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* Children’s Research Institute, Departments of
Pediatrics and
Molecular Virology, Immunology and Medical Genetics, and
Ohio State Biochemistry Program, Ohio State University, Columbus, OH 43205;
¶ Division of Rheumatology and Clinical Immunogenetics, University of Texas Health Science Center, Houston, TX 77225;
|| Department of Microbiology and Immunology, MCP Hahnemann University School of Medicine, Philadelphia, PA 19129;
# Pediatric Rheumatology Center, Department of Pediatrics, Texas Children’s Hospital, and
** Department of Immunology, Baylor College of Medicine, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induction (2, 15, 16).
Population studies revealed that deficiencies of C4A or C4B have a
combined frequency of 
2% (5, 17, 18). The presence of
a single C4A or C4B gene in one or both MHC
haplotypes and/or the isoexpression of C4A proteins only (or C4B
proteins only) from bimodular and trimodular RCCX structures constitute
complete or partial deficiencies of either C4A or C4B. Close to 30% of
the normal population have relatively lower levels of either C4A or C4B
proteins caused by the presence of only one C4A gene or only
one C4B gene in a diploid genome (5, 19, 20).
Also, in HLA A2 Cw3 B60 DR6 haplotypes with bimodular LS
structures for RCCX, the long C4A gene does not code for a
protein product whereas the short C4B gene codes for C4B1 or
C4B2 protein (21). The mutant C4A gene,
C4AQ0, is caused by frame-shift mutations due to a
dinucleotide insertion in exon 29 at the sequence for codon 1213. This
specific mutation has been detected in both healthy Caucasians and
those with autoimmune disease (5, 21, 22, 23, 24).
The great genetic diversity of human C4A and C4B genes leads to quantitative and qualitative variations of the polymorphic C4A and C4B proteins, which may be one of the intrinsic mechanisms in response to the selection pressure against a great variety of microbes or parasites. Deficiencies of C4A and/or C4B are associated with a variety of autoimmune or infectious diseases. For examples, deficiency of C4A or C4B is associated with type 1 diabetes (25, 26, 27). Complete or partial deficiency of C4A is linked to systemic lupus erythematosus (SLE) (28, 29, 30, 31), autoimmune hepatitis (32, 33), and disease manifestation and rapid progression of AIDS in HIV-infected patients (34, 35). Deficiency of C4B increases the vulnerability to bacterial (36, 37) and viral (38, 39) infections.
In contrast to the frequent partial or complete C4A or C4B deficiencies, complete deficiencies of both C4A and C4B in an individual is rare (23, 24, 40, 41, 42, 43). Complete C4 deficiency is almost invariably discovered first in individuals or family members with SLE or other autoimmune disease (17). Most completeC4-deficient patients regularly suffer recurrent bacterial or viral infections, are photosensitive, and have early onset lupus or lupus-like diseases (17). Studying the clinical symptoms in C4-deficient patients may lead to better understanding of the physiological roles of human C4A and C4B. Investigating the molecular basis leading to complete C4A and C4B deficiencies will help in creating diagnostic techniques to detect C4 mutations. Here we report the clinical case history of a complete C4A- and C4B-deficient patient and the characterization of the molecular basis of different mutations in the C4AQ0 and C4BQ0 genes leading to frame-shift mutations and premature stop codons.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human genomic DNA was isolated from the peripheral blood or from B cell lines transformed with EBV strain NPC-LC of normal individuals, SLE patients and families, and from the cultured cell line MOLT4 (T cell leukemia) using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN).
Six micrograms of genomic DNA were digested to completion with the
appropriate restriction enzyme, resolved on a 0.8% agarose gel,
blotted onto Hybond-N+ membrane (Amersham Life
Science, Arlington Heights, IL), and hybridized with appropriate
[
-32P]dCTP-labeled probe(s), as previously
described (5, 44).
