Thrombotic Microangiopathy in Inverted Formin 2-Mediated Renal Disease.

The demonstration of impaired C regulation in the thrombotic microangiopathy (TMA) atypical hemolytic uremic syndrome (aHUS) resulted in the successful introduction of the C inhibitor eculizumab into clinical practice. C abnormalities account for approximately 50% of aHUS cases; however, mutations in the non-C gene diacylglycerol kinase-ε have been described recently in individuals not responsive to eculizumab. We report here a family in which the proposita presented with aHUS but did not respond to eculizumab. Her mother had previously presented with a post-renal transplant TMA. Both the proposita and her mother also had Charcot-Marie-Tooth disease. Using whole-exome sequencing, we identified a mutation in the inverted formin 2 gene (INF2) in the mutational hotspot for FSGS. Subsequent analysis of the Newcastle aHUS cohort identified another family with a functionally-significant mutation in INF2 In this family, renal transplantation was associated with post-transplant TMA. All individuals with INF2 mutations presenting with a TMA also had aHUS risk haplotypes, potentially accounting for the genetic pleiotropy. Identifying individuals with TMAs who may not respond to eculizumab will avoid prolonged exposure of such individuals to the infectious complications of terminal pathway C blockade.

Chromosomal rearrangement-A rare cause of complement factor I associated atypical haemolytic uraemic syndrome.

Chromosomal rearrangements affecting the genes encoding complement factor H and the factor H related proteins have been described in aHUS patients. To date such disorders have not been described in other aHUS associated genes. We describe here a heterozygous 875,324bp deletion encompassing the gene (CFI) encoding complement factor I and ten other genes. The index case presented with aHUS and did not recover renal function. No abnormalities were detected on Sanger sequencing of CFI but a low factor I level led to a multiplex ligation-dependent probe amplification assay being undertaken. This showed a complete heterozygous deletion of CFI. The extent of the deletion and the breakpoint was defined. In the Newcastle aHUS cohort we have identified and report here 32 different CFI variants in 56 patients but to date this is the only deletion that we have identified. This finding although rare does suggest that screening for chromosomal rearrangements affecting CFI should be undertaken in all aHUS patients particularly if the factor I level is unexplainably low.

A De Novo Deletion in the Regulators of Complement Activation Cluster Producing a Hybrid Complement Factor H/Complement Factor H-Related 3 Gene in Atypical Hemolytic Uremic Syndrome.

The regulators of complement activation cluster at chromosome 1q32 contains the complement factor H (CFH) and five complement factor H-related (CFHR) genes. This area of the genome arose from several large genomic duplications, and these low-copy repeats can cause genome instability in this region. Genomic disorders affecting these genes have been described in atypical hemolytic uremic syndrome, arising commonly through nonallelic homologous recombination. We describe a novel CFH/CFHR3 hybrid gene secondary to a de novo 6.3-kb deletion that arose through microhomology-mediated end joining rather than nonallelic homologous recombination. We confirmed a transcript from this hybrid gene and showed a secreted protein product that lacks the recognition domain of factor H and exhibits impaired cell surface complement regulation. The fact that the formation of this hybrid gene arose as a de novo event suggests that this cluster is a dynamic area of the genome in which additional genomic disorders may arise.

Exome Sequencing of an Adult Pituitary Atypical Teratoid Rhabdoid Tumor.

Atypical teratoid rhabdoid tumors (AT/RTs) are rare pediatric brain tumors characterized by bialleic loss of the SMARCB1 tumor suppressor gene. In contrast to pediatric AT/RT that has a simple genome, very little is known about the adult AT/RT genomic landscape. Using a combination of whole-exome sequencing and high-resolution SNP array in a single adult pituitary AT/RT, we identified a total of 47 non-synonymous mutations, of which 20 were predicted to cause non-conservative amino acid substitutions, in addition to a subclone of cells with trisomy 8. We suggest that adult AT/RT may not be markedly dissimilar to other adult brain tumors where mutations in a range of genes, reflecting the functional specialization of different brain regions, but including SMARCB1 inactivation, may be required for its pathogenesis.

