and Immunity (2009) 10, 433–445 & 2009 Macmillan Publishers Limited All rights reserved 1466-4879/09 $32.00 www.nature.com/gene

ORIGINAL ARTICLE Molecular basis of complete complement C4 deficiency in two North-African families with systemic erythematosus

YL Wu1,2, G Hauptmann3, M Viguier4 and CY Yu1,2,5,6 1Center for Molecular and Human Genetics, The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA; 2Integrated Biomedical Science Graduate Program, The Ohio State University, Columbus, OH, USA; 3Laboratoire d’Immunogenetique Moleculaire, Universite Louis Pasteur, Strasbourg Cedex, France; 4Service de Dermatologie, Hoˆpital Saint-Louis, Assistance Publique-Hoˆpitaux de Paris, Paris, France; 5Department of Pediatrics, The Ohio State University, Columbus, OH, USA and 6Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH, USA

Complete deficiency of complement C4 is among the strongest genetic risk factors for human systemic lupus erythematosus (SLE). C4 is a constituent of the RP–C4–CYP21–TNX (RCCX) module in the human leukocyte antigen (HLA) that exhibits inter-individual copy-number and -size variations. Here, we studied two North-African families with complete C4 deficiency and SLE. The first included a Moroccan male SLE patient (1P) and a sibling, who were both homozygous for HLA-A*02 B*17 DRB1*07. The second had an Algerian female SLE patient (2P) homozygous for HLA-A*01 B*17 DRB1*13. Early SLE disease onset, the presence of photosensitive rashes, anti-Ro/SSA, renal disease and high titers of antinuclear antibodies were the common features of complete C4 deficiency. Southern blot analyses showed that 1P had monomodular RCCX with a long C4A, whereas 2P had bimodular RCCX with one long C4A and one short C4B. Genomic DNA fragments for these mutant genes were amplified and sequenced. A C4T transition that created the R540X nonsense mutation in C4A was found in 1P. An identical 4-bp insertion that generated the Y1537X nonsense mutation was discovered in both C4A and C4B of 2P. The high concordance of SLE and C4 deficiency among patients with non-DR3 and non-DR2 underscores the importance of C4 in the protection against SLE. Genes and Immunity (2009) 10, 433–445; doi:10.1038/gene.2009.10; published online 12 March 2009

Keywords: anti-Ro/SSA; complete C4 deficiency; copy-number variation; non-sense mutations; RCCX modules; systemic lupus erythematosus

Introduction autoantibodies that target multiple tissues and organs.5,6 Although the genetic basis for a majority of SLE cases is The is a group of plasma and polygenic,7,8 a homozygous deficiency in one of the early membrane proteins that are sequentially activated complement components alone can be strong enough to through proteolytic cleavages to defend against micro- cause the disease, a situation similar to a single-gene bial infections.1,2 In addition to immune defense, defect in an autosomal recessive disease. Of the patients complement components such as C1q, C4 and C3 are with complete C1q and C4 deficiencies, 93 and 78%, important as they bind to and facilitate the respectively, eventually develop SLE or a lupus-like clearance of immune complexes (IC) and apoptotic disease.4,9 In addition, the concordance rates for siblings debris, which would otherwise promote with homozygous deficiency of C1q or C4 to develop and autoimmune diseases. SLE are 90 and 80%, respectively, which are higher Complete deficiencies of the early complement com- than the rate in monozygotic twins (26–60%) with other ponents including C1q, C1r, C1s and C4 in the classical genetic defects. activation pathway are highly associated with human Genetically, the complement C4 gene located in the systemic lupus erythematosus (SLE).3,4 SLE is a systemic class III region of the major histocompatibility complex autoimmune disease characterized by the breakdown of (MHC) on 6p21.3 shows frequent inter- immunotolerance and the production of a wide range of individual copy-number variation (CNV). The C4 CNV is a result of segmental duplications known as RCCX modules. RCCX stands for genes coding for serine/ Correspondence: Professor CY Yu, Center for Molecular and threonine nuclear kinase RP (also known as Human Genetics, The Research Institute at Nationwide Children’s STK19), complement C4, cytochrome P450 21-hydroxy- Hospital, 700 Children’s Drive, Columbus, OH 43205, USA. E-mail: [email protected] lase CYP21 and extracellular matrix protein tenascin-X Received 22 December 2008; revised 3 February 2009; accepted 3 TNX. One to four RCCX modules can exist per February 2009; published online 12 March 2009 .10–13 In each duplicated module, there is Complete complement C4 deficiency and human SLE YL Wu et al 434 a gene fragment, known as RP2, (0.9 kb) that corresponds photosensitivity, chronic discoid lupus erythematosus to the last three exons of the RP1 gene, a full-length C4, (DLE) lesions on the lips, hands and feet, positivity for a full-length pseudogene CYP21A or a functional gene anti-Ro/SSA antibodies, high titers of antinuclear anti- CYP21B, and another gene fragment TNXA (4.5 kb) that bodies (1/1280), and renal involvement including mod- corresponds to intron 32 to exon 45 of TNXB. There is erate proteinuria and microscopic hematuria. Kidney one more layer of genetic structural variation: the C4 biopsies confirmed the presence of focal proliferative genes exhibit a dichotomous gene-size variation.14–16 glomerulonephritis with thickening of the capillary walls Each C4 gene is either 20.6 kb (long gene, C4L)or and a wire loop appearance. Immunofluorescence 14.2 kb (short gene, C4S) in length. The long C4 gene experiments showed mesangial deposits of IgG and contains an endogenous retrovirus, HERV-K(C4), in its C1q but not C3. Repeated laboratory measurements ninth intron. Approximately three-quarters of C4 genes showed no detectable C4 proteins or CH50 activity, but in Caucasian populations belong to the long form.17 serum C3 levels were normal.29 The brother of this About 40 protein variants for complement C4 have patient (1S) shared the same complement abnormalities. been documented.18 These proteins are segregated into He was reported to be healthy until the age of 12. At the two classes, the acidic C4A and the basic C4B. Each C4 age of 17, he presented with repeated infections of the gene codes for either a C4A or a C4B protein.19 The nasal cavity, and a severe sinusitis with infection of the isotypic sites for these two classes of proteins are left orbital cavity, which required a surgical intervention. determined by five single nucleotide polymorphisms He also presented with microscopic hematuria, but (SNP) in exon 26 of C4 genes, which contribute to four investigations to detect antinuclear antibodies yielded amino-acid changes at positions 1101, 1102, 1105 and negative results. HLA typing showed that these brothers 1106: PCPVLD in C4A and LSPVIH in C4B.20 The were homozygous for A*02 B*17 DRB1*07. The complete presence of His-1106 in C4B, in particular, catalyzes the absence of C4 proteins and CH50 activity but normal C3 formation of a covalent ester linkage with the target protein levels in these brothers suggested a genetic substrates through a highly reactive carbonyl group from deficiency of complements C4A and C4B. Patient 1P had the thioester bond in activated C4.21,22 This creates two other siblings who were heterozygous for the HLA differential functions for C4A and C4B proteins, includ- A*02 B*17 DRB1*07 . The younger sister had ing lower hemolytic activity for C4A, higher for C4B; relatively low C4 concentration (0.06 g l–1) and tested longer half-life of the activated product (C4b) for C4A, ANA-positive (1/320). shorter for C4B; and different substrate preferences. C4A preferentially binds to amino groups in IC, and is a more Case 2. Patient 2 was an Algerian girl who was among preferred ligand for 1 (CR1).23 one of the earliest complete C4 deficiency patients Activated C4B binds rapidly to the hydroxyl group in reported.30 She was a child of parents who were married substrates. Owing to such differences, C4A is believed to consanguineously (Figure 1, panel b). She presented with be important in the clearances of IC, and C4B is more SLE-related symptoms including lupus erythematous powerful in propagating the complement activation rash, high titers of speckled antinuclear antibodies (1/ cascades. The complete absence of both C4A and C4B 1024), anti-Ro and anti-Sm antibodies, moderate renal proteins may therefore lead to decreased ability of disease with mesangial proliferation plus IgM and C1q immune defense against microbes, as well as inefficient deposits. The patient also suffered from bacterial disposal of IC. meningitides and recurrent lung infections, developed To date, 28 individuals with complete C4 deficiency osteomyelitis of the left femur, and eventually died from from 19 families have been reported.3,4,24 Among them, cardiopulmonary complications at the age of 12. The 15 individuals developed SLE, 7 developed lupus-like patient was determined to be homozygous for HLA A*01 diseases, and 4 of the remaining 6 subjects were afflicted B*17 DRB1*13. TaqI restriction fragment length poly- by kidney diseases.24 Multiple complete C4 deficiency morphism (RFLP) analysis with a C4 50 probe showed the subjects also experienced severe or recurrent microbial presence of the 7.0 and 5.4 kb fragments representing the infections. At least 16 different HLA haplotypes have presence of long and short C4 genes.29,30 Patient 2P had been found in complete C4 deficiency individuals. four siblings, who were all heterozygous for the HLA Among the complete C4 deficiency cases, the molecular A*01 B*17 DRB1*13 haplotype, which had no functional basis of their defects have been elucidated in 12 C4 genes. All of her siblings tested ANA-positive, with individuals, but all were of European descent.25–28 In titers ranging from 1/160 to 1/640. this study, we present the clinical features and determine the molecular basis for the complete C4A and C4B Genotypic characterization of the RCCX modules and C4A deficiencies in two North-African SLE patients. and C4B gene copy-numbers and phenotypic characterization of complement proteins Family 1. From TaqI RFLP (Figure 2, left panel c), patient 1P and one of his siblings 1S shared identical patterns Results of RCCX with a TaqI restriction fragment of 7.0 kb, Case reports corresponding to a long C4 gene, followed by a 3.7-kb Two unrelated cases of complete C4 deficiency in which restriction fragment for CYP21B and a 2.5-kb restriction the patients developed SLE were studied fragment for TNXB. This restriction fragment pattern was indicative of a monomodular RCCX containing a Case 1. Patient 1P was a Moroccan male born to parents single long C4 gene on both the maternal and paternal of consanguineous marriage (Figure 1, panel a). He copies of chromosome 6. The homozygosity of the RCCX was diagnosed with SLE at the age of 6. His clinical was consistent with the homozygous HLA in these two presentation included malar rash with marked individuals. The same Southern blot for the patient’s

