[Show abstract][Hide abstract] ABSTRACT: Atypical hemolytic uremic syndrome (aHUS) is a rare, possibly life-threatening disease characterized by platelet activation, hemolysis and thrombotic microangiopathy (TMA) leading to renal and other end-organ damage. We originally conducted two phase 2 studies (26 weeks and 1 year) evaluating eculizumab, a terminal complement inhibitor, in patients with progressing TMA (trial 1) and those with long duration of aHUS and chronic kidney disease (trial 2). The current analysis assessed outcomes after 2 years (median eculizumab exposure 100 and 114 weeks, respectively). At all scheduled time points, eculizumab inhibited terminal complement activity. In trial 1 with 17 patients, the platelet count was significantly improved from baseline, and hematologic normalization was achieved in 13 patients at week 26, and in 15 patients at both 1 and 2 years. The estimated glomerular filtration rate (eGFR) was significantly improved compared with baseline and year 1. In trial 2 with 20 patients, TMA event-free status was achieved by 16 patients at week 26, 17 patients at year 1, and 19 patients at year 2. Criteria for hematologic normalization were met by 18 patients at each time point. Improvement of 15 ml/min per 1.73 m(2) or more in eGFR was achieved by 1 patient at week 26, 3 patients at 1 year, and 8 patients at 2 years. The mean change in eGFR was not significant compared with baseline, week 26, or year 1. Eculizumab was well tolerated, with no new safety concerns or meningococcal infections. Thus, a 2-year analysis found that the earlier clinical benefits achieved by eculizumab treatment of aHUS were maintained at 2 years of follow-up.Kidney International advance online publication, 4 February 2015; doi:10.1038/ki.2014.423.
Kidney International 02/2015; 87(5). DOI:10.1038/ki.2014.423 · 8.52 Impact Factor
[Show abstract][Hide abstract] 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 <0.01 to 0.10, at the age of 27 years from 0.16 to 0.29, at the age of 60 years from 0.39 to 0.51 and at the age of 70 years from 0.44 to 0.64. We found that the CFH haplotype on the allele not carrying the CFH mutation had a significant effect on disease penetrance. In this family, we did not find that the CD46 haplotypes had a significant effect on penetrance.
Journal of Medical Genetics 09/2014; 51(11). DOI:10.1136/jmedgenet-2014-102498 · 5.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Urofacial syndrome (UFS) is an autosomal recessive congenital disease featuring grimacing and incomplete bladder emptying. Mutations of HPSE2, encoding heparanase 2, a heparanase 1 inhibitor, occur in UFS, but knowledge about the HPSE2 mutation spectrum is limited. Here, seven UFS kindreds with HPSE2 mutations are presented, including one with deleted asparagine 254, suggesting a role for this amino acid, which is conserved in vertebrate orthologs. HPSE2 mutations were absent in 23 non-neurogenic neurogenic bladder probands and, of 439 families with nonsyndromic vesicoureteric reflux, only one carried a putative pathogenic HPSE2 variant. Homozygous Hpse2 mutant mouse bladders contained urine more often than did wild-type organs, phenocopying human UFS. Pelvic ganglia neural cell bodies contained heparanase 1, heparanase 2, and leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2), which is mutated in certain UFS families. In conclusion, heparanase 2 is an autonomic neural protein implicated in bladder emptying, but HPSE2 variants are uncommon in urinary diseases resembling UFS
Journal of the American Society of Nephrology 08/2014; 26(4). DOI:10.1681/ASN.2013090961 · 9.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Autoantibody formation against Factor H (FH) is found in 7-10% of patients who are diagnosed with atypical haemolytic uraemic syndrome (aHUS). These autoantibodies predominately target the C-terminal cell binding recognition domain of FH and are associated with absence of FHR1. Additional autoantibodies have also been identified in association with aHUS, for example autoantibodies to Factor I. Based on this, and that there are genetic mutations in other complement regulators and activators associated with aHUS, we hypothesised that other complement regulator proteins, particularly surface bound regulators in the kidney, might be the target for autoantibody formation in aHUS. Therefore, we assayed serum derived from 89 patients in the Newcastle aHUS cohort for the presence of autoantibodies to CD46 (membrane cofactor protein, MCP), CD55 (decay accelerating factor, DAF), CD35 (complement receptor type 1, CR1; TP10) and CD59. We also assayed 100 healthy blood donors to establish the normal levels of reactivity towards these proteins in the general population. Recombinant proteins CD46 and CD55 (purified from Escherichia coli) as well as soluble CR1 (CD35) and oligomeric C4BP-CD59 (purified from eukaryotic cell media) were used in ELISA to detect high responders. False positive results were established though Western blot and flow cytometric analysis. After excluding false positive responders to bacterial proteins in the CD46 and CD55 preparations, and responses to blood group antigens in CD35, we found no significant level of patient serum IgG reactivity with CD46, CD55, CD35 or CD59 above that detected in the normal population. These results suggest that membrane anchored complement regulators are not a target for autoantibody generation in aHUS.
