Identification of a mutation in complement factor H-related protein 5 in patients of Cypriot origin with glomerulonephritis

Introduction

Kidney disease is an important cause of morbidity and mortality worldwide. In many cases, renal injury results from damage caused by the immune system, either in response to microbial infection or because of inappropriate activation of defence mechanisms. The mechanisms that protect the kidney from immunological attack in healthy individuals—and that fail in disease—are not well understood.

The complement system is a key component of host defence, and variation in the genes that regulate complement activation is associated with disease, including age-related macular degeneration,^1,2 atypical haemolytic uraemic syndrome,^2–4 and glomerulonephritis.^2,5–7 The kidney is especially susceptible to the effects of complement activation, and glomerulonephritis (a leading cause of kidney failure worldwide) is generally characterised by presence of complement within the glomerulus. Typically, complement is accompanied by immunoglobulins, which activate it via the classical pathway. However, complement deposition can occur without immunoglobulin via the complement alternative pathway. This deposition occurs in dense-deposit disease, which is caused by genetic or acquired defects in complement regulation.⁵

Isolated glomerular C3 deposition and inflammation can also arise in the absence of dense-deposit disease. This heterogeneous entity has been termed C3 glomerulonephritis and is often associated with the histological appearance of membranoproliferative glomerulonephritis.⁷ Our aim was to investigate an inherited renal disease, which we show is endemic in Cyprus and is characterised by microscopic and synpharyngitic macroscopic haematuria, renal failure, and C3 glomerulonephritis.

Methods

Results

Renal disease segregated as an autosomal dominant trait in family 1 and family 2 (figure 1). Disease was characterised by persistent microscopic haematuria and episodes of synpharyngitic macroscopic haematuria (within 1–2 days of an upper respiratory tract infection). C3 glomerulonephritis was identified in renal biopsy specimens in affected male and female patients. Glomerular inflammation was variable, but subendothelial and mesangial deposits that were reactive with antibody to C3, but not to C1q, C4, or immunoglobulins, were always present in these patients. Subepithelial deposits were occasionally noted. Figure 2 shows a representative biopsy sample.

In family 1, a genome-wide SNP-based analysis that used an autosomal dominant model and scored all patients with haematuria as affected established linkage to an 18 cM (centimorgan) region of chromosome 1q31–32 (logarithm of the odds [LOD] score 2·22). With the addition of family 2, the combined LOD score was 3·40 (figure 3). A haplotype of 17 SNPs spanning 8·74 cM within the linked region was shared by all affected members of both families, which is consistent with inheritance of an allele at this locus from a common ancestor (figure 1). This haplotype (HapMap coordinates 192344247 to 201027281) included complement factor H (CFH) and CFH-related (CFHR) 1–5 genes. Sequencing of CFH and two of the CFH-related genes (CFHR1 and CFHR5) in patients V-4 and IV-5 (both family 1) did not reveal any mutations.

MLPA analysis detected a heterozygous deletion of CFHR1 and CFHR3 in three members of family 2 (figure 4), which did not segregate with renal disease and is a polymorphism present in 14·2% of healthy controls.¹⁸ MLPA analysis also showed heterozygosity for a previously unreported duplication of exons 2 and 3 of CFHR5 in affected individuals from family 1 (IV-5 and V-4) and family 2 (II-1 and III-2), but not in the unaffected individual in family 2 (II-2) (figure 4). The CFHR5 internal duplication was confirmed, and its size established, by Southern hybridisation of genomic DNA with CFHR5 exon 2. The boundary of the duplication was identified by resequencing a PCR product (figure 5). Figure 5 shows an additional 6·3 kbp fragment on Southern blot analysis in affected individuals from family 1 (IV-5 and V-4) and family 2 (II-1 and III-2). This band was not present in two unrelated control individuals or in an unaffected individual from family 2 (II-2), in whom only the expected 7·9 kbp band was seen. With a reverse primer positioned in CFHR5 exon 2 and a forward primer in intron 3, there was no amplification from wild-type DNA (as expected). The duplication of exons 2 and 3 in DNA with the mutation led to a product of 4·8 kbp with these primers. Sequencing of this product confirmed the position and size of the duplication. A diagnostic PCR test was then designed with three primers which gave a 298 bp product from the wild-type allele and an additional 222 bp product from the allele with the CFHR5 internal duplication. The 222 bp product was seen in affected but not unaffected members of families 1 and 2 (figure 6).

With this diagnostic PCR, we did not detect a CFHR5 internal duplication in 102 randomly selected participants from the UK 1958 birth cohort.⁸ The CFHR5 internal duplication was only detected in one individual of 1015 control participants in the MASTOS study (for whom no clinical or personal details were available).⁹ The CFHR5 internal duplication is therefore a rare allele in the Cypriot population.

