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JMG Online First, published on September 26, 2014 as 10.1136/jmedgenet-2014-102498 Phenotypes

ORIGINAL ARTICLE

Factors determining penetrance in familial atypical haemolytic uraemic syndrome Francis H Sansbury,1,2,3 Heather J Cordell,4 Coralie Bingham,2,5 Gilly Bromilow,1 Anthony Nicholls,5 Roy Powell,6 Bev Shields,2 Lucy Smyth,5 Paul Warwicker,7 Lisa Strain,8 Valerie Wilson,8 Judith A Goodship,4 Timothy H J Goodship,4 Peter D Turnpenny1,2 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jmedgenet-2014-102498). For numbered affiliations see end of article. Correspondence to Professor Tim Goodship, Institute of Genetic Medicine, Newcastle University, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK; [email protected] FHS, HJC, THJG and PDT contributed equally. Received 27 April 2014 Revised 14 August 2014 Accepted 27 August 2014

ABSTRACT Background Inherited abnormalities of complement are found in ∼60% of patients with atypical haemolytic uraemic syndrome (aHUS). Such abnormalities are not fully penetrant. In this study, we have estimated the penetrance of the disease in three families with a CFH mutation (c.3643C>G; p. Arg1215Gly) in whom a common lineage is probable. 25 individuals have been affected with aHUS with three peaks of incidence—early childhood (n=6), early adulthood (n=11) and late adulthood (n=8). Eighteen individuals who have not developed aHUS carry the mutation. Methods We estimated penetrance at the ages of 4, 27, 60 and 70 years as both a binary and a survival trait using MLINK and Mendel. We genotyped susceptibility factors in CFH, CD46 and CFHR1 in affected and unaffected carriers. Results and Conclusions We found that the estimates of penetrance at the age of 4 years ranged from G; p. Arg1215Gly) in two large families from the southwest of England. Since 1998, additional members of these families have been affected by the disease and unaffected individuals have been screened. We have also identified two other families in the same geographical area with the same mutation. We have used the information derived from the extensive screening undertaken in these families to estimate the penetrance of the mutation and genotyped the CD46, CFH and CFHR1 SNPs that

define the risk haplotypes in both affected and unaffected carriers.

METHODS Family history and pedigrees Family A was first described in 1978 by Edelsten and Tuck.6 At that time, there were four affected individuals in one family. Warwicker in 1998 described a further three affected individuals in family A. At the same time, he identified another family (family B) living in close proximity to family A with four affected individuals.5 Subsequently, we identified another family (family C) in the same area with a single affected individual carrying the same mutation. The pedigree for family A from 1978 (n=32) is shown in figure 1A, that from 1998 (n=53) in figure 1B and the current pedigree (n=167) is shown in figure 1C. The pedigree for family B from 1998 (n=15) is shown in figure 2A and the current pedigree (n=37) is shown in figure 2B. The current pedigree for family C is shown in figure 3 (n=22). We have not been able to establish a common lineage for families A, B and C, but members of the families live in close proximity in the county of Devon in England. For the purposes of analysing penetrance we considered them as three separate families, but in all likelihood they actually comprise a single family. There are 226 individuals within the three current pedigrees. For 150 of these individuals (family A, 124; family B, 20; family C, 6) further information was available as to whether they were affected by aHUS, whether CFH c.3643C>G; p. Arg1215Gly is present on screening, what if they have not been screened for CFH c.3643C>G; p. Arg1215Gly the risk is of them carrying this variant, what if they were affected by aHUS the age of diagnosis was, what if they are alive their current age is and what if they have deceased their age was, at the time of death (see online supplementary table S1). A total of 52 unaffected individuals within the families have been screened for CFH c.3643C>G; p. Arg1215Gly using direct fluorescent sequencing. Of these, 18 have been found to carry the mutation. One individual who had predictive testing, subsequently developed aHUS and was maintained on dialysis until the time of his death. One of the 18 unaffected confirmed mutation carriers died aged 94 years. The ages of the living unaffected mutation carriers ranges from 5 to 79

Sansbury FH, et al. J Med2014. Genet 2014;0:1–9. doi:10.1136/jmedgenet-2014-102498 Copyright Article author (or their employer) Produced by BMJ Publishing Group Ltd under licence.

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Sansbury FH, et al. J Med Genet 2014;0:1–9. doi:10.1136/jmedgenet-2014-102498

Figure 1

The pedigree of family A in (A) 1978, (B) 1998 and (C) 2012. The age at first presentation (in years) is given under the identifier for each affected family member.

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Phenotypes years. There are five obligate carriers within the pedigree. There are 45 individuals at a formal risk of 50% and 25 individuals at a formal risk of 25%. The study was approved by the Northern and Yorkshire Multi-Centre Research Ethics Committee, and informed consent obtained.

Mutation screening, genotyping and factor H autoantibodies

Figure 2 The pedigree of family B in (A) 1998 and (B) 2012. The age at first presentation (in years) is given under the identifier for each affected family member.