[Show abstract][Hide abstract] ABSTRACT: We present a case of haemolytic uraemic syndrome (HUS) triggered by Shigella flexneri. Of the Shigella species, only S. dysenteriae type 1 is said to produce Shiga toxin and consequently cause HUS. Investigation of the complement system in this patient
revealed a CD46 mutation. In individuals with mutations in complement genes incomplete penetrance of atypical HUS (aHUS) is seen, suggesting
that a trigger, such as infection, is required for disease to manifest. In an era of complement modulatory therapy for aHUS
it is important to be alert to unusual presentations of diarrhoeal-associated disease.
[Show abstract][Hide abstract] ABSTRACT: Introduction and Aims: According to classical nephrology practice, pure microscopic hematuria [MH] in early life is not considered a serious problem
and up to date diagnostic studies are not always carried out. Family urine studies to confirm or rule out a familial disease
should always be carried out early and if positive, molecular genetics should be used to study the whole family and establish
the underlying genetic defect.
Methods: During the last 30 years we have searched systematically in a homogeneous population of 650,000 people for familial MH and
some 120 such families are under study so far. Molecular genetic studies during the last 10 years have led to the correct
genetic diagnosis in 57 such families with a) 28 heterozygous COL4A3/A4 mutations and TBMN, b) 23 CFHR5 and C3 nephritis and
c) 6 families with Alport syndrome.
Results: Our most striking finding refers to the COL4A3/A4 heterozygous mutations that turn out to be the commonest cause of familial
MH and have been identified in 249 carriers in 30 families. Some of these families are very big indeed and two of these mutations,
COL4A3-G1334E and COL4A3-G871C, are particularly common. Mutation COL4A3-G1334E was found in 15 families and it accounts for
174 patients. C3 nephritis with the CFHR5 Cypriot mutation appears to be the 2nd commonest cause of familial MH with 150 carriers
in 23 families. Classical XLAS, COL4A5 Alport was identified in 3 families with 9 affected males and 8 female carriers, while
ARAS was also present in 3 additional families affecting 8 patients. Of the 249 patients with COL4A3/A4 heterozygous mutations
33 have reached ESKD and of the 150 CFHR5 patients 21 have reached ESKD.
Conclusions: Our results strengthen the great significance of the heterozygous COL4A3/A4 mutations, not really well understood until after
1996, less than 20 years ago. These mutations are common, lead to TBMN and are the commonest cause of familial MH. More importantly
however, these mutations lead much later in life to proteinuria, hypertension, CRF and ESKD and long term care is mandatory
with prompt attention to the addition of proteinuria that requires urgent treatment. Heterozygous mutations COL4A3/A4 cause
twice more patients to reach ESKD compared to classical XLAS and ARAS but fortunately at a much older age. Molecular genetics
should be used more widely for an early diagnosis of this entity and more renal attention should be given to these patients
to preserve kidney function and avoid ESKD.
[Show abstract][Hide abstract] ABSTRACT: Introduction and Aims: In hemodialysis (HD) patients low pre-HD serum sodium (SNa+) and high Na+ gradients have been associated with higher mortality. Increased pre-HD SNa+ variability has been linked to mortality, while stable SNa+ levels are associated with better survival. Here we investigated the joint relationship of SNa+ variability (expressed as standard deviation, SD) and rate of change of SNa+ (slope of SNa+) with the risk of all-cause death in incident HD patients.
Methods: We studied 20,193 incident HD patients (age 63.3±15.0 years, 59% males, 34% diabetic, relative interdialytic weight gain
(IDWG%) 2.9%±1.0%, BMI 26.9±7.5 kg/m2) from Europe (N=14,763) and the U.S. (N=5,430). During baseline (first 12 months on HD) mean SNa+, SNa+ slope and SNa+ SD were computed. Patient outcomes were noted in months 13 to 24 (follow-up). We investigated the joint effects of SNa+ and a) SNa+ variability and (b) SNa+ slopes, respectively, during baseline on probability of death during follow-up using logistic regression with smoothing spline
ANOVA models. Models were adjusted for age, gender, diabetes, IDWG%, serum albumin, phosphorus and BMI.