We then screened a cohort of 84 patients in Cyprus with advanced or end-stage renal failure. This screening identified one female and three male patients with the mutation, none of whom reported ancestry in the Troodos mountains. Families 3 and 4 were identified by testing Cypriot families with unexplained haematuria, and the CFHR5 duplication was identified in all seven individuals affected with microscopic or synpharyngitic macroscopic haematuria, but not in five unaffected relatives. A renal biopsy had been taken from one individual from these families and showed C3 glomerulonephritis. Neither of these families could trace their ancestry to the Troodos mountains region. The review of case records from two renal units in London followed by DNA testing identified three men and one woman of Cypriot descent with CFHR5 nephropathy. In one of these cases a brother with microscopic haematuria was also tested and found to have the CFHR5 mutation (family 5). Although not ostensibly related to family 1 or family 2, paternal ancestry was from the same valley in the Troodos Mountains.

The table summarises available clinical data for all the individuals we showed had the CFHR5 mutation or who were obligate carriers. In all affected men, there was progressive renal impairment with end-stage renal disease between the ages of 40 and 69 years. Renal impairment was less common in affected women than in affected men (p=0·0016; table).

These findings suggest that the newly identified CFHR5 duplication accounts for a substantial proportion of renal disease in Cyprus and is not confined to the Troodos mountain region. Further genotyping of SNPs showed a haplotype extending from 1·4 cM upstream to 4·7 cM downstream of the mutation that cosegregated with the CFHR5 internal duplication in the five families tested, which is consistent with inheritance of the mutation by all affected individuals from an original founder. By contrast, none of the 36 individuals with biopsies showing C3 glomerulonephritis from the UK and France had the CFHR5 mutation.

CFHR5 is a 65 kDa plasma protein consisting of nine short consensus repeat (SCR) domains (repeating units that are characteristic of CFH and CFHR proteins).¹⁶ Duplication of exon 2 (encoding SCR1) and exon 3 (encoding SCR2), predicted a novel CFHR5 protein with duplicated SCR domains 1 and 2 (figure 7). Western blotting of serum samples from affected individuals detected the normal CFHR5^123-9 (superscript numbers denote SCRs) protein, and a slow migrating protein that was consistent with the predicted molecular weight of CFHR5^12123-9 (figure 7).

Although the initial aminoacid sequence of the wild-type SCR1 is Glu-Gly-Thr-Leu-Cys-Asp (with the first glutamic acid encoded by exon 1), the duplicated SCR1 is encoded only by exon 2 and therefore is predicted not to have this glutamic acid. Splicing of exon 3 upstream of the exon 2 sequence results in a change in the initial codon of the duplicated SCR1 so that the second aminoacid in the sequence is arginine rather than glycine. Hence, the initial aminoacid sequence of the duplicated SCR1 is predicted to be Arg-Thr-Leu-Cys-Asp. The rest of the duplicated SCR1 aminoacid sequence and entire duplicated SCR2 aminoacid sequence is identical to wild-type CFHR5.

The physiological role of CFHR5 is not known; the plasma concentration is estimated to be 3–6 μg/mL,¹⁹ which is approximately 1% of that of the well characterised complement regulator CFH.²⁰ CFHR5 has been shown to co-localise with complement deposits in diseased human kidneys²¹ and binds to surface-bound activated C3 (termed C3b).¹⁶ We showed that mutant CFHR5^12123-9 protein bound less effectively than did wild-type protein to two sources of surface-bound complement; complement-lysed erythrocyte membranes (figure 8) and glomerular-bound mouse complement (webappendix p 1).

Previous in-vitro investigations have reported that CFHR5 shares some of the complement regulatory activities of CFH; CFHR5 can be a cofactor for the proteolytic inactivation of complement C3b by the plasma enzyme complement factor I, although its activity is weak by comparison with that of CFH.¹⁹ We confirmed that both the wild-type CFHR5 and mutant CFHR5^12123-9 proteins have complement factor I cofactor activity; in both cases the activity is substantially weaker than that reported for CFH. The mutant CFHR5^12123-9 protein did not have reduced activity compared with wild-type in this assay; perhaps surprisingly, complement factor I cofactor activity was increased (webappendix p 2).

Discussion

We provide evidence for an inherited renal disease, endemic in Cyprus, that is characterised by microscopic and synpharyngitic macroscopic haematuria, renal failure, and C3 glomerulonephritis, and show that affected individuals have an internal duplication within the gene for complement factor H-related protein 5 (CFHR5). We term this disease CFHR5 nephropathy.

Isolated microscopic haematuria is a common presentation that in the absence of urinary tract abnormalities, proteinuria, or renal impairment is usually believed to be benign, and with present guidelines is not investigated by renal biopsy.^22,23 However, this study shows that isolated microscopic haematuria can be the presenting feature of progressive renal disease, with implications for patients and their families. Furthermore, analysis of data suggests that familial isolated microscopic haematuria attributable to heterozygous mutations in COL4A3 and COL4A4 is also associated with progressive chronic kidney disease.¹⁰ Taken together, these findings underscore the importance of any history of renal disease in the family and the value of renal biopsy and genetic investigations in this setting.

