CFH Mutations Research Articles

#623 One-year outcome of kidney transplant recipients harbouring complement regulatory gene mutation

Abstract Background and Aims Thrombotic Microangiopathy (TMA) is associated with genetic abnormalities in complement regulatory pathways. The impact of complement regulatory gene (CRG) mutation on post-transplant TMA in Indian kidney transplant recipients (KTR) is unknown. This study was conducted to ascertain an association between post-transplant renal limited TMA and the risk of graft failure in KTRs harbouring mutations in genes encoding complement-regulatory proteins. Method A Single Centre, Prospective Cohort Study was undertaken to assess the incidence of post-transplant renal limited TMA in KTRs with mutations in genes encoding complement regulatory proteins. Patients enrolled over one year were followed up for one-year post-transplantation. Genetic testing was performed with MLPA and Clinical exome sequencing. Results 138 KTRs with a mean age of 36.39 ± 12.39 years were enrolled. CRG mutation was seen in 96/138 (69.6%) KTRs. Duplication of the CFHR gene (n = 72) was most prevalent followed by mutations in CFH (n = 6), CFI (n = 2), MCP (n = 2), and DGKE (n = 1). Mutations in CFB, C3, and THBD genes were not detected. The Incidence of Post transplant renal limited TMA in KTRs with CRG mutations is 9.37% (9/96). Mutation related to aHUS was seen in 9 out of 11 patients who developed TMA. There was no significant difference in eGFR of patients with or without CRG mutation at the end of 1 year. Conclusion The prevalence of CRG mutation in KTRs is high and the majority of patients with post-transplant de novo TMA had CRG mutations. The graft functions at the end of 1 year were similar in patients with or without CRG gene mutation. This study suggests that the presence of CRG mutation should not preclude the option of Kidney transplantation in a patient with ESRD.

Complement-mediated thrombotic microangiopathy treated with anticomplement protein 5 therapy, a retrospective study.

Complement-mediated thrombotic microangiopathy (CM-TMA), also called atypical hemolytic uremic syndrome (aHUS), is a difficult-to-diagnose rare disease that carries severe morbidity and mortality. Anti-C5 monoclonal antibodies (aC5-mab) are standard treatments, but large studies and long-term data are scarce. Here, we report our single institution experience to augment the knowledge of CM-TMA treated with aC5-mab therapy. We aimed to assess the short and long-term effects of aC5-mab in patients diagnosed with CM-TMA treated outside of a clinical trial. This was a retrospective study. We included all patients diagnosed with CM-TMA and treated with aC5-mab at our institution. There were no exclusion criteria. Endpoints included complete TMA response (CR) defined as normalization of hematological parameters and ≥25% improvement in serum creatinine (Cr) from baseline in patients with renal disease, relapse defined as losing the previously achieved CR, morbidity, adverse events, and survival. We found 28 patients with CM-TMA treated with aC5-mab. The median age was 50 years. Baseline laboratories: platelet counts 93 × 109 /L, hemoglobin 8.6 g/dL, lactate dehydrogenase 1326 U/L, serum Cr 4.7 mg/dL, and estimated glomerular filtration rate 19 mL/min. One individual was on renal replacement therapy (RRT) and 10 initiated RRT within 5 days of the first dose of aC5-mab. Genetic variants associated with CM-TMA included mutations in C3, CFB, CFH, CFHR1/3, CFI, and MCP. The mean duration of hospitalization was 24 days. The median time to initiation of aC5-mab was 10 days. Sixteen subjects received RRT. At the time of hospital discharge, 27 were alive, 14 remained on RRT, and 4 had a CR. At 6 months, 23 patients were alive, 18 continued aC5-mab, 8 remained on RRT, and 9 had a CR. At the last follow-up visit past 6 months, 20 were alive, 14 continued aC5-mab, 5 remained on RRT, 12 had a CR, and 1 was lost to follow-up. Our study provides real-world experience and insight into the long-term outcomes of CM-TMA treated with aC5-mab. Our findings validate that CM-TMA is an aggressive disease with significant morbidity and mortality, and confirm that aC5-mab is a relatively effective therapy for CM-TMA. Our study adds practical, real-world experience to the literature, but future research remains imperative.