Factors determining penetrance in familial atypical haemolytic uraemic syndrome.

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.

Characterization of a factor H mutation that perturbs the alternative pathway of complement in a family with membranoproliferative GN.

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.

Phenotypic variations of cartilage hair hypoplasia: granulomatous skin inflammation and severe T cell immunodeficiency as initial clinical presentation in otherwise well child with short stature.

We report a child with short stature since birth who was otherwise well, presenting at 2.8 years with progressive granulomatous skin lesions when diagnosed with severe T cell immunodeficiency. When previously investigated for short stature, and at the time of current investigations, she had no radiological skeletal features characteristics for cartilage hair hypoplasia, but we found a disease causing RMRP (RNase mitochondrial RNA processing endoribonuclease) gene mutation. Whilst search for HLA matched unrelated donor for haematopoietic stem cell transplantation (HSCT) was underway, she developed rapidly progressive EBV-related lymphoproliferative disorder requiring laparotomy and small bowel resection, and was treated with anti-B cell monoclonal antibody and eventually curative allogeneic HSCT. Screening for RMRP gene mutations should be part of immunological evaluation of patients with 'severe and/or combined' T cell immunodeficiency of unknown origin, especially when associated with short stature and regardless of presence or absence of radiological skeletal features.

Determining the population frequency of the CFHR3/CFHR1 deletion at 1q32.

In this study we have used multiplex ligation-dependent probe amplification (MLPA) to measure the copy number of CFHR3 and CFHR1 in DNA samples from 238 individuals from the UK and 439 individuals from the HGDP-CEPH Human Genome Diversity Cell Line Panel. We have then calculated the allele frequency and frequency of homozygosity for the copy number polymorphism represented by the CFHR3/CFHR1 deletion. There was a highly significant difference between geographical locations in both the allele frequency (X(2)  = 127.7, DF = 11, P-value = 4.97x10(-22)) and frequency of homozygosity (X(2)  = 142.3, DF = 22, P-value = 1.33x10(-19)). The highest frequency for the deleted allele (54.7%) was seen in DNA samples from Nigeria and the lowest (0%) in samples from South America and Japan. The observed frequencies in conjunction with the known association of the deletion with AMD, SLE and IgA nephropathy is in keeping with differences in the prevalence of these diseases in African and European Americans. This emphasises the importance of identifying copy number polymorphism in disease.

Factor H autoantibodies in membranoproliferative glomerulonephritis.

Factor H autoantibodies are found in ~10% of aHUS patients. Most are associated with complete deficiency of factor H related proteins 1/3 and bind to the C terminal recognition domain. MPGN, like aHUS, is characterised by complement activation. In this study we, therefore, examined the hypothesis that factor H autoantibodies are associated with MPGN. We screened sera from 16 MPGN patients and 100 normal controls using ELISA and detected strongly positive IgG factor H autoantibodies in 2 patients. One patient had type II (DDD) MPGN (male aged 24 yrs) with C3NeF and the other type I (female aged 26 yrs) with no detectable C3NeF. We identified the binding site of the autoantibodies using small SCR domain fragments in the ELISA and showed that the autoantibodies in both patients bound predominately to the N terminal complement regulatory domain of factor H. We measured CFHR 1/3 copy number using MLPA and showed that both patients had 2 copies of CFHR1 and 3. Finally, we examined the functionality of detected factor H autoantibodies using purified patient IgG and observed increased haemolysis when purified IgG from both patients was added to normal human sera prior to incubation with rabbit red blood cells. Thus, in a cohort of MPGN patients we have found a high titre of functionally significant factor H autoantibodies in two patients with MPGN. Antibody depleting therapy may have a role in such patients and we suggest that screening for factor H autoantibodies should be undertaken in all patients with MPGN.

Factor I autoantibodies in patients with atypical hemolytic uremic syndrome: disease-associated or an epiphenomenon?

