Dineke Westra

Atypical hemolytic uremic syndrome in children: complement mutations and clinical characteristics

BackgroundMutations in complement factor H (CFH), factor I (CFI), factor B (CFB), thrombomodulin (THBD), C3 and membrane cofactor protein (MCP), and autoantibodies against factor H (αFH) with or without a homozygous deletion in CFH-related protein 1 and 3 (∆CFHR1/3) predispose development of atypical hemolytic uremic syndrome (aHUS).MethodsDifferent mutations in genes encoding complement proteins in 45 pediatric aHUS patients were retrospectively linked with clinical features, treatment, and outcome.ResultsIn 47% of the study participants, potentially pathogenic genetic anomalies were found (5xCFH, 4xMCP, and 4xC3, 3xCFI, 2xCFB, 6xαFH, of which five had ∆CFHR1/3); four patients carried combined genetic defects or a mutation, together with αFH. In the majority (87%), disease onset was preceeded by a triggering event; in 25% of cases diarrhea was the presenting symptom. More than 50% had normal serum C3 levels at presentation. Relapses were seen in half of the patients, and there was renal graft failure in all except one case following transplant.ConclusionsPerforming adequate DNA analysis is essential for treatment and positive outcome in children with aHUS. The impact of intensive initial therapy and renal replacement therapy, as well as the high risk of recurrence of aHUS in renal transplant, warrants further understanding of the pathogenesis, which will lead to better treatment options.

Genetic disorders in complement (regulating) genes in patients with atypical haemolytic uraemic syndrome (aHUS)

BACKGROUND Atypical HUS (aHUS) is thought to be caused by predisposing mutations in genes encoding complement (regulating) proteins, such as Factor H (CFH), Factor I (IF), membrane co-factor protein (MCP) and Factor B (FB), or by auto-antibodies against CFH (alphaFH) in combination with a homozygous polymorphic deletion of the genes encoding Complement Factor H-related 1 and 3 (DeltaCFHR1/3). The clinical impact of this knowledge is high, as it might be a prognostic factor for the outcome of renal transplantations and kidney donations. METHODS Mutational screening, by means of PCR and DNA sequencing, is performed in the above-mentioned genes in a group of 72 aHUS patients. Also, the presence of alphaFH and DeltaCFHR1/3 was tested in patients and controls. RESULTS In 23 patients, a genetic aberration in at least one gene or the presence of alphaFH was found. A heterozygous mutation was observed in CFH in nine patients, in IF in seven patients and in MCP in three patients. No mutations were observed in FB. Seven patients presented alphaFH, of whom five also carried DeltaCFHR1/3. Three patients carried a combined mutation (two patients: IF and MCP; one patient: IF, alphaFH and DeltaCFHR1/3). A significant difference between patients and controls was detected for the presence of all three associated polymorphisms in CFH. CONCLUSIONS Genetic abnormalities or the presence of alphaFH were detected in 31.9% of the aHUS patients. Furthermore, bigenic mutations were present, indicating that routine DNA mutation analysis of all complement factors associated with aHUS is important.

Bigenic heterozygosity and the development of steroid-resistant focal segmental glomerulosclerosis

BACKGROUND Focal segmental glomerulosclerosis (FSGS) is a major cause of steroid-resistant nephrotic syndrome in childhood with a central role for the podocytes in the pathogenesis. Mutated proteins expressed in podocytes cause proteinuria. The role of combined gene defects in the development of FSGS is less clear. METHODS We analysed seven podocyte genes known to cause proteinuria and FSGS in a group of 19 non-familial childhood-onset steroid-resistant FSGS patients. These genes include NPHS1, NPHS2, ACTN4, CD2AP, WT-1, TRPC6 and PLCE1. We also screened for the mitochondrial A3243G DNA transition associated with the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), and occasionally FSGS. RESULTS No mutations were found in the ACTN4 and TRPC6 genes, and no mitochondrial A3243G DNA transition was found in our group of patients. Two patients showed mutations in the CD2AP gene, one combined with an NPHS2 mutation. A tri-allelic hit was found in a patient carrying compound heterozygous NPHS2 mutations and a heterozygous NPHS1 mutation. In another patient a de novo WT-1 mutation was found combined with a heterozygous NPHS1 mutation, and finally two patients showed three heterozygous PLCE1 mutations. CONCLUSIONS In our rather small group of 19 steroid-resistant FSGS patients, we found 11 mutations in podocyte genes in 6 patients. In four of them the found mutations could explain the pathology. Our data suggest that combined gene defects in podocyte genes may play a role in the development of FSGS.