Complement allotyping, assay of protein titers, and HLA typing
Complement C4A and C4B allotypes from EDTA-plasma samples were determined by immunofixation and immunoblot analyses as described previously (44, 45, 46). Ch and Rg phenotyping were performed on both RBC and plasma using standard serological methods. Complement C3 typing was performed by immunofixation of EDTA-plasma using a method similar to that of C4 except that the quantity of plasma was reduced by 5-fold. Goat antiserum for human C3 was purchased from Diasorin (Silverwater, MN).
C3 and C4 (C4A, C4B) levels were determined by radial immunodiffusion (47), ELISA (48), and total hemolytic complement in gels (Quantiplate; Kallestad, Beaumont, TX).
Typing of HLA-A, -B, and -DR Ags was performed by serology (49) at the University of Texas-Houston Medical School. HLA class II typings were by DNA oligotyping, as previously described (50).
Synthetic DNA primers
Oligonucleotides of human C4 and RCCX constituents were designed based on published DNA sequences (51, 52, 53) and synthesized by Life Technologies (Gaithersburg, MD) to facilitate DNA cloning and sequencing are as follows. For amplification of CYP21A for use as a probe: 21A5 TGT GGC CAT TGA GGA GGA A, 21A3 TGC CAC CGA TCA GGA GGT C. For amplification of TNXA for use as a probe: RDX-5 AAT TCA GTG AAA TCA GGG AGA CC, RDX-3 TTC CAG TGC AGC ACG GCG AA. For amplification of C4d-specific region for use as a probe: C4dB5 CCA AGG CTA CAT GCG GAT CCA GC, C4dB3 CAC ACT CAG GAT CCT AAG TCC CT. For amplification of the C4d region: C4E22.5 GAA GGG GCC ATC CAT AGA GA, C4E31.3 CTT CAG GGT TCC TTT GCT GT. For detection of 2-bp insertion in C4A or C4B: A-down AGG ACC CCT GTC CAG TGT TAG AC, B-down AGG ACC TCT CTC CAG TGA TAC AT, C4INS GCT CTG AGA ACC AGT GAC TAG AG. For C4 exon 13 deletion PCR: 13DELB CAT CAC CTG GCA CCC TCC TTT A, C4E14.3 CTT GC CCA TGT TGA GGG GCT. For C4 exon 20 deletion PCR: 20DELF AGT CCA GCT CCG GGT GTT CG, C423.3 GTA ACC CTG ACG TAG CTG TT. For cloning and/or sequencing of C4 gene, 52 forward or reverse primers were used for sequencing of the 41 exons in the entire short C4BQ0 gene. These primers span the 5' or 3' regions of exons 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 35, 37, 38, 39, 40, and 41. The primers were designed based on the genomic DNA sequence of C4A3a (51).
Amplification of genomic DNA via PCR
A 2.4-kb fragment corresponding to the C4d region was amplified with synthetic PCR primers C4E22.5 and C4E31.3 using the Expand High Fidelity PCR system (Boehringer Mannheim, Indianapolis, IN). PCR conditions were: 1 cycle at 94°C for 2 min; 10 cycles at 94°C for 15 s, 65°C to 56°C for 30 s, and 72°C for 1 min; 20 cycles at 94°C for 15 s, 56°C for 30 s, and 72°C for 1 min plus time extensions of 20 s; and 1 cycle at 72°C for 7 min. Similarly, a 6.8-kb fragment spanning from C4 exon 1 to the C4B-isotypic site in exon 26 was amplified with synthetic PCR primers C4E1.5 and B-up, whereas a 7.0-kb fragment spanning from the isotypic site in exon 26 to C4 exon 41 was amplified with synthetic PCR primers B-down and C4E41.3. PCR conditions were: 1 cycle at 94°C for 2 min; 10 cycles at 94°C for 15 s, 64°C to 55°C for 30 s, and 68°C for 8 min; 20 cycles at 94°C for 15 s, 55°C for 30 s, and 68°C for 8 min plus 5-s time extensions; and 1 cycle at 72°C for 7 min.
