Abstract
OBJECTIVE:
To perform a molecular characterization of the C1q, C2 and C4 genes in patients with juvenile systemic lupus erythematosus.
METHODS:
Patient 1 (P1) had undetectable C1q, patient 2 (P2) and patient 3 (P3) had decreased C2 and patient 4 (P4) had decreased C4 levels. All exons and non-coding regions of the C1q and C2 genes were sequenced. Mononuclear cells were cultured and stimulated with interferon gamma to evaluate C1q, C2 and C4 mRNA expression by quantitative real-time polymerase chain reaction.
RESULTS:
C1q sequencing revealed heterozygous silent mutations in the A (c.276 A>G Gly) and C (c.126 C>T Pro) chains, as well as a homozygous single-base change in the 3′ non-coding region of the B chain (c*78 A>G). C1qA mRNA expression without interferon was decreased compared with that of healthy controls (p<0.05) and was decreased after stimulation compared with that of non-treated cells. C1qB mRNA expression was decreased compared with that of controls and did not change with stimulation. C1qC mRNA expression was increased compared with that of controls and was even higher after stimulation. P2 and P3 had Type I C2 deficiency (heterozygous 28 bp deletion at exon 6). The C2 mRNA expression in P3 was 23 times lower compared with that of controls and did not change after stimulation. The C4B mRNA expression of P4 was decreased compared with that of controls and increased after stimulation.
CONCLUSIONS:
Silent mutations and single-base changes in the 3′ non-coding regions may modify mRNA transcription and C1q production. Type I C2 deficiency should be evaluated in JSLE patients with decreased C2 serum levels. Further studies are needed to clarify the role of decreased C4B mRNA expression in JSLE pathogenesis.
Juvenile systemic lupus erythematosus; Complement deficiency; C1q, C2 and C4 genes; Silent mutation; Non-coding region
INTRODUCTION
Inherited deficiencies of the complement components C1q, C2 and C4 are frequently
associated with the development of systemic lupus erythematosus (SLE) due to the
impaired clearance of apoptotic debris, a decreased capacity for immune-complex
handling, and/or their role in peripheral B-cell tolerance (11 Rahman A, Isenberg DA. Mechanisms of disease: Systemic lupus
erythematosus. N Engl J Med. 2008;358(9):929-39.
2 Carneiro-Sampaio M, Liphaus BL, Jesus AA, Silva CAA, Oliveira JB,
Kiss MH. Understanding systemic lupus erythematosus physiopathology in the light
of primary immunodeficiencies. J Clin Immunol. 2008;28:S34-41,
http://dx.doi.org/10.1007/s10875-008-9187-2.
http://dx.doi.org/10.1007/s10875-008-918...
3 Jesus AA, Liphaus BL, Silva CA, Bando SY, Andrade LEC, Coutinho
A, et al. Complement and antibody primary immunodeficiency in juvenile systemic
lupus erythematosus patients. Lupus. 2011;20(12):1275-84,
http://dx.doi.org/10.1177/0961203311411598.
http://dx.doi.org/10.1177/09612033114115...
4 Manderson AP, Botto M, Walport MJ. The role of complement in the
development of systemic lupus erythematosus. Ann Rev Immunol. 2004;22:431-56,
http://dx.doi.org/10.1146/annurev.immunol.22.012703.104549.
http://dx.doi.org/10.1146/annurev.immuno...
5 Pickering MC, Botto M, Taylor PR, Lachmann PJ, Walport MJ.
Systemic lupus erythematosus, complement deficiency, and apoptosis. Adv Immunol.
2001;76:227-324,
http://dx.doi.org/10.1016/S0065-2776(01)76021-X.
http://dx.doi.org/10.1016/S0065-2776(01)...
6 Liphaus BL, Bittencourt Kiss MH. The role of apoptosis proteins
and complement components in the eiophatogenesis of systemic lupus
erythematosus. Clinics. 2010;65(3):327-33,
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7 Jesus AA, Liphaus BL, Porta G, Kiss MH, Silva CA,
Carneiro-Sampaio M. C1q deficiency and juvenile-onset systemic lupus
erythematosus: report of two Brazilian cases. Clin Exp Immunol. 2008;154: 154
Suppl 1:1-224.
8 Truedsson L, Bengtsson AA, Sturfelt G. Complement deficiencies
and systemic lupus erythematosus. Autoimmunity. 2007;40(8):560-6,
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Homozygous C1q deficiency is the strongest genetic risk factor related to SLE (33 Jesus AA, Liphaus BL, Silva CA, Bando SY, Andrade LEC, Coutinho
A, et al. Complement and antibody primary immunodeficiency in juvenile systemic
lupus erythematosus patients. Lupus. 2011;20(12):1275-84,
http://dx.doi.org/10.1177/0961203311411598.
http://dx.doi.org/10.1177/09612033114115...
,1010 Schejbel L, Skattum L, Hagelberg S, Ahlin A, Schiller B, Berg S,
et al. Molecular basis of hereditary C1q deficiency-revisited: identification of
several novel disease-causing mutations. Genes Immunity. 2011;12(8):626-34,
http://dx.doi.org/10.1038/gene.2011.39.
http://dx.doi.org/10.1038/gene.2011.39...
11 Petry F. Molecular basis of hereditary C1q deficiency.
Immunobiol. 1998;199(2):286-94,
http://dx.doi.org/10.1016/S0171-2985(98)80033-8.
http://dx.doi.org/10.1016/S0171-2985(98)...
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hereditary complement C1q deficiencies. Immunogenetics. 2005;57(8):566-71,
http://dx.doi.org/10.1007/s00251-005-0023-z.
http://dx.doi.org/10.1007/s00251-005-002...
