To explore the clinical characteristics, treatment protocol and prognosis of children with anti-complement factor H (CFH) autoantibody (Ab)-associated hemolytic uremic syndrome (HUS). Clinical data of 8 patients with anti-CFH Ab-associated HUS who were admitted to Shandong Provincial Hospital from January 2011 to December 2020 were collected retrospectively. The age at disease onset ranged between 5.83 and 13.5 years, with a male: female ratio of 1.67:1. The time of onset was distributed from May to June and November to December. Digestive and upper respiratory tract infections were common prodromal infections. Positivity for anti-CFH Ab and reduced C3 levels were observed among all patients. Heterozygous mutation of the CHFR5 gene (c.669del A) and homozygous loss of the CFHR1 gene [loss2(EXON:2-6)] were found in two patients. All patients received early treatment with plasma exchange and corticosteroid therapy. Six patients were given immunosuppressive agents (cyclophosphamide and/or mycophenolate mofetil) for persistent proteinuria. The follow-up period was 12–114 months. Four of 8 patients achieved complete remission, 3 achieved partial remission, and 1 died. Relapse occurred in two patients. Children with anti-CFH Ab-associated HUS were mainly school-aged and predominantly male, with onset times of summer and winter. Digestive and upper respiratory tract infections were common prodromal infections. Plasma exchange combined with methylprednisolone pulse therapy in the acute phase and cyclophosphamide or mycophenolate mofetil treatment for maintenance can be utilized in children with anti-CFH Ab-associated HUS if eculizumab is not available.
Keywords: Hemolytic uremic syndrome; anti-CFH autoantibody; children; eculizumab; cyclophosphamide; mycophenolate mofetil
Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy (TMA) with three major clinical features: microvascular hemolytic anemia, acute kidney injury, and thrombocytopenia. HUS is divided into infection-related HUS, secondary HUS, and atypical HUS (aHUS) [[
Herein, we collected the clinical data of 8 cases of anti-CFH Ab-associated HUS in our hospital from 2011 to 2020 and retrospectively analyzed the clinical characteristics, treatment protocols and prognosis of these pediatric patients.
Clinical data of 8 patients with anti-CFH Ab-associated HUS who were admitted to Shandong Provincial Hospital from January 2011 to December 2020 were collected retrospectively. All patients were treated according to the same protocol. The diagnosis of HUS was based on the following criteria: acute hemolytic anemia, fragmented erythrocytes in a peripheral blood smear, thrombocytopenia (platelet count <150 × 109/L), elevated levels of lactate dehydrogenase (LDH), negative Coombs' test, and/or acute kidney injury [[
The inclusion criteria were as follows: (
The current study was approved by the ethics committee of our hospital with the approval number NO.2022-253, and all patients' legal guardians signed written informed consent.
Clinical and pathological data were collected, mainly consisting of general indexes (including sex, age and time of onset), symptoms of prodromal infections, clinical manifestations, laboratory tests (including hemoglobin, platelets, urea nitrogen, serum creatinine, 24 h urinary protein content, complement C3, C4, CFH and anti-CFH autoantibodies, etc.), kidney biopsy, genetic testing, treatment and prognosis.
Serum CFH levels were detected by ELISA [[
Plasma anti-CFH immunoglobulin G (IgG) titers were detected by ELISA [[
Kidney biopsy was performed on 3 of 8 patients. A spring-loaded needle biopsy kit was used to obtain kidney tissue samples under the real-time guidance of a B-ultrasonic detection system. After percutaneous kidney puncture biopsy, the kidney tissue was stained [hematoxylin-eosin (HE) staining, periodic acid-Schiff (PAS) staining, hexamine silver (PASM) staining and Masson staining], and electron microscopy and immunofluorescence were performed (IgA, IgG, IgM, Fib, C1q, C3).
Whole-exome sequencing (WES) was performed on 5 of 8 patients. Whole blood samples (2–5 mL) from the affected children and their parents were collected. DNA extraction was carried out, and the whole exome was captured and sequenced with the IDT xGen exome research panel v2.0 full exon capture chip. The variation classification adopts the three-factor classification system and the ACMG (American Medical Genetic Society) gene variation classification system. For suspected pathogenic mutations, Sanger sequencing was performed with an ABI3730 sequencer after PCR of the target sequence, and the verification results were obtained with sequence analysis software.
The response to therapy included complete remission, partial remission, and treatment failure [[
The statistical software SPSS version 18.0 (IBM Corp., Armonk, NY, USA) was used for the statistical analyses. The quantitative data are expressed as the median with interquartile range (IQR).