Screening for CFH c.3643C>G; p. Arg1215Gly was undertaken using direct fluorescent sequencing. Mutation screening of CD46, CFI, CFB, C3 and THBD was undertaken using direct fluorescent sequencing as described previously.7–11 Screening for genomic disorders affecting CFH, CFHR1, CFHR2, CFHR3 and CFHR5 was undertaken using multiplex ligation-dependent probe amplification12 (MLPA) using a kit from MRC Holland (www.mlpa.com) (SALSA MLPA kit P236-A1 ARMD). Copy number of CFHR3 and CFHR1 was derived from the same assay. The CD46 SNPs (−652A>G (rs2796267), −366A>G (rs2796268), IVS9 −78G>A (rs1962149), IVS12 +638G>A (rs859705) and c.4070T>C (rs7144)) which define the at-risk CD46GGAAC haplotype and the CFH SNPs −331C>T (rs3753394), c.184G>A; p.Val62Ile (rs800292), c.1204T>C p.Tyr402His (rs1061170), c.2016A>G; p.Gln672Gln (rs3753396), IVS15 −543G>A intron 15 (rs1410996), c.2808G>T; p.Glu936Asp (rs1065489)) which define the protective CFHCATAAG (known as CFH-H2) and at-risk CFHTGTGGT (known as CFH-H3) haplotypes were genotyped using direct sequencing. A polymorphic variant of CFHR1 has been described where the two alleles are denoted as CFHR1*A and CFHR1*B.13 The novel CFHR1*B allele differs from CFHR1*A by three nucleotides in exon 4 (at positions c.469, c.475 and c.523). These differences result in three amino acid changes, His157Tyr, Leu159Val and Glu175Gln in factor H related protein 1. The three nucleotide differences are the same as those that distinguish CFHR1 exon 4 from CFH exon 21 which suggests that CFHR1*B has arisen through gene conversion. The allele frequency of CFHR1*B is significantly increased in patients with aHUS particularly in those patients who are homozygous for CFHR1*B (B/B).13 These three SNPs were therefore genotyped by direct sequencing. Screening for factor H autoantibodies was undertaken using ELISA as described previously.14

Statistical analysis

Figure 3 The pedigree of family C in 2012. The age at first presentation (in years) is given under the identifier for each affected family member. Sansbury FH, et al. J Med Genet 2014;0:1–9. doi:10.1136/jmedgenet-2014-102498

We estimated penetrance using the software programmes MLINK15 16 (as implemented in the FASTLINK17 package) and Mendel.18 For analysis in MLINK, we assumed a dominant model with disease allele frequency 0.0001 and used the approach proposed by Schaid et al19 to perform a manual line-search for the penetrance estimates that maximise the retrospective likelihood (ie, that maximise the probability of the genotypes at the CFH mutation of all pedigree members, given their (binary) disease phenotypes). The advantage of this approach is that it implicitly accounts for ascertainment by conditioning on the event that caused the data to be obtained, without having to specify the ascertainment process (or the ‘proband’ individual that caused the pedigree to be ascertained) implicitly. Conditioning on the event that caused the data to be obtained is necessary in order for likelihoods to yield consistent estimates. For estimation of penetrance by ages 4, 27, 60 and 70 years, respectively, we considered individuals who had been diagnosed with aHUS by that age as ‘affected’, and individuals who were alive but had not been diagnosed with aHUS by that age as ‘unaffected’. 3

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Phenotypes For analysis in Mendel, we assumed a dominant model with disease allele frequency 0.0001. By default, Mendel fits a likelihood based on the joint probability of the genotypes at the CFH mutation of all pedigree members together with their disease phenotypes. We made use of Mendel’s facility for accounting for ascertainment through specifying ‘probands’ through which the family could be assumed to have been ascertained (rather than by conditioning on the phenotypes of all individuals, as in MLINK). We specified one individual from each of the three pedigrees as the index case or ‘proband’ (to be conditioned on). Estimation of penetrance for disease status considered as a binary trait was then carried out using the same definitions of ‘affected’ and ‘unaffected’ as for MLINK above, with penetrance modelled within Mendel via a generalised linear model using a binomial distribution with a logit link function. We also repeated the analysis with disease allele frequency estimated by Mendel (rather than fixed at 0.0001), but this made very little difference to the final results (data not shown). Additionally, we used Mendel to fit a parametric survival analysis model to the ages of onset of disease (assuming exponentially distributed failure times), while allowing for censoring. This analysis has the advantage of allowing information from unaffected individuals who had been alive for only a part of a period under study to contribute to the final estimate, unlike the binary trait analysis which ignores information from such individuals (and might, therefore, be expected to overestimate penetrance, particularly at older ages). In addition to calculating the penetrance of the CFH mutation, we also used Mendel to calculate (1) whether any significant effect on penetrance was conferred by the CD46GGAAC haplotype, while controlling for the effect of the CFH mutation; (2) the effect in CFH mutation carriers of alleles at CFHR1 exon 4 (CFHR1*A and CFHR1*B) present on the chromosome not carrying the CFH mutation; (3) the effect in CFH mutation carriers of CFH haplotypes (CFH-H2 and CFH-H3) on the chromosome not carrying the CFH mutation. These analyses were carried out by manually inferring haplotypes (where necessary) and then coding the resulting locus as a multiallelic marker for analysis in Mendel. The models were compared through a sequence of nested likelihood ratio tests, comparing a null model (in which the allele/haplotype of interest has no effect on penetrance) with an alternative model (in which the allele/haplotype of interest does have effect on penetrance) based on the likelihoods for these models as calculated by Mendel.

Figure 4 Age of first presentation of atypical haemolytic uraemic syndrome (aHUS) in the 25 affected individuals. 60 and 70 years) at which we estimated penetrance. Of the 25 affected individuals, 16 were male and 9 female. Eleven (42%) died at first presentation.

Estimated penetrance of aHUS in individuals carrying CFH c.3643C>G; p.Arg 1215Gly Table 1 shows the penetrance at the ages of 4, 27, 60 and 70 years estimated as a survival trait using Mendel, and as a binary trait using both Mendel and MLINK. The highest estimate at all four ages (4, penetrance=0.10; 27, penetrance=0.29; 60, penetrance=0.51 and 70, penetrance=0.64) was when estimated as a binary trait using Mendel.