Results: Risk of death was lowest with SNa+ around 138 to 141 mEq/L and SNa+ SD of 0 to 2 mEq/L. Deviations from this region in any direction were associated with increased risk. The increase in mortality
risk associated with higher SNa+ variability was steepest at SNa+ levels <135 mEq/L (Fig 1). Stable SNa+ conferred the lowest risk with SNa+ levels around 139 to 142 mEq/L (Fig 2). Mortality risk progressively increased outside of this domain. Of note, over the
range of our data, SNa+ departures from this range carried a steeper risk increase than departures in SNa+ slope. Lastly, while patients with SNa+ >136 mEq/L realized their best survival probability with SNa+ slopes around zero, in patients with SNa+ <136 mEq/L more positive SNa+ slopes were always linked to better survival along the entire range of SNa+ slopes (Fig 2).
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[Show abstract][Hide abstract] ABSTRACT: Complement C3 activation is a characteristic finding in membranoproliferative GN (MPGN). This activation can be caused by immune complex deposition or an acquired or inherited defect in complement regulation. Deficiency of complement factor H has long been associated with MPGN. More recently, heterozygous genetic variants have been reported in sporadic cases of MPGN, although their functional significance has not been assessed. We describe a family with MPGN and acquired partial lipodystrophy. Although C3 nephritic factor was shown in family members with acquired partial lipodystrophy, it did not segregate with the renal phenotype. Genetic analysis revealed a novel heterozygous mutation in complement factor H (R83S) in addition to known risk polymorphisms carried by individuals with MPGN. Patients with MPGN had normal levels of factor H, and structural analysis of the mutant revealed only subtle alterations. However, functional analysis revealed profoundly reduced C3b binding, cofactor activity, and decay accelerating activity leading to loss of regulation of the alternative pathway. In summary, this family showed a confluence of common and rare functionally significant genetic risk factors causing disease. Data from our analysis of these factors highlight the role of the alternative pathway of complement in MPGN.
Journal of the American Society of Nephrology 04/2014; 25(11). DOI:10.1681/ASN.2013070732 · 9.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atypical haemolytic uraemic syndrome (aHUS) is a severe renal disease caused by deregulation of the alternative complement pathway. The RCA (Regulators of Complement Activation) gene cluster contains several genes that have been implicated in the pathogenesis of aHUS, including Complement Factor H (CFH). The gene for CFH is in close proximity to the genes encoding the 5 Complement Factor H related proteins (CFHRs). These are thought to have arisen from several large genomic duplications and thus have a very high degree of sequence identity to CFH. This homology predisposes this area to gene conversions and genomic rearrangements through non-allelic homologous recombination (NAHR) and microhomology-mediated end joining (MMEJ).
In a sporadic case of aHUS, using multiplex ligation-dependent probe amplification, we describe a de novo deletion in the RCA cluster. Sequencing the PCR product across the deletion identified the breakpoint, with a 6.3 kb deletion between CFH intron 20 and the 3′UTR of CFH. Immediately adjacent to the breakpoint was a 7-bp region of microhomology. This is predicted to result in a novel hybrid CFH/CFHR3 gene. Western blotting confirmed 2 distinct anti-fH species in the sera of the affected individual. Using specific monoclonal antibodies, the higher molecular weight band gave a signal in keeping with the predicted CFH/CFHR3 hybrid protein.
In conclusion we describe a novel deletion which occurred through MMEJ in aHUS. This produced a CFH/CFHR3 hybrid protein predicted to have impaired cell surface complement regulation, predisposing to disease. This highlights the importance of undertaking copy number variation analysis when undertaking genetic screening of aHUS patients.
[Show abstract][Hide abstract] ABSTRACT: Twenty-five members of a family from the county of Devon in England have been affected by atypical haemolytic uraemic syndrome (aHUS) associated with a CFH mutation (c.3643C>G; p.Arg1215Gly). A 65-year-old male was diagnosed with aHUS after losing a renal transplant to a thrombotic microangiopathy. Subsequent mutation screening revealed the same CFH mutation without him being knowingly related to the local kindred. We designed a study to investigate the prevalence of this mutation in the local area. In addition, we examined the diagnoses of pre-existing haemodialysis patients to determine whether other patients might unknowingly be at risk of carrying the same CFH mutation.
The Exeter Ten Thousand (EXTEND) study aims to recruit 10,000 healthy volunteers over the age of 18 years living within 25 miles of Exeter in Devon. We genotyped DNA from 4,000 EXTEND subjects for CFH c.3643C>G; p.Arg1215Gly. We reviewed the diagnoses of 294 haemodialysis patients in the Devon area and genotyped 7 patients with either end-stage renal disease of unknown aetiology, malignant hypertension or renovascular disease.