There were 5 males and 3 females, with a male:female ratio of 1.67:1. The age at disease onset ranged between 5.83 and 13.5 years (median 6.67 years, 6.02–9.03 years). The time of onset was distributed from May to June and November to December. All patients had no other kidney diseases or family histories of thrombotic microangiopathy.
Prodromal infection symptoms were present in 87.5% (7/8) of patients, of whom 75% (6/8) had digestive symptoms such as vomiting and abdominal pain, 62.5% (5/8) had symptoms of upper respiratory tract infection such as fever and cough, and 50% (4/8) had coinfection of the respiratory system and digestive system. The symptoms of the digestive system mainly included nausea, vomiting, abdominal pain or diarrhea without blood stools.
All patients showed typical manifestations of HUS, such as hemolytic anemia, thrombocytopenia, and acute kidney injury. All patients showed edema, gross hematuria, foamy urine, and jaundice. Seven patients (87.5%) presented with bleeding points on the skin and mucous membranes. Additionally, gastrointestinal bleeding and/or nosebleed was present in 2 patients (25%). Oliguria or anuria was seen in 6 cases (75%). Five patients (62.5%) presented with liver involvement. Hypertension was present in 2 patients (25%). One patient (25%) presented with pancreatitis (Case 4). One patient (25%) died of pulmonary hemorrhage (Case 1). One patient (25%) presented with convulsions caused by hypertension encephalopathy (Case 8) (Table 1).
Table 1. Clinical features of 8 children with anti-CFH Ab-associated HUS.
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Age (years) 5.83 7 7.33 6.33 9.59 13.5 5.92 6.33 Sex Male Male Male Male Female Male Female Female Prodrome Vomiting Vomiting, abdominal pain, fever, cough Vomiting, fever, cough Vomiting, abdominal pain, diarrhea Vomiting, cough Cough None Vomiting, abdominal pain, fever, cough Time of onset November May December November May November June November Jaundice Yes Yes Yes Yes Yes Yes Yes No Petechia Yes Yes Yes Yes Yes Yes Yes No Hypertension No No No Yes No No No Yes Oliguria/Anuria Yes Yes No Yes No Yes Yes Yes Edema Yes Yes Yes Yes Yes Yes Yes Yes Hematuria Yes Yes Yes Yes Yes Yes Yes Yes Proteinuria Yes Yes Yes Yes Yes Yes Yes Yes Hepatic involvement No Yes Yes Yes No Yes Yes No Other extrarenal involvement Pulmonary hemorrhage, gastrointestinal hemorrhage, multiple organ failure no Epistaxis, gastrointestinal hemorrhage Pancreatitis No No No Convulsions, hypertensive encephalopathy, severe pneumonia, respiratory failure
In the acute phase, all patients tested positive for anti-CFH autoantibodies. The titers of CFH were normal in 2 patients (25%) and reduced in 6 patients (75%). All patients presented with moderate to severe anemia and thrombocytopenia, high levels of lactate dehydrogenase (LDH), a sharply increased ratio of reticulocytes, and severe acute kidney injury, with elevated levels of serum creatinine and urea nitrogen. Hypoalbuminemia and hyperbilirubinemia (mainly indirect bilirubin elevation) were present in 7 of 8 patients (87.5%). The serum levels of C3 were reduced, and serum C4 levels were normal in all 8 patients. The urinary protein quantification for all 24 h showed that all patients presented with gross proteinuria (Table 2, Figure 1).
PHOTO (COLOR): Figure 1. The laboratory results of 8 cases with anti CFH Ab associated HUS.
Table 2. Laboratory results of 8 children with anti-CFH Ab-associated HUS.