). Similarly, both Type I
and II C2 deficiency have been associated with SLE (44 Manderson AP, Botto M, Walport MJ. The role of complement in the
development of systemic lupus erythematosus. Ann Rev Immunol. 2004;22:431-56,
http://dx.doi.org/10.1146/annurev.immunol.22.012703.104549.
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Sturfelt G. Characterization of Type-I Complement C2 Deficiency MHC Haplotypes -
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16 Johnson CA, Densen P, Hurford RK, Colten HR, Wetsel RA. Type-I
Human-Complement C2 Deficiency - A 28-base pair Gene Deletion Causes Skipping of
Exon-6 During RNA Splicing. J Biol Chem.
1992;267(13):9347-53.-1717 Wetsel RA, Kulics J, Lokki ML, Kiepiela P, Akama H, Johnson CAC,
et al. Type II human complement C2 deficiency - Allele-specific amino acid
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). In addition, homozygous C4A deficiency is approximately 10
times more frequent in patients with SLE (44 Manderson AP, Botto M, Walport MJ. The role of complement in the
development of systemic lupus erythematosus. Ann Rev Immunol. 2004;22:431-56,
http://dx.doi.org/10.1146/annurev.immunol.22.012703.104549.
http://dx.doi.org/10.1146/annurev.immuno...
,1818 Yang Y, Lhotta K, Chung EK, Eder P, Neumair F, Yu CY. Complete
complement components C4A and C4B deficiencies in human kidney diseases and
systemic lupus erythematosus. J Immunol. 2004;173(4):2803-14,
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19 Rupert KL, Moulds JM, Yang Y, Arnett FC, Warren RW, Reveille JD,
et al. The molecular basis of complete complement C4A and C4B deficiencies in a
systemic lupus erythematosus patient with homozygous C4A and C4B mutant genes.
J Immunol. 2002;169(3):1570-8,
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20 Wu YL, Hauptmann G, Viguier M, Yu CY. Molecular basis of
complete complement C4 deficiency in two North-African families with systemic
lupus erythematosus. Genes Immunity. 2009;10(5):433-45,
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21 Yang Y, Chung EK, Wu YL, Savelli SL, Nagaraja HN, Zhou B, et al.
Gene copy-number variation and associated polymorphisms of complement component
C4 in human systemic lupus erythematosus (SLE): Low copy number is a risk factor
for and high copy number is a protective factor against SLE susceptibility in
European Americans. Am J Hum Genetics.
2007;80(6):1037-54.-2222 Andrade LEC, Pereira KMC, Faria AGA, Liphaus B, Jesus AA, Silva
C, et al. Low gene copy number for c4, c4a and c4b is a strong risk factor for
developing systemic lupus erythematosus in childhood. Arthitis Rheum.
2012;64(10):S965-S.).
Few studies have evaluated the non-coding regions and mRNA expression of complement
genes. Furthermore, complement deficiencies are rare among SLE patients but are
common among patients with juvenile SLE (JSLE) (2323 Aggarwal R, Sestak AL, D′Sousa A, Dillon SP, Namjou B,
Scofield RH. Complete complement deficiency in a large cohort of familial
systemic lupus erythematosus. Lupus. 2010;19(1):52-7,
http://dx.doi.org/10.1177/0961203309346508.
http://dx.doi.org/10.1177/09612033093465...
).
We previously showed an underlying primary complement deficiency in seven JSLE
patients (33 Jesus AA, Liphaus BL, Silva CA, Bando SY, Andrade LEC, Coutinho
A, et al. Complement and antibody primary immunodeficiency in juvenile systemic
lupus erythematosus patients. Lupus. 2011;20(12):1275-84,
http://dx.doi.org/10.1177/0961203311411598.
http://dx.doi.org/10.1177/09612033114115...
). We had access to DNA samples
and/or peripheral cells from four of these patients, and to better understand these
inherited deficiencies, we conducted a molecular characterization of the C1q, C2 and
C4 genes.
MATERIALS AND METHODS
Among 72 JSLE patients, 16 had primary immunodeficiency, and seven of the 16 were
determined to have complement deficiency based on a combination of low or
undetectable serum levels of C1q, or C1s, C1r, C2, C3 or C4 with normal levels of
the other components in at least two samples and low activity/inactive disease
(SLEDAI-2K score<4) (33 Jesus AA, Liphaus BL, Silva CA, Bando SY, Andrade LEC, Coutinho
A, et al. Complement and antibody primary immunodeficiency in juvenile systemic
lupus erythematosus patients. Lupus. 2011;20(12):1275-84,
http://dx.doi.org/10.1177/0961203311411598.
http://dx.doi.org/10.1177/09612033114115...
). Therefore, we
selected four patients: patient 1 (P1) had undetectable serum C1q, normal C3 and C4
levels, negative anti-C1q and available DNA and mononuclear cells; patient 2 (P2)
had decreased IgA, C2 and C4 serum levels and stored DNA but no cells available for
culture; patient 3 (P3) had decreased C2 and normal C3 and C4 levels; and patient 4
(P4) had repeated decreased C4 and normal C1q, C2 and C3 levels and peripheral cells
available for culture (Tables 1 and
2). Three healthy controls matched by
age and gender and with normal complement levels were also enrolled. Patients
fulfilled the revised American College of Rheumatology classification criteria, and
informed consent was obtained for all participants after the local ethics committee
had approved the study (2424 Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF,
et al. Special Article - The 1982 Revised Criteria for the Classification of
Systemic Lupus Erythematosus. Arthritis Rheum. 1982;25(11):1271-7,
http://dx.doi.org/10.1002/art.1780251101.
http://dx.doi.org/10.1002/art.1780251101...