BACKGROUND AND OBJECTIVES: Atypical hemolytic uremic syndrome is a disease associated with mutations in the genes encoding the complement regulators factors H and I. In addition, factor H autoantibodies have been reported in ∼10% of patients with atypical hemolytic uremic syndrome. This study searched for the presence of factor I autoantibodies in atypical hemolytic uremic syndrome. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This study screened 175 atypical hemolytic uremic syndrome patients for factor I autoantibodies using ELISA with confirmatory Western blotting. Functional studies using purified immunoglobulin from one patient were subsequently undertaken. RESULTS: Factor I autoantibodies were detected in three patients. In one patient with a high titer of autoantibody, the titer was tracked over time and was found to have no association with disease activity. This study found evidence of an immune complex of antibody and factor I in this patient, but purified IgG, isolated from current serum samples, had only a minor effect on fluid phase and cell surface complement regulation. Genetic analysis of the three patients with factor I autoantibodies revealed that they had two copies of the genes encoding factor H-related proteins 1 and 3 and therefore, did not have a deletion commonly associated with factor H autoantibodies in atypical hemolytic uremic syndrome. Two patients, however, had functionally significant mutations in complement factor H. CONCLUSIONS: These findings reinforce the concept of multiple concurrent risk factors being associated with atypical hemolytic uremic syndrome but question whether autoantibodies per se predispose to atypical hemolytic uremic syndrome.

A novel hybrid CFH/CFHR3 gene generated by a microhomology-mediated deletion in familial atypical hemolytic uremic syndrome.

Genomic disorders affecting the genes encoding factor H (fH) and the 5 factor H related proteins have been described in association with atypical hemolytic uremic syndrome. These include deletions of CFHR3, CFHR1, and CFHR4 in association with fH autoantibodies and the formation of a hybrid CFH/CFHR1 gene. These occur through nonallelic homologous recombination secondary to the presence of large segmental duplications (macrohomology) in this region. Using multiplex ligation-dependent probe amplification to screen for such genomic disorders, we have identified a large atypical hemolytic uremic syndrome family where a deletion has occurred through microhomology-mediated end joining rather than nonallelic homologous recombination. In the 3 affected persons of this family, we have shown that the deletion results in formation of a CFH/CFHR3 gene. We have shown that the protein product of this is a 24 SCR protein that is secreted with normal fluid-phase activity but marked loss of complement regulation at cell surfaces despite increased heparin binding. In this study, we have therefore shown that microhomology in this area of chromosome 1 predisposes to disease associated genomic disorders and that the complement regulatory function of fH at the cell surface is critically dependent on the structural integrity of the whole molecule.

Common genetic variants in complement genes other than CFH, CD46 and the CFHRs are not associated with aHUS.

It is well established that common genetic variants in CFH, CD46 and the CFHRs are additional risk factors for the development of aHUS. To examine the hypothesis that common variants in other complement genes have a similar effect we genotyped 501 SNPs in 47 complement genes in 94 aHUS patients from Newcastle, 126 aHUS patients from Paris, 374 UK controls and 165 French controls. We replicated the associations in CFH, CD46 and the CFHRs but found no association with any other complement gene. The strongest associations replicated in both cohorts were found for four SNPs within CD46 (p-value<10(-3)) and five SNPs within CFH (p-value<5×10(-3)). Significant replicable associations with single SNPs in CFHR2, CFHR4 and an intergenic SNP (CR1-CD46) were also found. Analysis of the Paris cohort showed that the association with CD46 SNPs was only present in those patients with complement mutations. Haplotype analysis showed at-risk and protective haplotypes in both CD46 and CFH. The CD46 haplotype was only disease-associated in those patients with mutations.

Successful simultaneous liver-kidney transplant in an adult with atypical hemolytic uremic syndrome associated with a mutation in complement factor H.