Mutation screening, genotyping and factor H autoantibodies

Twenty-five individuals have been affected by aHUS. Of these, we were able to find clinical information on seven that would fully satisfy the diagnostic criteria proposed by the UK aHUS Rare Disease Group (http://rarerenal.org/clinician-information/ haemolytic-uraemic-syndrome-atypical-ahus-clinician-information/). For the remainder, there was sufficient information available from published reports and family history to justify the diagnosis. In no case was pregnancy a precipitating factor. CFH mutation screening was undertaken in nine of the 25 affected individuals and in all, confirmed the presence of c.3643C>G; p. Arg1215Gly. There are five affected individuals alive and in all, c.3643C>G; p. Arg1215Gly was present on screening. There were three peaks of incidence (figure 4). Six individuals were affected between birth and the age of 3 years, 11 between the ages of 15 and 27 years and eight between 49 and 79 years. The age distribution determined the ages (4, 27,

We screened all known aHUS genes in seven affected individuals (family A 3;34, 4:37, 5:7, 6:12, 6:18, family B 3:5 and family C 3:2) by both sequencing and MLPA, and did not find any additional mutations. We examined four susceptibility ‘traits’ in affected and unaffected mutation carriers: CD46 haplotype, CFH haplotype, CFHR1/3 copy number and CFHR1*A/B. The genotyping results for CFH and CD46 in nine affected and 18 unaffected carriers are shown in tables 2 and 3. There are two major CD46 haplotypes CD46GGAAC (at risk) and CD46AAGGT. In control populations, the frequency of these two haplotypes has been reported to be 0.21–0.25 and 0.49– 0.54, respectively.4 20 There are also two minor CD46 haplotypes CD46GAGGT and CD46AGAAC with reported frequencies in control populations of 0.10–0.12 and 0.09–0.10, respectively.4 20 Of the nine affected individuals there were five who were CD46GGAAC homozygous and two CD46AAGGT homozygous. Using the information from those individuals who were homozygous for the two major CD46 haplotypes and the pedigree for family A, we were able to infer that 3:34 was CD46GGAAC/AAGGT. We were not able to categorically infer that 5:7 was CD46GGAAC/AAGGT but because of the above frequencies and the haplotypes in close family members, we assumed this for the analysis. Of the 18 unaffected carriers, there were seven who were CD46GGAAC homozygous and two CD46AAGGT homozygous. There were three who were CD46GGAAC/AGAAC. Using again the information from those individuals who were homozygous for either CD46GGAAC or CD46AAGGT we were able to infer that the remaining six individuals were all CD46GGAAC/AAGGT. The aHUS at-risk CD46GGAAC haplotype was present on 12/18 (67%) alleles in affected carriers and 23/36 (64%) in unaffected carriers (table 4). There was no significant effect of the CD46GGAAC haplotype on the risk of developing aHUS when analysed using Mendel both as a binary trait at the ages of 4 years ( p=0.891), 27 years ( p=0.975), 60 years (p=0.840) and 70 years ( p=0.700), and as a survival trait ( p=0.937). An unaffected individual 6:10 in pedigree was A was homozygous for all CFH genotypes from which we inferred that c.3643C>G; p. Arg1215Gly was present on the TGTAAG

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RESULTS Demography

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Phenotypes Table 1

Estimated penetrance of aHUS in individuals carrying CFH c.3643C>G

Method/software

Penetrance by age 4 years (95% CI)*

Penetrance by age 27 years (95% CI)*

Penetrance by age 60 years (95% CI)*

Penetrance by age 70 years (95% CI)*

MLINK (binary trait) Mendel (binary trait) Mendel (survival trait)

G; p. Arg1215Gly in affected and unaffected carriers. Of note, six of the 18 unaffected individuals carried the protective H2 haplotype compared with none of the nine affected individuals. Four of the affected individuals carried the risk H3 haplotype compared with one of the 18 unaffected individuals. The effect of the CFH haplotype on the allele not carrying c.3643C>G; p. Arg1215Gly on the risk of developing aHUS was analysed using Mendel both as a binary trait at the ages of 4, 27, 60 and 70 years, and as a survival trait. As a binary trait—by the age of 4 years the H2 haplotype lowered the odds of disease (OR=0.090), while H3 increased odds of disease (OR=1.920), ( p=0.00466 for a 2df test of the model where H2 and H3 are

allowed to influence the odds of disease compared with a model where they do not have any effect); by the age of 27 years, the H2 haplotype lowered the odds of disease (OR=9.15e-11), while H3 increased odds of disease (OR=13.295), (p=0.000114 for a test of the model where H2 and H3 are allowed to influence the odds of disease compared with a model where they do not have any effect); by the age of 60 years, H2 lowered odds of disease (OR=1.48e-07), while H3 also lowered odds of disease (OR=0.0373), (p=0.0167 for a test of the model where H2 and H3 are allowed to influence the odds of disease compared with a model where they do not have any effect); by the age of 70 years, H2 lowered the odds of disease (OR=5.95e-07) while H3 also lowered odds of disease (OR=0.263), ( p=0.134 for a test of the model where H2 and H3 are allowed to influence the odds of disease compared with a model where they do not have any effect). Although these

Table 2 CD46 genotypes in affected and unaffected carriers of c.3643C>G; p. Arg1215Gly Family Affected A A A A A B A C A Unaffected A A A A A A A A A A A A A A A A A A

ID

CD46 −652A>G (rs2796267)

CD46 −366A>G (rs2796268)

CD46 IVS9 −78G>A (rs1962149)

CD46 IV12 +638G>A (rs859705)

CD46 c.4070T>C (rs7144)

6:12 6:11 6:18 4:37 5:7 3:5 3:34 3:2 4:12

GG GG AA GG GA GG GA AA GG

GG GG AA GG GA GG GA AA GG

AA AA GG AA GA AA GA GG AA

AA AA GG AA GA AA GA GG AA

CC CC TT CC TC CC TC TT CC

5:43 6:10 6:9 5:35 5:34 5:27 5:19 5:14 5:24 5:16 5:13 5:3 4:35 4:21 4:18 4:17 4:16 3:16