Atipik Hemolitik Üremik Sendromlu Hastaların Uzun Dönem Sonuçları

Aim: Hemolytic uremic syndrome (HUS) is the clinical triad of anemia, thrombocytopenia and acute renal injury. Atypical hemolytic uremic syndrome (aHUS) is a rare disease of alternative complement pathways. It is aimed to evaluate long-term follow-up of patients with aHUS in the present study. Materials and methods: Eleven children diagnosed with aHUS were retrospectively evaluated. Demographic, clinical, and laboratory data and treatment details were reported. Results: A total of 11 patients were enrolled in the study. The mean age of patients at aHUS onset was 2.9±6 years. The mean follow-up time was 72 ± 4 months All patients had renal involvement. Extrarenal manifestations of aHUS were present in four patients. All patients had eculizumab treatment. Conclusion: Our study insight into diagnosing and managing aHUS, a very rare disease, in our pediatric patients. Genetic testing is used to improve the diagnosis of aHUS. We demonstrated the long-term efficacy and safety of eculizumab in our aHUS patients. Further studies are needed to determine the optimal time for discontinuation of eculizumab treatment.

Profile and Post-Kidney Retrieval Outcomes in Asymptomatic Kidney Donors With Genetic Mutations in Complement Factors–Related Genes

BackgroundAtypical hemolytic uremic syndrome (aHUS) is a rare disorder triggered by various stressors. Most of the time, stressors may not be identified in patients with aHUS. The disease may remain quiescent without manifestation throughout life. BackgroundTo assess the outcome of an asymptomatic carrier of genetic mutations of patients with aHUS who had undergone donor kidney retrieval surgery. MethodsWe retrospectively included the patients diagnosed with a genetic abnormality in complement factor H (CHF) or CHF-related (CFHR) genes without manifestation of the aHUS and who had undergone donor kidney retrieval surgery. The data were analyzed with descriptive statistics. ResultsAmong patients who were the kidney recipients from the prospective donors, 6 donors were screened for genetic mutations in CFH and CFHR genes. Four donors showed positive mutation for CFH and CFHR. The mean age was 54.5 years (range, 50-64 years). After over a year since donor kidney retrieval surgery, all prospective mother donors are alive without aHUS activation and with a normal kidney function on a single kidney. ConclusionAsymptomatic carriers of genetic mutations in CFH and CFHR can be the prospective donors for their first-degree family member who have active aHUS. A genetic mutation in an asymptomatic donor should not be a contraindication for refuting the prospective donor.

Overlap of C3 Glomerulopathy and Thrombotic Microangiopathy: A Case Series

Dysregulation of alternative complement pathway underlies the pathogenesis of both C3 glomerulopathy (C3G) and thrombotic microangiopathy (TMA). In this study, we describe both disease entities occurring in 5 patients. We identified 114 patients at our institution from 2007 to 2016 with C3G in native kidney biopsies and those with concurrent TMA were included. The median age at diagnosis was 58 years (range: 28-69); all were male. Median serum creatinine and proteinuria at presentation were 2.3 mg/dl and 2089 mg/d, respectively. Three cases presented with TMA-predominant phenotype and 2 with C3G-predominant phenotype. Immunofluorescence (IF) showed bright C3 staining in mesangium and/or capillary walls. Electron microscopy showed marked subendothelial expansion by fluffy material in the capillary loops without associated deposits. However, capillary wall deposits were present in other loops in 4 cases. Mesangial deposits were present in all cases. Four cases showed low C3, of which 2 showed low C4. Complement evaluation in 3 cases showed pathogenic CFH mutation in 1 case, and multiple variant of unknown significance along with factor B autoantibody and C4 nephritic factor in 1 case. One patient negative for complement abnormalities had a monoclonal gammopathy. Three cases were treated with steroids and/or immunosuppressants. One case progressed to end-stage renal disease (ESRD) at 38.3 months; the remaining showed median serum creatinine and proteinuria of 2.5 mg/dl and 1169 mg/d, respectively at median follow-up of 17.5 months. Overlap of C3G and TMA is rare and can clinically present as C3G-predominant or TMA-predominant phenotype. The significance of concurrent C3G/TMA findings on long-term renal survival remains to be explored.

HUS with mutations in CFH and STEC infection treated with eculizumab in a 4-year-old girl.

