Patients with Goodpasture's disease have two normal COL4A3 alleles encoding the NC1 domain of the type IV collagen
3 chain
Ulf Persson1,
Jens Michael Hertz2,
Malin Carlsson1,
Thomas Hellmark1,
Inger Juncker2,
Jörgen Wieslander3 and
Mårten Segelmark1
1 Department of Nephrology, Lund University, 3 Wieslab AB, Lund, Sweden and 2 Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark
Correspondence and offprint requests to: Mårten Segelmark, Department of Nephrology, University Hospital, 221 85 Lund, Sweden. Email: marten.segelmark{at}njur.lu.se
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Abstract
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Background. Goodpasture's disease (GP) is a rare but severe disease characterized by anti-glomerular basement membrane antibodies, rapidly progressive glomerulonephritis and lung haemorrhage. The autoantibodies are restricted to a narrow epitope region on the NC1 domain of the
3 chain of type IV collagen. GP is strongly associated with major histocompatibility complex (MHC) allele HLA DRB1-15. Recent research, however, has failed to identify a T-cell epitope with molecular characteristics that explain the relationship between the MHC class II molecule and the autoantibody generation. We hypothesized that an as yet unidentified sequence variant in exons 4852 of the COL4A3 gene that encodes the NC1 domain of the type IV collagen
3 chain could generate a new peptide sequence that, through interaction with specific MHC class II molecules, would increase the risk of developing GP.
Methods. All patients previously treated for GP at the Lund and Malmö University Hospitals, who were alive at the time of the study, were asked to participate. DNA was extracted from leukocytes and subjected to genomic tissue typing and sequencing of the COL4A3 gene exons 4852.
Results. All 15 patients in the study had a nucleotide sequence in the COL4A3 gene encoding a protein identical to GenBank entry NM_000091. HLA D allele distribution was in line with previous publications, showing a strong positive association between HLA DRB1-15, HLA DQB1-6 and GP (P<0.02). Of the 15 GP patients, 73% carried HLA DRB1-15 and 87% carried the HLA DQB1-6 antigen. Corresponding figures for the controls were 27 and 50%.
Conclusion. This study effectively falsifies the hypothesis that a minor alteration in the COL4A3 gene could be a major factor in the aetiology of GP. Scandinavian GP patients have an MHC distribution similar to that which has been described previously for Anglo-Saxon patients.
Keywords: COL4A3 gene/Goodpasture's disease
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Introduction
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Goodpasture's disease (GP) is characterized by the combination of rapidly progressive glomerulonephritis and anti-glomerular basement membrane (anti-GBM) antibodies, and is often accompanied by lung haemorrhage [1]. It usually results in permanent renal failure or death if not diagnosed and treated promptly. Transfer experiments have proven the toxicity of the antibodies in primates, and the removal of the antibodies is part of successful treatment of patients [2,3]. The antibodies are directed to the C-terminal non-collagenous domain (NC1) of type IV collagen. Most antibodies, maybe all disease-causing antibodies, react with a limited number of epitopes on the NC1 domain of the
3 chain [4,5]. Of special interest is an epitope region in the N-terminal third of the domain, where amino acid residues between the positions T1455 and S1469 together with Q1495 seem to be critical for the binding of the autoantibodies [6,7]. Antibodies reacting with other epitope regions and antibodies cross-reacting with other
-chains can also be found in many patients, but their pathogenic and diagnostic potentials remain unproven. Recent work regarding the three-dimensional structure of the NC1 domain and the quaternary organization of the
-chains has shown that GBM NC1 domains consist of two head-to-head-oriented
3,4,5 heterotrimers [8]. The putative major epitope is situated close to the collagen triple helix and facing the
5-chain within its own heterotrimer.