GA GG GA AA AA GA GG GA GA GG GA GA GG GG GA GG GA GG

GG GG GA AA AA GA GG GG GA GG GG GA GG GG GA GG GA GG

AA AA GA GG GG GA AA AA GA AA AA GA AA AA GA AA GA AA

AA AA GA GG GG GA AA AA GA AA AA GA AA AA GA AA GA AA

CC CC TC TT TT TC CC CC TC CC CC TC CC CC TC CC TC CC

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Phenotypes Table 3 CFH genotyping, CFHR1 copy number, CFHR3 copy number and CFHR1 exon 4 allele

Family

ID

Affected A 6:12 A 6:11 A 6:18 A 4:37 A 5:7 B 3:5 A 3:34 C 3:2 A 4:12 Unaffected A 5:43 A 6:10 A 6:9 A 5:35 A 5:34 A 5:27 A 5:19 A 5:14 A 5:24 A 5:16 A 5:13 A 5:3 A 4:35 A 4:21 A 4:18 A 4:17 A 4:16 A 3:16

CFH −331C>T (rs3753394)

CFH c.184G>A Val62Ile

CFH c.1204C>T His402Tyr (rs1061170)

CFH c.2016A>G Gln672Gln (rs3753396)

CFH IVS15 −543G>A (rs1410966)

CFH c.2808G>T Glu936Asp (rs1065489)

CFHR1 copy number

CFHR3 copy number

CFHR1 exon 4

TT TT TT CT TT CT CT CT CT

GG GG GG GG GG GG GG GG GG

TT TT TT CT TT CT TT TT CT

AG AG AG AA AG AA AA AG AA

GA GA GA GA GA GA GA GA GA

GT GT GT GG GT GG GG GT GG

1 1 1 1 1 1 1 1 n/a

1 1 1 1 1 1 1 1 n/a

CFHR1*B CFHR1*B CFHR1*B CFHR1*A CFHR1*B CFHR1*A CFHR1*A CFHR1*B n/a

TT TT TT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT

N/A GG GG GA GG GA GG GG GA GA GG GG GG GA GG GA GG GG

TT TT TT TT TT TT CT CT TT TT CT CT CT TT CT TT CT CT

AA AA AG AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA

AA AA GA AA AA AA GA GA AA AA GA GA GA AA GA AA GA GA

GG GG GT GG GG GG GG GG GG GG GG GG GG GG GG GG GG GG

0 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1

0 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1

CFHR1*B CFHR1*B CFHR1*B CFHR1*A CFHR1*A CFHR1*B CFHR1*B CFHR1*A CFHR1*A CFHR1*A CFHR1*B CFHR1*A CFHR1*B CFHR1*A CFHR1*A

N/A, not available.

ORs seem extreme, the (relatively) less extreme p values suggest that the CIs for these ORs (unfortunately not available from Mendel due to small sample issues) are likely to be very wide. Analysed as a survival trait, H2 increased the mean age of disease development by 15.75 years, while H3 increased the mean age of disease development by 2.22 years, p=0.000487 for a test of the model where H2 and H3 are allowed to influence the mean age of disease development compared with a model where they are not. The apparent contradiction between the effect of H3 seen here in the survival trait analysis (where it increases the mean age of disease development) compared with its effect at ages 4 years and 27 years (where it increases the odds of disease) may be due to it having a strong predisposing effect at younger ages but only a negligible effect at older ages; by definition, the survival analysis tries to model the effect of H3 over the entire age range. Table 3 shows the CFHR3 and CFHR1 copy number in both the affected and unaffected carriers. There are at least three mutation carriers (Family A 5:43, 6:10 and 5:34) who have zero copies of both CFHR1 and CFHR3 from which we infer that the chromosome carrying CFH c.3643C>G; p. Arg1215Gly has zero copies of CFHR1 and CFHR3. On the other allele, there was one copy of both CFHR1 and CFHR3 in 8/8 affected carriers and 15/18 unaffected carriers. In the eight affected, there were five with the risk CFHR1*B allele and three with the CFHR1*A allele. In the 15/18 unaffected carriers,

there were seven with the risk CFHR1*B allele and eight with the CFHR1*A allele. The effect of CFHR1 exon 4 (CFHR1*A and CFHR1*B) on the allele not carrying CFH c.3643C>G; p. Arg1215Gly on the risk of developing aHUS was analysed using Mendel both as a binary trait at the ages of 4, 27, 60 and 70 years, and as a survival trait. As a binary trait, CFHR1*A reduced the odds of disease by the age of 4 years compared with CFHR1*B (OR=2.21e-06, p=0.0061) and also reduced the odds of disease by the age of 27 years compared with CFHR1*B (OR=0.076, p=0.013). Although these ORs seem extreme, the (relatively) less extreme p values suggest that the CIs for these ORs (unfortunately not available from Mendel due to small sample issues) are likely to be very wide. There was no significant effect on the odds of disease development by the age of 60 years (OR=0.144, p=0.087) or 70 years (OR=0.301, p=0.317). When analysed as a survival trait, there was no significant increase in the mean age of development for CFHR1*A compared with CFHR1*B (CFHR1*A increased mean age of disease development by 2.92 years compared with CFHR1*B, p=0.123). It is of note that the four affected carriers and the one unaffected carrier who carry the risk H3 CFH haplotype also carry the CFHR1*B allele. Thus, in these five individuals, CFH H3 and CFHR1*B are on the same chromosome. Likewise in both groups, the CFH haplotype was always associated with only

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Phenotypes Table 4 Inferred CD46 and CFH haplotypes Family Affected A A A A A B A C A Unaffected A A A A A A A A A A A A A A A A A A

ID

CD46 haplotype

CFH haplotype

6:12 6:11 6:18 4:37 5:7 3:5 3:34 3:2 4:12

GGAAC/GGAAC GGAAC/GGAAC AAGGT/AAGGT GGAAC/GGAAC GGAAC/AAGGT GGAAC/GGAAC GGAAC/AAGGT AAGGT/AAGGT GGAAC/GGAAC

TGTAAG/TGTGGT TGTAAG/TGTGGT TGTAAG/TGTGGT TGTAAG/CGCAGG TGTAAG/TGTGGT TGTAAG/CGCAGG TGTAAG/CGTAGG TGTAAG/CGTGGT TGTAAG/CGCAGG