Hemolytic uremic syndrome secondary to Shiga-toxin-producing Escherichia coli infection (STEC-HUS) generally shows a favorable outcome. Few cases develop extra-renal complications, since neurological involvement is an important cause of morbidity and mortality. The role of complement in STEC-HUS has been recently highlighted, and the use of eculizumab in severe cases has been communicated. HUS results from environmental and genetic factors, but the simultaneous occurrence of STEC and complement mutations remains undetermined. A pediatric case with severe STEC-HUS carrying CFH mutations, with favorable response to eculizumab is analyzed. STEC-HUS was diagnosed in a 4-year-old girl with classic HUS, including low C3. Peritoneal dialysis was started due to hypertension, oligoanuria, and pleural effusion. She evolved with generalized tonic-clonic seizures and required mechanical ventilation. MRI reported multiple supra- and infratentorial ischemic lesions with laminar/striatal cortical necrosis and leukoencephalopathy. After two eculizumab doses, a significative stabilization in diuresis, blood pressure, creatinine, and C3 was achieved. At the third week, episodes of massive digestive bleeding and a life-threatening condition required a colectomy thus preserving the ileocecal valve. Due to atypical evolution, a genetic study was considered, identifying two heterozygous variants (CFH S1191L/V1197A). STEC-HUS in patients with a genetic predisposition has been previously reported, but the low frequency of occurrence makes it a rare disease. As in the present case, patients with atypical course might benefit from genetic analysis to evaluate early eculizumab initiation and to better understand its phenotype. A higher resolution version of the Graphical abstract is available as Supplementary information.

Retrospective evaluation of patients with diagnosis of atypical hemolytic uremic syndrome: Single center experience

Background To evaluate the demographic, clinical, laboratory findings, genetic results and final status of patients followed-up with the diagnosis of atypical hemolytic uremic syndrome (aHUS) Materials and methods Patients who were diagnosed and followed up in our pediatric nephrology center between January 2013 and June 2021 were included in the study retrospectively. Demographic data, history, age at diagnosis, physical examination, laboratory tests, organ involvement, genetic results, treatments, renal replacement therapies, follow-up and final status were evaluated. Results A total of 14 patients, 9 girls (64.3%) and 5 boys (35.7%) were included in our study over a period of 8 years. The mean age at presentation was 66.7±54.1 months (5.5±4.5 years) and the follow-up period in our center was 50.7±37.1 months. Recurrence was detected in 4 patients (28.6%) 18.5±20.4 months after diagnosis. In six of our patients (42.8%) neurological involvement was detected; 5 (35.7%) had hypertensive features and 1 (7.1%) had disease involvement. MCP mutation was detected in 4 patients (28.6%), CFH mutation in 3 patients (21.4%), and CFHR1-CFHR3 mutation in 3 patients (21.4%). While a total of 4 patients (28.6%) died in our center, 10 patients (71.4%) are still being followed up and treated. From these patients one is followed up with fresh frozen plasma therapy and the rest with eculizumab therapy. Conclusion: In conclusion we wanted to present our patients who were diagnosed with aHUS in our center in a period of eculizumab availability in our country. Satisfactory results were detected with eculizumab treatment who did not respond to plasmaphresis and/or plasma treatment. We think that eculizumab treatment may be the first choice considering with detailed genetic analysis (including antifactor H antibody) in the light of future multicenter studies with larger of patients.

Sex Disparities of Genomic Determinants in Response to Immune Checkpoint Inhibitors in Melanoma.

BackgroundDespite the acknowledged sex-related differences in immune response and immune checkpoint inhibitor (ICI) efficacy, little is known about the sex disparities in melanoma of novel genomic determinants for ICI therapies.MethodsPretreatment genomic profiles and clinical characteristics of 631 melanoma patients treated with ICIs (i.e., inhibitors of CTLA-4, PD-1/PD-L1, or both) were comprehensively curated. Genomic factors, i.e., significantly mutated genes (SMGs), mutational signatures, and molecular subtypes were identified, and their associations with ICI treatment efficacy in male and female patients were evaluated.ResultsOf the 15 SMGs identified in this study, three genes (i.e., CFH, DGKG, and PPP6C) were found to exhibit sex differences with respect to ICI efficacy. Among these, CFH mutations exhibited both response rate and survival benefits in male, but not in female patients. A total of four mutational signatures (i.e., signatures 1, 4, 7, and 11) were extracted. Male patients with signature 4 (also known as smoking-related signature) had an inferior ICI response rate and overall survival. However, this association was not significant in females. An immune subtype based on mutational activities was found to be significantly associated with poor ICI survival in female patients.ConclusionWe uncovered several sex-dependent genomic correlates of response to ICI treatment, such as male-biased CFH mutations and signature 4 and the female-biased immune resistance subtype. The findings derived from this research provide clues for exploring different immunotherapeutic approaches in male and female patients with melanoma.