Atypical hemolytic uremic syndrome was diagnosed in a 62-year-old man. Sequencing of the CFH gene, which encodes complement factor H, revealed a heterozygous adenine to guanine mutation at nucleotide 3550 of the complementary DNA, leading to a predicted substitution of alanine for threonine at amino acid position 1184 in the protein (c.3550A>G, p.Thr1184Ala). Three years later, he received a simultaneous liver-kidney transplant with plasmapheresis and intratransplant plasma infusion. The postoperative course was complicated by an anastomotic biliary stricture that was treated successfully using endoscopic stenting. One year later, he has excellent function of both transplants, emphasizing that simultaneous liver-kidney transplant is a valuable treatment option in the management of adult patients with atypical hemolytic uremic syndrome.

Complement factor h autoantibodies and age-related macular degeneration.

PURPOSE: In this case-control study, the hypothesis that factor H autoantibodies are associated with age-related macular degeneration (AMD) was examined. METHODS: One hundred AMD patients (median age, 78 years), 98 age-matched control subjects (median age, 78 years) known not to have AMD, and 100 healthy blood donors (median age, 43 years) were enrolled. An enzyme-linked immunosorbent assay (ELISA) was used to screen for complement factor H autoantibodies and either quantitative polymerase chain reaction (qPCR) or multiplex ligation-dependent probe amplification (MLPA) were performed to measure the copy number of the gene encoding complement factor H-related protein 3 (CFHR3). RESULTS: There was a significant difference in the median complement factor H autoantibody titer between the three groups (AMD patients, 196 reference units [RU]]; age-match control subjects, 316 RU; and blood donor control subjects, 121 RU; Kruskal-Wallis test, P < 0.001). Pair-wise comparison (Mann-Whitney test) showed that all three groups were significantly different from each other. Two different thresholds were used in the healthy blood donors to identify individuals with complement factor H autoantibodies. Both suggested that the prevalence of factor H autoantibodies was decreased in AMD patients. The CFHR3 copy number was measured as a surrogate for the deletion of the genes encoding complement factor H-related proteins 3 and 1 (CFHR3/1). The allele frequency of the deletion was significantly higher in the age-matched control subjects than in the AMD patients (22.2% vs. 8.2%). CONCLUSIONS: The level of factor H autoantibodies is lower in AMD patients than in age-matched control subjects.

Triggering of atypical hemolytic uremic syndrome by influenza A (H1N1).

We report a case of atypical hemolytic uremic syndrome (aHUS) triggered by influenza A (H1N1) in a 17-year-old boy with a mutation in the gene (CD46) encoding the transmembrane complement regulator membrane cofactor protein. The patient recovered completely following treatment with oseltamivir, plasma exchange, and hemodialysis. We describe the case and discuss this unusual association of diseases.

Maintenance of kidney function following treatment with eculizumab and discontinuation of plasma exchange after a third kidney transplant for atypical hemolytic uremic syndrome associated with a CFH mutation.

Kidney transplant in patients with atypical hemolytic uremic syndrome (aHUS) is associated with a poor outcome because of recurrent disease, especially in patients known to have a factor H mutation. Long-term prophylactic plasma exchange and combined liver-kidney transplant have prevented graft loss caused by recurrence. However, the mortality associated with liver transplant is not negligible, and prophylactic plasma exchange requires permanent vascular access and regular hospitalization and exposes the patient to potential allergic reactions to plasma. Eculizumab is a high-affinity humanized monoclonal antibody that binds to C5 and thus prevents generation of C5a and the membrane attack complex. We report the case of a 17-year-old girl with aHUS associated with a mutation in the gene for complement factor H (CFH; c.3572C>T, Ser1191Leu) who was highly dependent on plasma exchange. Because of severe allergic reactions to plasma after the third renal graft, eculizumab was introduced in place of plasma exchange without problems. This and other reports suggest that the promise of complement inhibitors in the management of aHUS is going to be fulfilled.

Association of factor H autoantibodies with deletions of CFHR1, CFHR3, CFHR4, and with mutations in CFH, CFI, CD46, and C3 in patients with atypical hemolytic uremic syndrome.