5:43 6:10 6:9 5:35 5:34 5:27 5:19 5:14 5:24 5:16 5:13 5:3 4:35 4:21 4:18 4:17 4:16 3:16

GGAAC/AGAAC GGAAC/GGAAC GGAAC/AAGGT AAGGT/AAGGT AAGGT/AAGGT AAGGT/GGAAC GGAAC/GGAAC GGAAC/AGAAC GGAAC/AAGGT GGAAC/GGAAC GGAAC/AGAAC GGAAC/AAGGT GGAAC/GGAAC GGAAC/GGAAC GGAAC/AAGGT GGAAC/GGAAC GGAAC/AAGGT GGAAC/GGAAC

TGTAAG/TGTAAG TGTAAG/TGTAAG TGTAAG/TGTGGT TGTAAG/CATAAG TGTAAG/CGTAAG TGTAAG/CATAAG TGTAAG/CGCAGG TGTAAG/CGCAGG TGTAAG/CATAAG TGTAAG/CATAAG TGTAAG/CGCAGG TGTAAG/CGCAGG TGTAAG/CGCAGG TGTAAG/CATAAG TGTAAG/CGCAGG TGTAAG/CATAAG TGTAAG/CGCAGG TGTAAG/CGCAGG

The risk CD46 and CFH haplotypes are shaded. The protective CFH haplotype is underlined.

one CFHR1 allele (table 6). CFH H1 was always associated with CFHR1*A and CFH H2 was always associated with CFHR1*B. We did not find factor H autoantibodies in any affected or unaffected carrier.

DISCUSSION In this study, we have examined the penetrance of aHUS in three families with multiple affected individuals carrying the same CFH mutation (c.3643C>G; p. Arg1215Gly). This mutation has been shown to decrease binding to both C3b/C3d and heparin.21–24 The close geographical proximity of these families

suggests a common lineage. This ‘extended’ family is one of the three that we originally studied to establish linkage at 1q.32.5 Mutation screening at that time revealed the first aHUS-associated CFH mutation. Since then, it has been shown that ∼30% of patients with aHUS will have a CFH mutation.1 A family history of aHUS is found in ∼10% of patients with a CFH mutation. However, mutation screening of unaffected relatives of those individuals without a family history of aHUS shows that most affected individuals have inherited the mutation. Once an unaffected family member has been found to be a carrier, the inevitable question raised is what is the risk of them developing the disease. Previous studies have addressed this question. Caprioli et al reported a penetrance of 59% for individuals carrying a CFH mutation.2 Sullivan et al studied affected individuals and their relatives. The penetrance in 18 affected index cases with a CFH mutation was 67% by the age of 40 years and 100% by the age of 65 years.25 Two of the index cases had a further family member who was affected by the disease and both were found to carry the same CFH mutation. Seventeen unaffected relatives of the remaining sporadic cases were found to carry the same CFH mutation as the index case. In the 19 mutation-positive relatives, the penetrance at age 40 years was 6%. In this study, we estimated penetrance at the ages of 4, 27, 60 and 70 years as a survival trait using Mendel and as a binary trait using both Mendel and MLINK. We found that the estimates of penetrance at the age of 4 years ranged from G; p. Arg1215Gly in affected (n=9) and unaffected (n=18) carriers

H1 H2 H3 H4a H4b H5 H6 H7 H8

CFH −331C>T (rs3753394)

CFH c.184G>A Val62Ile

CFH c.1204C>T His402Tyr (rs1061170)

CFH c.2016A>G Gln672Gln (rs3753396)

CFH IVS15 −543G>A (rs1410966)

CFH c.2808G>T Glu936Asp (rs1065489)

C C T C T T C T C

G A G G G G G G G

C T T T T C T T T

A A G A A A A A G

G A G A A G G G G

G G T G G G G G T

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Affected

Unaffected

3 0 4 0 0

8 6 1 1 2

1

0

1

0

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Phenotypes Table 6 The CFH haplotype and the CFHR1 allele (on the non-mutant chromosome) in both affected and unaffected carriers CFH haplotype