Genes and Immunity Complete complement C4 deficiency and human SLE YL Wu et al 435 Family 1 -Morocco

1M (1956)

a/b c/d C4 = 0.24 g/L ANA + 1/320, speckled

1P 1S (1976) (1978) (1980) (1986)

a/c b/c a/c a/d

C4 < 0.01 g/L C4 = 0.06 g/L C4 < 0.01 g/L C4 = 0.28 g/L ANA: 1/1280, speckled ANA : 1/320, speckled ANA: negative ANA: negative Anti-Ro/SSA positive ** SLE

HLA & complement haplotypes : a, c: A*02 B*17 DRB1*07 C2*C BF*F complete C4 deficiency b: A*02 B*21 DRB1*03 C2*C BF*F C4A*3 d: A*02 B*14 DRB1*11 C2*C BF*S C4A*2 C4B*1 C4B*2

Family 2 -Algeria

a/b c/d 2M (1932) (1944)

C4 = 0.12 g/L C4 = 0.11 g/L ANA : 1/160, ANA : 1/1280, homogeneous / speckled speckled Anti-Ro/SSA positive *

2S 2P (1963) (1966) (1971) (1975) (1997) + 1987

b/c a/d b/c a/c a/d

C4 = 0.13 g/L C4 = 0.08 g/L C4 = 0.17 g/L C4 < 0.01 g/L C4 = 0.12 g/L ANA: 1/320, ANA :1/640, ANA : 1/320, ANA: 1/1024, ANA : 1/160, speckled speckled speckled speckled speckled Anti-Ro, Anti-Sm SLE

HLA & complement haplotypes :

a, c : A*01 B*17 DRB1*13 C2*C BF*F complete C4 deficiency

b : A*30 B*17 DRB1*15 C2*C BF*F C4A*3 C4B*1

d : A*33 B*08 DRB1*15 C2*C BF*S C4B*1 Figure 1 Complete C4 deficiency in two North-African families. (a) The Moroccan family. Left panel, pedigree of the Moroccan family. Double horizontal lines represent consanguineous marriage. Two male subjects with complete C4 deficiency are shown in solid boxes. Haplotypes of the HLA and complement are italicized. Year of birth is shown in parentheses. Antinuclear antibodies were detected by immunofluorescence on Hep2 cells and titers of autoantibodies are shown. **Anti-Ro/SSA antibodies were detected through immunoprecipitation. C4 protein levels were measured by nephelometry (Beckman, Gagny, France). Right panel, photographs of patient 1P showing lesions on the lips, hands and feet. (b) The Algerian family. Left panel, pedigree of the Algerian family. The female subject (patient 2P) with complete C4 deficiency is shown in solid circle and the year of her death is marked by the þ sign. HLA haplotypes of the family are shown. Right panel, photograph of 2P showing malar rash on her face. Black and white photographs of Patient 2P were published earlier.4,30 mother 1M showed three sets of TaqI patterns, each with haplotype was a trimodular configuration with one C4L two restriction fragments of equal intensity: C4L linking gene and two C4S genes. PshAI–PvuII RFLP showed that to RP1 (7.0 kb) and C4S linking to RP2 (5.4 kb), CYP21B there were no C4B genes for 1P and 1S, with the absence (3.7 kb) and CYP21A (3.2 kb), and TNXB (2.5 kb) and of the 2.2-kb PshAI–PvuII restriction fragment specific for TNXA (2.4 kb). This phenomenon indicated that there C4B (Figure 2, left panel d). Equal intensities of bands were four copies of C4 genes (two of C4L and two of C4S) corresponding to C4A and C4B indicated that 1M had in the mother’s genome. As patient 1P and his sibling 1S equal numbers of C4A and C4B genes. Thus, the C4 inherited a monomodular RCCX from the mother, the genotypes of the mother were C4L (C4AQ0)/C4L–C4S– only possible interpretation for the mother’s other RCCX C4S (C4A–C4B–C4B).

Genes and Immunity Complete complement C4 deficiency and human SLE YL Wu et al 436 TaqI RFLP for RCCX modules and C4 CNV

kb 1P 1M 1S 2P 2M 2S2 RP1-C4L 7.0 Immunofixation of plasma C4 proteins RP1-C4S 6.4 RP2-C4L 6.0 1P 1M 1S C1 C2 2P C3 RP2-C4S 5.4 C4B1 C4B2 CYP21B 3.7 C4A2 CYP21A 3.2 C4A3 TNXB 2.5 TNXA 2.4

L/L L/LSS L/L LS/LS LS/S LS/LS Immunofixation of plasma C3 proteins PshAI-PvuII RFLP C4A and C4B genes 1P 1M 1S C1 C2 2P C3 1P 1M 1S 2P 2M 2S C3S C4B 2.2 C3F C4A 1.7

C4A:C4B 2:0 2:2 2:0 2:2 1:2 2:2

SSP-PCR to detect common mutations in C4 and C2

C4M C2M C2M C4M

+ -1P 1M 1S+ - m m 2P 2M 2S2 +-+ m

bp

908

174 146

Figure 2 Phenotypic analyses of plasma complement proteins and genotypic analyses of complement C4 in Family 1 and Family 2. (a) Immunofixation using EDTA-plasma showing the presence of C4A and C4B protein allotypes. In the left panel, C4 proteins were detectable in the mother (1M) of Family 1 and in the control sample ‘C1’. No C4 proteins were found in 1P and his sibling 1S. In the right panel, no C4 proteins were detectable for 2P. ‘C2’ and ‘C3’ were control plasma samples. (b) Immunofixation using the same EDTA-plasma samples as shown in (a), showing the presence of complement C3 proteins. Partial degradation of C3 proteins in 2P was probably a result of long-term storage. (c) TaqI RFLP Southern blot analyses showing the RP–C4–CYP21–TNX (RCCX) modular variations in families 1 and 2. 1P and 1S were homozygous for L/L RCCX, and 1M was heterozygous for L/LSS. 2P and 2S shared LS/LS structures, and 2M was heterozygous for LS/S. (d) PshAI–PvuII Southern blot analyses showing the ratio of C4A and C4B genes. Only the restriction fragment corresponding to the C4A gene was present in 1P and 1S. The restriction fragments for C4A and C4B were of equal intensity in 1M, 2P and 2S. In 2M, the intensity for the C4B restriction fragment was approximately twice as that for the C4A restriction fragment. (e) Multiplex sequence-specific primer-PCR to detect known mutations of the human C4 genes and C2 genes. The 908-bp fragment represent the presence of the 2-bp insertion in exon 29 of C4. The 174-bp fragment represent normal C2 genes, which also served as positive control for successful amplification of PCR. The 146-bp fragment represented the presence of the 28-bp deletion in the junction of exon 6–intron 6 of C2. Family 1 is shown on the left panel and Family 2 is shown on the right panel. DNA samples with complement C4 and C2 mutations (C4M þ and C2M þ ) as well as negative controls (À) are shown. m, 100-bp DNA ladder.