CFHR5 nephropathy has several noteworthy clinical features. First, the risk of progressive renal impairment is more common in men than in women. Second, since Berger's original description,²⁴ repeated episodes of synpharyngitic haematuria have been regarded as almost diagnostic of IgA nephropathy; CFHR5 nephropathy should now be thought of as a differential diagnosis. Third, although retinopathy is a well recognised feature of dense-deposit disease, with affected individuals developing ocular drusen at a young age, clinically significant visual impairment was not a feature of CFHR5 nephropathy. This finding is consistent with a study²⁵ that reported no association between CFHR5 polymorphisms and risk of age-related macular degeneration. However, because formal ophthalmological assessment was only done for two affected individuals in this study, we cannot exclude the presence of subclinical ocular disease in CFHR5 nephropathy.

An important pathological consideration was that most of the individuals who were biopsied initially had a histological diagnosis of membranoproliferative glomerulonephritis type 1. Review led to recognition that these abnormalities were C3 glomerulonephritis, which is an entity described in a French report⁷ of 19 patients that did not identify families with more than one affected individual. In that series,⁷ men were not more severely affected than were women, synpharyngitic macroscopic haematuria was not reported, and microscopic haematuria was absent in seven of 19 (37%) patients at diagnosis. By contrast, CFHR5 nephropathy is an inherited disease, characterised by synpharyngitic macroscopic haematuria (noted in seven [58%] of the affected individuals tested; see table) and microscopic haematuria (all affected individuals tested). Thus, CFHR5 nephropathy constitutes a distinct clinical entity that is associated with the histological category C3 glomerulonephritis. Abnormalities in other complement genes were identified in a subset of the patients in the French report,⁷ suggesting that different mechanisms of complement dysregulation can lead to much the same histological appearances.

The CFHR5^12123-9 mutation is a copy number variation (ie, a duplication or deletion of a segment of DNA), which is not detectable with standard exon-based sequencing. Copy number variations arise through non-allelic recombination events that are more common in regions of complex genomic architecture such as the CFH and CFHR1–5 gene cluster.

The extent of the shared haplotype suggests that affected members of the families inherited the mutation from a common ancestor about 16 generations ago,¹¹ and our identification of additional cases and affected families in the Cypriot population implies the existence of a substantial number of individuals with CFHR5^12123-9. The high penetrance of haematuria (all 22 mutation carriers tested; see table), the wide geographical distribution of ancestry within Cyprus, and the presence of affected individuals in the UK suggests that this disease will account for a substantial proportion of renal disease affecting inhabitants of the island and their descendants worldwide. Population-based studies need to be done to quantify this proportion; we examined only a small cohort of Cypriots with advanced or end-stage renal disease, either of unknown cause or attributed to presumed or incompletely characterised glomerulonephritis. In the US renal data system, about 25% of patients with advanced renal disease would fit into this category.

Although CFHR5 nephropathy seems to be a common cause of renal disease in the Cypriot population, we did not detect this specific CFHR5 mutation in 36 cases of sporadic C3 glomerulonephritis from France and the UK. Whether this geographical disparity is the effect of ascertainment bias, positive selection for the allele within Cyprus (perhaps because of the presence of an endemic infectious disease), or genetic drift within the island's population is not known. So far, mutations in other complement regulatory genes and the presence of C3 nephritic factor have been reported in a few cases of C3 glomerulonephritis;⁷ other genetic abnormalities of CFHR5 might exist.

The mutation we identified in CFHR5 provides a robust genetic marker for a novel hereditary nephritis, and screening for the mutation is a reliable clinical test. However, our understanding of how the mutation causes the disease is incomplete and we do not exclude the possibility that another mutation within the 6·1 cM-shared haplotype could exist. We propose that CFHR5 is important in complement processing within the kidney, and that the CFHR5^12123-9 mutation impairs the ability of CFHR5 to achieve this. Our model is consistent with the previous finding²¹ that wild-type CFHR5 co-localises with complement within the kidney in renal disease.

Our in-vitro functional studies show that the mutant CFHR5^12123-9 protein binds to surface-bound complement (on erythrocytes and mouse glomeruli) substantially less well than does wild-type CFHR5. Western blot analysis of serum from affected individuals showed greater intensity for mutant CFHR5^12123-9 protein than for wild-type CFHR5 (figure 7); an in-vivo finding that would be consistent with a defect in recruitment of the mutant protein from the circulation to interact with surface-bound complement. However, CFHR5^12123-9 is not a straightforward loss of functional allele, because it produces a circulating protein that shows enhanced complement factor I cofactor activity in vitro compared with wild-type CFHR5.

In laboratory models of C3 dysregulation, excessive production of inactivated C3b (iC3b) by complement factor I is important for the initiation of renal injury;⁵ complement factor I cofactor activity of CFHR5^12123-9 could lead to an increase in iC3b in the glomerulus. More studies will be necessary to elucidate fully the biological role of CFHR5 and the pathophysiology of C3 glomerulonephritis in CFHR5 nephropathy. The central role of abnormal complement deposition in this disease suggests that inhibition of the terminal complement pathway (for instance with the humanised anticomplement C5 monoclonal antibody eculizumab, which is effective for treatment of paroxysmal nocturnal haemoglobinuria²⁶) might be of therapeutic benefit in CFHR5 nephropathy and clinical studies are needed to address this issue.

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