,2525 Hochberg MC. Updating the American College of Rheumatology
revised criteria for the classification of systemic lupus erythematosus.
Arthritis Rheum. 1997;40(9):1725,
http://dx.doi.org/10.1002/art.1780400928.
http://dx.doi.org/10.1002/art.1780400928...
).
Clinical and laboratory findings from four juvenile systemic lupus erythematosus patients with primary complement deficiencies who were selected for molecular characterization (33 Jesus AA, Liphaus BL, Silva CA, Bando SY, Andrade LEC, Coutinho A, et al. Complement and antibody primary immunodeficiency in juvenile systemic lupus erythematosus patients. Lupus. 2011;20(12):1275-84, http://dx.doi.org/10.1177/0961203311411598.
http://dx.doi.org/10.1177/09612033114115... ).
We previously determined the C4 gene copy number by quantitative real-time polymerase
chain reaction (qRT-PCR), as reported (33 Jesus AA, Liphaus BL, Silva CA, Bando SY, Andrade LEC, Coutinho
A, et al. Complement and antibody primary immunodeficiency in juvenile systemic
lupus erythematosus patients. Lupus. 2011;20(12):1275-84,
http://dx.doi.org/10.1177/0961203311411598.
http://dx.doi.org/10.1177/09612033114115...
,2626 Szilagyi A, Blasko B, Szilassy D, Fust G, Sasvari-Szekely M,
Ronai Z. Real-time PCR quantification of human complement C4A and C4B genes. BMC
Genet. 2006;7:1, http://dx.doi.org/10.1186/1471-2156-7-1.
http://dx.doi.org/10.1186/1471-2156-7-1...
) (Table 1).
All exons and 3′ and 5′ non-coding regions (UTR) of the C1q and C2
genes (3 exons each for the C1q A, B, and C chains and 18 exons for the C2 gene)
were sequenced. Primers (Supplementary Table) were designed using the reference
sequences deposited in GenBank (NG_007282.1 for C1qA, NG_007283.1 for
C1qB, NG_007565.1 for C1qC and NG_011730.1 for C2) with the
Primer-BLAST program. DNA was obtained using the QIAamp DNA
Blood Midi kit (N°51104 Qiagen, Hilden, Germany), and purity and concentration
were determined with a Nanovue¯ spectrophotometer (GE
Healthcare, USA). Each PCR reaction was performed in a final volume of 25 µl
containing 100 ng of DNA, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.0 mM MgSO4, 100
µM each of dATP, dCTP, dGTP and dTTP, 0.5 µM of each primer and 1.5 U of
High-Fidelity Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA). The
amplification conditions included 35 cycles of 95°C for 30 s, 60°C for 30
s and 72°C for 2 min, with an initial denaturing step of 95°C for 5 min
and a final extension step of 72°C for 7 min. Products were purified using the
GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, UK).
Amplicons and primers were sent to Centro de Estudos do Genoma Humano for
sequencing. The BioEdit and ClustalW sequence alignment editors
(http://www.mbio.ncsu.edu/bioedit/bioedit.html) were used to align the nucleotide
sequences. The BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to
search variant sequences obtained from the human genomic database. The Chromas
software program was used for chromatogram visualization. Type I C2 deficiency was
evaluated by PCR as previously described and confirmed by gene sequencing (33 Jesus AA, Liphaus BL, Silva CA, Bando SY, Andrade LEC, Coutinho
A, et al. Complement and antibody primary immunodeficiency in juvenile systemic
lupus erythematosus patients. Lupus. 2011;20(12):1275-84,
http://dx.doi.org/10.1177/0961203311411598.
http://dx.doi.org/10.1177/09612033114115...
,2727 Boeckler P, Meyer A, Uring-Lambert B, Goetz J, Cribier B,
Hauptmann G, et al. Which complement assays and typings are necessary for the
diagnosis of complement deficiency in patients with lupus erythematosus?
A study of 25 patients. Clin Immunol. 2006;121(2):198-202,
http://dx.doi.org/10.1016/j.clim.2006.08.007.
http://dx.doi.org/10.1016/j.clim.2006.08...
).
The quantitative analysis of C1q, C2 and C4 mRNA was performed using peripheral
mononuclear cells obtained by centrifugation using Ficoll-Paque Plus (GE Healthcare,
Piscataway, NJ, USA) and cultured in supplemented DMEM medium (10% fetal bovine
serum, 10,000 U/ml penicillin and 100 g/ml streptomycin) at 37°C with 5%
CO2 for 36 hours. Parallel cultures were stimulated with 100
U/µl of recombinant human interferon gamma (IFNγ; N°300-02
Preprotech, Rocky Hill, NJ, USA) as previously described (2828 Vincent F, Delasalle H, Bohbot A, Bergerat JP, Hauptmann G,
Oberling F. Synthesis and Regulation of Complement Components by Human Monocytes
macrophages and by acute monocytic leukemia. DNA Cell Biol. 1993;12(5):415-23,
http://dx.doi.org/10.1089/dna.1993.12.415.
http://dx.doi.org/10.1089/dna.1993.12.41...
