Homozygous Hereditary C3 Deficiency Due to a ... - Springer Link

Journal of Clinical Immunology, Vol. 22, No. 6, November 2002 (©2002)

Homozygous Hereditary C3 Deficiency due to a Premature Stop Codon EDIMARA DA SILVA REIS,1 GISELE VANESSA BARACHO,1 ADRIANA SOUSA LIMA,1 CHUCK S. FARAH,2 and LOURDES ISAAC1,3

Accepted: June 17, 2002

convertases; (g) lysis mediated by membrane attack complex; and (h) formation of adjuvant molecules (C3d) (5). C3 is synthesized mainly by hepatocytes (6) but other cells such as macrophages and monocytes (7), polymorphonuclear leukocytes (8), fibroblasts (9), and endothelial cells (10) are able to secrete this protein. In the serum, C3 is found at a concentration of 1.0 –1.6 mg/ml (11) and the C3 gene (42 kb) is located in human chromosome 19 and contains 41 exons. Deficiency of this protein has been observed in only a few families and is quite often associated with a higher susceptibility to infections by microorganisms (i.e., Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae) and immune complex disorders (reviewed in ref. 12). C3 deficiency also is associated with the development of autoimmune diseases. For example, approximately 20% of C3-deficient patients suffer from systemic lupus erythematosus (13–16). The molecular basis of C3 deficiency so far has been investigated in only five families. Contrary to what is observed in many other genetic disorders, in each case a different mutation was responsible for the C3 deficiency: (a) a 5⬘-donor splice site mutation in exon 18 (17); (b) a gene deletion resulting in the exclusion of exons 22 and 23 (18); (c) another 5⬘-donor splice site mutation, causing aberrant splicing of exon 10 (19); (d) a mutation causing a D549N amino acid substitution in the C3 beta chain, resulting in impairment of C3 secretion (20); and (e) more recently a nonsense mutation at codon 1081 was observed in two C3-deficient sisters (16). A primary C3 deficiency found in a consanguineous Brazilian family was previously characterized by Grumach et al. (21) in which the homozygous proband presented recurrent and severe infections. Here, we investigated the molecular alterations responsible for the lack of this protein in this proband’s serum.

C3 deficiency in humans is a rare disorder characterized by severe recurrent infections. We identified the mutations responsible for a complete homozygous C3 deficiency. Sequencing of the proband C3 cDNA (5067 bp) revealed the following alterations: (a) a silent G3 A transition at nucleotide 972; (b) a T3 C substitution at nucleotide 1001 resulting in a L314P transition; and (c) a stop codon in exon 13 caused by a G3 A substitution at position 1716. The presence of the same premature termination codon was confirmed in approximately half the clones obtained from the proband’s paternal and maternal genomic DNAs. Finally, the proband produced ⬃20-fold less C3 mRNA than the normal control. Therefore, in addition to the fact that no functional protein will be synthesized in the deficient cells, this nonsense mutation may be associated with the low C3 mRNA levels. KEY WORDS: Complement; immunodeficiency; complement C3; nonsense-mediated decay.

INTRODUCTION

C3 is a key component for all three pathways of activation of the complement system and several biologically functional molecules are generated after its cleavage (reviewed in ref. 1): (a) opsonization mediated by C3b and iC3b; (b) B lymphocyte activation followed by increased immunoglobulin production (2– 4); (c) degranulation of mast cells and basophils (C3a); (d) solubilization of immune complexes; (e) clearance of immune complexes (C3b); (f) formation of C3- and C51

Departamento de Imunologia, Instituto de Cieˆncias Biome´dicas, Universidade de Saõ Paulo, Sao Paulo, Brazil. Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Saõ Paulo, Sao Paulo, Brazil. 3 To whom correspondence should be addressed at Departamento de Imunologia, Instituto de Cieˆncias Biome´dicas, Universidade de Saõ Paulo, Av. Prof. Lineu Prestes, 1730, CEP 05508-900, Saõ Paulo, SP, Brazil. Tel: (55 11) 30917390; Fax: (55 11) 30917224; e-mail: [email protected]. 2