Atypical hemolytic uremic syndrome in the Colombian Caribbean: its particular characteristics.

IntroductionAtypical hemolytic uremic syndrome (aHUS) is a rare and genetically mediated systemic disease most often caused by uncontrolled and chronic complement activation that leads to systemic thrombotic microangiopathy, renal and extra-renal damage.Materials and methodsThis is descriptive, retrospective and multicenter study, which reports demographic, clinical, laboratory, and genetic characteristics, as well as their treatment response and outcome of 20 aHUS patients diagnosed between 2014 and 2018.ResultsMost patients were female adults (75%) and 30% were associated to pregnancy/postpartum, 15% to autoimmune disease, and 65% to infections. Gastrointestinal involvement (75%) was the most frequent extra-renal organ damage. Antenatal mortality and mortality rate were 5% and 10%, respectively. 25% of the patients progressed to end-stage renal disease. In 4/8 of patients treated within 1 week of presentation, eculizumab treatment restored multi-organ function after 4 weeks of treatment. CFH (37%) and CFI (25%) mutations were the most frequent.ConclusionThis is the first series of aHUS cases of Colombian Caribbean region which reports the clinical and epidemiological characteristics of this condition in this region.

The Whole Exome Analysis and Clinical Relevance in Patients with Atypical Hemolytic Uremic Syndrome

INTRODUCTION:Atypical hemolytic uremic syndrome (aHUS) is a rare, genetic, progressive, life-threatening form of thrombotic microangiopathy (TMA). A genetic mutation has been found as an explanation for constitutive complement activation in 50-60% of cases. In this abstract, we present whole exome analysis data results and their clinical relevance in 12 patients with aHUS.MATERIALS AND METHOD:We have enrolled 24 patients in this study who were diagnosed as aHUS and followed in our clinic since 2012. For exome sequencing, libraries were prepared using SeqCap EZ Human Exome Library v3.0 kit. Real Time Analysis software version with standard parameters was used for real-time image analysis and base calling. Raw sequencing data were aligned to the human genome hg19 reference (Homo sapiens (hg19) sequence) using CLC Genomic Workbench V10 (Qiagen) with standard parameters in paired-end mode. All variants were filtered according to the following criteria: not in dbSNP144 and also 1000 genome MAF <0.01, homozygous in the affected patients and heterozygous in the carrier parents, Genotype Quality (GQ) >30 and coverage (DP) >20 and, in homozygous region.FINDINGS AND RESULTS:We performed whole exome sequencing in all 24 patients but during raw data analysis 12 patients were found to be eligible for further analysis according to QC value. We didn't find any mutation in one of the patients (case 10) who had presented with diarrhea and had very good response to plasmapheresis only. We detected 32 different complement mutations in 4 different genes including CC3, CFI, CFH and membrane MCP in all other 11 patients. These mutations are summarized in the table.Four of the patients have responded to plasmapheresis and eculizumab was not needed. Of the remaining 20 patients eculizumab was aimed to be used. However, two of them died due to acute respiratory distress and thrombosis prior to eculizumab therapy. Case 9 was one of them and we found 21 different mutation including nine in CC3, five in CFH and 7 in MCF. One of our patients died in her 3rd month of eculizumab therapy (Case 2) and she had also seven mutations mostly in CFH gene. We used eculizumab in 18 of the patients and got complete response for thrombotic microangiopathy (TMA) in all. We had very good response to eculizumab in all these patients; even hemodialysis need disappeared in nine of them during eculizumab therapy. We experienced recurrence of renal failure in one patient (Case11) under eculizumab treatment in two years. She had also two different mutations in CFI gene four of the seven mutations in MCP gene and four of the 15 mutations in CC3 gene. This patient was lost due to multiple organ failure.We decided to stop eculizumab at the ninth month of treatment in two of the patients (Case 1 and Case 3) who had complete hematologic and renal recovery. Case 1 relapsed 16 months later and successfully retreated with eculizumab. She had 15 different mutations as eight mutations in CC3, six mutations in CFH, and 1mutation in MCP gene. Case 3 is still under control for three years after stopping eculizumab treatment. But as an interesting finding, this patient was 80 years old and had the heaviest mutation load with 22 different mutations (6 of the 7 mutations in MCP gene, 11 of the 15 mutation in CC3 gene, 4 of the 8 mutation in CFH gene).AHUS has been diagnosed during pregnancy in two of the patients and they had a successful delivery with healthy babies without any complication including ESRD. We also found 14 different mutations mostly in CFH gene in one of them (Case 8).DISCUSSION AND CONCLUSION:We found at least four different mutations in all patients except one case. Although MCP mutations accounts for only 15% of the aHUS cases we also found at least one MCP mutation in 91% of our cases. We found the rarest mutations in CFI gene. Although there are no clinical predictors of outcome, knowledge of the underlying genetic defect is helpful in predicting prognosis. But the patient who had the most number of mutations also had the best prognosis in our cohort. Therefore it is hard to use mutation data for management and prognosis of the syndrome. But the presence of a mutation is clearly important for the ESRD before transplantation decision and also would encourage patients to continue treatment. [Display omitted] DisclosuresYenerel:Alexion: Honoraria. Caliskan:Alexion: Honoraria. Yildiz:Alexion: Honoraria. Turkmen:Alexion: Honoraria.