). RNA was obtained from cultures using an RNeasy Midi Kit
(N°74104, Qiagen, Hilden, Germany). RNA (500 ng) was reverse-transcribed to
produce complementary DNA (cDNA) using the SuperScript III First-Strand
Synthesis System for RT-PCR (N°18080-051, Invitrogen, Carlsbad,
CA, USA). The qRT-PCR was performed using 2 µl of cDNA, 1 µM each of
forward and reverse primers and 1X SYBR Green Mix QuantiFast PCR
Kit (N°204054, Qiagen, Hilden, Germany) in a StepOnePlus Real-Time
PCR System (Applied Biosystems, Forrest City, CA, USA). The cycling parameters
included an initial 15 min hot start at 95°C, followed by 50 cycles of 15 s at
95°C and 30 s at 60°C. C4B primers were designed based on a GenBank
reference sequence (NG_011638, Supplementary Table). Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was used as an endogenous reference gene for qRT-PCR reaction
normalization. Relative expression (RE) was determined by the standard curve method,
and mean values are presented here as relative variation expression (RVE) obtained
from RVE = target gene RE - GAPDH RE.
A t-test was used to compare the control and untreated samples and the patient treated and untreated samples. p<0.05 was considered significant.
RESULTS
C1q gene sequencing (P1) revealed a heterozygous silent mutation in exon 3 of the A chain (c.276 A>G Gly), a homozygous single-base change in the 3′ UTR of the B chain (c*78 A>G), and a heterozygous silent mutation in exon 2 of the C chain (c.126 C>T Pro) (Figures 1,2,3). The qRT-PCR analysis revealed that C1qA mRNA expression was decreased compared with that of healthy controls (p<0.05) and was also decreased after IFNγ stimulation (treated cells) compared with that of non-treated cells; C1qB mRNA expression was decreased compared with that of controls and did not change with stimulation, and C1qC mRNA expression was increased compared with that of controls and was even higher after IFNγ stimulation (Figure 5A).
C1qA gene sequencing from juvenile systemic lupus erythematosus patient 1 (P1) and P1's mother. A - Nucleotide sequence alignment for P1 and P1's mother; B - Amino acid sequence for P1 and P1's mother; C and D - Nucleotide chromatograms of P1 and P1's mother, respectively. NG_007282.1 is the reference codified sequence for C1qA. The arrows indicate the variation sites (exon 3, c.276 A>G Gly; c.295 A>C Ile-Leu).
C1qB gene sequencing from juvenile systemic lupus erythematosus patient 1 (P1) and P1's mother. A - Nucleotide sequence alignment for P1 and P1's mother; B and C - Nucleotide chromatograms of P1 and P1's mother, respectively. NG_007283.1 is the reference codified or mRNA sequence for C1qB. The arrow indicates the variation site (3′ UTR c*78 A>G).
C1qC gene sequencing from juvenile systemic lupus erythematosus patient 1 (P1) and P1's mother. A - Nucleotide sequence alignment for P1 and P1's mother; B and C - Nucleotide chromatograms of P1 and P1's mother, respectively. NG_007565.1 is the reference codified sequence for C1qC. The arrow indicates the variation site (exon 2, c.126 C>T Pro).
P1's healthy mother had an identical but homozygous silent mutation in the A chain, a homozygous single-base change in the B chain, a heterozygous silent mutation in the C chain, and an additional missense mutation in the A chain (c. 295 A>C Ile-Leu) (Figure 1). The peripheral cells of the mother were not available for mRNA expression.
P2 and P3 had a heterozygous 28 bp deletion at exon 6, characterizing Type I C2 deficiency, as shown in Figure 4. The C2 mRNA expression (P3) was 23 times lower compared with that of controls and did not change after IFNγ stimulation (Figure 5B).
Type I C2 deficiency in juvenile systemic lupus erythematosus patients 2 (P2) and 3 (P3). A - Nucleotide sequence alignment for P2 and P3; B and C - Nucleotide chromatograms for P2 and P3, respectively. NG_11730.1 is the reference DNA sequence for C2, and NG_11730 is the codified sequence for C2 (final region of exon 6). The blue arrow indicates the last nucleotide of exon 6, and the red arrow indicates the initial deletion site; D - Agarose gel electrophoresis showing the heterozygous 28 bp deletion in exon 6 of the C2 gene. Lane 1: 100 bp DNA ladder; lane 2: P2; lane 3: P3; lane 4: positive control; lane 5: negative control. The blue and red arrows indicate the 174 bp and 146 bp fragments, respectively.
Relative expression of mRNA in cells from juvenile systemic lupus erythematosus patients and controls and after in vitro IFN-γ stimulation (treated). A - The relative expression of the C1q A, B and C chains in P1's cells; B - The relative expression of C2 in P3's cells; C - The relative expression of C4B in P4's cells. RVE - relative variation expression. *indicates a significant difference, p<0.05.
The C4B mRNA expression in P4 was significantly decreased compared with that of controls and increased after IFNγ stimulation (Figure 5C). The C4A mRNA expression was not technically adequate, and it was therefore not considered for analysis.
DISCUSSION
For the first time in JSLE patients, we showed heterozygous silent mutations, a homozygous single-base change in the 3′ UTR and decreased mRNA expression for the C1q gene; a heterozygous Type I deficiency and decreased mRNA expression for the C2 gene; and decreased mRNA expression for the C4B gene.
Although complement deficiencies were the first identified genetic risk factors for
SLE, it remains unclear how these deficiencies should be assessed (22 Carneiro-Sampaio M, Liphaus BL, Jesus AA, Silva CAA, Oliveira JB,
Kiss MH. Understanding systemic lupus erythematosus physiopathology in the light
of primary immunodeficiencies. J Clin Immunol. 2008;28:S34-41,
http://dx.doi.org/10.1007/s10875-008-9187-2.
http://dx.doi.org/10.1007/s10875-008-918...
3 Jesus AA, Liphaus BL, Silva CA, Bando SY, Andrade LEC, Coutinho
A, et al. Complement and antibody primary immunodeficiency in juvenile systemic
lupus erythematosus patients. Lupus. 2011;20(12):1275-84,
http://dx.doi.org/10.1177/0961203311411598.
http://dx.doi.org/10.1177/09612033114115...