321 0271-9142/02/1100-0321/0 © 2002 Plenum Publishing Corporation

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MATERIALS AND METHODS

The patient (CA) is a 19-year-old Brazilian male whose C3 deficiency was first reported by Grumach et al. (21). He had suffered from recurrent infections including bronchopneumonia, meningococcal meningitis, otitis media, osteomyelitis, pyodermitis, urinary tract infection, arthritis, and fever of unknown origin. There is consanguinity in his family: the parents are first cousins and the maternal great-grandparents were related. The patient had two clinically healthy brothers. A third brother died at 5 months of age after prolonged diarrhea (21). The concentration of C3 was measured by capture ELISA as described (22) and by competitive solid-phase radioimmunoassay (23) with previously described modifications (24). Fibroblasts were obtained by skin biopsy (25, 26) from the patient and a normal control. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (inactivated at 56°C for 30 min) containing 2 mM glutamine and maintained at 37°C in a 5% CO2 atmosphere. Genomic DNA was obtained from peripheral blood cells as described (27). DNA (10 ␮g) was digested to completion with 40 – 60 U of restriction endonucleases EcoR1, BamH1, or KpnI at 37°C. After digestion, precipitation, and resolubilization, the DNA samples and markers were loaded on a 0.8% agarose gel and electrophoresed overnight followed by transfer onto Hybond-N membrane (Amersham Pharmacia Biotech). The membrane was hybridized overnight at 65°C with a fulllength C3 cDNA radioactive probe (pSVC3, kindly donated by Dr. Aiko Taniguchi-Sidle and Dr. David E. Isenman, University of Toronto, Canada) prepared using “T7Quick Prime Kit” (Amersham Pharmacia Biotech). The membranes were washed with 300 mM NaCl, 30 mM sodium citrate (pH 7.2), and 0.1% sodium dodecyl sulphate twice at room temperature (5 min), followed by a 15 min wash at 65°C. The membranes were prepared for autoradiography and exposed to BioMax MR-1 film (Kodak) for 2 days at ⫺70°C. Normal and proband fibroblasts were grown to confluence in flasks and stimulated with 1 ␮g/ml lipopolysaccharide (LPS) from E. coli (Sigma) overnight. Approximately 2 ⫻ 106 cells were lysed and total RNA was harvested and extracted using the “Total RNA Isolation System” (Promega) according to the manufacturer’s protocol. RT-PCR was performed with “SuperScript One-Step RT-PCR System” (Life Technologies) and 200 ng of total RNA in 50 ␮l containing 1 ⫻ Reaction Mix, 20

DA SILVA REIS, BARACHO, LIMA, FARAH, AND ISAAC

pmoles of each primer and 1 ␮ l RT/Taq Mix. The RT-PCR was carried out as follows: 50°C for 30 min; 94°C for 2 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min and a final incubation at 72°C for 7 min. To amplify genomic DNA by PCR we used 50 ng DNA, 50 ng of each primer, 2.5 U Taq DNA Polymerase (Life Technologies), in the following buffer: 20 mM Tris-HCl pH 8.4, 50 mM KCl plus 1.5 mM MgCl2, 200 ␮M dNTPs and water to complete up to 50 ␮l. The products were amplified using 35 cycles of denaturing (95°C for 1 min), annealing (50°C for 30 s), and extension (72°C, 1 min). The products were analyzed after electrophoresis in agarose gel followed by ethidium bromide staining. The sequences and positions of the oligonucleotides primers used are listed in Table I. Some of these oligonucleotides were a kind gift of Dr. Bernard J. Morley, Imperial College School of Medicine, Hammersmith Hospital, London, England. The RT-PCR products were purified with “Concert Rapid Gel Extraction System” (Life Technologies) according to manufacturer’s protocol. These products were cloned into the pGEM威 vector (Promega). The ligation reaction was used to transform competent DH10B E. coli cells. The bacteria were grown in Luria-Bertani agar medium containing 10 mM MgCl2, 50 ␮g/ml carbenicilin, 32 ␮ g/ml 5-bromo-4-chloro-3-indolyl ␤ -Dgalactopyranoside, and 32 ␮g/ml isopropyl ␤-D-thio galactopyranoside. White colonies containing the DNA inserts were picked and grown for small-scale preparation of plasmid DNA (27). The presence of the correct inserts was confirmed by restriction digest analysis. Insert DNA was sequenced according to the standard protocol of the “BigDye Terminator Cycle Sequencing Kit” (PE Applied Biosystems). The nucleotide sequences were compared (28) to the wild-type human C3 cDNA sequence (29). In some cases, RT-PCR was performed using 5, 10, 20, and 200 ng of total RNA and specific C3 primers to amplify nucleotides 582–1553 of the C3 mRNA. A segment of GAPDH mRNA also was amplified as an internal control of total mRNA. After electrophoresis in 1% agarose gels and ethidium bromide staining, the amounts of RT-PCR products were quantitatively determined by densitometric scanning of the gels using “␣-Innotech Image System” (␣-Innotech Corporation). The results were expressed in arbitrary units as the integrated density value (IDV). RESULTS

