of DNA synthesis. III: double‐stranded DNA molecules with labeled primers and one newly synthesized DNA strand do not contain heteroduplexes. (B ) Psh AI RFLP of the DNA mixtures. The original genomic DNA sample has two C4A and two C4B genes. A Psh AI site is present in the C4A isotypic sequence in exon 26. Notice the upper panel shows false results with higher band intensity for C4B than C4A in the ethidium bromide–stained gel. This is because C4A / C4B heteroduplexes were resistant to Psh AI enzyme digestion and therefore yielded misleading result with higher C4B band intensity. The lower panel with autoradiography of radioactively labeled images faithfully shows equal intensities for C4A and C4B genes.

View Image

Figure 13.8.6 Restriction fragment length polymorphism (RFLP) analyses of labeled‐primer single‐cycle DNA polymerization (LSP) products from six human subjects with two to six C4 genes. PCR products spanning exons 26 to 29 were subjected to LSP and restriction enzyme digests. Deviations of data obtained via ethidium bromide–stained gel and via phosphorimaging (or autoradiography) from expected results (closed circles) are plotted. (A ) LSP‐ Psh AI RFLP to demonstrate the quantitative variations of C4A and C4B dosages. (B ) LSP‐ Xcm I RFLP to demonstrate the quantitative variation of C4 genes associated with Rg1 and with Ch1 antigenic determinants. Note that LSP data are closer to expected results in both cases. The asterisk on the 520‐bp band indicates the Rg1‐DNA fragment that carries the labeled primer after the Xcm I digest. This fragment is shown in the autoradiograph in the middle panel. Lanes 1 to 5 are from human subjects identical to those described in Figures and . These five subjects have zero, one, three, four, and five C4A genes, respectively. Subjects 1 and 2 each have two C4B genes. Subjects 3, 4, and 5 each have one C4B gene. Lane 6 is from an individual with two C4A genes and two C4B genes (modified from Chung et al., ).

View Image

Figure 13.8.7 Psh AI and Xcm I restriction fragment length polymorphism (RFLP) analyses of labeled‐primer single‐cycle DNA polymerization (LSP) PCR products. After 1% (w/v) agarose gel electrophoresis of the restriction enzyme–digested products, the gel portion in front of the fast blue (purple) dye can be excised and discarded to reduce the background noise generated by the labeled primer and free [γ‐³² P]ATP. It is also helpful not to let these radioactive, low‐molecular‐weight molecules migrate into the electrophoresis chambers, which could lead to high background noise on the entire gel.

View Image

Figure 13.8.8 Deleterious mutations leading to nonexpression of C4A and C4B proteins. (A ) Locations of mutations (diamonds) in long and short C4 genes (see Fig. for additional gene descriptions). 13, exon 13; 20, exon 20; i28, intron 28; 29, exon 29. (B ) HLA haplotypes, RCCX structures, and molecular basis of mutations leading to C4A and C4B protein deficiencies (modified from Yang et al., ). The number in parentheses represents the number of human subjects with complete C4A and C4B deficiencies demonstrated to have such a mutation. The mutant C4AQ0 or C4BQ0 genes with the described mutations are shown in bold fonts. The 2‐bp insertion at exon 29 is also frequently observed in Caucasian and African‐American subjects with C4A deficiency.

View Image

Figure 13.8.9 Multiplex PCR to detect common mutations in complement components C2 and C4 . Lanes 1 to 3 are from subjects with heterozygous C2 deficiency that is characterized by a 28‐bp deletion spanning exon 6 to intron 6. Lanes 5 to 7 are from subjects with a 2‐bp insertion in exon 29 of C4A (Mutation 5).

View Image

Figure 13.8.10 Phenotyping of complement C4A and C4B by immunofixation. (A ) Effects of neuraminidase and carboxyl peptidase digestions of EDTA‐plasma samples from three human subjects (1: C4A3 B1/C4A3 B1; 2: C4A3 BQ0/C4A3 BQ0; 3: C4AQ0 B1/C4AQ0 B1) for C4 allotyping. The plasma samples were resolved based on the gross differences of electric charges by high‐voltage agarose gel electrophoresis (HVAGE). An arrow represents direction of migration. The gel on the left was run for 4.5 hr. The gel on the right was run for 3.5 hr. The heterogeneities of C4 protein samples were the results of complex glycosylations and incomplete processing of the α‐ and β‐chain carboxyl termini. Notice that neuraminidase and carboxyl peptidase B digests greatly reduced the complexity of C4A and C4B allotypes (Sim and Cross, ). (B ) C4 allotyping of EDTA‐plasma samples (after neuraminidase and carboxyl peptidase B digestions) from five subjects with two to six C4 genes as described in Figures , , and . (C ) Southern blot analysis of Psh AI‐ Pvu II‐digested genomic DNA from the same subjects (modified from Chung et al., ).

