Patent Application: US-201013382831-A

Abstract:
the invention relates to methods of regulating complement . in particular , the inventors have identified a relationship between a particular gene , cfhr5 , and irregularities in complement regulation . the invention provides a method for diagnosing a complement related disease , comprising identifying a mutation in the cfhr5 gene in a sample obtained from a subject .

Description:
the study was approved by the local research ethics committees . family 1 . the index case ( v - 4 in fig1 a ) presented aged 17 with hypertension , microscopic hematuria and episodic macroscopic hematuria coinciding with upper respiratory tract infections . renal biopsy demonstrated mild expansion of the mesangial matrix and increased glomerular cellularity , segmental capillary wall thickening and focal tubular atrophy . electron microscopy showed subendothelial and mesangial electron dense deposits with infrequent subepithelial deposits . there was positive staining for complement c3 but not for immunoglobulins or c1q in these areas ( fig1 b ). serum complement c3 and c4 levels were normal ( c3 1 . 05 g / l , normal range 0 . 7 - 1 . 7 and c4 0 . 24 g / l , normal range 0 . 16 - 0 . 54 ) and there was no clinical or serological evidence of chronic infection , c3 nephritic factor or microangiopathy . over the next 10 years proteinuria (& lt ; 1 g per day ) and renal impairment developed , with a fall in 51cr - edta clearance from & gt ; 100 ml / min at original presentation to 70 ml / min at age 26 . a repeat biopsy showed increased tubular atrophy and glomerular obsolescence . there was a family history of renal disease and , where performed , findings on biopsy were essentially identical ( fig1 a ). retinal photography in individuals v - 4 and iv - 6 was normal with no evidence of drusen and circulating factor h levels were normal ( 548 and 631 mg / l respectively ). family 2 . an ostensibly unrelated individual ( iii - 2 in fig1 a ) presented aged 38 with microscopic hematuria , episodic macroscopic hematuria coincident with upper respiratory tract infections , proteinuria ( 0 . 5 - 1 g per day ), hypertension , renal impairment ( serum creatinine 1 . 8 mg / dl ) and a family history of renal disease ( fig1 a ). renal biopsy demonstrated c3 glomerulonephritis . both families originated from the same valley of the troodos mountains in cyprus . genome - wide linkage study . dna was extracted from blood or saliva ( oragene , dna genotek , canada ). genotypes and haplotypes of 6008 single nucleotide polymorphisms ( snps ) ( linkage iv panel , illumina , calif .) in the families were analyzed using easylinkage , 18 pedcheck , 19 genehunterv2 . 1 20 and haplopainter . 21 bidirectional sequencing of the exons of candidate genes was performed following polymerase chain reaction ( pcr ) amplification ( primers available on request ). cfhr5 internal duplication . multiplex ligation - dependent probe amplification ( mlpa ) was performed on unamplified genomic dna using the p236 a1 armd mix 1 from mrc - holland ( amsterdam , the netherlands ). full details are available at www . mrc - holland . com ). southern blotting was performed on genomic dna digested with ecori ( new england biolabs , ma ). membranes were probed with a 32 p - labelled 371 base pair sequence containing exon 2 of cfhr5 . pcr amplification of the cfhr5 duplication insertion point used primers : 5 ′- tggaagcctgtggtataaatga - 3 ′ and 5 ′- tccggcacatccttctctat - 3 ′ ( fig2 b ). pcr amplifying both cfhr5 alleles in a single reaction used primers 5 ′- gattccatttgtcaaatattg - 3 ′, 5 ′- tcttctccaaaactatctaatgtcaa - 3 ′ and 5 ′- tttgaatgctgttttagctcg - 3 ′. detection of cfhr5 in sera and functional analysis . cfhr5 was detected by the western blotting of serum using rabbit antiserum 17 ( a gift from j . mcrae , immunology research centre , melbourne , australia ). wild - type and mutant cfhr5 functional tests were performed with washed chicken erythrocytes ( ce ) suspended 1 : 10 ( v / v ) in 25 μl saline and incubated at 37 ° c . overnight with 50 μl of serum and 425 μl of saline , with or without 10 mm edta to inhibit complement activation . 17 after centrifugation at 13000 rpm cfhr5 in the fractions was assayed by western blotting . binding of cfhr5 to heparin was assayed as previously described . 22 in view of the previous association of mutations in ap components with c3gn , we sequenced the exons of the genes bf , c3 , cfh , cfi , cd46 and the cfh - related genes cfhr1 and cfhr5 in individuals v - 4 and iv - 6 from family 1 . no coding or splice site mutations were detected in these genes . a genome wide snp - based analysis established linkage in family 1 to an 18 cm region of chromosome 1q31 - 32 ( lod 2 . 