De Novo and recurrent thrombotic microangiopathy (TMA) after renal transplantation: current concepts in management

Thrombotic microangiopathy (TMA) is a well-recognized complication of kidney transplantation that leads frequently to allograft failure. This serious outcome depends greatly on the underlying etiology as well as the timing of therapeutic interventions. TMA syndromes may occur with no previous history of TMA, i.e., de novo TMA, mostly due to medications or infection, or more frequently recurs after kidney transplantation i.e., recurrent TMA in patients with ESRF due to the atypical hemolytic uremic syndrome (aHUS). On the other hand, patients with shiga-toxin induced HUS (classic HUS), particularly in childhood has a favorable prognosis. One of the fundamental tools of management of this disease is the genetic screening for abnormal mutations, determination of which will recognize the tools of therapy and consequently outcome of the disease to a large extent. While patients with CFH and CFI mutations have a worse prognosis, other patients with MCP mutations-for example- have a more favorable prognosis. Accordingly, plan of therapy can be thoroughly drawn with a better chance of cure. Unfortunately, the successful use of the biological agent “eculizumab”, an anti-C5 agent, in some of these syndromes is largely impeded by its high cost linked to its use as a life-long therapy. However, a new therapeutic option has been recently admitted ameliorating this drawback and improve the cost-effectiveness balance.

1371P - Genomic landscape of primary malignant melanoma of esophagus

BackgroundAnti-PD-1 monoclonal antibody monotherapy achieves poor outcomes in mucosal melanoma, but our previous study has demonstrated that primary malignant melanoma of esophagus (PMME) has a dramatically high response rate (ORR 75%). The genomic landscape of PMME and reasons for this finding have not been extensively investigated. MethodsWhole exome sequencing were performed in seven treatment-naive PMME tumor samples and matched normal tissues, and genomic data of eight mucosal melanomas was obtained from the International Cancer Genome Consortium. Comprehensive profiling was performed to analyze the of genetic differences of PMME with mucosal melanoma. ResultsThe mean depth of coverage was 268X (ranged from 100X to 400X), 1203 SNV/InDels were detected in seven PMME samples, with a mean value of 172 mutations. The median tumor mutation burden (TMB) was 3.58 mutations/Mb (range 0.19-9.58 mutations/Mb), which was significantly than mucosal melanoma (P=0.021). The mutation spectrum of SNVs displayed more frequency of T>A transition while less frequency of C>T transversion compared to mucosal melanoma. There were 36 genes found to be mutated in more than two PMME tumor samples, and the most significantly mutated genes were CFH (42.9%, 3/7) and MUC16 (42.9%, 3/7). Both BRAF and NRAS were only found in one sample, and no NF1, KIT and SF3B1 mutation were detected. In total, 671 CNVs were detected, and the most frequently amplification chromosomal regions were 10q and 1p, while most frequently deletion in 1q and 8q. Genetic aberrations were mainly enriched in RTK/RAS and WNT pathways. ConclusionsThe TMB is higher in PMME compared to mucosal melanoma, with the highest mutation frequency in CFH and MUC16. CFH has a critical role in the regulation of complement activation, and MUC16 has been reported to be associated with higher tumor mutation load and outcomes in gastric cancer. In summary, our study reveals that the genomic landscape of PMME is different from mucosal melanoma originates from other mucosal membrane sites, and indicates that CFH and MUC16 mutation might be associated with better response to anti-PD-1 monoclonal antibody in PMME patients. Legal entity responsible for the studyPeking University Cancer Hospital-Beijing Cancer Hospital. FundingBeijing Talents Foundation (2016000021223ZK18), National Natural Science Foundation of China (81672696, 81402264). DisclosureAll authors have declared no conflicts of interest.