While the B-cell epitopes are well characterized, less is known about T-cell involvement. The IgG subclass distribution of the autoantibodies, with a preponderance for subclasses IgG1 and IgG4, suggests an antigen-driven and T-cell-dependent process [9]. Studies of the functional affinity, however, have been unsuccessful in establishing evidence for affinity maturation during the course of the disease [10]. The strongest indirect evidence for T-cell involvement in the pathogenesis stems from the skewed distribution of major histocompatibility complex (MHC) class II alleles among patients with GP, compared with controls. Several studies have shown strong positive association with the DR alleles HLA DRB1-15 and DRB1-4, and negative associations with HLA DRB1-7 and DRB1-1 [11]. HLA DRB1-15 alleles have been found in 7090% of patients of Western European descent, compared with 2030% in the background population. Phelps and co-workers have launched extensive studies in order to elucidate specific T-cell epitopes of importance [1214]. They found that none of the overlapping peptides they constructed bound HLA DRB1-15 with much higher affinity than for both DRB1-1 and DRB1-7. This included peptides previously identified as being expressed by HLA DRB1-15-positive antigen-presenting cells (APCs) after processing of recombinant
3(IV) [13]. Instead, the tendency was in the other direction, and they proposed that DRB1-1/7 might protect from anti-GBM disease by capturing peptides in heterozygotic APCs. In a recent article from the same group, it was shown that T cells from patients, in contrast to controls, showed reactivity towards certain
3(IV)-derived peptides presented by HLA DRB1-15 [14]. However, the relationship between HLA DRB1-15 and GP still remains unexplained.
It is well established that inherited defects in the COL4A3 gene can cause renal disease. A homozygotic state of non-functioning alleles or alleles coding for non-functioning proteins gives rise to the autosomal recessive form of Alport syndrome, while a heterozygotic state usually manifests as thin basement membrane disease [15]. After renal transplantation, some patients with Alport syndrome develop anti-GBM antibodies, which can lead to transplant failure [16]. Thus, defects in type IV collagen genes may under certain circumstances predispose to anti-GBM production.
While HLA DR1-15 is common and GP is rare, other factors, genetic or environmental, must be critical for the development of anti-GBM antibodies. We hypothesized that there might exist sequence variants in the COL4A3 gene leading to alterations in the primary amino acid sequence of the NC1 domain. This could subsequently lead to the generation of new T-cell epitopes having the ability to interact unfavourably with specific MHC alleles, increasing the risk of developing anti-GBM antibodies. In order to test this hypothesis, we sequenced exons 4852 of the COL4A3 gene in 15 patients who previously had been treated for GP and determined their MHC class II genotype. The findings did not support the hypothesis.
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Patients and methods
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Databases of biopsy registries at the University Hospitals of Lund and Malmö, and the Wieslab AB serological database were searched for patients with GP and anti-GBM antibodies. Patients were included if they had circulating anti-GBM antibodies that could be confirmed by retesting stored sera, and had a clinical history indicating rapidly progressive glomerulonephritis such as haematuria, casts in the urinary sediment and a rising serum creatinine concentration. All patients alive at the time of the study were contacted and asked to participate. After informed consent was granted, clinical data were retrieved from the patients hospital records. The local ethics committee approved the protocol.
HLA typing
Leukocytes were prepared from whole blood by density centrifugation, and DNA was extracted as described by Miller et al. [17]. HLA DRB1, DRB3-5 and DQB1 typing was done at the local tissue-typing laboratory using polymerase chain reaction (PCR)-based standard clinical procedures. Results were compared with previously established allele frequencies based on the local population of blood donors.
PCR amplification of genomic DNA
Primers for COL4A3 exons 4852 were synthesized according to published sequences [GenBank accession no. NM_000091 (http://www.ncbi.nlm.nih.gov/)] (Table 1). Genomic DNA was amplified in a reaction volume of 25 µl containing 200 ng of DNA, 200 µM dNTP, 20 pmol of each primer and 1 U of Taq DNA polymerase (Boehringer Mannheim). The buffer used for PCR contained 10 mM TrisHCl, pH 8.3, 50 mM KCl and 1.5 mM MgCl2. The PCR conditions were as follows: initial denaturation at 94°C for 4 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing for 1 min at the temperatures as indicated in Table 1, and extension at 72°C for 2 min. The final extension was 4 min at 72°C.