The C3 deficiency present in this family was previously characterized by Grumach et al. (21) using radial


MOLECULAR BASIS OF HUMAN

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Table I. Primers Used in RT-PCR, PCR and Sequencing, Their Sense and Sequence Primersa Primers used in RT-PCR 1 87 134 582 1300 1338 1553 2797 2887 3839 5042 5067 GAPDH GAPDH Primers used in PCR 1338 1540 1746 1779 Primers used in sequencing 582 834 1000 1300 1338 1553 1841 2240 2797 2887 3433 3839 3960 4425 5042 PUC/MP13 forward PUC/MP13 reverse a

Sense

Sequence (5⬘-3⬘)

F F R F R F R F R F R R F R

CTC CTC CCC ATC CTC TCC CTC GCT GCT CCT GCT ATC AAC CCA ATG GGA CTC CCC AGA GCC AGG CCA ACA GGG AGT TCA AGT CAG AAA AGG TGG GTG TGT TGA TGC TGA GTT TGG GAA GAA GCA GGA GCT CTC GG CCC TTG TTC ATG ATC AGG TAG G AAG GCT GCC GTC TAC CAT CAT T CCA CAG TTT TGT TGA TTC TGA TTC GAT ACT ACG GTG GTG GCT ATG CAC CCA AAG ACA ACC ATG CTC GGA ATG GGG GTG TGG TCA GTT TCT CTG CTC CTC CTG TTC GAC GGA TCT CGC TCC TGG AAG ATG

F F R R

GAA GAA GCA GGA GCT CTC GG ATC ATG AAC AAG GGC AGG CTG AGC CCA CGA AGG AGT TCC TTG CCG GTC TTC TGA CTG GCC G

F F R R F R F R F R R F R R R F R

CCA ACA GGG AGT TCA AGT CAG AAA AGG TGG GTT CCT CTA CGG GAA GAA AGT GGT TCT GCA CCC CGT CCA GCA GTG TGT TGA TGC TGA GTT TGG GAA GAA GCA GGA GCT CTC GG CCC TTG TTC ATG ATC AGG TAG G TCA TGG TGG CCG TGG GCA GTC CAG GAA GAC ATT CTT AAG GCT GCC GTC TAC CAT CAT T CCA CAG TTT TGT TGA TTC TGA TTC TCA CGG GCG CAT CCT CCT GGA GAT ACT ACG GTG GTG GCT ATG CAG TTG GAG GGA CAC ATC AAG CTC AGA GTG TGA GAC CTT GTC C CAC CCA AAG ACA ACC ATG CTC GTT TTC CCA GTC ACG AC GTC ATA GCT GTT TCC TG

cDNA numbering.

