Human Molecular Genetics Advance Access originally published online on January 20, 2005
Human Molecular Genetics 2005 14(5):703-712; doi:10.1093/hmg/ddi066
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Human Molecular Genetics, Vol. 14, No. 5 © Oxford University Press 2005; all rights reserved
Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32
1Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain, 2Unidad de Hemostasia i Trombosis, Hospital de la Santa Creu i Sant Pau, Sant Antoni Ma Claret 167, 08025 Barcelona, Spain, 3Servicio de Nefrología, Hospital Universitario de Bellvitge, Feixa Llarga s/n, 08907 Barcelona, Spain and 4Unidad de Inmunología, Hospital Universitario La Paz, Paseo de la Castellana 261, 28046 Madrid, Spain
* To whom correspondence should be addressed. Tel: +34 918373112; Fax: +34 915360432; Email: srdecordoba{at}cib.csic.es
Received November 4, 2004; Revised December 7, 2004; Accepted January 17, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The efficiency of the complement system as an innate immune defense mechanism depends on a fine control that restricts its action to pathogens and prevents non-specific damage to host tissues. Genetic and functional analyses have shown that this critical control of complement activation may be impaired in atypical hemolytic uremic syndrome (aHUS) patients. Mutations in HF1, MCP or FI have been found in aHUS patients, but incomplete penetrance of the disease in individuals carrying these mutations is relatively frequent and no genetic defect has yet been found in a majority of aHUS patients. We report here the identification of a specific SNP haplotype block, spanning the MCP gene in the regulators of complement activation gene cluster, which is over-represented in aHUS patients and strongly associates with the severity of the disease. Linkage disequilibrium analyses suggest that this SNP haplotype also includes the CR1, DAF and C4BP genes. Initial studies identified two SNPs in the haplotype that influence the transcription activity of the MCP promoter in transient transfection experiments. Notably, the SNP haplotype block was found to be particularly frequent among patients who carry mutations in HF1, MCP or FI. These findings and the identification of aHUS patients carrying mutations in two complement regulatory genes provide an important insight into the etiology of aHUS. Together, they suggest that complement regulatory molecules act as a protein network and that multiple hits, involving plasma- and membrane-associated complement regulatory proteins, are necessary to impair protection to host tissues and to confer significant predisposition to aHUS.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hemolytic uremic syndrome (HUS) is a major cause of acute renal failure in children. HUS is a microvasculature disorder clinically defined by thrombocytopenia, microangiopathic hemolytic anemia and acute renal failure. In ‘typical’ HUS, the etiologic agent is frequently an infection of the Escherichia coli strain O157:H7, which secretes a potent toxin that inhibits protein synthesis and damages endothelial cells in the microvasculature (1
). Most of these HUS patients evolve satisfactorily, and complete recovery of the renal function is achieved in 2–3 weeks. A poorer prognosis is found in 5–10% of patients with HUS (2). This ‘atypical’ form of HUS (aHUS) shows no particular relationship with infection, but is frequently associated to immunosuppressive drugs, cancer therapies, oral contraceptives, pregnancy or postpartum. Genetic studies performed during the last 5 years have shown linkage of aHUS to the regulators of complement activation (RCA) gene cluster in chromosome 1q32 and identified patients with mutations in the genes for factor H (HF1) and membrane cofactor protein (MCP) (3–8). Factor H is essential for regulating complement activation in plasma and for restricting the action of complement to activating surfaces (reviewed in 9). MCP protects the cell surfaces from damage by complement activation (reviewed in 10). HF1 mutations associated to aHUS rarely result in hypocomplementemia or decreased factor H plasma levels. These mutations tend to cluster within SCR19 and SCR20 and normally result in mutant factor H molecules that have a normal complement regulatory activity in fluid phase, but show impaired capacity to control complement activation on surfaces. It is thought that these factor H mutations limit the capacity of aHUS patients to protect their cells from complement lysis (7,8,11). Similarly, MCP mutations associated with aHUS result in functionally inactive molecules or in decreased levels of MCP on the cell surfaces which become potentially less protected from complement lysis (12–13). On the basis of these findings, we have proposed that aHUS results from the combination of both an active complement system and a defective protection of cellular surfaces from complement activation due to an improper function of complement regulatory proteins (7,9). The conclusion that aHUS results from a defective control of complement activation is further supported by the recent finding that some aHUS patients carry mutations in the factor I gene (14).