Genes and Immunity Complete complement C4 deficiency and human SLE YL Wu et al 437 Allotyping of C4 proteins by immunofixation using Screening for the most common C4 and Type I C2 mutation EDTA-plasma samples from this family showed that in Family 1 and Family 2 there was no trace of C4 proteins for 1P and 1S (Figure 2, To determine the molecular basis of complete comple- left panel a). There were three C4 protein variants ment C4A and C4B deficiencies, we screened patients 1 present in 1M: C4A2, C4B1 and C4B2. However, despite and 2 and their family members for the 2-bp insertion at 1M having two copies of C4A genes, the intensity of the codon 1213 from exon 29 of both C4A and C4B genes.31 C4A2 protein was the same as those for C4B1 and C4B2. This mutation is relatively common, with a frequency In addition, C3 allotyping showed the presence of 41% even among healthy subjects of European ances- normal levels of C3 proteins in all three subjects (Figure 2, try.17 A multiplex sequence-specific primer (SSP)-PCR panel b). Such a phenomenon suggests that the absence along with an amplicon to detect the common 28-bp of C4 proteins in 1P and 1S was unlikely to be a deletion at the exon 6–intron 6 junction of the comple- consequence of degradation or consumption of comple- ment C2 gene was used. None of the members in these ment proteins. These protein allotyping data were two families were positive for the common mutations consistent with the constantly undetectable levels of C4 present in the C4 and C2 genes (Figure 2, panel e). proteins for 1P and 1S by repeated clinical measures. Negative results in both patients 1 and 2 were also Given the results from both phenotypic and genotypic obtained for other known C4 nonsense mutations in exon analyses, the null allele of C4 was determined to be a 13, exon 20 and exon 2828 (data not shown). Thus, we single copy of long C4A in a monomodular RCCX linking proceeded to determine the sequence anomalies con- to HLA A*02 B*17 DRB1*07. Both 1P and 1S were tributing to the non-expression of C4 proteins in these homozygous for this haplotype, and their mother 1M patients. was heterozygous, with monomodular L for a mutant C4A gene, and trimodular LSS coding for C4A2, A C to T transition in exon 13 of the long C4A gene in C4B1 and C4B2, which was linked to HLA A*02 B*14 HLA-A*2 B*17 DRB1*07 haplotype resulting in a nonsense DRB1*11. mutation in codon 540 The long C4A gene from 1P was amplified into five Family 2. TaqI RFLP was performed in patient 2P, her overlapping DNA fragments that ranged from 2.1 to mother 2M and one of her siblings 2S (Figure 2, right 3.5 kb in length by PCR (Figure 3, panel a). Each panel c). In 2P and 2S, we observed the presence of TaqI fragment was sequenced to completion using multiple restriction fragments with equal intensities for the 7.0 C4 specific primers in both sense and anti-sense and 5.4-kb fragments corresponding to C4L and C4S, orientations.16 Sequence comparisons to known C4A respectively; 3.7- and 3.2-kb fragments corresponding to gene sequences showed that there were no insertions CYP21B and CYP21A, respectively; and 2.5- and 2.4-kb or deletions in the long C4A gene in 1P. Sequences fragments corresponding to TNXB and TNXA, respec- distinct from that of the C4 reference in the NCBI tively. The patient’s mother 2M showed a distinctive 6.4- database (NC_000006.10) SNPs are shown in Table 1. Of kb TaqI fragment that is characteristic of a short C4 gene the two novel SNPs, one is a single nucleotide change linking to RP1. This 6.4-kb fragment was of the same from G to A at nt-20 055 located in intron 39; the other is a intensity as the 7.0-kb fragment for RP1–C4L and the 5.4- C to T transition at nt-10 142 of codon 540 from exon 13 kb fragment for RP2–C4S. On the other hand, the 3.7-kb that replaces an arginine residue with a stop codon. fragment for CYP21B was twice that of the 3.2-kb To verify that the 10 142 C4T (R540X) mutation was fragment for CYP21A, and the 2.5-kb fragment for TNXB not an artifact introduced by PCR but a genuine change was twice as intense as the 2.4-kb TNXA fragment. Thus, in the genomic sequences of 1P, a new SSP PCR RFLP it is concluded that 2M had bimodular long-short (LS) was designed to interrogate genomic DNA samples of 1P and monomodular-short (S) RCCX haplotypes (LS/S), and his family members. In this SSP-PCR, an HhaI whereas patients 2P and 2S were both homozygous for restriction enzyme cleavage site specifically recognizing LS/LS. the normal C allele at nt-10 142 was introduced during The PshAI–PvuII Southern blot (Figure 2, right panel c) PCR by the reverse primer, resulting in a smaller showed that the numbers of C4A and C4B genes were the fragment after HhaI restriction digest and gel electro- same for 2P and 2S, whereas those of 2M had a ratio of phoresis. If the T-allele was present instead of the 1:2. Combining the results from the two sets of Southern C-allele, this restriction site was not present and the blots, the RCCX haplotypes for 2M were LS (C4A–C4B) PCR product would be resistant to digestion by HhaI. A and S (C4B), and those for 2P and her siblings were LS single fragment of 159 bp was observed for 1P and 1S, (C4A–C4B)/LS (C4A–C4B). but two fragments, one of 159 bp and the other of 132 bp, Stored EDTA-plasma sample from patient 2P was were observed for 1M (Figure 3, panel c). The results available for complement C4 and C3 protein allotyping confirmed that both 1P and 1S were homozygous for the experiments. No C4 protein was detectable (Figure 2, 10 142 C4T mutation, and that their mother 1M was right panel a), while proteins corresponding to the slow heterozygous. and fast variants of C3 were present (Figure 2, panel b). Clinically measurable C4 concentrations were observed An identical 4-bp insertion in exon 36 of the long C4A in all family members, but were repeatedly undetectable and short C4B in the HLA-A*01 B*17 DRB1*13 haplotype for 2P. With this information, plus the homozygous HLA Patient 2P was homozygous for a bimodular RCCX, with haplotypes and the consanguineous marriage of her a long C4A mutant gene and a short C4B mutant gene on parents, patient 2P was deduced to be homozygous for each copy of her chromosome 6. Using primers specific the bimodular-LS haplotype containing a null allele for for the long terminal repeat (LTR) of the endogenous C4A and a null allele for C4B, that is, LS (C4AQ0– retrovirus HERV-K (C4) in the C4A gene, and for the C4BQ0), linking to HLA-A*01 B*17 DRB1*13. C4A/C4B isotypic site in exon 26 as anchors, the first 35

Genes and Immunity Complete complement C4 deficiency and human SLE YL Wu et al 438 PCR Strategy to amplify the mutant C4A gene in monomodular-long RCCX HLA A2 B17 DR7

CYP21B

RP1 C4A TNXB K(C4)

1 2 3 kb

Exon no. 5 10 15 20 25 30 35 40 RP C4 3' 5' LTR LTR env AT pol gag

HERV-K(C4) E1.5 E9.3 E10.5 E21.3 E28.5 I33.3

A: 2.1 kb B: 3.3 kb D: 2.6 kb E19.5 E29.32 E33.5 E41.3

C: 2.8 kb E: 3.5 kb

SSP-PCR and HhaI RFLP to detect the 10142 C>T (R540X) mutation C to T transition in exon 13 of the C4A mutant m 1P 1M 1S -