Factor H autoantibodies have been reported in approximately 10% of patients with atypical hemolytic uremic syndrome (aHUS) and are associated with deficiency of factor H-related proteins 1 and 3. In this study we examined the prevalence of factor H autoantibodies in the Newcastle cohort of aHUS patients, determined whether the presence of such autoantibodies is always associated with deficiency of factor H-related proteins 1 and 3, and examined whether such patients have additional susceptibility factors and/or mutations in the genes encoding complement regulator/activators. We screened 142 patients with aHUS and found factor H autoantibodies in 13 individuals (age 1-11 years). The presence of the autoantibodies was confirmed by Western blotting. By using multiplex ligation-dependent probe amplification we measured complement factor H-related (CFHR)1 and CFHR3 copy number. In 10 of the 13 patients there were 0 copies of CFHR1, and in 3 patients there were 2. In 3 of the patients with 0 copies of CFHR1 there was 1 copy of CFHR3, and these individuals exhibited a novel deletion incorporating CFHR1 and CFHR4. In 5 patients mutations were identified: 1 in CFH, 1 in CFI, 1 in CD46, and 2 in C3. The latter observation emphasizes that multiple concurrent factors may be necessary in individual patients for disease manifestation.

Prophylactic plasma exchange in CD46-associated atypical haemolytic uremic syndrome.

Patients with atypical haemolytic uremic syndrome (aHUS) with a mutation in the gene encoding membrane cofactor protein (CD46) are known to have a better prognosis than those with mutations in factor H (CFH) or factor I (CFI), but a small number of the former still proceed to end-stage renal failure. Plasma therapy (PE) is the recommended approach to treat both acute episodes and prevent recurrences in aHUS, but studies have yet to show PE efficacy in aHUS associated with a CD46 mutation. The factors determining failure to treatment are not clear and may be related to the mutation involved or to insufficient treatment. Our experience of PE in a family of three sisters with CFH-associated aHUS suggests that intensive and prophylactic PE allows renal function to be maintained in both native kidneys and allografts. The success of this strategy has led us to use it in all cases of aHUS. Here, we describe the effect of this strategy in a child with aHUS and a CD46 mutation. The initial episode was treated with daily PE, resulting in the recovery of renal function. However, over the next 4 years, there was a progressive decline in renal function to end-stage renal failure, with evidence of an on-going thrombotic microangiopathy despite continuous prophylactic PE. Prophylactic PE does not influence the natural course of aHUS and CD46 mutation.

Is complement factor H a susceptibility factor for IgA nephropathy?

There is substantial evidence to suggest that complement activation plays a pivotal role in the pathogenesis of IgA nephropathy. Mesangial C3 deposition is seen in approximately 90% of patients and polymeric IgA has been shown to activate the alternative and lectin pathways. In addition there have been reports of deficiency and mutations in the serum complement regulator factor H (CFH) in association with IgA nephropathy. In this study we have examined the hypothesis that CFH is a susceptibility factor for IgA nephropathy. In 46 IgA nephropathy patients we undertook genotyping of three CFH SNPS (rs3753394, rs3753396 and rs1065489). There was no significant difference in the allele frequency of these 3 SNPs between the patients and normal controls. In the same group of patients we undertook mutation screening of CFH exons 18-23 using direct sequencing and found no abnormalities. All the patients had a normal serum factor H concentration. In this small cohort of IgA nephropathy patients we have not found evidence to support the hypothesis that factor H is a major susceptibility factor for the disease.

Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a disease of complement dysregulation. In approximately 50% of patients, mutations have been described in the genes encoding the complement regulators factor H, MCP, and factor I or the activator factor B. We report here mutations in the central component of the complement cascade, C3, in association with aHUS. We describe 9 novel C3 mutations in 14 aHUS patients with a persistently low serum C3 level. We have demonstrated that 5 of these mutations are gain-of-function and 2 are inactivating. This establishes C3 as a susceptibility factor for aHUS.