Cloning and sequencing of C4d region and C4 exons 1–41
Genomic PCR products were purified (Qiagen, Chatsworth, CA) and cloned into the TA vector (Invitrogen, San Diego, CA). The presence of the correct size insert was verified via EcoRI restriction digests. Furthermore, to separate C4d clones into C4A and C4B isotypes, the C4d insert was amplified again via PCR, and the purified PCR product was restriction digested with NlaIV. A 467-bp fragment represents a C4B whereas two fragments (276 and 191 bp) represent C4A (19). To verify that the amplified PCR products from the C4E1.5 to B-up and B-down to C4E41.3 reactions correspond to C4, the EcoRI-digested clones were subjected to Southern blotting and hybridized to a C4d-specific probe. Clones showing highlighted fragments corresponding to the correct size insert restriction fragments were considered positive.
DNA samples were sequenced on an automated system (ABI Prism, Foster City, CA). Comparison of DNA sequences with national databases was performed with the GCG FASTA program from the Pittsburgh Supercomputer Center (Pittsburgh, PA). DNA alignments were performed with the SeqManII program.
Sequence-specific PCR to detect C4 mutations
Detection of 2-bp insertion in C4 exon 29. The presence of a 2-bp insertion in C4A exon 29 (21) or C4B exon 29 (24) was detected via PCR using synthetic PCR primer pairs A-down and C4INS, and B-down and C4INS, respectively. PCR was performed with Taq DNA polymerase (Life Technologies). PCR conditions were: 1 cycle at 94°C for 5 min; 25 cycles at 94°C for 30 s, 65°C for 45 s, and 72°C for 1 min; and 1 cycle at 72°C for 10 min.
Detection of C4 exon 13 deletion via PCR. The existence of the 1-bp deletion in C4 exon 13 was confirmed via genomic PCR using synthetic PCR primers 13DELB and C4E14.3. PCR was performed with Taq DNA polymerase (Life Technologies). Conditions were: 1 cycle at 94°C for 5 min; 25 cycles at 94°C for 30 s, 65°C for 45 s, and 72°C for 1 min; and 1 cycle at 72°C for 10 min.
Multiplex PCR. A multiplex PCR strategy was created to simultaneously report existence of all known C4 mutations in one DNA sample. Synthetic PCR primers 13DELB and C4E14.3 amplify a 400-bp fragment corresponding to the 1-bp deletion in exon 13; primers 29INSR and C4E26.5 amplify a 860-bp fragment corresponding to the 2-bp insertion in exon 29; positive control primers 21A5 and 21A3 amplify a 757-bp fragment corresponding to the gene CYP21. PCR was performed with Taq DNA polymerase, and conditions were: 1 cycle at 94°C for 5 min; 25 cycles at 94°C for 30 s, 60°C for 45 s, and 72°C for 1 min; and 1 cycle at 72°C for 10 min.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The proband was a 23-year-old white man born in 1978 who had recurrent viral and bacterial infections as a child, as well as fever, arthralgias, and rashes. In 1985, he was found to be totally deficient for C4. Between 1988 and 1989, fevers became higher and he developed dry eyes and dry mouth accompanied by a unilateral parotitis. Soon thereafter, he evolved a nondeforming polyarthritis, Raynaud’s phenomenon, a photosensitive, maculopapular rash over the malar areas and arms, and multiple vasculitic lesions of the fingers and toes. An antinuclear Ab (ANA) assay was positive at 1:10,240, and other positive serologies included anti-Sm (Smith), anti-U1-ribonucleoprotein, and anti-cardiolipin Abs. Abs to dsDNA were consistently negative. He was diagnosed with SLE and Sjögren’s syndrome and treated variously with prednisone, hydroxychloroquine, and cyclophosphamide. A renal biopsy in 1992 was read as WHO class III mild lupus nephritis. Since 1999, the proband declined further medication and did not consult a physician for >2 years. In October 2001, he was hospitalized after several months of increasing incoordination of gait, loss of fine motor control of arms and hands, slowed mentation, and somnolence. Magnetic resonance imaging of the brain showed multiple nonenhancing lesions throughout the cerebrum and cerebellum. Two brain biopsies were performed 1 month apart and showed multiple arterial walls infiltrated with mononuclear cells compatible with vasculitis. Cultures were negative. Despite high doses of corticosteroids (up to 1000 mg daily), the patient’s neurological status continued to deteriorate, and he died.