-44 Manderson AP, Botto M, Walport MJ. The role of complement in the
development of systemic lupus erythematosus. Ann Rev Immunol. 2004;22:431-56,
http://dx.doi.org/10.1146/annurev.immunol.22.012703.104549.
http://dx.doi.org/10.1146/annurev.immuno...
,77 Jesus AA, Liphaus BL, Porta G, Kiss MH, Silva CA,
Carneiro-Sampaio M. C1q deficiency and juvenile-onset systemic lupus
erythematosus: report of two Brazilian cases. Clin Exp Immunol. 2008;154: 154
Suppl 1:1-224.,88 Truedsson L, Bengtsson AA, Sturfelt G. Complement deficiencies
and systemic lupus erythematosus. Autoimmunity. 2007;40(8):560-6,
http://dx.doi.org/10.1080/08916930701510673.
http://dx.doi.org/10.1080/08916930701510...
,2727 Boeckler P, Meyer A, Uring-Lambert B, Goetz J, Cribier B,
Hauptmann G, et al. Which complement assays and typings are necessary for the
diagnosis of complement deficiency in patients with lupus erythematosus?
A study of 25 patients. Clin Immunol. 2006;121(2):198-202,
http://dx.doi.org/10.1016/j.clim.2006.08.007.
http://dx.doi.org/10.1016/j.clim.2006.08...
,2929 Pickering MC, Walport MJ. Links between complement abnormalities
and systemic lupus erythematosus. Rheumatology (Oxford). 2000;39(2):133-41,
http://dx.doi.org/10.1093/rheumatology/39.2.133.
http://dx.doi.org/10.1093/rheumatology/3...
).
Studies have described nonsense and missense mutations in the C1q gene, with a total
of 12 causative mutations (33 Jesus AA, Liphaus BL, Silva CA, Bando SY, Andrade LEC, Coutinho
A, et al. Complement and antibody primary immunodeficiency in juvenile systemic
lupus erythematosus patients. Lupus. 2011;20(12):1275-84,
http://dx.doi.org/10.1177/0961203311411598.
http://dx.doi.org/10.1177/09612033114115...
4 Manderson AP, Botto M, Walport MJ. The role of complement in the
development of systemic lupus erythematosus. Ann Rev Immunol. 2004;22:431-56,
http://dx.doi.org/10.1146/annurev.immunol.22.012703.104549.
http://dx.doi.org/10.1146/annurev.immuno...
-55 Pickering MC, Botto M, Taylor PR, Lachmann PJ, Walport MJ.
Systemic lupus erythematosus, complement deficiency, and apoptosis. Adv Immunol.
2001;76:227-324,
http://dx.doi.org/10.1016/S0065-2776(01)76021-X.
http://dx.doi.org/10.1016/S0065-2776(01)...
,1010 Schejbel L, Skattum L, Hagelberg S, Ahlin A, Schiller B, Berg S,
et al. Molecular basis of hereditary C1q deficiency-revisited: identification of
several novel disease-causing mutations. Genes Immunity. 2011;12(8):626-34,
http://dx.doi.org/10.1038/gene.2011.39.
http://dx.doi.org/10.1038/gene.2011.39...
11 Petry F. Molecular basis of hereditary C1q deficiency.
Immunobiol. 1998;199(2):286-94,
http://dx.doi.org/10.1016/S0171-2985(98)80033-8.
http://dx.doi.org/10.1016/S0171-2985(98)...
-1212 Petry F, Loos M. Common silent mutations in all types of
hereditary complement C1q deficiencies. Immunogenetics. 2005;57(8):566-71,
http://dx.doi.org/10.1007/s00251-005-0023-z.
http://dx.doi.org/10.1007/s00251-005-002...
,2323 Aggarwal R, Sestak AL, D′Sousa A, Dillon SP, Namjou B,
Scofield RH. Complete complement deficiency in a large cohort of familial
systemic lupus erythematosus. Lupus. 2010;19(1):52-7,
http://dx.doi.org/10.1177/0961203309346508.
http://dx.doi.org/10.1177/09612033093465...
,3030 Gaboriaud C, Juanhuix J, Gruez A, Lacroix M, Darnault C, Pignol
D, et al. The crystal structure of the globular head of complement protein C1q
provides a basis for its versatile recognition properties. J Biol Chem.
2003;278(47):46974-82,
http://dx.doi.org/10.1074/jbc.M307764200.
http://dx.doi.org/10.1074/jbc.M307764200...
31 Kishore W, Ghai R, Greenhough TJ, Shrive AK, Bonifati DM,
Gadjeva MG, et al. Structural and functional anatomy of the globular domain of
complement protein C1q. Immunol Lett. 2004;95(2):113-28,
http://dx.doi.org/10.1016/j.imlet.2004.06.015.
http://dx.doi.org/10.1016/j.imlet.2004.0...
32 Sellar GC, Blake DJ, Reid KBM. Characterization and organization
of the genes encoding the A-, B- and C-chains of human complement subcomponent
C1q. The complete derived amino acid sequence of human C1q. Biochem J.
1991;274(Pt 2):481-90.
33 Petry F, Le DT, Kirschfink M, Loos M. Nonsense and Missense
Mutations in the Structural Genes of Complement Component C1q A-Chains and
C-Chains are Linked with 2 Different Types of Complete Selective C1q
Deficiencies. J Immunol. 1995;155(10):4734-8.
34 Petry F, Berkel AI, Loos M. Multiple identification of a
particular type of hereditary C1q deficiency in the Turkish population: Review
of the cases and additional genetic and functional analysis. Hum Genet.