Treatment of hemolytic uremic syndrome related to Bordetella pertussis infection \u2014is plasma exchange or eculizumab use necessary?

BackgroundBordetella pertussis infection is a known trigger of atypical hemolytic uremic syndrome (HUS). For patients suspected of having atypical HUS, prompt plasma exchange/infusion (PE/PI) or eculizumab (ECZ) treatment is recommended.Case presentationWe report a 1-month-old female infant who was admitted with a severe cough and a B. pertussis-positive sputum culture. She was born at 38 weeks gestation and did not have a family history of renal diseases. Hemophagocytic syndrome was suspected and she was transferred to our hospital 17 days after her initial admission. One day later, she developed acute kidney injury and was diagnosed with HUS triggered by B. pertussis infection. Her plasma complement levels were low and her kidney function continued to worsen over the next few days. However, prior to starting ECZ treatment, her kidney function improved spontaneously; she did not receive PE/PI or ECZ. She was discharged 46 days after her initial hospitalization, without complications. A genetic workup revealed no mutations in CFH, CFI, CFB, C3, MCP, THBD, or DGKE.ConclusionsThis case demonstrates that B. pertussis infection-related HUS may resolve spontaneously. The decision to treat during the acute phase is challenging because B. pertussis often affects infants suspected of having atypical HUS. However, ECZ may not be the first treatment option for patients with B. pertussis infection-related HUS unless they show an indicated genetic abnormality; if ECZ is used, early discontinuation should be considered.

A retrospective study of pregnancy-associated atypical hemolytic uremic syndrome

Pregnancy-associated atypical hemolytic uremic syndrome (aHUS) refers to the thrombotic microangiopathy resulting from uncontrolled complement activation during pregnancy or the postpartum period. Pregnancy-associated aHUS is a devastating disease for which there is a limited clinical understanding and treatment experience. Here we report a retrospective study to analyze the clinical and prognostic data of 22 cases of pregnancy-associated aHUS from the Spanish aHUS Registry under different treatments. Sixteen patients presented during the first pregnancy and as many as nine patients required hemodialysis at diagnosis. Identification of inherited complement abnormalities explained nine of the 22 cases, with CFH mutations and CFH to CFHR1 gene conversion events being the most prevalent genetic alterations associated with this disorder (66%). In thirteen of the cases, pregnancy complications were sufficient to trigger a thrombotic microangiopathy in the absence of genetic or acquired complement alterations. The postpartum period was the time with highest risk to develop the disease and the group shows an association of cesarean section with pregnancy-associated aHUS. Seventeen patients underwent plasma treatments with a positive renal response in only three cases. In contrast, ten patients received eculizumab with an excellent renal response in all, independent of carrying or not inherited complement abnormalities. Although the cohort is relatively small, the data suggest that pregnancy-associated aHUS is not different from other types of aHUS and suggest the efficacy of eculizumab treatment over plasma therapies. This study may be useful to improve prognosis in this group of aHUS patients.

AHUS in children: A severe disease with increasingly better diagnostic and treatment options