The proband’s parents and only sibling were examined in 1985 and 1988 and followed up by phone in 1998. The family had resided in Louisiana for several generations and was of French descent. Any consanguinity was denied. The deceased maternal grandfather had been diagnosed with discoid lupus. Both parents were healthy and had negative tests for ANA.
The proband’s brother had developed biopsy-proved lupus rash of the
face in 1981 at age 21 years, as well as recurrent oral ulcers
and a transient polyarthritis. Residual rash of the face was
present in 1988, but his examination was otherwise normal. Serum
testing in 1988 showed negative tests for ANA, anti-dsDNA,
anti-Ro (SS-A), anti-La (SS-B), anti-Sm (Smith), and
anti-U1-ribonucleoprotein. Subsequently, he has remained healthy
except for a persistent facial rash. He has not had an unusual
frequency of infections. A diagram of the family pedigree is shown in
Fig. 1
A.
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EDTA-plasma samples from the proband (Fig. 1
, lanes 1),
his brother (Fig. 1, lanes 2), and parents (Fig. 1, lanes 3 and 4) were used to show the polymorphism
of complement C3 (Fig. 1B) and C4 (Fig. 1C) by
immunofixation. Except for the patient’s father who was
homozygous for the slow-migrating variant of C3, C3S (Fig. 1B, lane 3), the other three members of the
family were all heterozygous for the fast- and slow-migrating variants
of complement C3. Positive results of the C3 allotyping experiment
suggested that the EDTA-plasma samples were not degraded.
No C4 proteins were detected in the samples of the proband and his
brother, by both immunofixation (Fig. 1C) and immunoblot
(Fig. 1D) analyses or by serological methods. A relative
prominent band corresponding to C4A3 but not C4B was detected in the
patient’s father (Fig. 1C, lane 3). As expected,
this C4A3 allotype did not interact with the anti-Ch1 mAb (Fig. 1D, lane 3). Both C4B1 and C4A3 protein were
detectable in the mother (Fig. 1C, lane 4). The
intensity of the C4B1 protein was considerably higher than that of
C4A3. As shown in Fig. 1D, the C4B1 protein strongly reacted
with the Ch1-monoclonal. Negative results for C4A and C4B proteins in
the proband and his brother were obtained from blood samples obtained
on multiple occasions using immunofixation, ELISA, and serological
methods.
Genomic TaqI RFLP analysis of the family shows that the patient and his brother had homozygous, bimodular RCCX structures each with a long and a short C4 gene, i.e., LS/LS. In other words, four C4 genes present in each of their diploid genomes. The parents were heterozygous with two long C4 genes on one chromosome 6 (i.e., LL), and one long C4 gene and one short C4 gene on the other chromosome 6 (i.e., LS). In other words, the bimodular LS chromosomes were transmitted to the proband and his sibling. There were no unusual patterns for the neighboring genes RP, CYP21, and TNX.
Genomic PshAI RFLP experiments were performed to detect the
quantitative variations of C4A and C4B genes
(Fig. 1F). Equal intensities of the 8.2-kb and 4.35-kb
PshAI corresponding to C4B and C4A
genes, respectively, were observed in the patient (Fig. 1F,
lane 1), the sibling (Fig. 1F, lane 2)
and their mother (Fig. 1F, lane 4). Because each
member of the family had four C4 genes, there are two
C4A genes and two C4B genes in the proband, the
sibling, and their mother. On the contrary, the band intensity of the
C4A fragment was 3 times greater than that of the
C4B fragment in the patient’s father (Fig. 1F,
lane 3), suggesting the presence of three C4A
genes and one C4B gene.