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Anti-CFH IgG Positive Positive Positive Positive Positive Positive Positive Positive CFH titer (µg/ml) 103 ↓ 117.1 ↓ 122 ↓ 213.3 ↓ 161 ↓ 281.9 287 167.1 ↓ Hb (g/L) 41 61 51 69 25 67 47 58 Plt (x10^9/L) 16 23 53 19 18 54 3 82 Ret (%) 6.38 31.97 16.42 16.88 31.13 24.39 31.66 12.74 Serum albumin (g/L) 27.7 31.1 26.9 20.7 29.8 33 35.2 29.2 TBIL (μmol/L) 79.51 56.2 26.47 76.71 37.9 58.73 63.74 14.98 IBIL (μmol/L) 65.3 47.7 23.4 66.57 32.65 51.14 54.27 11.96 DBIL (μmol/L) 14.21 8.5 3.07 10.14 5.25 7.59 9.47 3.02 BUN (mmol/L) 70.7 41.3 49.75 60.8 49.4 39.2 24.1 29.2 Scr (μmol/L) 730.13 238.75 441.05 252.73 345.67 801 146 542.7 LDH (U/L) 4115 4416 2223 3267 2698 2584 2995 1753 C3 (g/L) 0.39 0.4 0.6 0.68 0.61 0.41 0.67 0.75 C4 (g/L) 0.23 0.22 0.16 0.22 0.1 0.18 0.16 0.13 24 h Urinary protein content (mg/kg) – 207 261 118 194 71 115 50 Renal biopsy No No Yes Yes No Yes No No Gene mutation – – No definite pathogenic mutation – No definite pathogenic mutation No definite pathogenic mutation CFH R5 heterozygous mutation CFHR1 homozygous loss
1 CFH: complement factor H; Hb: hemoglobin; Plt: platelet; Ret: reticulocyte; TBIL: total bilirubin; IBIL: indirect bilirubin; DBIL: direct bilirubin; BUN: blood urea nitrogen; Scr: serum creatinine; LDH: lactate dehydrogenase; C3: complement 3; C4: complement 4.
A correlation matrix between CFH titer and regular parameters (hemoglobin, platelets, ratio of reticulocytes, albumin, total bilirubin, indirect bilirubin, direct bilirubin, urea nitrogen, serum creatinine, LDH, complement C3, C4, 24 h urinary protein content) has been made. However, no significant correlation between CFH titer and the above parameters was observed (Figure 2).
Graph: Figure 2. A correlation matrix scatter plot between CFH titer and other laboratory parameters of 8 cases with anti CFH Ab associated HUS.
Three of 8 patients underwent kidney biopsy (Table 2). Under a light microscope, mild to moderate segmental proliferation of the mesangial region and shrinkage of the basement membrane were observed. Glomerular capillary tuft collapse was observed in 1 case. Moreover, segmental glomerulosclerosis was observed in 1 or 2 locations in 2 patients. Two or 3 balloon adhesions were found in 2 patients. Swollen arteriolar endothelial cells were observed in 1 patient, and microthrombi were found in 1 patient. Direct immunofluorescence analysis revealed that one patient had positive staining for IgM (2+) and F (2+), one patient had positive staining for IgM (3+), and one patient had no immune complex deposition. In addition, the immune complexes were distributed in granular form along the capillary wall. Under an electron microscope, swollen endothelial cells of glomerular capillaries, widened subendothelial space, and fused foot process cells were observed. One patient appeared to have cellulose-like substances deposited under the endothelium.
Five of the 8 patients underwent genetic testing (Table 2). Heterozygous mutation of the CHFR5 gene (c.669del A) and homozygous loss of the CFHR1 gene [loss2 (EXON:2-6)] were found in two patients (Cases 7 and 8), and no disease-related mutations were found in the other three patients.
All patients received early treatment with plasma exchange and corticosteroid therapy. One patient (case 7) received treatment with plasma exchange and 2 courses of methylprednisolone pulse (MPP) therapy. One patient (case 8) received treatment with plasma exchange 9 times, hemodialysis and continuous kidney replacement therapy (CKRT) therapy with MPP therapy (1 d, stopped for severe infection). Five patients received treatment with plasma exchange 5–7 times at first, and 1–3 courses of MPP were adopted later due to poor control of hemolysis. One patient (case 1) received treatment with plasma infusion and plasma exchange and two rounds of high-dose MPP on the 4th day after admission. However, he unfortunately died of pulmonary hemorrhage on the 9th day. The remaining 7 patients continued to receive oral corticosteroid (prednisone) treatment. Six patients were given immunosuppressive agents for persistent proteinuria. Among them, 3 patients received cyclophosphamide (CTX) pulse therapy after the acute phase. The other 3 patients were treated with mycophenolate mofetil (MMF) within six months after the acute phase. Two patients received MMF after CTX therapy for persistent proteinuria and relapse after 14 months (Table 3).
Table 3. Treatment and prognosis of 8 children with anti-CFH Ab-associated HUS.