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Table 1. Primer sequences and conditions for PCR amplification of the 3' end of COL4A3 encoding the NC1 domain of the (3) chain of type IV collagen
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Sequencing of PCR products
Sequencing was performed by cycle sequencing of double-stranded DNA using an ABI PRISM Big Dye Terminator Labelling Cycle Sequencing Kit (PE Applied Biosystems) as recommended by the manufacturer, and run on an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems). Obtained sequences were analysed by Sequence Navigator (Applied Biosystems) and compared with control sequences from a normal individual. Nucleotides are numbered starting from the A of the ATG initiator codon.
ELISA
An enzyme-linked immunosorbent assay for circulating antibodies against
3(IV) NC1 (anti-GBM), proteinase 3 [PR3-anti-neutrophil cytoplasmic antibody (ANCA)] and myeloperoxidase (MPO-ANCA) was carried out as described previously [9,18]. In brief, antigen was coated in microtitre plates at a concentration of 0.5 µg/ml for
3(IV) NC1, and at 1 µg/ml for PR3 and MPO in carbonate buffer. After washing, plates were incubated for 1 h with sera diluted 1:100 in ELISA buffer. In the next step, alkaline phosphatase-conjugated goat anti-human IgG (Sigma, St Louis, MO) was incubated on the plates at a dilution of 1:20 000. Finally, a substrate buffer (1 M diethanolamine, 0.5 mM MgCl2, pH 9.8) with p-nitrophenyl phosphate was added and the optical density (OD) at 405 nm was read with a spectrophotometer. All samples were run in triplicate and the results were expressed as arithmetic means. A positive result was defined as being above the mean results of healthy controls +3SD (0.19 for GP, 0.16 for PR3 and 0.13 for MPO).
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Results
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For this study, 15 patients were identified who had been treated previously for anti-GBM-mediated renal disease in Lund or Malmö. All patients agreed to participate in the study. Of the 15 patients, seven were men and eight were women. The mean age at diagnosis was 42.8 years (range 1868, Table 2). All patients had circulating anti-GBM antibodies at diagnosis, and in 11 of the patients kidney-reactive antibodies were also detected by direct immunofluorescence. There was a wide range in kidney function at the time of diagnosis as reflected by serum creatinine concentrations ranging from 181 to 1240 µmol/l (median value 608).
Sequencing of exons 4852 of the COL4A3 gene yielded a sequence identical to the published sequence in 14 of the 15 patients. In patient no. 9, a single base substitution at position 5301 was identified (5301 C>T). This is 288 bp 3' from the TGA stop codon in exon 52 and does not result in any change in the amino acid composition.
Tissue typing of the MHC locus HLA D revealed a significantly skewed pattern (Tables 3 and 4). Eleven of the 15 patients (73%) were positive for DRB1-15 compared with 27% in the background population (P<0.001). All 11 DRB1-15-positive patients were also positive for DQB1-06. In total, 13 out 15 patients (87%) were positive for DQB1-06 as compared with 50% among the blood donors (P<0.01). The two DQB1-06-negative patients were both positive for DRB1-04 and DQB1-03. No patients were found with any of the following alleles: DRB1-07, 08, 09, 10, 11 or 12. Individually, none of these negative associations is statistically significant.
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Table 3. COL4A3 gene NC1 domain sequence (as compared with GenBank accession number NM_000091) and HLA MHC class II allele determination among 15 patients with GP together with results from anti-GBM and ANCA ELISA
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Five patients were ANCA positive in ELISA, four in MPO-ANCA ELISA and one in PR3-ANCA ELISA. ANCA-positive patients were older than ANCA-negative patients, median age 65 vs 26 years. This was highly significant; the MannWhitney U-test yielded a P-value of 0.0013. There were no trends suggesting a different MHC genotype among ANCA-positive patients. Four of the five ANCA-positive patients were DRB1-15 positive, and both DQB1-06-negative patients were ANCA negative. MHC genes did not seem to influence the anti-GBM titre. The mean OD value for the 11 DRB1-15-positive patients was similar to the OD value for the four DRB1-15-negative patients (1.8 vs 1.6, P = 0.47).