CFHR1 allele

Number of alleles

H1 H2 H3 H4a H4b H5 H6 H7 H8

CFHR1*A CFHR1*B CFHR1*B ΔCFHR3-CFHR1 ΔCFHR3-CFHR1

10 6 5 1 1

CFHR1*A

1

CFHR1*B

1

Barrow et al27 allowed appropriately in their analyses for the non-independence between observations from the same pedigree due to familial relationships, an issue that becomes increasingly important when analysing a small number of large pedigrees, as done here. de Snoo et al28 estimated carrier risk in first-degree relatives from their relationship to known carriers and the age and melanoma status of that person and their relatives using an adapted version of Mendel, similar to the method used here. The reasons why non-penetrance is a feature of aHUS are complex. There are thought to be at least three factors. First, in most patients, there is a rare genetic variant (mutation). Second, there is also a trigger. Infection and pregnancy are frequently described triggers.1 29 Third, a further genetic variant (modifier) can increase the risk of developing the disease. This can be either an additional mutation30 and/or a common at-risk genetic variant. Common risk genetic variants (SNPs and haplotype blocks) in CFH, CD46 and CFHR1 have been shown to act as susceptibility factors for the development of the disease.4 13 31 Caprioli et al32 first reported this for CFH and EsparzaGordillo et al33 for CD46 (at risk CD46GGAAC). Subsequently, protective (H2) and at risk (H3) CFH haplotypes have been reported.34 35 The SNPs that determine these haplotypes are show in table 5. A polymorphic variant of CFHR1 has been described where the two alleles are denoted as CFHR1*A and CFHR1*B.13 The novel CFHR1*B allele differs from CFHR1*A by three nucleotides in exon 4 (at positions c.469, c.475 and c.523). These differences result in three amino acid changes, His157Tyr, Leu159Val and Glu175Gln in factor H related protein 1. The allele frequency of CFHR1*B is significantly increased in patients with aHUS particularly those who are homozygous for CFHR1*B.13 We examined the risk of developing aHUS using Mendel as both a binary trait at the ages of 27, 60 and 70 years, and as a survival trait. We did not find any significant effect of the CD46GGAAC haplotype on the risk of developing aHUS. However, the CFH haplotype of the non-mutated copy of the gene had a significant effect on the risk of developing aHUS. The results suggested that the protective H2 haplotype had the most significant effect in keeping with 6/18 unaffected individuals carrying the protective H2 haplotype versus 0/9 affected. We also found that the two CFHR1 exon 4 alleles (CFHR1*A, protective vs CFHR1*B, at risk) were associated with the risk of developing aHUS, but this relationship was not as strong as for the CFH haplotypes. How might the CFH haplotype on the non-mutant allele be modulating the penetrance of the disease? Factor H levels are known to show a high degree of heritability.36 A recent study has shown that the CFHR3/1 deletion (CNP147) is strongly correlated with factor H concentrations, with higher factor H 8

concentrations being associated with the deletion.37 In the family we describe here, the mutant allele also carries the CFHR3/1 deletion and, therefore, the concentration of mutant factor H may be higher, This alone, however, would not account for non-penetrance, as both the affected and unaffected carriers possess the same allele. That the concentration of mutant factor H might be higher could, however, be a factor that explains why in this family so many individuals who carry the mutation have been affected. How might the H2 and H3 alleles on the non-mutated allele be respectively protective and risks? There is no information as to whether the H2 haplotype modulates factor H levels. However, the H2 allele encodes isoleucine at position 62, whereas, the H3 encodes valine. It has been shown that the Ile62 factor H variant binds better to C3b than the Val62 variant and also has increased cofactor activity.38 In this family, we found no evidence that CD46 haplotypes modify disease penetrance. This is at odds with previous reports for both cohorts of UK patients4 39 and individual families from Spain.40 This suggests that modifiers identified in cohorts and families may not be universally applicable to unaffected mutation carriers. We would, therefore, urge caution in using the data derived from CD46 and CFH genotyping to provide an individualised risk for the development of aHUS in unaffected carriers. That all the affected and unaffected carriers in this family carry either one or zero copies of CFHR3 and CFHR1 is unusual given that the reported allele frequency of the CFHR3/ 1 deletion in the UK is ∼18%.41 Thus, the observations derived from this family may also not be universally applicable. In conclusion, we have determined age-related penetrance in three families with a probable common lineage. Interpretation is compounded by the peak of incidence later in life which leaves many of the unaffected carriers still at risk. Can the information on penetrance that we have derived from this family be used when counselling the relatives of other individuals with aHUS who are found to have a CFH mutation? Screening of families where there is only one affected individual with a C terminal pathogenic factor H mutation often reveals unaffected family members carrying the same variant. Thus, we think that the estimate of 64% penetrance by the age of 70 years derived from this extended family should be given as a ‘worst case’ risk. Understanding why the penetrance of the disease is higher in families such as the one we describe here may provide information useful for the development of further therapies for aHUS. Author affiliations 1 Peninsula Clinical Genetics Service, Royal Devon & Exeter NHS Foundation Trust, Royal Devon & Exeter Hospital (Heavitree), Exeter, UK 2 University of Exeter Medical School, University of Exeter, Exeter, UK 3 Bristol Clinical Genetics Service, University Hospitals Bristol NHS Foundation Trust, Clinical Genetics, St. Michael’s Hospital, Bristol, UK 4 Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK 5 Department of Renal Medicine, Royal Devon & Exeter NHS Foundation Trust, Royal Devon & Exeter Hospital (Wonford), Exeter, UK 6 Research Design Service South West, Royal Devon & Exeter NHS Foundation Trust, Royal Devon & Exeter Hospital (Wonford), Exeter, UK 7 Lister Renal Units, East and North Hertfordshire NHS Trust, Stevenage, UK 8 Northern Molecular Genetics Service, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK Acknowledgements Funding for this study was provided by the UK Medical Research Council (G0701325). FHS would like to thank the National Institute for Health Research for an Academic Clinical Fellowship. The research leading to these results has received funding from the European Union’s Seventh Framework Programme (FP7/2007–2013) under grant agreement number 305608 (EURenOmics). Contributors All authors contributed to the design of the study and the analysis/ interpretation of the results. Competing interests None. Sansbury FH, et al. J Med Genet 2014;0:1–9. doi:10.1136/jmedgenet-2014-102498

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Phenotypes Ethics approval Northern and Yorkshire Multi-Centre Research Ethics Committee. Provenance and peer review Not commissioned; externally peer reviewed.