Combined Pulmonary Hypertension and Renal Thrombotic Microangiopathy in Cobalamin C Deficiency

Pulmonary arterial hypertension (PAH) and renal thrombotic microangiopathy (rTMA) are rare diseases in childhood, frequently leading to death and end-stage renal disease, respectively. Their combined occurrence has been reported anecdotally. We investigated the clinical, biochemical, and genetic aspects of 5 children with the rare combination of PAH and rTMA. Onset of disease ranged from 1.5 to 14 years of age. The 2 youngest patients presented with concomitant pulmonary and renal disease; in the older patients, PAH was preceded by rTMA from age 2.5 to 7 years. Three patients presenting at ≤3 years of age died of right ventricular failure secondary to progressive PAH. In 2 patients, cobalamin C (cblC) deficiency was diagnosed postmortem. Three patients were treated with hydroxocobalamin; 1 died 2 weeks after diagnosis, 1 patient exhibited progressive pulmonary vasculopathy, and 1 patient is currently in stable condition. cblC deficiency was diagnosed biochemically 2 days to 18 years after initial presentation. Genetic analysis confirmed mutations in MMACHC in all patients; 4 patients were compound heterozygous, with all having base-pair substitutions (G>A or G>T) at nucleotide 276 in addition to frame-shift mutations. One patient had homozygous nonsense mutations of MMACHC. We established cblC deficiency as the denominator in the rare combination of PAH and rTMA in these children. Early recognition of cblC deficiency and vigorous treatment with hydroxocobalamin may beneficially affect the course of this devastating disease.

Novel C3 mutation p.Lys65Gln in aHUS affects complement factor H binding.

BackgroundAtypical hemolytic uremic syndrome (aHUS) is associated with mutations affecting complement proteins and regulators and with autoantibodies against complement factor H (CFH). Approximately half of the aHUS patients progress to end-stage renal disease. DNA analysis of the risk factor genes is important for prognosis of aHUS recurrence after renal transplantation.MethodsMutational screening of C3 encoding the central complement component was performed by Sanger sequencing in 70 aHUS patients. Mutated and wild type recombinant C3b proteins were produced and their affinity to CFH was analyzed by ELISA.ResultsA single novel missense change p.Lys65Gln in C3 was found in 3 aHUS patients. The alteration leads to decreased binding of C3b to CFH in vitro. All three patients acquired the illness as adults and had a first aHUS episode after renal transplantation or suffered recurrence of the disease after transplantation.ConclusionsThe novel C3 change was found in 3 aHUS patients. It results in decreased C3b binding to CFH and thus might lead to impaired C3b inactivation in vivo. The p.Lys65Gln is likely to be associated with aHUS after kidney transplantation and, therefore, might be an important prognostic factor.

ATYPICAL HEMOLYTIC UREMIC SYNDROME AND GENETIC ABERRATIONS IN THE COMPLEMENT FACTOR H RELATED 5 GENE

Atypical hemolytic uremic syndrome (aHUS) is a severe renal disorder that is associated with mutations in genes encoding proteins of the alternative complement pathway. Previously, we identified pathogenic variations in genes encoding complement regulators (CFH, CFI and MCP) in our aHUS cohort. In this study, we screened for mutations in the alternative pathway regulator CFHR5 in 65 aHUS patients by means of PCR on genomic DNA and sequence analysis. Potential pathogenicity of genetic alterations was determined by published data on CFHR5 variants, evolutionary conservation and in silico mutation prediction programs. Detection of serum CFHR5 was performed by western blot analysis and enzyme-linked immunosorbent assay. A potentially pathogenic sequence variation was found in CFHR5 in three patients (4.6%). All variations were located in short consensus repeats that might be involved in binding to C3b, heparin or C-reactive protein. The identified CFHR5 mutations require functional studies to determine their relevance to aHUS, but they might be candidates for an altered genetic profile predisposing to the disease.