Despite these advances in our understanding of the molecular basis of aHUS, no genetic defect has yet been found in two-thirds of aHUS patients, and incomplete penetrance of the disease in individuals carrying factor H, MCP or factor I mutation is relatively frequent, suggesting the existence of additional genetic factors that predispose to aHUS. Within the RCA gene cluster, genetic factors potentially predisposing to aHUS are the membrane proteins MCP, DAF and CR1, which, together with the plasma proteins factor H and C4BP, confer protection to cellular surfaces when complement is activated (reviewed in 15). In some pedigrees, the inheritance of decreased plasma levels of factor H in addition to factor H mutations provides an explanation for the incomplete penetrance of aHUS in carriers of factor H mutations (4,7). Recently, an association between three relatively frequent HF1 alleles (–257T, c.2089G and c.2881T) and aHUS has been reported (16), suggesting that, in addition to mutations in factor H, MCP or factor I, other genetic variations at the RCA gene cluster also predispose to aHUS.
To search for additional susceptibility factors to aHUS within the RCA gene cluster, we have performed an association study in our aHUS series (n=41) using a gene-based approach that includes 21 haplotype-tagging SNPs identifying most of the common variants of the RCA genes HF1, C4BP, MCP, DAF and CR1. Our results identify novel aHUS predisposition factors in the RCA gene cluster and demonstrate that concurrence of different susceptibility alleles is likely to be required to confer significant predisposition to aHUS.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Although aHUS is rare, our study sample consisted of 41 Spanish patients with a clinical history of HUS not associated to E. coli related diarrhea. These aHUS patients have been studied in detail regarding complement profiles and have been analyzed for the presence of mutations in the HF1, DAF, MCP and FI genes. Briefly, nine of our patients carry mutations in HF1 that specifically impair the capacity of factor H to protect host cells (Table 1). Six aHUS patients in our series present novel mutations or rare polymorphisms in MCP, some of them associated with low expression levels of MCP in peripheral blood lymphocytes (PBLs), and one patient carries a novel mutation in the FI gene (Table 1). No mutations were found in the DAF gene. It was particularly interesting to find out that one of our aHUS patients carries mutations in both MCP (P165S, a missense mutation that associates with decreased MCP levels) and FI (c.1610insAT; an insertion that causes a frameshift in exon 13 and decreased factor I levels). Moreover, another of our patients also carries very rare polymorphisms in both MCP (c.573A>G) and HF1 (c.3221A>T) and presents low levels of factor H. No genetic defect was identified in as many as 27 of our 41 patients (66%), suggesting that additional aHUS susceptibility factors remain to be identified.
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In this regard, our first objective was to obtain a detailed knowledge of genetic variation in different subregions of the RCA gene cluster. To this end we sequenced functionally relevant DNA fragments of the HF1, C4BPA, C4BPB, DAF, CR1 and MCP genes in a sample of at least 40 unrelated Spanish chromosomes. These analyses, as well as SNP database searches, provided us with a large set of SNPs distributed along the RCA gene cluster (Fig. 1A). Most of these SNPs organize haplotype blocks with strong linkage disequilibrium (LD) and low haplotype diversity in three subregions of the RCA gene cluster including HF1, C4BP and MCP (Fig. 1B). To minimize the genotyping effort we selected ‘haplotype tagging SNPs’ that captured most of the genetic variation within each of these subregions (Fig. 1B). In addition to the HF1, C4BP and MCP haplotype tagging SNPs, we have included in these studies five SNPs in the CR1 promoter region, one in the DAF gene and three in the CD34 gene, as markers flanking the RCA gene cluster.
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We genotyped our aHUS cohort and a matched control population of 73 individuals for the 21 haplotype tagging SNPs selected within the RCA gene cluster. The allelic frequencies of each of these polymorphisms were determined and compared between the two groups (Table 2). A strong and significant association at the required level for the multiple comparison correction (see Materials and Methods) was found between aHUS and the MCP alleles –547G, –261G, IVS9–78A, IVS12+638A and c.2232C (Table 2). These MCP alleles are in strong LD and organize one of the two major MCP haplotypes (Fig. 2). We refer to this haplotype as the MCPggaac haplotype. We used the frequencies of the c.2232C allele to estimate that the MCPggaac haplotype increases 2-fold in aHUS compared with controls [44% in cases; 23% in controls; P=0.0014; odds ratio (OR)=2.68; 95% CI=1.43–5.05]. Interestingly, this association is due almost exclusively to the aHUS patients with mutations in HF1, MCP or FI (61% in cases; 23% in controls; P=0.00016; (OR)=5.25; 95% CI=2.08–13.79), whereas aHUS patients without identified mutations in HF1, MCP or FI show no significant differences with the control group (35% in cases; 23% in controls; P=0.1) (Table 3). The association of the MCPggaac haplotype with aHUS in individuals carrying mutations is not the result of prevalent HF1, MCP or FI mutation in this group. As illustrated in Table 1, there are many different HF1 and MCP mutations and most of them are present in one single patient.