Atypical haemolytic uraemic syndrome (aHUS) accounts for around 10% of children presenting with haemolytic uremic syndrome, a triad of thrombocytopenia, haemolytic anaemia and renal failure. As opposed to diarrhoeal causes usually associated with E. coli infection producing the toxin STEC, aHUS generally presents without diarrhoea and is frequently associated with defects in the complement pathway leading to over-activation of complement.The major cause of paediatric aHUS is due to defects in complement factor H, a regulatory protein that limits complement activation. Mutations in CFH that are both dominant and recessive have been described. There are now over 10 other genes that have been described in aHUS with around 40% of sporadic cases of aHUS without an identified genetic cause.Clinically patients often present acutely with an infectious prodrome, and may require supportive haemodialysis and transfusion. Children occasionally have extra renal manifestations involving GI and CNS. Therapy involves plasma exchange to replace complement factors and more recently the use of C5 inhibitors that have been highly effective.In those who progress to end-stage renal failure, transplantation carries a high risk of disease recurrence and this is highest with CFH mutations.Exclusion of infectious HUS and of TTP, a similar adult disease due to defects in ADAMTS13, is important. Genetic diagnosis has been significantly aided by massively parallel sequencing that can sequence the 10 known genes in one run. Further analysis using MLPA to identify rearrangements and deletions is also increasingly of use. Genetic identification assists in risk stratification and in identifying those children who will benefit from C5 inhibitors. Atypical haemolytic uraemic syndrome (aHUS) accounts for around 10% of children presenting with haemolytic uremic syndrome, a triad of thrombocytopenia, haemolytic anaemia and renal failure. As opposed to diarrhoeal causes usually associated with E. coli infection producing the toxin STEC, aHUS generally presents without diarrhoea and is frequently associated with defects in the complement pathway leading to over-activation of complement. The major cause of paediatric aHUS is due to defects in complement factor H, a regulatory protein that limits complement activation. Mutations in CFH that are both dominant and recessive have been described. There are now over 10 other genes that have been described in aHUS with around 40% of sporadic cases of aHUS without an identified genetic cause. Clinically patients often present acutely with an infectious prodrome, and may require supportive haemodialysis and transfusion. Children occasionally have extra renal manifestations involving GI and CNS. Therapy involves plasma exchange to replace complement factors and more recently the use of C5 inhibitors that have been highly effective. In those who progress to end-stage renal failure, transplantation carries a high risk of disease recurrence and this is highest with CFH mutations. Exclusion of infectious HUS and of TTP, a similar adult disease due to defects in ADAMTS13, is important. Genetic diagnosis has been significantly aided by massively parallel sequencing that can sequence the 10 known genes in one run. Further analysis using MLPA to identify rearrangements and deletions is also increasingly of use. Genetic identification assists in risk stratification and in identifying those children who will benefit from C5 inhibitors.

Genetic analysis and functional characterization of novel mutations in a series of patients with atypical hemolytic uremic syndrome

Atypical hemolytic uremic syndrome (aHUS) is a rare disorder caused by dysregulation of the complement alternative pathway, and associated with mutations in genes of complement components and regulators. In the recent years several studies have been published describing these mutations, however, no data is available from the Central and Eastern European region. In this study we present a detailed genetic analysis of our 30 patients, hospitalized with the diagnosis of aHUS in the past 7 years. We analyzed the genetic variants of genes CFH, CFI, CD46, THBD, CFB and C3; furthermore the possible effect of mutations that may alter the function or level of factor H protein was also investigated. We identified 27 (12 novel and 15 previously described) potentially disease-causing mutations in the candidate genes in 23 patients. Genetic analysis of family members revealed that in most cases the disease develops in individuals with multiple genetic risk factors, which may explain the low penetrance of the mutations. Here we showed that two novel mutations (p.W198R, p.P1161T) and a previously reported one (p.R1215Q) in CFH caused impaired regulation as indicated by increased lysis in hemolytic test, while four CFH mutations (p.V609D, p.S722X, p.T1216del and p.C448Y) were associated with decreased factor H protein level in serum as determined by allele-specific immunoassay. These results further point to the necessity of complete genetic workup of patients with aHUS and to the importance of functional characterization of novel variations.

The role of ADAMTS‐13 activity and complement mutational analysis in differentiating acute thrombotic microangiopathies

Essentials Molecular diagnostics has improved the differentiation of acute thrombotic microangiopathys (TMAs). Atypical hemolytic uremic syndrome may have features mimicking thrombotic thrombocytopenic purpura. We identified novel complement mutations and a high incidence of CD46, with favorable long term outcomes. Complement mutation analysis in TMA where the diagnosis is unclear and ADAMTS‐13 activity is >10%. SummaryBackgroundDifferentiation of acute thrombotic microangiopathy (TMA) at presentation has historically been dependent on clinical parameters. Confirmation of thrombotic thrombocytopenic purpura (TTP) is increasingly reliant on demonstrating deficient ADAMTS‐13 activity. The identification of alternative complement pathway abnormalities in atypical hemolytic uremic syndrome (aHUS), along with the proven efficacy of terminal complement inhibitors in treatment, has increased the need for rapid differentiation of TTP from aHUS.ObjectivesWe describe the clinical phenotype and nature of complement mutations in a cohort of aHUS patients referred as acute TMAs.Patients/methodsFourteen consecutive aHUS patients were screened for mutations in C3,CD46,CFH,CFI, and CFB, as well as factor H (FH) antibodies. All aHUS patients had ADAMTS‐13 activity > 10%.ResultsOf 14 aHUS patients, 11 (79%) had platelet counts < 30 × 109/L during the acute phase. Median presenting creatinine level was 295 μmol L−1, while five (36%) of 14 presented with a serum creatinine level < 200 μmol L−1. Alternative complement pathway mutations were detected in 9 (64%) of 14 patients, including CD46 mutations in five (36%) of 14 patients. Patients were identified with novel mutations in CFB and C3 that have not been previously reported.ConclusionsWe demonstrate that diagnostic differentiation based on platelet count and renal function is insufficient to predict an underlying complement mutation in some aHUS cases. Specifically, we demonstrate a high frequency of functionally significant CD46 mutations which may mimic TTP. ADAMTS‐13 activity > 10% in a patient with a TMA should necessitate genetic screening for complement abnormalities.