Ref Seq

bp 159 1P 132

The C to T transition in the long-C4A gene introduces a premature stop codon ...... Exon 13. . . gctgggggggttcaaggctgaggctgtcccatgaagaggcaaccactcttgtccctcccattcttggcccagATCCTATCCCGAGGGCAGATCGTGTTCA C4L: 10019 (490) I L S R G Q I V F M 499 ...... TGAATCGAGAGCCCAAGAGGACCCTGACCTCGGTCTCGGTGTTTGTGGACCATCACCTGGCACCCTCCTTCTACTTTGTGGCCTTCTACTACCATGGAGA C4L: 10119 N R E P K R T L T S V S V F V D H H L A P S F Y F V A F Y Y H G D 532

. 10142 T ...... CCACCCAGTGGCCAACTCCCTGCGAGTGGATGTCCAGGCTGGGGCCTGCGAGGGCAAGgtgaccggggtcaggagagatggcacttgtgccgagggggtt C4L: 10219 H P V A N S L R V D V Q A G A C E G K (551)

Figure 3 Elucidation of the molecular basis of complete C4 deficiency in Patient 1P. (a) Map of monomodular RCCX, C4A gene structure and PCR strategy to amplify the mutant C4A gene associated with the HLA A2 B17 DR7 haplotype. Exons are shown as solid boxes. Transcription directions for RP, C4 and the orientation of the endogenous retrovirus HERV-K(C4) are shown by arrows above intron–exon structures. Arrows below the exon–intron structures represent PCR primers used to amplify the C4 gene into fragments A–D. (b) Sequence chromatogram showing the C to T transition (in exon 13) at nucleotide 10 142 of the C4A mutant gene in 1P. (c) Sequence-specific primer (SSP)-PCR and HhaI RFLP to detect the C-T transition of the C4A gene in Family 1. The 159-bp fragment represented the presence of the normal C allele. The 132-bp band represented the presence of the T mutation. Both 1P and 1S were homozygous, and 1M was heterozygous for the C to T mutation. m,100-bp DNA ladder. À, negative control. (d) DNA and derived amino acid at exon 13 showing the C to T mutation at nucleotide 10 142 that changed codon 540 from an Arg to a stop codon (R540X).

exons for the long-C4A gene and for the short-C4B gene coding SNPs and two novel intronic SNPs were found. were each amplified into three large PCR fragments. Of the novel coding SNPs, one was a synonymous C to T A fragment covering from exon 20 to exon 31 and transition at nt-4659 (codon 661) from exon 16 of the another fragment from exon 35 to the last exon, exon 41, short-C4B genes, whereas the other was a 4bp insertion, were indiscriminately amplified for both C4A and C4B GACT, into exon 36 that created frame-shift mutations (Figure 4, panels b). Direct DNA sequencing of the PCR and changed the tyrosine residue at codon 1537 to a stop fragments showed multiple polymorphisms in the codon TGA (Y1537X) (Figure 4, panel e). The latter was coding and non-coding regions (Table 2). Two novel observed in a PCR fragment spanning exons 35–41,

Genes and Immunity Complete complement C4 deficiency and human SLE YL Wu et al 439 Table 1 Sequence variations of the long C4A gene in patient 1P presence of the 4-bp insertion in C4. The 79-bp fragment compared with Reference sequence NC_00006 was detectable in all family members (Figure 4, panel d), confirming the presence of the GACT insertion into exon Exon/Intron Nucleotide Nucleotide change Amino Remarks 36 of the C4 genes in the Algerian family. location positiona acid change

Exon 9 2296 C-A(TCTtoTAT) S328Y Discussion Exon 11 9468 C-T (GAC to GAT) 407D Intron 11 9538 c-t In this study, we have characterized the RCCX structures Intron 12 9882 c-a and elucidated the molecular basis of complete comple- Exon 13 10 142 C-T(CGA to TGA) R540X Novel ment C4A and C4B deficiencies in two North-African nonsense b families. The first case is a Moroccan subject (the parents mutation came from Algeria and their children were born in Intron 19 11 965 c-t Intron 21 12 750 a-g Morocco) who has homozygous HLA-A*02 B*17 Intron 30 15 340 c-a DRB1*07 haplotype with a monomodular RCCX struc- Intron 30 15 410 t-c ture containing a single long C4A mutant gene. A C4T Intron 31 16 668 t-c mutation at nucleotide 10 142 in exon 13 changed the Intron 35 18 448 t-g - Arg-540 codon to a stop codon, which was the only Intron 38 19 589 c t molecular defect that knocked out the expression of C4 Intron 39 20 055 g-a Novel change protein in this patient. By newly designed SSP-PCR and HhaI RFLP analysis, the same mutation was found in the patient’s sibling with complete C4 deficiency, and in his Sequence variations from the reference human C4 sequence in the GenBank are shown. Novel sequence variations found in this work mother, who was heterozygous for the HLA-A*02 B*17 are listed in bold; changes which were also found in other C4 DRB1*07 haplotype. Nonsense mutations in exon 13 had polymorphic variants are shown in normal font; the polymorphic or been found in two other different HLA haplotypes. One mutant nucleotide sequence within a codon is italicized.19,20 was a 2-bp deletion in codon 497 of a long-C4A mutant Ref Seq: Reference sequence NC_000006.10|NC_000006:32 090 549– gene in HLA A24 Cw7 B38 DR13 with a monomodular 28 3 2111173, total size 20 624 bp. RCCX structure. The other is a 1-bp deletion at codon aNucleotide position starts from ATG in long C4 genes. 522 of the short-C4B gene in the HLA A2 B12 DR6 bMutation responsible for the non-expression of C4 genes. R540X, a haplotype with a bimodular LS structure containing one mutation changed Arg-540 to a stop codon. long-C4A and one short-C4B mutant genes.27 Thus, exon 13 seems to be one of the hotspots for C4 mutations which was common for the long-C4A and the short-C4B (Figure 5). genes. It was thus essential to determine whether the In the second case, an Algerian girl homozygous with GACT insertion was present in C4A, C4B or both. Careful HLA-A*01 B*17 DRB1*13 was found to have both C4A examinations of the DNA sequence chromatograms of and C4B genes but no C4 proteins. Southern blot analysis the 3.6-kb AB2-PCR fragment from patient 2P showed no showed the presence of one long C4 and one short C4 on sign of heterozygosity of this 4-bp insertion (Figure 4, each chromosome 6. PshAI–PvuII RFLP showed that both panel c), suggesting that this mutation was present in C4A and C4B genes were present. Sequence-specific PCR both C4A and C4B genes. suggested that the short gene was C4B and therefore the To eliminate the possibility of an unlikely bias of the long gene was C4A. PCR amplification and DNA PCR for AB2-fragment that selectively amplified one C4 sequencing of specific DNA fragments covering exons gene but not the other, the same PCR-primer sets were 1–35 for C4A, and for C4B, did not show any sequence used to amplify genomic regions covering exons 35–41 defects. However, sequencing of the 3.6-kb DNA frag- from DNA samples of the patient’s mother (2M) and one ment corresponding to exons 33–41, which was common of the patient’s siblings (2S). Direct DNA sequencing of to both C4A and C4B genes in 2P, showed a GACT the PCR fragments from these two family members insertion in exon 36. Such 4-bp insertion led to frameshift showed double sequences at the corresponding regions, and created the Y1537X nonsense mutation. The implying that 2M and 2S were heterozygous for the 4-bp homogeneity of the sequencing chromatograms in the insertion (Figure 4, panel c). Thus, it was concluded that patient but not in her mother 2M or sibling 2S (panel c, patient 2P possessed the identical GACT insertions into Figure 4), who were heterozygous for HLA A*01 B*17 exon-36 of both C4A and C4B genes. It was also of DRB1*13 and RCCX modules, suggested that the GACT interest to note that not only the 4-bp insertion to exon insertion was present in both mutant C4A and C4B 36, but also other polymorphic variants between intron genes of 2P. Sequence-specific PCR further confirmed the 31 and exon 41 seemed to be identical in the C4A and presence of this insertion in the patient’s mother and C4B mutant genes of patient 2P (Table 2). siblings. A multiplex SSP-PCR combining the Type I C2- Identical molecular defects in both C4 genes from mutation screening primers and specific forward primer bimodular RCCX attributed to complete C4 deficiency to recognize the 4-bp insertion in the insertion site and had been observed in two other HLA haplotypes. One thus amplify the mutant C4 genes was developed to was present in a female Finnish SLE patient who was screen for this GACT insertion in the genomic DNA from homozygous for HLA haplotype A2 Cw7 B39 DR15 with all members in Family 2. The presence of a 174-bp PCR LS RCCX modules containing one C4A and one C4B.An product indicates the absence of the Type I C2-mutation, identical 2-bp insertion at codon 1213 of exon 29 was as well as the successful amplification of the PCR. The present in both the C4A gene and the C4B gene.26 The presence of a 79-bp PCR product corresponds to the other one was present in HLA haplotype A*30 B*18