22 ), which contains the genes for cfh and cfhr1 - 5 ( termed the regulators of complement activation ( rca ) cluster ). with the addition of family 2 ( scoring female relatives with microscopic hematuria as affected ) the combined lod score at this locus was 3 . 40 ( fig1 c ). in addition , a haplotype comprising 15 snps and spanning 8 . 74cm within the linked region and including the rca cluster was shared between all affected members of both families ( fig1 a ), consistent with inheritance of an allele at this locus from a single common ancestor . other loci containing complement genes were excluded ( lod & lt ;- 2 ). mlpa analysis in v - 4 ( c3gn ) and iv - 6 ( c3gn ) from family 1 and iii - 2 ( c3gn ), ii - 2 ( microscopic hematuria ) and ii - 1 ( unaffected ) from family 2 showed heterozygosity for a duplication of exons 2 and 3 of cfhr5 in affected individuals but not the unaffected family member ( fig2 a ). in addition , deletion of the cfhr1 and cfhr3 genes was noted in all 3 members of family 2 tested but was not observed in either member of family 1 ( fig2 a ). southern blot of genomic dna probed with cfhr5 exon 2 revealed an additional 6 . 3 kbp band in individuals with renal disease and the boundary of the duplication was identified by resequencing a pcr product ( fig2 b ). a pcr reaction demonstrated the presence of the internal duplication in all individuals in both families with c3gn and in obligate carriers ( fig2 c ). one individual ( v - 2 in family 1 ) was noted to have microscopic hematuria on a single occasion but did not have the duplication . to exclude the possibility that this allele is a normal variant we showed that it was not present in 102 unrelated individuals from the uk 1958 birth cohort . to explore its possible frequency in cyprus we screened dna from individuals collected as control subjects in the mastos study 23 . heterozygosity for the mutation was identified in a single individual amongst the 1015 that we screened , implying that this is a rare allele in the cypriot population . the ethical approval under which this investigation was performed did not allow demographic or clinical details to be obtained , so we do not know if the individual has associated clinical manifestations . in order to identify whether any additional affected individuals exist in cyprus we screened a cohort of 84 greek - cypriot patients with sporadic renal disease for the presence of the mutation and identified 3 males and one female patient with the mutation ( 4 . 8 %). none of these individuals reported ancestry in the troodos mountains . a histological diagnosis was not available in 41 of these ( of which one male and one female were affected ). the diagnoses for the remaining 43 patients are recorded in table 1 . in addition , 2 further separate families in whom microscopic haematuria segregated as an autosomal dominant trait and direct exon sequencing had excluded known mutations in the genes col4a3 - col4a4 24 were investigated . both families comprised 5 affected individuals , all of whom carried the cfhr5 duplication which was not identified in any unaffected relatives . a renal biopsy had been performed in a single individual from one of these families and this showed c3gn . neither of these families could trace their ancestry to the troodos mountains . we conclude that this cfhr5 duplication accounts for a significant proportion of renal disease in cyprus and is not confined to the troodos region of the island . cfhr5 consists of nine short consensus repeat ( scr ) domains ( cfhr5 123 - 9 , superscript numbers denoting scrs ). duplication of exons 2 and 3 predicts a cfhr5 protein containing two additional scrs ( denoted by cfhr5 12123 - 9 ) since scrs 1 and 2 of cfhr5 are encoded by exons 2 and 3 respectively ( fig3 a ). western blot of sera from affected individuals demonstrated , in addition to the normal cfhr5 protein , the presence of a slower migrating protein , consistent with the predicted molecular weight of the cfhr5 12123 - 9 protein ( fig3 a ). functional assays demonstrated that the binding of the cfhr5 12123 - 9 in patient sera to complement - lysed chick erythrocytes was markedly reduced compared to cfhr5 123 - 9 ( fig3 b ). binding of both cfhr5 proteins to erythrocyte surfaces was inhibited by 10 mm edta indicating that it was dependent on complement activation . furthermore , heparin affinity chromatography demonstrated that the cfhr5 12123 - 9 protein had reduced affinity for heparin since it was eluted at a lower concentration ( 300 mm nacl ) compared to cfhr5 123 - 9 ( which eluted at 350 mm , data not shown ). taken together these data show inheritance of a defective cfhr5 allele which causes c3gn . cfhr5 is a member of the cfh gene family which comprises cfh and the 5 cfh related genes ( cfhr1 - 5 ). the in vivo importance of cfh as the major regulator of plasma ap activity is illustrated by the severe ap dysregulation , resulting in secondary depletion of plasma c3 , which occurs in individuals with complete genetic cfh deficiency . 