HLA typing of the class I and class II alleles revealed that the
parents shared one common haplotype, A2 B12 and
DR6 (DRB1*1302, DQA1*0102,
DQB1*0604) (haplotype a), which were both
transmitted to the proband and his sibling (Fig. 1, A and
G). This haplotype has the LS structure for the bimodular
RCCX and carries one nonexpressing C4A mutant gene
(C4AQ0) and one nonexpressing C4B mutant
gene (C4BQ0). Segregation analysis revealed that the other
HLA haplotype (haplotype b) in the proband’s father was
A2 B16, C4A3 C4A3, and DR6
(DRB1*1302, DQA1*0102, DQB1*0604) in
the class I, III, and II regions, respectively. The nontransmitted
haplotype c in the mother was HLA A2 B12 C4A3 C4B1
DR6 (DRB1*1302, DQA1*0102,
DQB1*0604). Haplotypes b and c both
had the bimodular LL structure in the RCCX.
Mutation analyses at the C4d region via sequence-specific PCR and DNA sequencing
To determine whether the C4A and C4B mutant
genes in the proband were the result of the TC/GA dinucleotide
insertion into the sequence for codon 1213 in exon 29 of the
C4A and C4B genes, sequence-specific PCR and DNA
sequencing were performed. Genomic DNA from the patient’s mother and
father, the patient and his brother, positive controls F-M and F-F
(24), and unrelated negative controls K1 and C1 were
subjected to PCR using primers A-down and C4INS. The A-down primer
selectively hybridizes to the C4A-isotypic site, and the
C4INS primer anneals and primes DNA synthesis only to the sequence
bearing the 2-bp insertion. The resulting 780-bp fragment represents
the 2-bp insertion in C4A only. As shown in Fig. 2A, all members of the
proband’s family (Fig. 2, lanes 2–5), exhibited the 780-bp
fragment along with positive controls F-M and F-F (Fig. 2, lanes
6 and 7, respectively), previously described as
possessing the 2-bp insertion in C4A. No amplification product was
observed in the unrelated negative controls K1 and C1 (Fig. 2, lanes 8 and 9, respectively). To prove that the
780-bp fragment represents the exon 29 of C4, the gel was
Southern blotted and hybridized to a C4 exon 29-specific
probe. As shown in Fig. 2B, the probe hybridizes to the
780-bp fragment in lanes 2 through 7.
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To characterize the sequence polymorphisms of the C4 genes,
the 2.4-kb genomic DNA fragments spanning from exon 22 to exon 31
corresponding to the patient’s C4d was amplified via a high fidelity
PCR, cloned into TA vector and sequenced (2). The
C4A and C4B clones were differentiated via a
NlaIV digest (19). The patient’s C4d region of
the C4B gene was sequenced to completion to verify the
C4B nature of the clone. Accordingly, the clones contained
the C4B-isotypic, derived amino acid sequence LSPVIH at positions
1101–1106, the C4B-associated amino acid sequence ADLR for the Ch1 Ag
at positions 1188–1191, S1157 that is associated with Ch6, and D1054
associated with Ch5 (54, 55). However, congruent with
results obtained by the PCR, the patient’s C4B C4d region
did not contain the 2-bp insertion in exon 29. A comparison of
sequencing electropherograms at the exon 29 regions of both the
patient’s C4A and C4B is shown in Fig. 2C. The 2-bp insertion is evident in the electropherogram of
the patient’s C4A clone (Fig. 2C, left),
supporting the above described PCR results. This mutation was not
observed in sequencing results from the patient’s cloned C4d of the
C4B gene (Fig. 2C, right).
The recent publication of a 1-bp deletion (C) in sequence for codon 811 from exon 20 of C4A in another complete C4-deficient SLE patient (with HLA A30 B18 and DR3) (23) prompted us to investigate the presence of this mutation in the C4B of the patient, mother, and father. The region from intron 19 to exon 21 was amplified via PCR, cloned into TA vector, and sequenced. Again, the sequence for exon 20 was intact in each family member with no mutation found (data not shown).