immunodiffusion. In the present work, we assayed C3 in the proband’s serum by ELISA and radioimmunoassay and confirmed the results of Grumach et al. Both methods were unable to detect C3 in the proband’s serum (data not shown). Southern blotting analysis was performed using genomic DNA extracted from blood leukocytes from the C3-deficient proband, his mother, and a normal control. Genomic DNAs were digested with EcoR1, BamH1, or KpnI restriction endonucleases. The DNA fragments were probed with radiolabelled plasmid pSVC3 (30), which contains the full-length human C3 cDNA. No differences were detected between the C3 gene fragments from the proband, mother, and control (data not shown). This result indicates that no large insertions, deletions, or rearrangements of C3 gene could explain the absence of this protein in the C3-deficient serum.


Several regions that fully cover the C3 cDNA were amplified using the primers listed in Table I. RT-PCR was performed using mRNA purified from proband and normal control fibroblasts. The amplified C3 mRNA sequences are shown in Fig. 1: (a) nucleotides 1 to 134 (134 bp); (b) nucleotides 87 to 1300 (1213 bp); (c) nucleotides 582 to 1553 (971 bp); (d) nucleotides 1338 to 2887 (1549 bp); (e) nucleotides 2797 to 5042 (2245 bp); and (f) nucleotides from 3839 to 5067 (1228 bp). Amplification products from both the proband and control RNAs gave bands of the expected sizes and no size differences were detected between samples from the proband and control. Furthermore, most RT-PCR products cover mRNA sequences coded by more than one exon, eliminating the possibility of amplification of contaminating genomic DNA.

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Fig. 1. Amplification of C3 cDNA by RT-PCR. Top: Location of RT-PCR fragments within the Pro-C3 cDNA. The 5⬘ positions of the C3 specific primers are indicated in bold (nucleotide numbering as per ref. (29). The sizes (bp) of the RT-PCR products (A–F) are indicated in parenthesis. Bottom: Agarose gel electrophoresis of RT-PCR products (A–F) derived from control (C) and proband (P) C3 cDNA.

While there were no size differences between the proband and control C3 cDNA or genomic DNA, we clearly observed different sample-specific expression of C3 mRNA by LPS-stimulated fibroblasts (Figs. 1 and 2). Quantitative determinations by densitometry showed that LPS-stimulated fibroblasts from the C3-deficient proband expressed at least 20 times less C3 mRNA than fibroblasts from the healthy control (Fig. 2 and Table II). As an internal control, the expression of the constitutive human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was quantitatively determined in both proband’s and control’s LPS-stimulated fibroblasts. No

significant differences in the amount of this product (350 bp) were detected (Fig. 2). This result indicates that the lower expression of C3 mRNA by the proband’s fibroblasts are not due to large-scale metabolic differences between proband and control fibroblasts, nor due to any general impairment in the proband transcription machinery. Nucleotide sequencing analysis of the proband’s C3 cDNA was carried out in order to attempt to identify a molecular basis for both the complete lack of serum C3 and the low expression levels of the C3 mRNA. We subcloned the RT-PCR products (Fig. 1) into the pGEM威 vector. Insert DNA was then sequenced using primers



C3

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Fig. 2. Comparison of C3 mRNA levels from the control (C) and proband (P) LPS-stimulated fibroblasts. Fragments were amplified by RT-PCR with C3specific (582-1553) and GAPDH-specific primers. The reactions were performed using total RNA from control (200 –5 ng) or proband (200 ng) and the products separated by agarose gel electrophoresis.

indicated in Table I. We also amplified, cloned, and sequenced C3 cDNA from normal control human fibroblasts. Each RT-PCR was carried out at least twice and several independent clones from each product were sequenced on both strands. The nucleotide sequences from both proband and control samples were compared to those of normal human C3 cDNA (29) and the following differences were found: (a) all clones obtained from the proband C3 cDNAs contained a 972G3 A transition, which corresponds to a silent mutation in the Ala304 codon (Fig. 3A, B) and which differs from that previously reported (29); (b) all sequences obtained from the proband and control C3 cDNAs contained a previously reported polymorphic variation at nucleotide 1001 Table II. Densitometry Values Obtained after Measuring the RT-PCR Products from Control and Proband, using C3 specific (582 ⫹ 1553) and GAPDH primers with different RNA inicial concentrations. IDV C3 (oligos 582 ⫹ 1553)

GAPDH

RNA (ng)

Proband

Control

Proband

Control

200 20 10 5

5760 – – –

11360 6400 5280 5760

25536 – – –

25200 – – –

IDV, Integrated density value (arbitrary units).