Genes and Immunity Complete complement C4 deficiency and human SLE YL Wu et al 440 DR*07 with bimodular RCCX modules containing two sequences between duplicated genes is intriguing but not short C4B genes (SS, C4B–C4B). A G4A mutation at the uncommon, as we observed considerably high frequen- donor site of intron 28 in both C4B genes probably led to cies of bimodular RCCX haplotypes with C4A–C4A or alternative splicing through a cryptic splice site and C4B–C4B instead of C4A–C4B configurations among incorporation of seven additional nucleotides to the healthy subjects, the presence of CYP21B–CYP21B in transcript and the generation of a stop codon at codon bimodular RCCX haplotypes among many Asian 1208. Such a phenomenon of homogenization of DNA Indians, and the presence of two mutant genes

Bimodular Long-short (LS) RCCX haplotype with a mutant C4A gene and a mutant C4B gene

HLA A1 B17 DR13 CYP21A CYP21B

RP1 C4A C4B TNXB K(C4)

PCR strategy to amplify the long-C4A and short C4B mutant genes 1 2 3 kb 1 2 3 kb C4A - long gene C4B - short gene

Exon no. 5 10 15 20 25 30 35 40 Exon no. 5 10 15 20 25 30 35 40

RP C4 3' 5' RP C4 Intron 9 LTR LTR env AT pol gag

HERV-K(C4) RPIN7F C4L-3LTR-R C4A-down E35.3 RPIN7F E10.3 C4B-down E35.3 A3: 3.4 kb A1: 3.0 kb E10.5 C4A-up B1: 3.0 kb B3: 3.4 kb C4Fin95 C4B-up A2: 4.9 kb B2: 5.0 kb E20.5 E31.3 E33.5 E41.3 E20.5 E31.3 E33.5 E41.3 AB1: 3.5 kb AB2: 3.6 kb AB1: 3.5 kb AB2: 3.6 kb

DNA sequencing to show the GACT- insertion in exon 36 SSP-PCR to detect the 4-bp insertion in exon 36 Sense 5’→3’ m - 2P 2M 2S Ref Seq

2P

Antisense 3’→5’

Ref Seq

2P

* bp

2M 174

* 79

2S

The 4-bp insertion introduced a stop codon in exon 36 in both C4 genes

...... Exon 36 . . C4L: 19020 ggccaaggaaacccagtacagggggctgcagggcccagggagtgggtccctcatctctcctccccacgcttggccagGTCCCCACCTCCCGGGAGTGCGT C4S: 12600 (1507) V P T S R E C V 1514

+GACT (insertion) ...... ↓ . . . C4L: 19120 GGGCTTTGAGGCTGTGCAGGAAGTGCCGGTGGGGCTGGTGCAGCCGGCCAGCGCAACCCTGTACGACTACTACAACCCCGgtgagcactgcaggacaccc C4S: 12700 G F E A V Q E V P V G L V Q P A S A T L Y D Y Y N P E (1541)

...... C4L: 19220 tgaaattcaggagaactttggcataggtgccctcctatgggacaatggacaccggggtagtgagggggcagagagccctggggctccctgggactgagga C4S: 12800

Genes and Immunity Complete complement C4 deficiency and human SLE YL Wu et al 441 CYP21A–CYP21A in many congenital adrenal complex-related diseases, irrespective of the HLA DR3 hyperplasia patients.17,32,33 or DR2 haplotypes that have been linked to SLE.34,35 The complete deficiencies of complements C4A and Twenty-two out of 28 patients with complete C4 C4B serve as natural ‘knock-out’ models yielding in- deficiency from 16 different HLA haplotypes developed sights into the function of C4 proteins. The pathological SLE or lupus-like diseases, and 4 others had renal consequence for the complete absence of C4 protein diseases including glomerulonephritis.4 Inefficient clear- highlights its importance in microbial defense, as most ance immune complexes caused by genetic C4 deficien- C4-deficient subjects experienced recurrent infections. cies is a likely etiologic mechanism that promotes Another important outcome in most complete C4 inflammatory and autoimmune conditions. Among the deficiency subjects is the onset of SLE or immune- complete C4 deficiency patients who developed SLE,

Table 2 Sequence variations in the C4A and C4B genes of patient 2P compared with the GenBank C4 reference sequences

Exon-intron location Nucleotide positiona Nucleotide change Amino acid change Remark

(A) Sequence variations found in the long C4A genesb Exon 9 2296 C-A(TCTtoTAT) S328Y Exon 11 9468 C-T (GAC to GAT) 407D Intron 28 14 559 g-c 14 563 +c Intron 30 15 410 t-c Share c allele with S-C4B Intron 38 19 589 c-t Share t allele with S-C4B

(B) Sequence changes found in the short C4B genesc Exon 16 4659 C-T (AAC to AAT) 661N Novel change Intron 19 5653 a-g 5666 g-a 5753 a-g 5761 t-c Intron 20 5985 g-a Intron 21 6307 g-a Novel change Intron 31 9576 g-a

(C) Sequence changes found in both the long C4A genesb and the short C4B genesc Intron 11 3119 (9538) c-t Intron 12 3463 (9882) c-a Intron 19 5546 (11965) t-c Novel change Exon 20 5793 (12212) T-C (GTT to GTC) 806V Exon 21 6150 (12569) A-G(ACC to GCC) T888A Intron 21 6331 (12750) a-g 6419 (12838) t-c Exon 26 7535 (13954) C-A (GGC to GGA) 1076G Intron 30 8920 (15340) c-a Intron 31 10248 (16668) t-c Intron 35 12028 (18448) t-g Exon 36 12664 (19084) +GACT Y1537X Novel nonsense mutationd Intron 38 13286 (19706) c-t Novel change

Sequence variations from the reference human C4 sequence in the GenBank are shown. Novel sequence variations found in this work are listed in bold; changes which were also found in other C4 polymorphic variants are shown in normal font; the polymorphic or mutant nucleotide sequence within a codon is italicized.19,20 aNucleotide position starts from ATG in short (and in long) C4 genes. bRef Seq: NC_000006.10|NC_000006:32 090 549–3 211173, total 20 624 bp (a long C4A). cRef Seq: AL049547 (contains a short C4B). dMutation responsible for the non-expression of C4 genes. Y1537X, a mutation that changed Tyr-1537 to a stop codon.

Figure 4 Elucidation of the molecular basis of complete C4 deficiency in Patient 2P. (a) Map of the bimodular RCCX containing a long C4A gene and a short C4B gene associated with the HLA A1 B17 DR13 haplotype. (b, c) Gene structures of the long and short C4 genes and PCR strategies to amplify the long-C4A gene and the short-C4B gene in 2P. Exons are shown as solid boxes. Transcription directions for RP, C4 and the endogenous retrovirus HERV-K(C4) are shown by arrows above exon–intron structures. Arrows below the exon–intron structures represent the PCR primers used to amplify the C4 genes. Note that exons 33–41 were amplified indistinguishably for both C4A genes and C4B genes. (d) Sequence determination of the 4-bp insertion in exon 36 of the mutant long C4A and short C4B genes. The first panel shows the sense-strand DNA sequence for 2P. The second, third and fourth panels shows the antisense strand sequences for 2P, 2M and 2S, respectively. Note that double sequences appeared after the 4-bp insertion in both 2M and 2S, which are each marked by an asterisk. (e) SSP-PCR to detect the 4-bp insertion ( þ GACT) in exon 36 in the genomic DNA samples from Family 2. The 79-bp fragment represented the presence of a 4-bp insertion. The 174-bp fragment represented the normal complement C2 genes, and was used as a positive control for PCR. m, 100-bp DNA ladder. À, negative control. (f) The DNA and derived amino acid sequence of exon 36 showing the location of the 4-bp insertion that caused a frame-shift mutation, which introduced a stop codon (TGA) at codon 1537 of the mutant C4 genes.