1997;100(1):51-6, http://dx.doi.org/10.1007/s004390050464.
http://dx.doi.org/10.1007/s004390050464...
35 Sun-Tan C, Oezguer TT, Kilinc G, Topaloglu R, Gokoz O,
Ersoy-Evans S, et al. Hereditary C1q deficiency: a new family with C1qA
deficiency. Turkish J Pediatr. 2010;52(2):184-6.
36 Rafiq S, Frayling TM, Vyse TJ, Graham DSC, Eggleton P. Assessing
association of common variation in the C1Q gene cluster with systemic lupus
erythematosus. Clin Exp Immunol. 2010;161(2):284-9.-3737 Namjou B, Keddache M, Fletcher D, Dillon S, Kottyan L, Wiley G,
et al. Identification of novel coding mutation in C1qA gene in an
African-American pedigree with lupus and C1q deficiency. Lupus.
2012;21(10):1113-8, http://dx.doi.org/10.1177/0961203312443993.
http://dx.doi.org/10.1177/09612033124439...
). Single nucleotide polymorphisms and silent
mutations have also been described (1212 Petry F, Loos M. Common silent mutations in all types of
hereditary complement C1q deficiencies. Immunogenetics. 2005;57(8):566-71,
http://dx.doi.org/10.1007/s00251-005-0023-z.
http://dx.doi.org/10.1007/s00251-005-002...
,3838 Racila DM, Sontheimer CJ, Sheffield A, Wisnieski JJ, Racila E,
Sontheimer RD. Homozygous single nucleotide polymorphism of the complement C1QA
gene is associated with decreased levels of Clq in patients with subacute
cutaneous lupus erythematosus. Lupus. 2003;12(2):124-32,
http://dx.doi.org/10.1191/0961203303lu329oa.
http://dx.doi.org/10.1191/0961203303lu32...
). However, although there were no amino acid
changes due to the identified base changes, the mRNA expression of the C1qA, B and C
chains was altered. There is evidence that silent mutations can influence gene
expression via alternative splicing by altering the exonic splicing enhancer or
silencer sites (3939 Cartegni L, Chew SL, Krainer AR. Listening to silence and
understanding nonsense: Exonic mutations that affect splicing. Nat Rev Genet.
2002;3(4):285-98, http://dx.doi.org/10.1038/nrg775.
http://dx.doi.org/10.1038/nrg775...
). There is no evidence
that heterozygous mutations of the C1q gene can lead to lupus if the other allele is
normal. Of note, our patient presented photosensitivity, which is a characteristic
associated with a single-nucleotide change in the C1qA gene (3838 Racila DM, Sontheimer CJ, Sheffield A, Wisnieski JJ, Racila E,
Sontheimer RD. Homozygous single nucleotide polymorphism of the complement C1QA
gene is associated with decreased levels of Clq in patients with subacute
cutaneous lupus erythematosus. Lupus. 2003;12(2):124-32,
http://dx.doi.org/10.1191/0961203303lu329oa.
http://dx.doi.org/10.1191/0961203303lu32...
). In addition, Rafiq reported allelic variance in the
3′ UTR of the C1qB chain associated with low C1q serum levels (3636 Rafiq S, Frayling TM, Vyse TJ, Graham DSC, Eggleton P. Assessing
association of common variation in the C1Q gene cluster with systemic lupus
erythematosus. Clin Exp Immunol. 2010;161(2):284-9.). The fact that the 3′ UTR
single-base change in C1qB was homozygous implies that the mother and father each
have one allele with the same sequence variants. P1's mother had normal C1q
levels and did not present any symptoms of autoimmune disease; however, she harbored
silent mutations with an additional missense mutation in the A chain. Because the
C1q protein was present in P1's mother, it becomes questionable whether the
observed variants are, by themselves, pathogenic (4040 Walport MJ, Davies KA, Botto M. Clq and systemic lupus
erythematosus. Immunobiology. 1998;199(2):265-85,
http://dx.doi.org/10.1016/S0171-2985(98)80032-6.
http://dx.doi.org/10.1016/S0171-2985(98)...
,4141 Berkel AI, Birben E, Oner C, Oner R, Loos M, Petry F. Molecular,
genetic and epidemiologic studies on selective complete C1q deficiency in
Turkey. Immunobiol. 2000;201(3-4):347-55,
http://dx.doi.org/10.1016/S0171-2985(00)80089-3.
http://dx.doi.org/10.1016/S0171-2985(00)...
). Peripheral cells from the
patient's father and mother were not available for mRNA expression analysis,
which could have addressed on the latter speculation.
An intriguing aspect of these results was the observation of mRNA expression from the
C1q chains after IFNγ stimulation. IFNγ is able to induce and
up-regulate complement production by monocytes (2828 Vincent F, Delasalle H, Bohbot A, Bergerat JP, Hauptmann G,
Oberling F. Synthesis and Regulation of Complement Components by Human Monocytes
macrophages and by acute monocytic leukemia. DNA Cell Biol. 1993;12(5):415-23,
http://dx.doi.org/10.1089/dna.1993.12.415.
http://dx.doi.org/10.1089/dna.1993.12.41...
,4242 Lappin DF, Birnie GD, Whaley K. Modulation by Interferons of the
Expression of Monocyte Complement Genes. Biochem J. 1990
1;268(2):387-92.,4343 Lipsker DM, Schreckenberg-Gilliot C, Uring-Lambert B, Meyer A,
Hartmann D, Grosshans EM, et al. Lupus erythematosus associated with genetically
determined deficiency of the second component of the complement. Arch Dermatol.