A novel quantitative hemolytic assay coupled with restriction fragment length polymorphisms analysis enabled early diagnosis of atypical hemolytic uremic syndrome and identified unique predisposing mutations in Japan.

For thrombotic microangiopathies (TMAs), the diagnosis of atypical hemolytic uremic syndrome (aHUS) is made by ruling out Shiga toxin-producing Escherichia coli (STEC)-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP), often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs) and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH) monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45) had moderate-to-severe (≥50%) hemolysis, whereas the remaining 76% (34/45) patients had mild or no hemolysis (<50%). The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34), which were identified by restriction fragment length polymorphism (RFLP) analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45) of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients.

Chronic Glomerulonephritis Causing Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is characterized by hemolytic anemia, thrombocytopenia and kidney failure. Unlike typical HUS which is usually caused by infection with certain strains of E.Coli, a-HUS is caused by abnormalities in complement system, triggered by certain environmental factors or genetic mutations. aHUS can be genetic, acquired, or idiopathic. Mutations in at least 10 genes identified so far (C3, CD46, CFB, CFH, CFHR1, 3, 4 and 5, CFI and THBD) appear to increase the risk of developing aHUS. CFH mutation is the most common accounting for approximately 30 percent of all cases. Pathophysiology is proposed to be microangiopathic in nature, where platelet thrombi occlude microcirculation resulting in organ dysfunction. It is believed that aHUS causes renal dysfunction but we propose that either condition can precede the other. Lucio et al (2013) followed patients who had biopsy-proven diagnosis of glomerulonephritis and found that significant number of them developed aHUS over a period of time.A 26-year old male with no past medical history presented to E.D. with malaise for one week. He denied any fever or chills, recent or remote diarrheal illness or any upper respiratory symptoms. Initial diagnostics revealed hemoglobin 6.8, hematocrit 19.7, WBC 9.2, platelets 79 with peripheral blood smear showing schistocyte, BUN 109, creatinine 28.6, LDH 813, and haptoglobin<10 consistent with hemolytic anemia, thrombocytopenia and renal insufficiency prompting the presumptive diagnosis of TTP/HUS. He was treated with emergent plasmapheresis and hemodialysis. ADAMTS13, complement C3and C4 levels were normal. Renal ultrasound discovered atrophic kidneys consistent with severe chronic kidney disease and kidney biopsy revealed focal segmental collapsing glomerulosclerosis without any evidence of microthrombi. Testing for HIV and hepatitis A, B, C and D were also negative. Given the absence of any preceding diarrheal illness, patient’s age at presentation, and triad of thrombocytopenia, hemolytic anemia and renal insufficiency prompted the diagnosis of a-HUS. Genetic studies for CFH, C3, CFB and CFI mutations were sent, which are still pending. Patient condition remained critical with ongoing hemolysis needing regular plasma exchange therapy and hemodialysis for over period of one month, consistent with aHUS refractory or dependent to plasma therapy. Patient was subsequently referred to a specialized center for treatment with Eculizumab and possible renal transplant.The sequence of events and kidney biopsy findings for our patient suggest that he had an underlying chronic glomerulonephritis which was complicated by aHUS, which has been reported previously. We propose that some common genetic mutations may predispose patients to glomerulonephritis and development of aHUS. Till now it has been postulated that underlying pathophysiology in aHUS is chronic, uncontrolled activation of the complement system. In this case normal complement levels and underlying chronic glomerulonephritis suggests an alternate mechanism for development of aHUS. We emphasizes the importance of further testing and research into the mechanism of aHUS and possible genetic linkage of chronic glomerulonephritis to aHUS. DisclosuresNo relevant conflicts of interest to declare.

Factors determining penetrance in familial atypical haemolytic uraemic syndrome

BackgroundInherited 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...