Search for novel mutation in the proband’s mutant C4B gene
To determine whether the molecular basis of the patient’s C4B
deficiency was the result of a novel mutation in the C4B
coding sequence, a selective PCR strategy was designed to allow for
sequencing of only the C4B gene. To selectively clone
C4B, two PCRs were designed: one using synthetic primers
C4E1.5 and B-up, which amplified exon 1 through the C4B-isotypic site
of exon 26, and the other using primers B-down and C4E41.3, which
amplified from the C4B-isotypic site of exon 26 to exon 41 (Fig. 3A). The resulting PCR
amplification products were purified, cloned into TA vector, and
sequenced. To verify that the PCR products corresponded to
C4, the EcoRI-digested clones were subjected to
Southern blotting and hybridized to a C4d-specific probe (data not
shown).
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C).
Sequence electropherograms derived from both forward and reverse
primer positions are shown in Fig. 3B. Assuming correct
splicing of the exon 13-intron 14 junction, this deletion resulted in a
frame-shift mutation and generated a premature stop codon (TGA) at aa
568 (Fig. 3D).
A summary of the characteristic nucleotide and derived amino acid
sequences (2) are shown in Table I. The C>G767
mutation from exon 3 led to the L122V amino acid
substitution that has not been reported in any other known
C4 sequences. It has the A>C2296
polymorphism from exon 9 that is responsible for the Y327S
substitution, C>T5056 from exon 17 that
contributes to the L707P polymorphism, and a silent
C>T7308 mutation. Similar to the sequence for
C4A3a, it has DNA sequence that codes for D1478.
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A simple PCR strategy was devised to rapidly screen genomic DNA
samples for the herein described C4 mutations in one
reaction. As illustrated in Fig. 3A, PCR primers 13DELB and
C4E14.3 amplify a 400-bp fragment corresponding to the 1-bp deletion in
exon 13; primers 29INSR and C4E26.5 amplify a 860-bp fragment
corresponding to the 2-bp insertion in exon 29; and positive control
primers 21A5 and 21A3 amplify a 757-bp fragment corresponding to the
gene CYP21. All three primers were added to the multiplex
genomic DNA PCR.
Fig. 3E shows that all members of the proband’s family
(Fig. 3E, lanes 1–4) possess both the 400-bp and 860-bp
fragments, corresponding to the 1-bp deletion in exon 13 and the 2-bp
insertion in exon 29, respectively. The F-M and F-F of family F possess
only the 860-bp fragment corresponding to the exon 29 two-bp insertion.
Negative controls Y1 and M1 do not have any C4 frame-shift
amplification products, suggesting intact C4A and
C4B genes for these regions. However, the positive control
band corresponding to 757-bp CYP21 is visible, confirming
the presence of genomic DNA in the reaction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Similar to our patient, complete C4-deficient patients may experience early onset SLE with fulminate and sometimes life-threatening infections (56, 57). Besides the crippling effect of the broken link between the Ab-Ag (or MBL-Ag) interaction and the formation of the membrane attack complex, complete C4A and C4B deficiencies in humans impair the clearance of immune complexes by the erythrocytes, the phagocytosis of immune complex and apoptotic materials by macrophages, and the chemotaxis of inflammatory cells (58, 59). It may also weaken adaptive immune response as a complete C4-deficient patient may have abnormal immunological memory and fail to switch from IgM to IgG during secondary response (56). A similar phenomenon in the failure of secondary immune response had been observed in C4-deficient guinea pigs (60, 61, 62).
The redundancy of the immune defense system cannot be overlooked,
however, because the male sibling of the SLE patient reported here with
identical HLA haplotypes and complete C4A and C4B deficiencies
experienced only mild lupus-like disease. In a previous case, a female
Finnish SLE patient with complete C4 deficiency also had a male
C4-deficient sibling who had only photosensitivity (Table II; Ref. 24).