2000;136(12):1508-14.). C1qA and B expression was decreased, and C1qC was
increased, which suggests deregulated production that could influence the
heterotrimeric assembly of protein. Nanjou et al. demonstrated that only one defect
in any C1q chain can compromise the entire synthesis and secretion of the C1q
protein (3737 Namjou B, Keddache M, Fletcher D, Dillon S, Kottyan L, Wiley G,
et al. Identification of novel coding mutation in C1qA gene in an
African-American pedigree with lupus and C1q deficiency. Lupus.
2012;21(10):1113-8, http://dx.doi.org/10.1177/0961203312443993.
http://dx.doi.org/10.1177/09612033124439...
). Because mRNA was still
expressed, it is unlikely that the observed base changes could completely block
protein expression, and alternative causes must be considered. Therefore, further
work assessing mRNA stability and translation efficiency will be needed.
Type I C2 deficiency is due to a 9 bp deletion from the 3′ end of exon 6 and a
19 bp deletion from the 5′ region of the following intron, leading to a frame
shift and a premature stop codon (22 Carneiro-Sampaio M, Liphaus BL, Jesus AA, Silva CAA, Oliveira JB,
Kiss MH. Understanding systemic lupus erythematosus physiopathology in the light
of primary immunodeficiencies. J Clin Immunol. 2008;28:S34-41,
http://dx.doi.org/10.1007/s10875-008-9187-2.
http://dx.doi.org/10.1007/s10875-008-918...
,1313 Truedsson L, Sturfelt G, Nived O. Prevalence of the Type-I
Complement C2 Deficiency Gene in Swedish Systemic Lupus Erythematosus Patients.
Lupus. 1993;2(5):325-7,
http://dx.doi.org/10.1177/096120339300200509.
http://dx.doi.org/10.1177/09612033930020...
,1515 Truedsson L, Alper CA, Awdeh ZL, Johansen P, Sjoholm AG,
Sturfelt G. Characterization of Type-I Complement C2 Deficiency MHC Haplotypes -
Strong Conservation of the Complotype/HLA-B-Region and Absence of Disease
Association due to Linked Class-II Genes. J Immunol.
1993;151(10):5856-63.,1616 Johnson CA, Densen P, Hurford RK, Colten HR, Wetsel RA. Type-I
Human-Complement C2 Deficiency - A 28-base pair Gene Deletion Causes Skipping of
Exon-6 During RNA Splicing. J Biol Chem.
1992;267(13):9347-53.,2929 Pickering MC, Walport MJ. Links between complement abnormalities
and systemic lupus erythematosus. Rheumatology (Oxford). 2000;39(2):133-41,
http://dx.doi.org/10.1093/rheumatology/39.2.133.
http://dx.doi.org/10.1093/rheumatology/3...
). More than half of individuals with primary C2 deficiency
develop SLE or lupus-like disease (22 Carneiro-Sampaio M, Liphaus BL, Jesus AA, Silva CAA, Oliveira JB,
Kiss MH. Understanding systemic lupus erythematosus physiopathology in the light
of primary immunodeficiencies. J Clin Immunol. 2008;28:S34-41,
http://dx.doi.org/10.1007/s10875-008-9187-2.
http://dx.doi.org/10.1007/s10875-008-918...
,2929 Pickering MC, Walport MJ. Links between complement abnormalities
and systemic lupus erythematosus. Rheumatology (Oxford). 2000;39(2):133-41,
http://dx.doi.org/10.1093/rheumatology/39.2.133.
http://dx.doi.org/10.1093/rheumatology/3...
). C2 gene sequencing was performed because
most Type I deficiencies associated with SLE are homozygous and because Zhu et al.
have reported a compound heterozygous Type I and II C2 deficiency in an
African-American family (1414 Zhu ZB, Atkinson TP, Volanakis JE. A novel type II complement C2
deficiency allele in an African-American family. J Immunol.
1998;161(2):578-84.
15 Truedsson L, Alper CA, Awdeh ZL, Johansen P, Sjoholm AG,
Sturfelt G. Characterization of Type-I Complement C2 Deficiency MHC Haplotypes -
Strong Conservation of the Complotype/HLA-B-Region and Absence of Disease
Association due to Linked Class-II Genes. J Immunol.
1993;151(10):5856-63.-1616 Johnson CA, Densen P, Hurford RK, Colten HR, Wetsel RA. Type-I
Human-Complement C2 Deficiency - A 28-base pair Gene Deletion Causes Skipping of
Exon-6 During RNA Splicing. J Biol Chem.
1992;267(13):9347-53.,2929 Pickering MC, Walport MJ. Links between complement abnormalities
and systemic lupus erythematosus. Rheumatology (Oxford). 2000;39(2):133-41,
http://dx.doi.org/10.1093/rheumatology/39.2.133.
http://dx.doi.org/10.1093/rheumatology/3...
).
Although the Type I C2 deficiency was heterozygous, it resulted in a 23-fold reduction in mRNA expression. Lipsker et al. observed decreased but still detectable C2 levels in SLE patients with a heterozygous Type I C2 deficiency (4343 Lipsker DM, Schreckenberg-Gilliot C, Uring-Lambert B, Meyer A, Hartmann D, Grosshans EM, et al. Lupus erythematosus associated with genetically determined deficiency of the second component of the complement. Arch Dermatol. 2000;136(12):1508-14.). Our findings raise both (1) the possibility that heterozygous C2 mutations alone or in combination with other factors could lead to SLE and (2) the question of why C2 mRNA expression was so low when the patient still had one functional C2 gene. Therefore, further JSLE patients with decreased C2 serum levels should be evaluated for Type I C2 deficiency to better understand the relationship of this condition with the disease.