25 cfh also appears to play a physiological role in protecting renal endothelium since mutations that impair binding to this surface predispose to ahus . 8 recently the association of genetic variants in cfhr genes with disease suggested they are also important . thus deletion of cfhr1 and cfhr3 genes is a common polymorphism that appears to confer altered susceptibility to amd and ahus ( reviewed in 8 ) and certain common cfhr5 variants preferentially associate with ahus 26 and ddd . 27 cfhr5 is unique among cfhr proteins in having detectable complement regulatory activity in vitro . 28 , 29 furthermore , in vitro , cfhr5 can bind to heparin and to activated c3 , functions that would enable interaction with complement deposited in tissues . 28 consistent with this , cfhr5 has been detected in kidney when there is complement deposition . 26 cfhr5 is a 65 kda protein with 9 scr domains 17 ; the internal duplication we identified adds an additional two scr domains . our functional studies tested the ability of the cfhr5 12123 - 9 protein to mediate two of the known functions of the normal cfhr5 123 - 9 protein : the ability to bind to complement on a surface and heparin binding . in both instances the cfhr5 12123 - 9 protein demonstrated reduced affinity compared with normal cfhr5 123 - 9 protein , suggesting that the cfhr5 12123 - 9 protein is a ‘ loss of function ’ variant although we do not exclude that it may also exert a dominant negative effect . western blot analysis of serum from individuals with the cfhr5 12123 - 9 protein variant showed that the intensity of the normal cfhr5 123 - 9 protein band was reduced compared to control samples . in part this is likely to be a dose effect , reflecting heterozygosity for the wild type cfhr5 123 - 9 allele but would also be consistent with greater sequestration of wild type cfhr5 protein by complement deposits within the c3gn lesions . the fact that this allele was identified in 2 ostensibly unrelated families ( in whom it can be inferred from the size of their shared haplotype that the most recent common ancestor at the locus in question was most likely to have lived approximately 10 generations ago 30 ) suggested that a larger population of affected individuals existed . this supposition was supported firstly by the detection of the allele in an individual in a large , independently obtained sample of the cypriot population and secondly by the identification of multiple additional affected individuals and families among cypriot patients with renal disease . the high penetrance of haematuria and wide geographical distribution of the ancestry within cyprus of these individuals and families suggests that this disease may account for a significant proportion of renal disease affecting inhabitants of the island and their descendants across the world . the current study provides a molecular explanation for these cases of c3gn . we did not detect this specific cfhr5 mutation in 36 other sporadic cases from france and the uk ( data not shown ). although mutations in other complement regulatory genes and / or the presence of c3nef have been identified in c3gn , in the majority of cases no molecular cause has yet been identified . 13 our data suggest that other genetic mechanisms impairing cfhr5 function may be present in these individuals . in summary , we describe a novel genetic cause for c3gn which is endemic in cyprus and provide direct evidence that cfhr5 plays an important physiological role in the processing of complement within the kidney ( fig3 c ). the inventors have identified the cfhr5 mutation in over 100 people from across cyprus , 90 % of whom have evidence of kidney disease . they have noted that the disease recurs following a transplant . this proves that the disease results from a circulating abnormality and thus implies that therapeutic targeting of the circulation to increase the level of functional cfhr5 is likely to be of benefit . plasma exchange resulted in immediate cessation of macroscopic haematuria and reduction in serum creatinine . this was associated with a relative reduction in levels of circulating mutant protein ( assessed by western blotting ) and suggests that manipulating activity of complement in the circulation may be of therapeutic benefit . 1 . zarkadis i k , mastellos d , lambris j d : phylogenetic aspects of the complement system . dev comp immunol 2001 ; 25 : 745 - 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