REFERENCES 1 2

3 4

5

6 7

8

9

10

11

12

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15 16 17 18 19

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Noris M, Remuzzi G. Atypical hemolytic-uremic syndrome. N Engl J Med 2009;361:1676–87. Caprioli J, Noris M, Brioschi S, Pianetti G, Castelletti F, Bettinaglio P, Mele C, Bresin E, Cassis L, Gamba S, Porrati F, Bucchioni S, Monteferrante G, Fang CJ, Liszewski MK, Kavanagh D, Atkinson JP, Remuzzi G. Genetics of HUS: the impact of MCP, CFH and IF mutations on clinical presentation, response to treatment, and outcome. Blood 2006;108:1267–79. Rodriguez de Cordoba S. aHUS: a disorder with many risk factors. Blood 2010;115:158–60. Ermini L, Goodship THJ, Strain L, Weale ME, Sacks SH, Cordell HJ, Fremeaux-Bacchi V, Sheerin NS. Common genetic variants in complement genes other than CFH, CD46 and the CFHRs are not associated with aHUS. Mol Immunol 2012;49:640–8. Warwicker P, Goodship THJ, Donne RL, Pirson Y, Nicholls A, Ward RM, Goodship JA. Genetic studies into inherited and sporadic haemolytic uraemic syndrome. Kidney Int 1998;53:836–44. Edelsten AD, Tuck S. Familial haemolytic uraemic syndrome. Arch Dis Child 1978;53:255–6. Richards A, Buddles MR, Donne RL, Kaplan BS, Kirk E, Venning MC, Tielemans CL, Goodship JA, Goodship THJ. Factor H mutations in hemolytic uremic syndrome cluster in exons 18–20, a domain important for host cell recognition. Am J Hum Genet 2001;68:485–90. Richards A, Kemp EJ, Liszewski MK, Goodship JA, Lampe AK, Decorte R, Muslumanoglu MH, Kavukcu S, Filler G, Pirson Y, Wen LS, Atkinson JP, Goodship THJ. Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome. Proc Natl Acad Sci USA 2003;100:12966–71. Kavanagh D, Kemp EJ, Mayland E, Winney RJ, Duffield JS, Warwick G, Richards A, Ward R, Goodship JA, Goodship TH. Mutations in complement factor I predispose to development of atypical hemolytic uremic syndrome. J Am Soc Nephrol 2005;16:2150–5. Kavanagh D, Kemp EJ, Richards A, Burgess RM, Mayland E, Goodship JA, Goodship TH. Does complement factor B have a role in the pathogenesis of atypical HUS? Mol Immunol 2006;43:856–9. Fremeaux-Bacchi V, Miller EC, Liszewski MK, Strain L, Blouin J, Brown AL, Moghal N, Kaplan BS, Weiss RA, Lhotta K, Kapur G, Mattoo T, Nivet H, Wong W, Gie S, Hurault de Ligny B, Fischbach M, Gupta R, Hauhart R, Meunier V, Loirat C, Dragon-Durey MA, Fridman WH, Janssen BJ, Goodship THJ, Atkinson JP. Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome. Blood 2008;112:4948–52. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002;30:e57. Abarrategui-Garrido C, Martinez-Barricarte R, Lopez-Trascasa M, de Cordoba SR, Sanchez-Corral P. Characterization of complement factor H-related (CFHR) proteins in plasma reveals novel genetic variations of CFHR1 associated with atypical hemolytic uremic syndrome. Blood 2009;114:4261–71. Moore I, Strain L, Pappworth I, Kavanagh D, Barlow PN, Herbert AP, Schmidt CQ, Staniforth SJ, Holmes LV, Ward R, Morgan L, Goodship THJ, Marchbank KJ. Association of factor H autoantibodies with deletions of CFHR1, CFHR3, CFHR4 and with mutations in CFH, CFI, CD46, and C3 in patients with atypical haemolytic uraemic syndrome. Blood 2010;115:379–87. Lathrop GM, Lalouel JM. Efficient computations in multilocus linkage analysis. Am J Hum Genet 1988;42:498–505. Lathrop GM, Lalouel JM, Julier C, Ott J. Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA 1984;81:3443–6. Cottingham RW Jr, Idury RM, Schaffer AA. Faster sequential genetic linkage computations. Am J Hum Genet 1993;53:252–63. Lange K, Papp JC, Sinsheimer JS, Sripracha R, Zhou H, Sobel EM. Mendel: the Swiss army knife of genetic analysis programs. Bioinformatics 2013;29:1568–70. Schaid DJ, McDonnell SK, Riska SM, Carlson EE, Thibodeau SN. Estimation of genotype relative risks from pedigree data by retrospective likelihoods. Genet Epidemiol 2010;34:287–98. Servais A, Noel LH, Roumenina LT, Le Quintrec M, Ngo S, Dragon-Durey MA, Macher MA, Zuber J, Karras A, Provot F, Moulin B, Grunfeld JP, Niaudet P, Lesavre P, Fremeaux-Bacchi V. Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies. Kidney Int 2012;82:454–64. Ferreira VP, Herbert AP, Cortes C, McKee KA, Blaum BS, Esswein ST, Uhrin D, Barlow PN, Pangburn MK, Kavanagh D. The binding of factor H to a complex of physiological polyanions and C3b on cells is impaired in atypical hemolytic uremic syndrome. J Immunol 2009;182:7009–18. Jozsi M, Heinen S, Hartmann A, Ostrowicz CW, Halbich S, Richter H, Kunert A, Licht C, Saunders RE, Perkins SJ, Zipfel PF, Skerka C. Factor H and atypical