Genes and Immunity Complete complement C4 deficiency and human SLE YL Wu et al 442 1 2 3 kb Long C4 gene, 20.6 kb

Exon no. 5 10 15 20 25 30 35 40 RP C4 5' 3' LTR LTR envAT pol gag

HERV-K(C4)   * * 13 20 i28, 29 36 Short C4 gene, 14.2 kb 

Intron 9

Exon 13 (9)

1-bp del, codon 522 HLA A2 B12 DR6 RCCX: LS; C4AQ0-C4BQ0

2-bp del, codon 497 HLA A24 Cw7 B38 DR13 RCCX: mono-L; C4AQ0 C T mutation, codon 540 HLA A2 B17 DR7 RCCX: mono-L; C4AQ0

Exon 20 (1) 1-bp del, codon 811 HLA A30 B18 DR3 RCCX: mono-L; C4AQ0

Intron 28 donor site (4) G A mutation HLA A30 B18 DR7 RCCX: SS; C4BQ0-C4BQ0

Exon 29 (5) 2-bp ins, codon 1213 HLA A2 Cw3 B40 DR6 RCCX: mono-L; C4AQ0

HLA A2 B12 DR6 RCCX: LS; C4AQ0-C4BQ0

HLA A2 Cw7 B39 DR15 RCCX: LS; C4AQ0-C4BQ0 Exon 36 (1) 4-bp ins, codon 1537 HLA A1 B17 DR13 RCCX: LS; C4AQ0-C4BQ0 Figure 5 A summary of known deleterious mutations in the long and short C4 genes leading to C4A and/or C4B deficiencies. The locations of the earlier determined mutations28 are marked by } and those determined in this study are marked by asterisks. The mutations, associated HLA and RCCX haplotypes as well as the mutant C4A or C4B containing the defects are listed below the exon–intron structures.

there are also common features that include early disease triggers such as hormonal fluctuations and infections. onset, severe photosensitive skin lesions, and the The average female-to-male ratio in SLE is approxi- presence of anti-Ro/SSA and high titers of antinuclear mately 9:1. However, such a female dominance effect, antibodies.4 The prevalence of anti-Ro/SSA is of parti- which is at least in part contributed by female hormones, cular interest, as they are among the earliest autoanti- is completely masked by the condition of complete C4 bodies detectable in sera and before manifestations of deficiency. Complete C4 deficiency patients developed SLE clinical criterion or diagnosis.36 It has been observed SLE with a female-to-male ratio of 1:1 and most that anti-Ro is associated with neonatal lupus, photo- developed the disease before puberty. The higher sensitive rash, subacute cutaneous lupus erythematosus susceptibility of these individuals to infections poten- and Sjogren’s syndrome.37 Whether the genetic and/or tially lowers the threshold to trigger the autoimmune acquired deficiency of complement C4 plays a role in disease. Although rare in prevalence in human popula- initiating the generation of anti-Ro has not been tions, clinical presentations in subjects of complete investigated. complement C4 deficiency underscore the importance Systemic lupus erythematosus is a complex disease of C4 proteins in the protection against SLE. Such a that requires both genetic risk factors and environmental notion is reiterated in mouse complement C4 knockout

Genes and Immunity Complete complement C4 deficiency and human SLE YL Wu et al 443 models that showed an impaired humoral immune USA) for the visualization of C4 protein allotypes. response, high prevalence in the generation of anti- Complement C3F (fast) and C3S (slow) protein allotypes nuclear antibodies, anti-double-stranded DNA antibo- were determined similarly, except that five times less of dies and glomerulonephritis.38,39 In humans, common plasma samples and goat anti-human C3 polyclonal inter-individual CNV of C4 genes leads to the generation of antibodies were used. two isotypes of polymorphic proteins, C4A and C4B, and a quantitative trait in plasma C4 protein concentrations Genomic RFLP to determine RCCX modular variations and ranging from 10 to 70 mg dl–1 (that is, 0.1–0.7 g l–1).33,40,41 copy-number of C4A and C4B genes The low gene copy-number of C4A has been shown to Two genomic RFLP and Southern blot analyses were be strongly associated with SLE in human subjects of employed to determine the RCCX modular variations European ancestry.17 Thus, the absence or the low and copy-numbers of C4A and C4B genes. Genomic expression of complement C4 or, more specifically, C4A DNA samples from studied subjects were digested by seems to be an important genetic risk factor for SLE. TaqI restriction enzyme and resolved by agarose gel Among the SLE patients, concurrent consumptions or electrophoresis as described earlier.44 After hybridization fluctuations of plasma protein levels for C4 and C3, as with RP-, CYP21- and TNX-specific probes and auto- well as the deposition of C4 inactivation product C4d on radiography, the relative intensities of the bands were erythrocytes, are relevant biomarkers for SLE disease quantified. Along with specific patterns, the numbers of activities.41–43 Although genetic deficiency of comple- long and short C4 genes that linked to RP1 and RP2,as ment C4 is a probable causative factor for SLE disease well as the ratio of CYP21B to CYP21A genes and the pathogenesis, transient or acquired phenotypic defi- ratio of the TNXB genes to TNXA gene fragments were ciency of serum/plasma C4 proteins has been thought determined. The relative copy-numbers of C4A and C4B to be a result of SLE disease activity. We propose that genes were determined by PshAI–PvuII Southern blot. human subjects with low total C4 or C4A gene copy- After hybridization using a probe specific to the C4d number would be more likely to experience transient C4 region in both C4A and C4B genes, the relative band protein deficiency under an acute infection or other intensity yielded the ratio of C4A to C4B. Together with pathogenic conditions, which could trigger aberrant TaqI RFLP, which elucidated the total number of C4 immune responses and initiate a path cumulating in genes, the individual numbers of C4A and C4B genes autoimmune diseases. The observations of antinuclear were determined. antibodies in multiple members of Families 1 and 2 (Figure 1), who had low copy-numbers or the absence of PCR amplification and sequencing of C4 genes functional C4A and/or C4B genes, are in keeping with 1P. The long C4A gene was amplified into five over- the notion for a link between the quantitative variation of lapping DNA fragments (Fragments A–E) with sizes human C4 and immunotolerance or autoimmunity. ranging from 2.1 to 3.5 kb by polymerase chain reaction (PCR) (Figure 3, panel a). PCR primers for human C4 were designed on the basis of published sequences.14,16 Materials and methods Fragment A spanning exons 1–9 was amplified by primers E1.5 (that is, primers corresponding to the 50 Genomic DNA samples and plasma samples end of exon 1) and E9.3 (that is, reverse primer DNA samples were prepared from peripheral blood corresponding to the 30 end of exon 9). Skipping the lymphocytes from frozen blood samples of subjects of endogenous retroviral element in intron 9, HERV-K(C4), Family 1 and patient 2P using a commercial DNA isolation Fragment B spanning exons 10–21 was amplified kit (Puregene, Gentra Systems/QIAGEN, Valencia, CA, by primers E10.5 and E21.3. Fragment C spanning exons USA) following the manufacturer’s instructions. DNA 19–29 was amplified by primers E19.5 and E29.32. samples for subjects 2S and 2M of Family 2 were prepared Fragment D spanning exons 28–33 was amplified by from frozen blood samples using a Blood and Cell culture primers E28.5 and I33.3. Fragment E spanning exons DNA Maxi kit (QIAGEN). 33–41 was amplified by primers E33.5 and E41.3.