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Discussion
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This study effectively falsifies the hypothesis that minor alterations in the Goodpasture antigen are a major factor in the aetiology of GP. We reasoned that if a sequence variant would cause a new T-cell epitope, forming a specific interaction with a particular MHC antigen, this variant had to be present in >30% of the DRB1-15 cases in order to be considered a major factor. We found no alteration in the gene encoding the primary amino acid sequence among 11 DRB1-15 carriers. The probability that such an association still exists in spite of our findings is very low (P<0.02). A second possibility is that a certain variant may make DRB1-15 non-carriers susceptible to anti-GBM production. Even though we found no variants among the four non-carriers in the present study, we cannot exclude the possibility that such variants exist in a majority of the non-DRB1-15 cases, but it is unlikely (P<0.0625). A recent study concerning experimental autoimmune glomerulonephritis in rats came to a similar conclusion [19]. There was no linkage between disease susceptibility and the rat Col4a3 gene.
ANCA was found in five of our 15 patients at diagnosis. This is in the same range as that which has been reported previously [20]. Despite many studies, no distinct MHC association has been found for ANCA or any of the ANCA-associated vasculitides [21]. Our study was not powered to find associations between ANCA and certain MHC alleles among the patients with GP. It is of interest to note, however, that four out of five ANCA-positive patients carried the DRB1-15 allele. Our findings make it unlikely that differences in the distribution of DRB1 alleles explain why some GP patients also make ANCA. Instead, age was a factor associated with ANCA among GP patients. Even in this small study, the age difference between ANCA-positive and ANCA-negative patients was highly significant.
MHC class II molecules are also present on B cells. Direct interaction between the degraded basement membrane fragments, HLA DRB1 molecules and the B-cell receptors might amplify autoantibody production. In this study, we found no significant difference between mean levels of circulating anti-GBM antibodies between DRB1-15 carriers and non-carriers. Thus it is unlikely that the DRB1-15 molecule affects the anti-GBM production directly.
To our knowledge, this study is the first to investigate MHC allele distribution among Scandinavian GP patients. Our results are very similar to results from studies performed in England, France, the USA and Australia [11]. The strong positive correlation with DRB1-15 together with a negative correlation for DRB1-1 and DRB1-7 are evident also in the present study. We also find a very strong association with DQB6, which is in linkage disequilibrium with DRB1-15. If anti-GBM generation is a consequence of interaction between HLA DRB1-15 and a specific environmental factor, this factor is present not only in England and France, but also in Sweden, the USA and Australia.
Basement membrane components are synthesized, secreted and assembled in a highly organized fashion, and defects in one component often affect the appearance of other components in the GBM. Inherited defects in the ability to coordinate the production of collagen type IV chains might generate increased amounts of antigenic breakdown fragments of the immune system. However, if GP is not associated with inborn variants in the COL4A3 gene, it is also unlikely that inborn alterations of other major basement membrane components would contribute to disease susceptibility. However, the absence of COL4A3 mutations in genomic DNA from peripheral blood leukocytes does not exclude the presence of somatic mutations in the kidneys and lungs where the
3(IV) chain is highly expressed, nor does it exclude the presence of aberrant COL4A3 splice variants.
It has been proposed that the GP epitope is not normally exposed to the immune system and that environmental factors such as hydrocarbon exposure may unmask the epitope [22]. It has also been suggested that reactive oxygen species can alter the structural integrity of the GBM and thereby increase its autoimmunicity [23]. Even though these theories might have bearing on the aetiology of GP, they all fail to explain the strong MHC association. Recent results from Wong and co-workers showing that the
3(IV) and
5(IV) chains are expressed in the thymus (without incorporation into a basement membrane) [24] open up another line of thinking. This can be interpreted as if GP epitope-reactive T cells normally are kept under control by central tolerance, and that the MHC association might stem from a defective clonal deletion due to the low affinity between DRB1-15 and NC1 peptides [12]. The production of NC1 by non-basement membrane-producing cells also opens up the possibility that other cells, including those of a haematopoietic origin, under pathological conditions might transcribe the COLA43 gene, eventually leading to autoantibody production.
In conclusion, we found that GP among Scandinavian patients is strongly linked to HLA DRB1-15, and that this association is not a consequence of interaction with certain genetic variants of the GP autoantigen.
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Acknowledgments
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This work was supported by grants from the Swedish Scientific Research Council and by the Swedish Kidney Foundation.
Conflict of interest statement. None declared.
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Received for publication: 28.11.03
Accepted in revised form: 31. 3.04