CFH c.3643C>G; p.Arg1215Gly was not detected in any of the 7 haemodialysis patients or the 4,000 individuals within the EXTEND study.
We conclude that CFH c.3643C>G; p.Arg1215Gly is not endemic in Devon. This reinforces our existing practice of genotyping only patients with kidney disease and evidence of a thrombotic microangiopathy for this mutation. This is the first study looking at the prevalence of CFH mutations in the general population.
[Show abstract][Hide abstract] ABSTRACT: A 2-year-old patient with a neuroblastoma developed haemolytic uraemic syndrome (HUS) following treatment with cisplatin and
carboplatin. Following treatment with eculizumab, there was a substantial improvement in renal function with the recovery
of the platelet count and the cessation of haemolysis. Subsequent investigations showed a novel, heterozygous CD46 splice site mutation with reduced peripheral blood neutrophil CD46 expression. Withdrawal of eculizumab was followed by the
recurrence of disease activity, which resolved with re-introduction of therapy. Abnormal regulation of complement may be associated
with other cases of cisplatin-induced HUS and treatment with eculizumab may be appropriate for other affected individuals.
[Show abstract][Hide abstract] ABSTRACT: We report a male infant who presented at 8 months of age with atypical hemolytic uremic syndrome (aHUS) responsive to plasma therapy. Investigation showed him to have complement factor H (CFH) deficiency associated with a homozygous CFH mutation (c.2880delT [p.Phe960fs]). Mutation screening of the child's parents revealed that the father was heterozygous for this change but that it was not present in his mother. Chromosome 1 uniparental isodisomy of paternal origin was confirmed by genotyping chromosome 1 SNPs. CD46 SNP genotyping was undertaken in this individual and another patient with CFH deficiency associated with chromosome 1 uniparental isodisomy. This showed a homozygous aHUS risk haplotype (CD46GGAAC) in the patient with aHUS and a homozygous glomerulonephritis risk haplotype (CD46AAGGT) in the patient with endocapillary glomerulonephritis. We also showed that FHL-1 (factor H-like protein 1) was present in the patient with aHUS and absent in the patient with glomerulonephritis. This study emphasizes that modifiers such as CD46 and FHL-1 may determine the kidney phenotype of patients who present with homozygous CFH deficiency.
American Journal of Kidney Diseases 07/2013; 62(5). DOI:10.1053/j.ajkd.2013.05.020 · 5.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atypical hemolytic-uremic syndrome is a genetic, life-threatening, chronic disease of complement-mediated thrombotic microangiopathy. Plasma exchange or infusion may transiently maintain normal levels of hematologic measures but does not treat the underlying systemic disease.
We conducted two prospective phase 2 trials in which patients with atypical hemolytic-uremic syndrome who were 12 years of age or older received eculizumab for 26 weeks and during long-term extension phases. Patients with low platelet counts and renal damage (in trial 1) and those with renal damage but no decrease in the platelet count of more than 25% for at least 8 weeks during plasma exchange or infusion (in trial 2) were recruited. The primary end points included a change in the platelet count (in trial 1) and thrombotic microangiopathy event-free status (no decrease in the platelet count of >25%, no plasma exchange or infusion, and no initiation of dialysis) (in trial 2).
A total of 37 patients (17 in trial 1 and 20 in trial 2) received eculizumab for a median of 64 and 62 weeks, respectively. Eculizumab resulted in increases in the platelet count; in trial 1, the mean increase in the count from baseline to week 26 was 73×10(9) per liter (P<0.001). In trial 2, 80% of the patients had thrombotic microangiopathy event-free status. Eculizumab was associated with significant improvement in all secondary end points, with continuous, time-dependent increases in the estimated glomerular filtration rate (GFR). In trial 1, dialysis was discontinued in 4 of 5 patients. Earlier intervention with eculizumab was associated with significantly greater improvement in the estimated GFR. Eculizumab was also associated with improvement in health-related quality of life. No cumulative toxicity of therapy or serious infection-related adverse events, including meningococcal infections, were observed through the extension period.
Eculizumab inhibited complement-mediated thrombotic microangiopathy and was associated with significant time-dependent improvement in renal function in patients with atypical hemolytic-uremic syndrome. (Funded by Alexion Pharmaceuticals; C08-002 ClinicalTrials.gov numbers, NCT00844545 [adults] and NCT00844844 [adolescents]; C08-003 ClinicalTrials.gov numbers, NCT00838513 [adults] and NCT00844428 [adolescents]).
New England Journal of Medicine 06/2013; 368(23):2169-81. DOI:10.1056/NEJMoa1208981 · 54.42 Impact Factor