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Treatment Plasma infusion Yes No No No No No No No Plasma exchanges (times) 1 2 5 9 8 5 7 9 CKRT/hemodialysis Yes Yes No Yes No Yes No Yes Steroids MPP MPP MPP MPP MPP MPP MPP MPP Immunosuppressants none MMF CTX, MMF CTX MMF MMF CTX, MMF CTX Time of follow-up (months) – 114 70 57 65 72 62 12 Prognosis death CR PR CR CR CR PR PR Time to recovery of blood parameters and renal function (days) – 13 29 24 41 23 25 – Time to recovery of urine protein (months) – 20 – 3 19 10 – – Time to recovery of C3 (days) – 25 28 7 – – 33 – Relapse – Yes, 2 years later No No No No Yes, 1 year later No
2 CKRT: continuous kidney replacement therapy; MPP: methylprednisolone pulse therapy; MMF:mycophenolate mofetil; CTX:cyclophosphamide; CR: complete remission; PR: partial remission.
The follow-up period was 12–114 months (median 65 months, 57–72 months). Four of 8 patients achieved complete remission, 3 achieved partial remission, and 1 died (Table 3). During the recovery period, the levels of hemoglobin, platelets, LDH, reticulocyte ratio, serum creatinine and urea nitrogen completely returned to normal in 6 patients after 13–41 days of treatment. However, mild anemia was still present in case 8 after 12 months of therapy. The level of complement 3 returned to normal in four patients after 7–33 days of treatment, with 1 patient (case 5) still showing a reduced level. Proteinuria completely disappeared in 4 patients after 3-20 months of follow-up, with occasional reappearance in 2 patients (case 3, case 7) and a partial decrease in 1 patient (case 8).
Relapse occurred in two patients (cases 2 and 7). Case 2 was discharged after partial improvement because of the unwillingness of his parents to continue treatment, and he irregularly received treatment with Chinese medicine. Relapse occurred after 2 years, and the boy received treatment in our hospital again. MMF was adopted to maintain remission after the disease was under control. No recurrence occurred after follow-up during the following 90 months. Case 7 relapsed at 5 months after steroid withdrawal, and the total time of complete remission was 14 months. She received plasma exchange, MPP therapy and later, MMF. No recurrence occurred within the following 42 months.
The CFH family is composed of seven distinct proteins. They include CFH, factor H-like protein 1 (FHL-1), a splice derivative of the CFH gene, and five CFH-related (CFHR) proteins. Anti-CFH Ab-associated HUS is also called autoimmune HUS (AI-HUS), which accounts for 6–56% of aHUS cases [[
Anti-CFH Ab-associated aHUS predominantly presents in childhood [[
The main seasons of onset in this study were summer (from May to June) and winter (from November to December). However, Puraswani M et al. reported that the peak incidence of onset for anti-CFH Ab-associated HUS in children occurs from December to April [[
In this study, all 8 patients presented with severe hemolytic anemia, thrombocytopenia, acute kidney injury, hematuria and gross proteinuria. Extrarenal symptoms such as pancreatitis, pulmonary hemorrhage, gastrointestinal hemorrhage, and hypertension encephalopathy were also observed in these patients. Serum positivity for anti-CFH Ab was present in these 8 children, and 6 patients showed reduced serum CFH levels. Reduced serum C3 levels were shown in 8 patients. Aditi Sinha reported complement C3 levels lower than 70 mg/dl in 62.0% of aHUS children [[
Kidney pathological changes in children with aHUS may help predict patient prognosis. Morphological features of HUS have been traditionally divided into early changes and late changes on light microscopic kidney evaluation [[
Homozygous loss of the CFHR1 gene [loss2(EXON:2-6)] and heterozygous mutation of the CHFR5 gene (c.669del A) were found in two patients in this study. Previous work has also shown a correlation between the occurrence of anti-CFH aHUS and homozygous deletions in CFHR1/CFHR3 [[
Eculizumab is a recombinant, fully humanized hybrid IgG2/IgG4 monoclonal antibody directly that directly blocking blocks human complement component C5. The drug was first used in aHUS cases in 2009, and it has been used as a successful treatment for both adult and pediatric aHUS patients [[
In summary, children with anti-CFH Ab-associated HUS were mainly school-aged and predominantly male, with onset times of summer and winter. Digestive and upper respiratory tract infections were common prodromal infections. CFHR1 and CHFR5 mutations were related to the pathogenesis of aHUS. Plasma exchange combined with MPP therapy in the acute phase and CTX or MMF treatment for maintenance can be utilized in children with anti-CFH Ab-associated HUS if eculizumab is not available.
No potential conflicts of interest are reported by the author(s).
The data used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
By Qian Li; Xinxin Kong; Minle Tian; Jing Wang; Zhenle Yang; Lichun Yu; Suwen Liu; Cong Wang; Xiaoyuan Wang and Shuzhen Sun
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