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In addition to demonstrating association between aHUS and the MCPggaac haplotype, we found a moderate but nominally significant increase (0.49 in cases; 0.33 in controls; P=0.0316; (OR)=1.89; 95% CI=1.05–3.45) in the frequency of the –257T HF1 allele in our aHUS series (Table 2). This result is not significant at the required level for the multiple comparison correction but, being a replication of a previous result (16
), we consider it real. Similarly to the MCPggaac haplotype, the association between the HF1 –257T allele and aHUS is likely due to patients carrying mutations or rare polymorphisms in HF1, MCP or FI, but the size of this sample is too small to show significant differences (Table 3).
Although the remaining HF1 SNPs (c.2089A>G and c.2881G>T) and all the C4BPB, DAF, CR1 and CD34 SNPs included in our study showed similar frequencies in the case and control groups, demonstrating no association with the disease in our aHUS series (Table 2), we noticed that the patterns of LD for the RCA SNPs show important differences between the aHUS and control groups (Fig. 2). In controls, LD is limited to SNPs within the same gene, whereas in aHUS additional LD is observed between SNPs in the C4BP, DAF, CR1, MCP and CD34 genes. These findings suggest that the aHUS-associated MCPggaac haplotype extends over a large portion of the RCA gene cluster including the C4BP, DAF, CR1 and MCP genes. Strong LD across such large distances has been observed earlier in the RCA gene cluster. In fact, the cluster was in part discovered because of the complete LD between the rare C4BP*2 allele and the CR1*B allele (17).
To investigate the functional characterization of SNPs in the aHUS-associated RCA haplotype, we focused in the CR1L1–MCP intergenic region because this region should contain elements that regulate expression of the MCP gene (Fig. 3). We have used the MatInspector and Tess software tools to search the Transfact 4.0 and other databases for transcription factor binding sites that are modified by the MCP SNPs associated to aHUS. These analyses revealed that the MCP allele –261G within the CR1L1–MCP intergenic region disrupts a potentially functional CBF-1/RBP-Jk-binding site in the MCP promoter. CBF-1/RBP-Jk is a member of the Notch signaling pathway that is involved in multiple aspects of vascular development (18). Moreover, CBF-1/RBP-Jk plays a role in the transcriptional regulation of CR2, another RCA gene evolutionary related to MCP (19).
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65% identity; black boxes) in the homologous region of the mouse genome (vertical axis), suggesting that are functionally relevant. (B) MCP extended haplotypes. We amplified and sequenced these evolutionary conserved DNA segments in 10 individuals (patients and controls) homozygous for each of the two MCP haplotypes. These analyses demonstrated very little genetic variability within this region and only detected three additional SNPs: –13274G>C, –13246A>G and –12319G>T. Alleles at these three sites show association with the two MCP haplotypes. The horizontal line represents the CR1L1–MCP genomic region in humans, with exons indicated as black vertical rectangles. Numbered grey rectangles represent the conserved regions between human and mouse genomes. Thin lines indicate the location of the polymorphic sites in the region and grey horizontal bars show the two major MCP haplotypes. The location of the consensus CBF-1/RBP-Jk binding sequence disrupted by the –261A>G SNP is also indicated. (C) Effect of the –547A>G and –261A>G MCP SNPs on transcriptional activity. The MCP promoter region with the location of the –547A>G and –261A>G SNPs, the transcription start site (+1) and the initiation codon (ATG) is shown at the top of the figure. pXP2_–547A/–261A is the construct with the –547A and –261A alleles cloned upstream the LUC gene in the pXP2 plasmid. pXP2_–547G/–261G is the construct with the –547G and –261G alleles. pXP2 is the plasmid with no insert (negative control) and pTK.LUC is the plasmid with the TK promoter (positive control). Mean and standard deviation of the normalized luciferase activity is shown for each construct. P<0.001 refers to a Student's t-test used to compare luciferase activity between the pXP2_–547A/–261A and the pXP2_–547G/–261G plasmid constructs.To determine whether the –261A>G variation in the MCP promoter has an effect on transcription, we amplified the DNA region spanning from –688 to +99 of the MCP gene using genomic DNA from individuals who are homozygous for the –547G, –261G alleles or homozygous for the –547A, –261A alleles. The resulting PCR products were cloned upstream of a LUC reporter gene in the pXP2 plasmid vector and the capacity of these constructs to drive transcription was tested in transient transfection experiments in human embryonic kidney (HEK293) cells. For each experiment, the LUC values were corrected for transfection efficiency using the ßGAL activities obtained from cotransfecting equal amounts of a CMV-ßGAL plasmid. In these experiments, we transfected in parallel constructs containing the LUC gene under the control of the promoter of the herpes simplex virus (HSV) thymidine kinase (TK) and an empty pXP2 plasmid vector. The results of these experiments are represented in Figure 3C, which summarizes the combined data of three independent experiments performed with six plasmid preparations of each construct. Both alleles of the MCP promoter showed a strong promoter activity in HEK293 cells, which was similar to that of the TK promoter construct. Interestingly, however, the –547G, –261G construct persistently showed a 25% lower transcriptional activity than the –547A, –261A construct, suggesting that these two nucleotide changes are functionally relevant and they result in different levels of expression of MCP on the cell surface. This 25% is not a negligible difference if one considers that most MCP mutations associated with aHUS are heterozygous and represent a 50% reduction in MCP levels on the cell surface (12
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The efficiency of the complement system as an innate defense mechanism against microbial infections depends on a fine control that avoids the wasteful consumption of its components and restricts its activation to the surface of microorganisms, thus preventing non-specific damage to host tissues. Complement regulation is performed by a set of plasma- and membrane-associated proteins, many of them encoded by genes in the RCA gene cluster in human chromosome 1q32 (20
). Genetic and functional analyses have shown that this critical control of complement activation may be impaired in aHUS patients. Accordingly, it is generally accepted that the mutations in HF1, MCP or FI found in many patients predispose to aHUS because they generate a situation of haploinsufficiency unable to provide efficient protection to the host cellular surfaces in the case of complement activation (3–8,11–14,16,21). We report here the identification of a specific SNP haplotype block, spanning the MCP gene in the RCA gene cluster, that is highly over-represented in aHUS patients and strongly associates with the severity of the disease. Moreover, we show that the frequent HF1 allele –257T also associates with aHUS. These data demonstrate that, in addition to mutations in the complement regulatory genes, common genetic variations at the RCA gene cluster also predisposed to aHUS.
Transient transfection experiments showed that the MCP allele –261G, disrupting a potentially functional CBF-1/RBP-Jk-binding site in the MCP promoter, may have an effect on MCP transcription (Fig. 3C). Similarly, the HF1 allele –257T is thought to influence transcription by disrupting a putative NFkB binding site in the HF1 promoter region (16,22). Although no association between these HF1 –257T and MCP –261G alleles and decreased levels of factor H or MCP has been found yet in plasma or PBLs, they may play a role and influence the expression levels of these complement regulators locally or in conditions of infection or inflammation (23,24).
We have provided evidence that the ‘SNP haplotype block’ including the MCP allele –261G spans the MCP gene and most likely extends until the C4BPB gene (>800 kb upstream the MCP gene) also including the CR1, DAF and C4BPA genes. This is interesting because we have previously mapped to this RCA region loci responsible for quantitative variations in factor H (25), C4BP (26) (Esparza-Gordillo et al., unpublished data) and CR1 (27). On the basis of these findings, it is tempting to speculate that the MCPggaac haplotype described here forms part of an extended RCA haplotype that influences simultaneously the expression levels of different RCA genes encoding complement regulators. Analyses are being performed to further characterize this RCA extended haplotype and to identify additional functionally relevant SNPs that help to explain its association with aHUS.
A remarkable finding of our study is that the MCPggaac haplotype and the HF1–257T allele do not significantly associate with aHUS in the absence of mutations in the complement regulatory genes HF1, MCP or FI. In fact, our data suggest that these novel genetic predisposition factors are, instead, needed for manifestation of the disease in carriers of HF1, MCP or FI mutations. This finding would agree with the idea that complement regulatory molecules act as a protein network and that multiple hits, probably involving plasma- and membrane-associated complement regulatory proteins, are required to impair protection to host tissues significantly. In this context it does not seems coincidental that we have identified patients with mutations in two complement regulatory genes.