(T3 C), which changes the Leu314 codon to Pro (Fig. 3A,B); and (c) the most significant difference between proband and normal cDNA sequences was the G3 A transition at nucleotide 1716 which changes the Lys552 codon (TGG) to a stop codon (TAG) (Fig. 4A,B). This codon is found in exon 13, which codes for part of the 70-kDa C3 ␤-chain. This mutation was found in all proband C3 cDNA sequences analyzed and was not detected in any of the control cDNAs. A stop codon at this position would most surely produce a nonfunctional polypeptide. In order to determine whether the proband was homozygous or heterozygous for the stop codon mutation, we amplified, cloned, and sequenced all of exon 13 from the proband’s genomic DNA (Fig. 4D). Exon 13 codes for nucleotides 1540 to 1746 in the cDNA. We also sequenced exon 13 from the normal control DNA (Fig. 4C) and the proband’s father and mother (Fig. 4E,F). The 1716G3 A mutation was found in 100% of the 12 genomic clones from the C3 deficient patient, in three of seven sequences of his father’s C3 (Fig. 4E) and two of nine clones from his mother’s (Fig. 4F) and in none of the control DNA. Therefore, we conclude that the proband is homozygous for the nonsense mutation at codon 552, leading to a truncated and nonfunctional protein that is probably rapidly degraded by the cell’s proteolytic machinery before secretion.

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Fig. 3. DNA sequencing results from control and proband cDNA. Sequence chromatographs from control and proband showing the silent G 3 A substitution at position 972 in proband cDNA and the T 3 C substitution at position 1001, which results in a L314P amino acid substitution in both control and proband cDNA. The chromatographs show sequencing of non-coding strand of the control and proband cDNA.

DISCUSSION

Deficiency of complement component C3 results in recurrent severe pyogenic infections and is inherited as an autosomal recessive trait (13, 14, 17, 21, 26,

31– 41). This deficiency is quite often associated with high morbidity and only few cases have had their causes elucidated at the molecular level. To date, in each case, the deficiency has been shown to be the result of a different mutation in the C3 gene.



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Fig. 4. Sequence results from control, proband, and proband’s parents. Sequence chromatographs from control cDNA (A) and control genomic DNA (C) showing the 1716G; proband cDNA (B) and proband genomic DNA (D) showing 1716G3 A, which creates a stop codon (TGA); proband’s father’s (E) and mother’s (F) genomic DNA showing 1716G3 A.

The absence of C3 was observed in the proband’s serum by radial immunodiffusion (21). The proband was


not able to produce complement-dependent opsonins or hemolytic activity (21). LPS-stimulated blood leukocytes