Sensitive, reliable and easy-performed laboratory monitoring of eculizumab therapy in atypical hemolytic uremic syndrome

Complement C5 inhibitor eculizumab treatment in atypical hemolytic uremic syndrome is effective, but associated with high costs. Complement inhibition monitoring in these patients has not been standardized. In this study we evaluated novel functional assays for application in routine follow-up. We documented that the Wieslab® complement screen assay showed a sensitivity of 1-2% of C5 activity by adding purified C5 or normal human serum to a C5 deficient serum. All the patient samples obtained during the treatment course, were completely blocked for terminal complement pathway activity for up to four weeks after the eculizumab infusion. Levels of complexes between eculizumab and C5 were inversely correlated to the complement activity (p=0.01). Moreover, titrating serum from eculizumab-treated patients into normal serum revealed that eculizumab was present in excess up to four weeks after infusion. Thus, we demonstrate sensitive, reliable and easy-performed assays which can be used to design individual eculizumab dosage regimens.

Complement activation patterns in atypical haemolytic uraemic syndrome during acute phase and in remission.

Atypical haemolytic uraemic syndrome (aHUS) is associated with (genetic) alterations in alternative complement pathway. Nevertheless, comprehensive evidence that the complement system in aHUS patients is more prone to activation is still lacking. Therefore, we performed a thorough analysis of complement activation in acute phase and in remission of this disease. Complement activation patterns of the aHUS patients in acute phase and in remission were compared to those of healthy controls. Background levels of complement activation products C3b/c, C3bBbP and terminal complement complex (TCC) were measured using enzyme‐linked immunosorbent assay (ELISA) in ethylenediamine tetraacetic acid (EDTA) plasma. In vitro‐triggered complement activation in serum samples was studied using zymosan‐coating and pathway‐specific assay. Furthermore, efficiencies of the C3b/c, C3bBbP and TCC generation in fluid phase during spontaneous activation were analysed. Patients with acute aHUS showed elevated levels of C3b/c (P < 0·01), C3bBbP (P < 0·0001) and TCC (P < 0·0001) in EDTA plasma, while values of patients in remission were normal, compared to those of healthy controls. Using data from a single aHUS patient with complement factor B mutation we illustrated normalization of complement activation during aHUS recovery. Serum samples from patients in remission showed normal in vitro patterns of complement activation and demonstrated normal kinetics of complement activation in the fluid phase. Our data indicate that while aHUS patients have clearly activated complement in acute phase of the disease, this is not the case in remission of aHUS. This knowledge provides important insight into complement regulation in aHUS and may have an impact on monitoring of these patients, particularly when using complement inhibition therapy.

Complement Factor H Serum Levels Determine Resistance to Pneumococcal Invasive Disease

Streptococcus pneumoniae is a major cause of life-threatening infections. Complement activation plays a vital role in opsonophagocytic killing of pneumococci in blood. Initial complement activation via the classical and lectin pathways is amplified through the alternative pathway amplification loop. Alternative pathway activity is inhibited by complement factor H (FH). Our study demonstrates the functional consequences of the variability in human serum FH levels on host defense. Using an in vivo mouse model combined with human in vitro assays, we show that the level of serum FH correlates with the efficacy of opsonophagocytic killing of pneumococci. In summary, we found that FH levels determine a delicate balance of alternative pathway activity, thus affecting the resistance to invasive pneumococcal disease. Our results suggest that variation in FH expression levels, naturally occurring in the human population, plays a thus far unrecognized role in the resistance to invasive pneumococcal disease.

Whole Exome Sequencing in Patients with the Cuticular Drusen Subtype of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly people worldwide. Cuticular drusen (CD) is a clinical subtype of AMD, which typically displays an earlier age at onset, and has a strong genetic component. Genetic studies support a role for rare sequence variants in CD susceptibility, and rare sequence variants in the CFH gene have been identified in 8.8% of CD cases. To further explore the role of rare variants in CD, we performed whole exome sequencing (WES) in 14 affected members of six families and 12 sporadic cases with CD. We detected rare sequence variants in CFH and FBLN5, which previously were shown to harbor rare variants in patients with CD. In addition, we detected heterozygous rare sequence variants in several genes encoding components of the extracellular matrix (ECM), including FBLN1, FBLN3/EFEMP1, FBLN5, FBLN6/HMCN1, FBN2, and COL15A1. Two rare pathogenic variants were identified in the COL15A1 gene: one in a sporadic case and another was found to segregate in a family with six affected individuals with CD. In addition, two rare pathogenic variants were identified in the FGL1 gene in three unrelated CD cases. These findings suggest that alterations in the ECM and in the coagulation pathway may play a role in the pathogenesis of CD. The identified candidate genes require further analyses in larger cohorts to confirm their role in the CD subtype of AMD. No evidence was found of rare sequence variants in a single gene that segregate with CD in the six families, suggesting that the disease is genetically heterogeneous.