The C4A and C4B genes each have 41 exons and differ from each other by only five
nucleotides. The number of gene copies ranges from two to eight, and deficiencies in
these two genes have been associated with SLE (1818 Yang Y, Lhotta K, Chung EK, Eder P, Neumair F, Yu CY. Complete
complement components C4A and C4B deficiencies in human kidney diseases and
systemic lupus erythematosus. J Immunol. 2004;173(4):2803-14,
http://dx.doi.org/10.4049/jimmunol.173.4.2803.
http://dx.doi.org/10.4049/jimmunol.173.4...
19 Rupert KL, Moulds JM, Yang Y, Arnett FC, Warren RW, Reveille JD,
et al. The molecular basis of complete complement C4A and C4B deficiencies in a
systemic lupus erythematosus patient with homozygous C4A and C4B mutant genes.
J Immunol. 2002;169(3):1570-8,
http://dx.doi.org/10.4049/jimmunol.169.3.1570.
http://dx.doi.org/10.4049/jimmunol.169.3...
-2020 Wu YL, Hauptmann G, Viguier M, Yu CY. Molecular basis of
complete complement C4 deficiency in two North-African families with systemic
lupus erythematosus. Genes Immunity. 2009;10(5):433-45,
http://dx.doi.org/10.1038/gene.2009.10.
http://dx.doi.org/10.1038/gene.2009.10...
,4444 Blanchong CA, Chung EK, Rupert KL, Yang Y, Yang ZY, Zhou B, et
al. Genetic, structural and functional diversities of human complement
components C4A and C4B and their mouse homologues, Slp and C4. Int Immunoph.
2001;1(3):365-92,
http://dx.doi.org/10.1016/S1567-5769(01)00019-4.
http://dx.doi.org/10.1016/S1567-5769(01)...
,4545 Ittiprasert W, Kantachuvesiri S, Pavasuthipaisit K,
Verasertniyom O, Chaomthum L, Totemchokchyakarn K, et al. Complete deficiencies
of complement C4A and C4B including 2-bp insertion in codon 1213 are genetic
risk factors of systemic lupus erythematosus in Thai populations.
J Autoimmun. 2005;25(1):77-84,
http://dx.doi.org/10.1016/j.jaut.2005.04.004.
http://dx.doi.org/10.1016/j.jaut.2005.04...
). P4 had
persistently low C4 serum levels, with two copies of C4A and three of C4B; the C4B
mRNA expression in this individual was significantly decreased compared with
controls and increased after IFNγ stimulation. Although this was an
interesting finding, attributing the abnormal C4B mRNA expression to a gene mutation
requires presuming that there were mutations in most or all of her three copies,
which is possible but unlikely. Additionally, we would presume that there was a
simultaneous problem with C4A transcription, which could not be evaluated. Thus,
further work is needed to clarify the role of decreased C4B mRNA expression in JSLE
pathogenesis.
This study was unprecedented because it involved the sequencing of all exons and non-coding regions of the C1q and C2 genes as well as the evaluation of mRNA expression. The limitations included the small number of patients, which created the need for experimental replicates, and the need for fresh mononuclear cells for mRNA expression analysis.
In conclusion, silent mutations and single-base changes in the 3′ non-coding region may modulate mRNA transcription and C1q production; Type I C2 deficiency should be evaluated in JSLE patients with decreased C2 serum levels; and further studies are needed to clarify the role of decreased C4B mRNA expression in JSLE pathogenesis.
Acknowledgments
The authors gratefully acknowledge all of the patients for their kind participation. The authors also acknowledge Luis EC Andrade and Estela M Novak for their assistance with laboratory tests and mRNA expression data analysis.
Financial support: This study was supported by São Paulo Research Foundation (FAPESP) grant 2008/58238-4.
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29Pickering MC, Walport MJ. Links between complement abnormalities and systemic lupus erythematosus. Rheumatology (Oxford). 2000;39(2):133-41, http://dx.doi.org/10.1093/rheumatology/39.2.133.
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30Gaboriaud C, Juanhuix J, Gruez A, Lacroix M, Darnault C, Pignol D, et al. The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties. J Biol Chem. 2003;278(47):46974-82, http://dx.doi.org/10.1074/jbc.M307764200.
» http://dx.doi.org/10.1074/jbc.M307764200 -
31Kishore W, Ghai R, Greenhough TJ, Shrive AK, Bonifati DM, Gadjeva MG, et al. Structural and functional anatomy of the globular domain of complement protein C1q. Immunol Lett. 2004;95(2):113-28, http://dx.doi.org/10.1016/j.imlet.2004.06.015.
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32Sellar GC, Blake DJ, Reid KBM. Characterization and organization of the genes encoding the A-, B- and C-chains of human complement subcomponent C1q. The complete derived amino acid sequence of human C1q. Biochem J. 1991;274(Pt 2):481-90.
-
33Petry F, Le DT, Kirschfink M, Loos M. Nonsense and Missense Mutations in the Structural Genes of Complement Component C1q A-Chains and C-Chains are Linked with 2 Different Types of Complete Selective C1q Deficiencies. J Immunol. 1995;155(10):4734-8.
-
34Petry F, Berkel AI, Loos M. Multiple identification of a particular type of hereditary C1q deficiency in the Turkish population: Review of the cases and additional genetic and functional analysis. Hum Genet. 1997;100(1):51-6, http://dx.doi.org/10.1007/s004390050464.
» http://dx.doi.org/10.1007/s004390050464 -
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Publication Dates
-
Publication in this collection
Mar 2015
History
-
Received
14 Aug 2014 -
Reviewed
1 Oct 2014 -
Accepted
5 Jan 2015