Sansbury FH, et al. J Med Genet 2014;0:1–9. doi:10.1136/jmedgenet-2014-102498

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

hemolytic uremic syndrome: mutations in the C-terminus cause structural changes and defective recognition functions. J Am Soc Nephrol 2006;17:170–7. Lehtinen MJ, Rops AL, Isenman DE, van der Vlag J, Jokiranta TS. Mutations of factor H impair regulation of surface-bound C3b by three mechanisms in atypical hemolytic uremic syndrome. J Biol Chem 2009;284:15650–8. Herbert AP, Uhrin D, Lyon M, Pangburn MK, Barlow PN. Disease-associated sequence variations congregate in a polyanion recognition patch on human factor H revealed in three-dimensional structure. J Biol Chem 2006;281:16512–20. Sullivan M, Rybicki LA, Winter A, Hoffmann MM, Reiermann S, Linke H, Arbeiter K, Patzer L, Budde K, Hoppe B, Zeier M, Lhotta K, Bock A, Wiech T, Gaspert A, Fehr T, Woznowski M, Berisha G, Malinoc A, Goek ON, Eng C, Neumann HP. Age-related penetrance of hereditary atypical hemolytic uremic syndrome. Ann Hum Genet 2011;75:639–47. Evans DG, Shenton A, Woodward E, Lalloo F, Howell A, Maher ER. Penetrance estimates for BRCA1 and BRCA2 based on genetic testing in a Clinical Cancer Genetics service setting: risks of breast/ovarian cancer quoted should reflect the cancer burden in the family. BMC Cancer 2008;8:155. Barrow E, Alduaij W, Robinson L, Shenton A, Clancy T, Lalloo F, Hill J, Evans DG. Colorectal cancer in HNPCC: cumulative lifetime incidence, survival and tumour distribution. A report of 121 families with proven mutations. Clin Genet 2008;74:233–42. de Snoo FA, Bishop DT, Bergman W, van Leeuwen I, van der Drift C, van Nieuwpoort FA, Out-Luiting CJ, Vasen HF, ter Huurne JA, Frants RR, Willemze R, Breuning MH, Gruis NA. Increased risk of cancer other than melanoma in CDKN2A founder mutation ( p16-Leiden)-positive melanoma families. Clin Cancer Res 2008;14:7151–7. Fakhouri F, Roumenina L, Provot F, Sallee M, Caillard S, Couzi L, Essig M, Ribes D, Dragon-Durey MA, Bridoux F, Rondeau E, Fremeaux-Bacchi V. Pregnancy-associated hemolytic uremic syndrome revisited in the era of complement gene mutations. J Am Soc Nephrol 2010;21:859–67. Bresin E, Rurali E, Caprioli J, Sanchez-Corral P, Fremeaux-Bacchi V, Rodriguez de Cordoba S, Pinto S, Goodship TH, Alberti M, Ribes D, Valoti E, Remuzzi G, Noris M, European Working Party on Complement Genetics in Renal D. Combined complement gene mutations in atypical hemolytic uremic syndrome influence clinical phenotype. J Am Soc Nephrol 2013;24:475–86. Martinez-Barricarte R, Pianetti G, Gautard R, Misselwitz J, Strain L, Fremeaux-Bacchi V, Skerka C, Zipfel PF, Goodship THJ, Noris M, Remuzzi G, de Cordoba SR. The complement factor H R1210C mutation is associated with atypical hemolytic uremic syndrome. J Am Soc Nephrol 2008;19:639–46. Caprioli J, Castelletti F, Bucchioni S, Bettinaglio P, Bresin E, Pianetti G, Gamba S, Brioschi S, Daina E, Remuzzi G, Noris M. Complement factor H mutations and gene polymorphisms in haemolytic uraemic syndrome: the C-257T, the A2089G and the G2881T polymorphisms are strongly associated with the disease. Hum Mol Genet 2003;12:3385–95. Esparza-Gordillo J, Goicoechea de JE, Buil A, Carreras BL, Lopez-Trascasa M, Sanchez-Corral P, Rodriguez de CS. Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32. Hum Mol Genet 2005;14:703–12. Pickering MC, de Jorge EG, Martinez-Barricarte R, Recalde S, Garcia-Layana A, Rose KL, Moss J, Walport MJ, Cook HT, de Cordoba SR, Botto M. Spontaneous hemolytic uremic syndrome triggered by complement factor H lacking surface recognition domains. J Exp Med 2007;204:1249–56. de Cordoba SR, de Jorge EG. Translational mini-review series on complement factor H: genetics and disease associations of human complement factor H. Clin Exp Immunol 2008;151:1–13. Esparza-Gordillo J, Soria JM, Buil A, Almasy L, Blangero J, Fontcuberta J, Rodriguez de CS. Genetic and environmental factors influencing the human factor H plasma levels. Immunogenetics 2004;56:77–82. Ansari M, McKeigue PM, Skerka C, Hayward C, Rudan I, Vitart V, Polasek O, Armbrecht AM, Yates JR, Vatavuk Z, Bencic G, Kolcic I, Oostra BA, Van Duijn CM, Campbell S, Stanton CM, Huffman J, Shu X, Khan JC, Shahid H, Harding SP, Bishop PN, Deary IJ, Moore AT, Dhillon B, Rudan P, Zipfel PF, Sim RB, Hastie ND, Campbell H, Wright AF. Genetic influences on plasma CFH and CFHR1 concentrations and their role in susceptibility to age-related macular degeneration. Hum Mol Genet 2013;22:4857–69. Tortajada A, Montes T, Martinez-Barricarte R, Morgan BP, Harris CL, de Cordoba SR. The disease-protective complement factor H allotypic variant Ile62 shows increased binding affinity for C3b and enhanced cofactor activity. Hum Mol Genet 2009;18:3452–61. Fremeaux-Bacchi V, Kemp EJ, Goodship JA, Dragon-Durey MA, Strain L, Loirat C, Deng HW, Goodship TH. The development of atypical haemolytic-uraemic syndrome is influenced by susceptibility factors in factor H and membrane cofactor protein: evidence from two independent cohorts. J Med Genet 2005;42:852–6. Esparza-Gordillo J, Goicoechea de Jorge E, Garrido CA, Carreras L, Lopez-Trascasa M, Sanchez-Corral P, Rodriguez de Cordoba S. Insights into hemolytic uremic syndrome: Segregation of three independent predisposition factors in a large, multiple affected pedigree. Mol Immunol 2006;43:1769–75. Holmes LV, Strain L, Staniforth SJ, Moore I, Marchbank K, Kavanagh D, Goodship JA, Cordell HJ, Goodship TH. Determining the population frequency of the CFHR3/CFHR1 deletion at 1q32. PLoS ONE 2013;8:e60352.

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Factors determining penetrance in familial atypical haemolytic uraemic syndrome Francis H Sansbury, Heather J Cordell, Coralie Bingham, et al. J Med Genet published online September 26, 2014

doi: 10.1136/jmedgenet-2014-102498

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References

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