Our aHUS cohort has been exhaustively screened for mutations in HF1, MCP, FI and DAF genes and tested for quantitative variations of factor H, factor I, C4BP, MCP and DAF in plasma or in PBLs. It is, therefore, unlikely that in our cohort of aHUS patients other mutations in the RCA genes remain undetected.
aHUS is considered a multifactorial disease that results from the interaction of genetic predisposition (impaired function of complement regulatory proteins) and environmental factors (such as infection or cytotoxic and immunosuppressive drugs) that activate the complement system and trigger the onset of the disease. In this regard, it is striking that there is no significant association between the HF1 and MCP polymorphisms and aHUS in patients without mutations in HF1, MCP or FI. In this regard, we believe that this lack of association is related to heterogeneity within this group of patients. We have noted that, as a whole, the disease in patients without mutations is less severe than in patients with mutations. Thus, while disease recurrence and chronic renal failure affect 43 and 93% of patients with mutations, respectively, among patients without mutations, only 15% have had disease recurrence and 56% present chronic renal failure. Interestingly, among the 15 patients who developed chronic renal failure in this last group, eight are homozygous for the MCP or the HF1 risk alleles. In conclusion, despite the lack of a significant association between the MCPggaac haplotype or the HF1 –257T allele and aHUS without mutations in HF1, MCP or FI, these polymorphisms distinguish those patients with more severe disease.
Additional studies will be needed to determine whether there are other genetic factors predisposing to aHUS in patients without HF1, MCP or FI mutations and who are not carriers of the HF1 and MCP risk alleles. This may be difficult because the lower severity of the disease in these patients suggests that perhaps HUS in this group is determined by a strong environmental component with only a marginal contribution of genetic predisposition factors.
Our report is the first case–control study that systematically analyzes the genetic variability at different regions of the RCA gene cluster in relation to aHUS. Independent series of aHUS patients, fully characterized for mutations and quantitative variations in the RCA genes, and the corresponding controls should be tested for these HF1 and MCP SNPs. This will help to understand whether the –257T HF1 and the –261G MCP alleles are directly involved in predisposition to aHUS, or they both associate to aHUS because they are in LD with other variations within the RCA gene cluster, which are the true susceptibility factors to aHUS.
Different reports have already indicated the therapeutic importance of identifying whether the HUS patients carry mutations in HF1 or MCP, as it determines the outcome of the kidney transplants in these individuals. Consequently, there is a high risk of recurrence in patients with HF1 mutations, whereas in patients without HF1 mutations a significant percentage of grafts survive (28). Our data provide further insight into the genetic complexity of aHUS. They demonstrate that, in addition to mutations in the HF1 and MCP genes, there are common polymorphic variants of these genes that predispose to aHUS and strongly associate with the severity of the disease. As a whole, our data consolidate the hypothesis that an inefficient protection of the cellular surfaces in the case of complement activation is a general feature of patients with aHUS. Accordingly, we believe that the implementation of complement inhibition therapies to prevent or reduce tissue damage by complement activation, as a general strategy for the treatment of aHUS patients, is fully justified.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients and controls
Forty-one unrelated aHUS patients were included in the series. Of them, 13 have been described previously (4
,7). The remaining 28 are novel cases and will be described in detail elsewhere. All patients presented a history of HUS (clinical findings of microangiopathic hemolytic anemia and thrombocytopenia linked to acute renal failure) not associated to diarrhea of E. coli related origin. Chronic renal failure and/or unsuccessful renal transplantation led most of them to hemodialysis. Plasma C3, C4, factor H, factor I, C4BP, CH50 and AP50 were measured by standard procedures as previously described (4,25,29,30). Expression levels of MCP (CD46) and DAF (CD55) in PBLs have been analyzed by flow cytometry in most of these patients. Patients have also been screened for mutations in HF1, MCP, FI and DAF genes. A summary of the mutations and polymorphisms found in these patients is shown in Table 1. Seventy-three unrelated healthy subjects were included in the study as a matched reference group. All protocols have been approved by national and/or local institutional review boards, and all subjects gave their informed consent.
SNP identification and genotyping
The identification and selection of the RCA polymorphisms to be included in the association study was performed as follows. We selected a group of 20 genomic DNA samples from unrelated control individuals and searched for polymorphisms within candidate genes by PCR followed by automatic DNA sequencing. The complete coding sequence (including the flanking intronic sequences) and at least 500 bp of the promoter region were analyzed for the HF1, C4BPB, DAF and MCP genes. We also analyzed 500 bp of the C4BPA and CR1 promoter region. Additional SNPs were obtained from the NCBI SNP database. We used the SNPHAP software to identify and estimate the frequencies of SNP haplotypes for each of the regions genotyped within the RCA gene cluster. Haplotype tagging SNPs that capture most of the genetic variation were selected within each region. Genotyping was performed on 10 ng of genomic DNA by allelic discrimination using TaqMan probes (Applied Biosystems; Foster City, CA, USA) and a real time PCR equipment (PE7700; Applied Biosystems) following the manufacturer's specifications.