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from the proband also showed impairment in the synthesis/secretion of C3, since this protein was not detected by radioimmunoassay in the supernatant of these cells (data not shown). This strongly suggests that the deficiency was not exclusively limited to the hepatocyte environment, the major source of C3 in humans. In order to initiate investigating the molecular basis of this deficiency, we analyzed the integrity of the C3 gene in the proband by Southern blotting. After digesting proband and control genomic DNA with three different restriction enzymes, we found no evidence of large structural rearrangements (deletions, insertions, or inversions). Although the proband’s C3 cDNA was of a normal size, after amplification by RT-PCR we observed that C3 mRNA in the proband’s fibroblasts was at least 20-fold less abundant when compared to the normal control. We did not sequence the proband’s genomic DNA upstream of the transcription start site nor perform other experiments to define the mechanism responsible for the low mRNA levels. Therefore, we cannot rule out the possibility of a mutation in the C3 regulatory region that results in suppressed transcription of the C3 gene. However, the fact that the proband’s C3 mRNA encodes a nonsense codon at position 552 suggests the possibility that a nonsense-mediated decay (NMD) mechanism may be responsible for the reduced mRNA levels observed. A correlation between the presence of premature termination codons and lower amounts of mRNA previously has been noted in a number of investigations of other human genetic disorders (42) including ataxia telangiectasialike disorder (43), but not in complement deficiencies. A NMD mechanism also is suggested by the fact that we have previously analyzed another unrelated C3deficient patient who also had low amounts of C3 mRNA (26). At that time, the genetic lesion responsible for the deficiency had not been identified. Recently we have determined that a premature termination codon in the patient’s C3 mRNA is responsible for the C3 deficiency (manuscript in preparation). Furthermore, another family that we recently have studied presented a complement factor I deficiency caused by the presence of a premature termination codon with a concomitantly lower amount of factor I mRNA (44). These facts strongly suggest those complement deficiencies caused by premature stop codons often may be associated with NMD in order to reduce cellular levels of truncated, nonfunctional, and possibly toxic polypeptides. The accelerated decay of mRNAs containing nonsense codons is part of a RNA surveillance process, which eliminates aberrant transcripts, avoiding the translation and production of truncated proteins (45). The importance of NMD has been demonstrated in one dominantly


inherited form of human ␤ thalassemia caused by the presence of premature termination codon in the ␤ globin gene. Here, a nonsense mutation in exon 1 or 2 triggers ␤ globin mRNA decay and the affected individuals present ⬃50% of normal ␤ globin chain synthesis (46). However, a more severe condition is observed if the premature termination codon is present in exon 3. In this case, the transcript is known to be NMD-resistant and insoluble truncated proteins are found in the cytoplasm which probably contributes to ineffective erytropoiesis (46). The presence of a premature termination codon was reported in the first molecular study of human C3 deficiency (17), however, C3 mRNA levels were not quantified for comparison with normal controls. No mutation was found in the C3 gene of a Laotian patient who presented approximately 0.3% of normal serum concentration of C3 and low levels of C3 mRNA (20). The only other study to date that documented both the presence of a premature termination codon and reduced mRNA levels (observed by Northern blotting) was that of Huang and Lin (19). More recently, a brief report (16) mentioned the presence of a stop codon and low amounts of C3 mRNA in two C3-deficient sisters. However, no correlation between the presence of premature termination codon and lower concentration of C3 mRNA so far has been suggested in the literature, nor has NMD been suggested as its possible cause in C3 deficiencies. The L314P substitution was detected in all the sequences tested (proband and control DNAs) and previously has been identified as a polymorphic variation of C3 that segregates with reactivity to monoclonal antibody HAV 4–1 (47). In 45 studied normal individuals the frequency of HAV 4–1⫹ was 0.21. This suggests that the variation L314P found in the proband’s C3 gene cannot be used as a marker for this deficiency. The silent mutation present at residue R304 in the proband C3 cDNA does not affect the C3 structural and functional properties. Understanding the molecular basis of immunodeficiencies is a necessary step to pursue more advanced protocols of genetic therapy. Deficiency of C3 quite often is associated with morbidity and the very few cases that have had their genetic causes elucidated were each found to be unique. In addition, this area of investigation has the potential to aid in illuminating structure–function relationships in this important complement protein. ACKNOWLEDGMENTS

We would like to thank CA’s family and Dr. Paulo Andrade for providing materials for this research. We are indebted to Dr. Cesar Isaac for the biopsies and Marlene P. C. Florido and the laboratory of Dr. Carlos Alberto



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Moreira Filho for technical assistance. This work was sponsored by Fundaç a˜ o de Amparo a` Pesquisa do Estado de Sa˜ o Paulo (FAPESP). E.S. Reis and A.S. Lima received a FAPESP fellowship, and G.V. Baracho and L. Isaac received a fellowship from Conselho Nacional de Pesquisa e Desenvolvimento Tecnolo´ gico.

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