Determination of complement protein allotypes 2P. Three overlapping fragments specific for the Complement C4A and C4B protein allotypes were long-C4A genes were amplified (Figure 4, left panel b): determined by immunofixation after standard protocol,44 Fragment A1, covering the first nine exons, was using EDTA-plasma samples stored at –80 1C. Briefly, amplified using a forward primer (RPIN7F) that 4–6 ml of neat plasma was diluted to a final volume of 8 ml anchored at intron 7 of the RP gene and a reverse primer with 1 Â phosphate-buffered saline (PBS). Samples were (C4L–3LTR–R) that anchored at the 30LTR of HERV- digested by neuraminidase overnight at 4 1C to remove K(C4). Fragment A2 spanning exons 10–21 was amplified post-translational irregularities caused by glycosylation using primers C4E10.5 and C4A-up, which was specific on complement C4 proteins,45 followed by digestion with for the C4A isotypic site in exon 26. Fragment A3, carboxypeptidase B for 30 min at room temperature to covering from the C4A isotypic site to exon 35, was eliminate heterogeneities caused by incomplete proteo- amplified using primers C4A-down and E35.3. lytic processing of the b- and a-chain carboxyl termini of Similarly, three large fragments specific for the short the three-chain polypeptide structures.46 Digested pro- C4B genes were generated (Figure 4, right panel b): teins were subjected to high-voltage gel electrophoresis, Fragment B1, covering up to the first 10 exons, was and then to immunofixation with goat anti-human C4 amplified using primers RPIN7F and E10.3. Fragment B2 polyclonal antibodies (DiaSorin, Stillwater, MN, USA). spanning from intron 9 to the isotypic site for C4B in After multiple blottings and washings, gels were stained exon 26 was amplified using primers C4Fin95 and C4B- with SimplyBlue Safestain (Invitrogen, Carlsbad, CA, up. Fragment B3 spanning from the C4B isotypic site

Genes and Immunity Complete complement C4 deficiency and human SLE YL Wu et al 444 through exon 35 was amplified using primers C4B-down Acknowledgements and E35.3. Two common fragments from both the long-C4A genes We are indebted to the patients and their family and the short-C4B genes were amplified: Primers E20.5 members who contributed to this study. This work was and E31.3 were used to amplify exons 20–31 (Fragment supported by Grants 1R01 AR050078, 1R01 AR054459 AB1), and primers E33.5 and E41.3 were used to amplify from the NIAMS, NIAID, NIDDK and the Office of the exons 33–41 (Fragment AB2) of the long-C4A gene and Director, the National Institutes of Health, and by a pilot short-C4B gene indiscriminately. grant from the Lupus Foundation of America. All PCRs were carried out using the Failsafe PCR System (Epicentre Technologies, Madison, WI, USA). PCR cycles were 94 1C for 2 min followed by 33 to 36 cycles at 94 1C for 45 s, 58 1C for 45 s and 72 1C for 3 min, References and one step at 72 1C for 10 min. PCR fragments were 1 Reid KBM, Porter RR. The proteolytic activation systems of subjected to sequencing reaction using C4-specific complement. Ann Rev Biochem 1981; 50: 433–464. primers and the ABI Big Dye v3.1 kit (Part No.: 2 Walport MJ. Complement-part I. New Engl J Med 2001; 344: 4336917, Warrington, UK). Sequencing products were 1058–1066. purified using a gel filtration cartridge (Cat No.: 42453) 3 Hauptmann G, Tappeiner G, Schifferli J. Inherited deficiency from EDGE Biosystems (Gaithersburg, MD, USA) and of the fourth component of human complement. Immunodefic analyzed using the 3130 Â l Genetic Analyzer (Applied Rev 1988; 1: 3–22. Biosystems, Foster City, CA, USA) operated by the 4 Yu CY, Hauptmann G, Yang Y, Wu YL, Birmingham DJ, Rovin sequencing core at the Research Institute at Nationwide BH et al. Complement deficiencies in human systemic lupus Children’s Hospital. Sequences were aligned to a short- erythematosus (SLE) and SLE nephritis: epidemiology and C4B gene (AL049547) using Lasergene SeqMan v7.0 pathogenesis. In: Tsokos GC, Gordon C, Smolen JS (eds). Systemic Lupus Erythematosus: A Companion to Rheumatology. (DNASTAR Inc., Madison, WI, USA). Novel changes Elsevier: Philadelphia, 2007, pp 203–213. were defined as those not found in dbSNP Build 129 5 Rahman A, Isenberg DA. Systemic lupus erythematosus. (http://www.ncbi.nlm.nih.gov/SNP) or in the published N Engl J Med 2008; 358: 929–939. literature.16,19,47 6 Tsokos GC, Gordon C, Smolen JS. Systemic Lupus Erythemato- sus: A Companion to Rheumatology, 1st edn. Mosby-Elsevier: Sequence-specific primer (SSP-) PCR to screen complement Philadelphia, 2007. C2 and C4 mutations in the genomic sequences 7 Tsao BP, Wu H. The genetics of human lupus. In: Wallace LC, 2-bp insertion in exon 29 of C4 and Type I C2 mutations.A Hahn BH (eds). Dubois’ Lupus Erythematosus, 7th edn. multiplex SSP-PCR was developed earlier to screen for Lippincott Williams & Wilkins: Philadelphia, 2007, pp 54–81. 8 Vyse TJ, Kotzin BL. Genetic susceptibility to systemic lupus the most common C4 mutation, a 2-bp insertion in codon erythematosus. Annu Rev Immunol 1998; 16: 261–292. 1213 from exon 29, and the type I C2 mutation, a 28-bp 9 Botto M, Walport MJ. C1q, autoimmunity and apoptosis. deletion in the junction of exon 6 and intron 6.48 Primers Immunobiology 2002; 205: 395–406. C4E26.5 and C4E29insR were used to amplify the 10 Shen LM, Wu LC, Sanlioglu S, Chen R, Mendoza AR, Dangel mutated C4 gene. Primers 5C2 and 3C2 were used to A et al. Structure and genetics of the partially duplicated gene amplify the C2 exon 6–intron 6 junction. PCR cycles were RP located immediately upstream of the complement C4A 94 1C for 3 min followed by 30 cycles at 94 1C for 30 s, and C4B genes in the HLA class III region: molecular cloning, 60 1C for 1 min and 72 1C for 1 min, and one step at 72 1C exon-intron structure, composite retroposon and breakpoint of for 10 min. gene duplication. J Biol Chem 1994; 269: 8466–8476. 11 Yang Z, Mendoza AR, Welch TR, Zipf WB, Yu CY. Modular variations of HLA class III genes for serine/threonine kinase C to T mutation in exon 13 of C4. For the C to T mutation, RP, complement C4, steroid 21-hydroxylase CYP21 and primers E13–FY1 and E13–RY2 were used to amplify tenascin TNX (RCCX): a mechanism for gene deletions and exon 13 with a thermal cycle of 94 1C for 3 min followed disease associations. J Biol Chem 1999; 274: 12147–12156. by 35 cycles at 94 1C for 30 s, 59 1C for 1 min and 72 1C for 12 Chung EK, Yang Y, Rennebohm RM, Lokki ML, Higgins GC, 2 min, and one step at 72 1C for 5 min. PCR product Jones KN et al. Genetic sophistication of human complement was subjected to digestion by HhaI enzyme. In the C4A and C4B and RP-C4-CYP21-TNX (RCCX) modules in the presence of the normal C allele the PCR fragment will be major histocompatibility complex (MHC). Am J Hum Genet subjected to enzymatic digestion, whereas in the 2002; 71: 823–837. presence of the mutant T allele the PCR fragment will 13 Wu YL, Savelli SL, Yang Y, Zhou B, Rovin BH, Birmingham DJ et al. Sensitive and specific real-time PCR assays to accurately not be cut. determine copy-number variations (CNVs) of human comple- ment C4A, C4B, C4-Long, C4-Short and RCCX modules: 4-bp insertion in exon 36 of C4. The 4-bp insertion in exon Elucidation of C4 CNVs in 50 consanguineous subjects with 36 was screened by PCR using primers 4 bp-InsF2 and defined HLA genotypes. J Immunol 2007; 179: 3012–3025. 4 bp-InsR1 with PCR cycles of 94 1C for 3 min followed 14 Dangel AW, Mendoza AR, Baker BJ, Daniel CM, Carroll MC, by 35 cycles at 94 1C for 30 s, 57 1C for 1 min and 72 1C for Wu L-C et al. The dichotomous size variation of human 1 min, and one step at 72 1C for 5 min. Primers 5C2 and complement C4 gene is mediated by a novel family of 3C2 were also added in the reaction mix as positive endogenous retroviruses which also establishes species- amplification control. specific genomic patterns among Old World primates. Immunogenetics 1994; 40: 425–436. The Failsafe PCR System (Epicentre Technologies, 15 Chu X, Rittner C, Schneider PM. Length polymorphism of the Madison, WI, USA) was used for all three SSP-PCR human complement component C4 gene is due to an ancient assays. PCR products were resolved by gel electrophor- retroviral integration. Exp Clin Immunogenet 1995; 12: 74–81. esis. Specific sequences of PCR primers are listed in 16 Yu CY. The complete exon-intron structure of a human Supplementary Table 1. complement component C4A gene: DNA sequences, poly-