Statistical analysis
Differences in the allelic frequencies between the aHUS and control groups were analyzed by the two-sided Fisher exact test. The ORs for these comparisons were also calculated. A P-value of 0.05 was considered to be statistically significant at the nominal level but, as we are testing many SNPs, we applied a correction for multiple comparisons. The 21 SNPs show high LD between them, that is, they are not independent variables. Therefore, the application of the Bonferroni correction for 21 independent tests would be too conservative. On the basis of the evaluation of the Eigen structure of the correlation matrix among markers (31), the effective number of SNPs is 16. Thus, the 21 SNPs behave statistically like 16 independent variables. Therefore, if we apply Bonferroni correction for 16 independent variables, the required P-value for significance is 0.003. We used the Student's t-test to compare the transcription activity of the different constructs used in the transfection experiments. LD between each pair of SNPs was estimated by the pairwise correlation (
) among genotypes. Working with SNPs, this measure is equivalent to the correlation of Weir (32) [see Appendix A in Meng et al. (33)]. Calculations and graphs were made with the computer program SOLAR.
Cell culture and transfection experiments
HEK293 cells were maintained as monolayer cultures and grown in Iscove's modified dulbecco's medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal calf serum (Life Technologies). Transient transfections were performed using Lipofectine (Invitrogen) as recommended by the manufacturer. Cells were plated in 24-well plates 1 day prior to transfection at 5x104 cells per well. Transfections were carried out with 650 ng of the pXP2-based constructs and 1.5 µl of Lipofectine in a total volumen of 500 µl of medium per well. In total, 20 ng of pCMV5-ßGAL plasmid (Clontech) was included in all transfections as an internal control to correct for transfection efficiency.
Plasmid constructs
A DNA fragment of 787 bp of the MCP gene, from –688 to +99, was amplified from genomic DNA using the 5'-ggtgttgcttaataaattagattcg-3' and 5'-gaaataacagcgtcttccgcg-3' primers. Individuals with genotype –547G/G and –261G/G or –547A/A and –261A/A were used to generate two different constructs with opposite genotypes. The PCR fragments were cloned upstream of the luciferase gene of the pXP2 vector (34). All constructs were tested by DNA sequencing. A TK.LUC control construct, containing the luciferase gene under the control of the promoter of HSV TK gene, was also used (35).
Luciferase and ß-GAL assays
Forty-eight hours after transfection, cells were lysed with 100 µl of Cell Culture Lysis Reagent (Promega). Twenty microliters of cellular lysates were mixed with 50 µl of luciferase substrate (Promega) and luciferase activity was measured on a TD-20/20 Turner Designs luminometer. The ßGAL activity was assayed by chemiluminescence, using the Tropix Galacto-LightTM Plus System (Applied Biosystems). The luciferase activities were normalized with respect to the ßGAL activities. Transfection and reporter assays were repeated in three independent experiments, each experiment being performed in triplicate with six different plasmid preparations of each construct.
URLs for databases and public software
- dbSNP (http://www.ncbi.nlm.nih.gov/SNP/)
- Mendelian Inheritance in Man (OMIM) (http://www.ncbi.nlm.nih.gov/Omim/)
- SNPHAP, a program for estimating frequencies of large haplotypes of SNPs (http://archimedes.well.ox.ac.uk/pise/snphap.html)
- MatInspector and Tess software available at (http://www.genomatix.de/cgi-bin/matinspector/matinspector.pl and http://www.cbil.upenn.edu/tess/, respectively)
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ACKNOWLEDGEMENTS |
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We are grateful to the patients with HUS, the control individuals and the collaborating clinicians for their participation in this study. We also thank Dr L. Botella, Dr A. Corbi and the members of the DNA sequencing laboratory at the Centro de Investigaciones Biológicas, for invaluable help. These studies were performed with funds provided by the Ministerio de Educación y Cultura (SAF2002-01085 and SAF2003-03485) and from the Fondo de Investigaciones Sanitarias (C03/05, G03/054, G03/011, FIS 01/3029 and FIS 01/A046). J.E.-G. was supported by a grant from the Comunidad Autónoma de Madrid.
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