It is likely that in both of these cases the alternative and the MBL
complement activation pathways were intact, and they fulfilled the
effector arm of the humoral immune response. Under extreme situations
with large quantities of Ag-Ab complexes and activated C1, the
complement lytic pathway could be activated, bypassing C4, albeit
inefficiently, using components of the alternative pathway (63, 64). Moreover, these C4-deficient siblings might have protective
genetic factors outside the MHC that render them less vulnerable to the
full-blown disease. Alternatively, they might lack the additional
genetic risk factor(s) for the diathesis of the complex disease, and/or
they were not assaulted by the triggering environmental factors at a
critical stage of development. The possible environmental factor could
be a bad infection, hormonal stress, exposure to UV irradiation, or
others (65, 66, 67, 68).
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The HLA haplotype A2 Cw3 B40 (DR6) with monomodular long (mono-L) RCCX structure and a mutant C4A gene has been detected in six Caucasian patients from five independent families (17, 23, 24, 40, 41, 42, 43, 71). This haplotype might have originated from the HLA A2 Cw3 B60 DR6 which has a mutant C4A gene and the short RCCX module with a C4B gene was lost through an unequal crossover. The 2-bp insertion might have spread to the C4A and C4B genes in other haplotypes including HLA A2 Cw7 B39 DR15 (24) and the C4A gene of the HLA A2 B12 DR6 described in this work. HLA A2, Cw3 and DR6 appear multiple times in patients with the identical defects in the C4AQ0 mutant genes. A different mutation leading to nonexpression of C4A by a 1-bp deletion at the sequence for codon 811 of exon 20 was observed in HLA A30 B18 DR3. In the current work, we discovered a novel mutation, i.e., 1-bp deletion in exon 13 at the sequence for codon 522, in a C4B gene that led to frame-shift mutation and premature termination. The patient is homozygous with this HLA haplotype with a long C4AQ0 gene and a short C4BQ0 gene. Another family with complete C4 deficiency also shared the same HLA A2 B12 haplotype, which might have the same molecular defects in the C4A and C4B genes (42).
There are at least 11 additional HLA haplotypes with complete C4
deficiency. HLA A30 B18 DR7 is another common haplotype with
complete C4 deficiency that has been detected in six individuals from
three different families (41, 59, 72, 73). By elucidating
the sequence defects leading to the complete C4A and C4B deficiencies
in these haplotypes, it may be possible to develop sequence-specific
PCR technology, as illustrated in Fig. 3E, to detect the
presence of mutant C4A and C4B genes. Such a
technique would help epidemiological studies of diseases associated
with complement C4A and C4B deficiencies.
To date 28 complete C4-deficient individuals have been reported; all were typed for the HLA haplotypes, except the 3 subjects identified during a large scale medical survey of apparently healthy individuals (n = 42,000) in Japan more than 30 years ago (23, 24, 40, 41, 42, 56, 57, 59, 63, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80). It is particularly striking that all HLA-typed, C4-deficient individuals but one (41) experienced symptoms related to immune complex or clearance disorders such as lupus or lupus-like disease, nephritis or kidney disease, and/or photosensitivity. Depending on the inclusion criteria, the disease penetrance of complete C4 deficiency ranges between 75 and 96%. Besides the genetic deficiency of complement C1q, there is probably no other known genetic deficiency that confers such a high penetrance of human systemic autoimmune disease as complete C4 deficiency (30, 81).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. C. Yung Yu, Children’s Research Institute, 700 Children’s Drive, Columbus, OH 43205-2696. E-mail address: cyu{at}chi.osu.edu
3 Abbreviations used in this paper: RCCX, serine/threonine nuclear kinase RP, complement component C4, steroid 21-hydroxylase CYP21, and extracellular matrix protein tenascin TNX; Ch, Chido; SLE, systemic lupus erythematosus; MBL, mannose-binding lectin; Rg, Rodgers; ANA, antinuclear Ab.
Received for publication March 26, 2002. Accepted for publication May 22, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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