Genes and Immunity Complete complement C4 deficiency and human SLE YL Wu et al 445 morphism, and linkage to the 21-hydroxylase gene. J Immunol RCCX genetic diversity on MHC-associated disease. J Exp Med 1991; 146: 1057–1066. 2000; 191: 2183–2196. 17 Yang Y, Chung EK, Wu YL, Savelli SL, Nagaraja HN, Zhou B 33 Saxena K, Kitzmiller KJ, Wu YL, Zhou B, Esack N, Hiremath L et al. Gene copy number variation and associated polymorph- et al. Great genotypic and phenotypic diversities associated isms of complement component C4 in human systemic with copy-number variation of complement C4 and RP-CY- erythematosus (SLE): low copy number is a risk factor for CYP21-TNX (RCCX) modules: a comparison of Asian Indian and high copy number is a protective factor against European and European American populations. Mol Immunol 2009 American SLE disease susceptibility. Am J Hum Genet 2007; 80: (in press; e-pub ahead of print 8 January; PMID: 19135723). 1037–1054. 34 Castano-Rodriguez N, Diaz-Gallo LM, Pineda-Tamayo R, 18 Mauff G, Luther B, Schneider PM, Rittner C, Strandmann- Rojas-Villarraga A, Anaya JM. Meta-analysis of HLA-DRB1 Bellinghausen B, Dawkins R et al. Reference typing report for and HLA-DQB1 polymorphisms in Latin American patients complement component C4. Exp Clin Immunogenet 1998; 15: with systemic lupus erythematosus. Autoimmun Rev 2008; 7: 249–260. 322–330. 19 Yu CY, Chung EK, Yang Y, Blanchong CA, Jacobsen N, 35 Fernando MM, Stevens CR, Sabeti PC, Walsh EC, McWhinnie Saxena K et al. Dancing with complement C4 and the RP-C4- AJ, Shah A et al. Identification of two independent risk factors CYP21-TNX (RCCX) modules of the major histocompatibility for lupus within the MHC in United Kingdom families. complex. Progr Nucl Acid Res Mol Biol 2003; 75: 217–292. PLoS Genet 2007; 3: e192. 20 Yu CY, Belt KT, Giles CM, Campbell RD, Porter RR. Structural 36 Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, basis of the polymorphism of human complement component Dennis GJ, James JA et al. Development of autoantibodies C4A and C4B: gene size, reactivity and antigenicity. EMBO J before the clinical onset of systemic lupus erythematosus. 1986; 5: 2873–2881. N Engl J Med 2003; 349: 1526–1533. 21 Dodds AW, Ren X-D, Willis AC, Law SKA. The reaction 37 Shmerling RH. Autoantibodies in systemic lupus erythema- mechanism of the internal thioester in the human complement tosus—there before you know it. N Engl J Med 2003; 349: component C4. Nature 1996; 379: 177–179. 1499–1500. 22 Isenman DE, Young JR. The molecular basis for the differences 38 Prodeus AP, Goerg S, Shen LM, Pozdnyakova OO, Chu L, in immune hemolysis activity of the Chido and Rodgers Alicot EM et al. A critical role for complement in maintenance isotypes of human complement component C4. J Immunol of self-tolerance. Immunity 1998; 9: 721–731. 1984; 132: 3019–3027. 39 Chen Z, Koralov SB, Kelsoe G. Complement C4 inhibits 23 Schifferli JA, Hauptmann G, Pierre-Paccaud J. Complement- systemic autoimmunity through a mechanism independent of mediated adherence of immune complexes to human ery- complement receptors CR1 and CR2. J Exp Med 2000; 192: throcytes. Difference in the requirements for C4A and C4B. 1339–1351. FEBS Lett 1987; 213: 415–418. 40 Yang Y, Chung EK, Zhou B, Blanchong CA, Yu CY, Fu¨ st G et al. 24 Yang Y, Chung EK, Zhou B, Lhotta K, Hebert LA, Birmingham Diversity in intrinsic strengths of the human complement DJ et al. The intricate role of complement C4 in human systemic system: serum C4 protein concentrations correlate with C4 lupus erythematosus. Curr Direct Autoimmun 2004; 7: 98–132. gene size and polygenic variations, hemolytic activities and 25 Fredrikson GN, Gullstrand B, Schneider PM, Witzel-Schlomp body mass index. J Immunol 2003; 171: 2734–2745. K, Sjoholm AG, Alper CA et al. Characterization of non- 41 Wu YL, Higgins GC, Rennebohm RM, Chung EK, Yang Y, expressed C4 genes in a case of complete C4 deficiency: Zhou B et al. Three distinct profiles of serum complement C4 identification of a novel point mutation leading to a premature proteins in pediatric systemic lupus erythematosus (SLE) stop codon. Hum Immunol 1998; 59: 713–719. patients: tight associations of complement C4 and C3 protein 26 Lokki M-L, Circolo A, Ahokas P, Rupert KL, Yu CY, Colten levels in SLE but not in healthy subjects. Adv Exp Med Biol HR. Deficiency of human complement protein C4 due to 2006; 586: 227–247. identical frameshift mutations in the C4A and C4B genes. 42 Hebert LA, Cosio FG, Neff JC. Diagnostic significance J Immunol 1999; 162: 3687–3693. of hypocomplementemia. Kidney Int 1991; 39: 811–821. 27 Rupert KL, Moulds JM, Yang Y, Arnett FC, Warren RW, 43 Manzi S, Navratil JS, Ruffing MJ, Liu CC, Danchenko N, Reveille JD et al. The molecular basis of complete C4A and Nilson SE et al. Measurement of erythrocyte C4d and C4B deficiencies in a systemic lupus erythematosus (SLE) in systemic lupus erythematosus. patient with homozygous C4A and C4B mutant genes. Arthritis Rheum 2004; 50: 3596–3604. J Immunol 2002; 169: 1570–1578. 44 Chung EK, Wu YL, Yang Y, Zhou B, Yu CY. Human 28 Yang Y, Lhotta K, Chung EK, Eder P, Neumair F, Yu CY. complement components C4A and C4B genetic diversities: Complete complement components C4A and C4B deficiencies complex genotypes and phenotypes. Curr Protoc Immunol in human kidney diseases and systemic lupus erythematosus. 2005, Chapter 13: Unit 13.8. J Immunol 2004; 173: 2803–2814. 45 Awdeh ZL, Alper CA. Inherited structural polymorphism of 29 Uring-Lambert B, Mascart-Lemone F, Tongio M-M, Goetz J, the fourth component of human complement. Proc Natl Acad Hauptmann G. Molecular basis of complete C4 deficiency. a Sci USA 1980; 77: 3576–3580. study of three patients. Hum Immunol 1989; 24: 125–132. 46 Sim E, Cross S. Phenotyping of human complement 30 Dumas R, Hauptmann G, Chayon E, Bascoul S, Serre A, Baldet component C4, a class III HLA antigen. Biochem J 1986; 239: P et al. Hereditary deficiency of the fourth component of 763–767. complement (C4) in a child with a lupus-like disorder. Arch Fr 47 Blanchong CA, Chung EK, Rupert KL, Yang Y, Yang Z, Zhou B Pediatr 1986; 43: 267–269. et al. Genetic, structural and functional diversities of 31 Barba G, Rittner C, Schneider PM. Genetic basis of human human complement components C4A and C4B and their complement C4A deficiency. Detection of a point mutation mouse homologs, Slp and C4. Int Immunopharmacol 2001; 1: leading to nonexpression. J Clin Invest 1993; 91: 1681–1686. 365–392. 32 Blanchong CA, Zhou B, Rupert KL, Chung EK, Jones KN, 48 Johnson CA, Densen P, Hurford Jr RK, Colten HR, Wetsel RA. Sotos JF et al. Deficiencies of human complement component Type I human complement C2 deficiency. A 28- gene C4A and C4B and heterozygosity in length variants of RP-C4- deletion causes skipping of exon 6 during RNA splicing. J Biol CYP21-TNX (RCCX) modules in Caucasians: the load of Chem 1992; 267: 9347–9353.

Supplementary Information accompanies the paper on Genes and Immunity website (http://www.nature.com/gene)

Genes and Immunity