View Image

Videos

Literature Cited

	Awdeh, Z.L. and Alper, C.A. 1980. Inherited structural polymorphism of the fourth component of human complement. Proc. Natl. Acad. Sci. U.S.A. 77:3576‐3580.
	Barba, G., Rittner, C., and Schneider, P.M. 1993. Genetic basis of human complement C4A deficiency: Detection of a point mutation leading to nonexpression. J. Clin. Invest. 91:1681‐1686.
	Blanchong, C.A., Zhou, B., Rupert, K.L., Chung, E.K., Jones, K.N., Sotos, J.F., Rennebohm, R.M., and Yu, C.Y. 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 MHC‐associated disease. J. Exp. Med. 191:2183‐2196.
	Bristow, J., Tee, M.K., Gitelman, S.E., Mellon, S.H., and Miller, W.L. 1993. Tenascin‐X: A novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B. J. Cell. Biol. 122:265‐278.
	Chung, E.K., Yang, Y., Rennebohm, R.M., Lokki, M.L., Higgins, G.C., Jones, K.N., Zhou, B., Blanchong, C.A., and Yu, C.Y. 2002a. Genetic sophistication of human complement C4A and C4B and RP‐C4‐CYP21‐TNX (RCCX) modules in the major histocompatibility complex (MHC). Am. J. Hum. Genet. 71:823‐837.
	Chung, E.K., Yang, Y., Rupert, K.L., Jones, K.N., Rennebohm, R.M., Blanchong, C.A., and Yu, C.Y. 2002b. Determining the one, two, three or four long and short loci of human complement C4 in a major histocompatibility complex haplotype encoding for C4A or C4B proteins. Am. J. Hum. Genet. 71:810‐822.
	Dangel, A.W., Mendoza, A.R., Baker, B.J., Daniel, C.M., Carroll, M.C., Wu, L.‐C., and Yu, C.Y. 1994. The dichotomous size variation of human complement C4 gene is mediated by a novel family of endogenous retroviruses which also establishes species‐specific genomic patterns among Old World primates. Immunogenetics 40:425‐436.
	Dodds, A.W., Ren, X.‐D., Willis, A.C., and Law, S.K.A. 1996. The reaction mechanism of the internal thioester in the human complement component C4. Nature 379:177‐179.
	Feinberg, A.P. and Vogelstein, B. 1984. Addendum to “a technique for radiolabelling DNA restriction endonuclease fragments to high activity.” Anal. Biochem. 137:266‐267.
	Feucht, H.E., Schneeberger, H., Hillebrand, G., Burkhardt, K., Weis, M., Riethmuller, G., Land, W., and Albert, E. 1993. Capillary deposition of C4d complement fragment and early renal graft loss. Kidney Int. 43:1333‐1338.
	Fredrikson, G.N., Gullstrand, B., Schneider, P.M., Witzel‐Schlomp, K., Sjoholm, A.G., Alper, C.A., Awdeh, Z., and Truedsson, L. 1998. Characterization of non‐expressed C4 genes in a case of complete C4 deficiency: Identification of a novel point mutation leading to a premature stop codon. Hum. Immunol. 59:713‐719.
	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.
	Giles, C.M., Davies, K.A., and Walport, M.J. 1991. In vivo and in vitro binding of C4 molecules on red cells: A correlation of numbers of molecules and agglutination. Transfusion 31:222‐228.
	Johnson, C.A., Densen, P., Hurford, R.K. Jr., Colten, H.R., and Wetsel, R.A. 1992. Type I human complement C2 deficiency. J. Biol. Chem. 267:9347‐9353.
	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.
	Manzi, S., Navratil, J.S., Ruffing, M.J., Liu, C.C., Danchenko, N., Nilson, S.E., Krishnaswami, S., King, D.E., Kao, A.H., and Ahearn, J.M. 2004. Measurement of erythrocyte C4d and complement receptor 1 in systemic lupus erythematosus. Arthritis Rheum. 50:3596‐3604.
	Mauff, G., Luther, B., Schneider, P.M., Rittner, C., Strandmann‐Bellinghausen, B., Dawkins, R., and Moulds, J.M. 1998. Reference typing report for complement component C4. Exp. Clin. Immunogenet. 15:249‐260.
	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.
	Shen, L., Wu, L.‐C., Sanlioglu, S., Chen, R., Mendoza, A.R., Dangel, A.W., Carroll, M.C., Zipf, W.B., and Yu, C.Y. 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. 269:8466‐8476.
	Sim, E. and Cross, S. 1986. Phenotyping of human complement component C4, a class III HLA antigen. Biochem. J. 239:763‐767.
	Stewart, C.A., Horton, R., Allcock, R.J., Ashurst, J.L., Atrazhev, A.M., Coggill, P., Dunham, I., Forbes, S., Halls, K., Howson, J.M., Humphray, S.J., Hunt, S., Mungall, A.J., Osoegawa, K., Palmer, S., Roberts, A.N., Rogers, J., Sims, S., Wang, Y., Wilming, L.G., Elliott, J.F., de Jong, P.J., Sawcer, S., Todd, J.A., Trowsdale, J., and Beck, S. 2004. Complete MHC haplotype sequencing for common disease gene mapping. Genome Res. 14:1176‐1187.
	Uejima, H., Lee, M.P., Cui, H., and Feinberg, A.P. 2000. Hot‐stop PCR: A simple and general assay for linear quantitation of allele ratios. Nat. Genet. 25:375‐376.
	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.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

关于丁香通

公司信息

个人用户

企业机构

无忧采购轻松科研

提问

扫一扫

实验小助手

扫码领资料

反馈

TOP

打开小程序

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited

关于丁香通

公司信息

个人用户

企业机构