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- Material and Methods
Hemolytic uremic syndrome (HUS) is a rare disease characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure, with a worldwide incidence of 0.7–2 cases/year/100 000 people .
About 90% of patients develop typical diarrhea-associated HUS, most commonly triggered by entero hemorrhagic strains of E. coli, mainly of serotype O157:H7, producing Shiga-like toxins (STEC) [2, 3]. STEC-HUS occurs primarily in children . However, a large outbreak (>4000 cases) of gastroenteritis and HUS, caused by an unusual enteroaggregative STEC strain (O104:H4) and predominantly affecting adults, occurred in Germany in 2011 . More than 25% of infected subjects developed HUS, a proportion much higher than those recorded in outbreaks of enterohemorrhagic STEC .
In STEC-associated HUS, binding of Shiga-like toxin (Stx) to globotrialosyl ceramide receptors, highly expressed on glomerular endothelial cells, generates a cascade of signals resulting in expression of chemokines and adhesion molecules, complement activation, leukocyte recruitment and thrombus formation [3, 4, 6-8].
More than 90% of childhood cases of STEC-HUS fully recover from acute disease, although long-term renal sequelae have been reported in up to 25% of patients . The reasons why only a small fraction of STEC-infected subjects develops a severe form of HUS is not clear, but a role of genetic predisposition has been hypothesized .
On the other hand, 10% of HUS cases are not associated to STEC infections (atypical HUS, aHUS). Mutations in genes encoding for proteins of the complement system have been shown to predispose to aHUS . The complement system is involved in the immediate defense against pathogens through three different pathways, the classical, the lectin and the alternative pathways, converging at the cleavage of C3. About 60% of patients with aHUS carry mutations in genes encoding regulatory proteins of the alternative pathway (AP) of complement, like factor H (CFH), factor I (CFI) and membrane cofactor protein (MCP), or gain-of-function mutations in the genes of the two components of the AP C3 convertase, C3 and factor B (CFB) . Genetic abnormalities of the endothelial anticoagulant and complement-inhibitory protein thrombomodulin (THBD) have also been reported in aHUS . In addition, anti-CFH autoantibodies have been described mostly in children that lack CFHR1 and CFHR3 (CFH-related proteins 1 and 3) due to a deletion of the corresponding genes [13-16]. All the above abnormalities result in uncontrolled complement activation on endothelial cell surface [11, 17-19].
The clinical outcome of aHUS is usually unfavorable. About 50% of cases progress to end stage renal disease (ESRD) and up to 25% may die during the acute phase .
Here we describe two patients with a clinical history of STEC-HUS, selected among 41 STEC-HUS patients of the International Registry of Hemolytic Uremic Syndrome/Thrombotic Thrombocytopenic Purpura (HUS/TTP). Both patients developed ESRD soon after disease onset and received a kidney graft, one from a deceased donor and one from a living-related donor. Disease recurrence is uncommon in patients transplanted after STEC-HUS , and indeed the other three STEC-HUS transplanted patients of the Registry had an excellent graft outcome. At variance both cases here presented lost the kidney graft for HUS recurrence, a complication more commonly observed in aHUS [19, 20]. Thus, before planning a second renal transplantation, the patients underwent genetic screening for aHUS-associated genes. We found a heterozygous CFI mutation in patient #1 and a heterozygous MCP mutation in patient #2 and also in her mother, who donated the kidney.
We argue that the two cases originally diagnosed as STEC-HUS had indeed aHUS triggered by STEC infection on a genetic background of impaired complement regulation, which predisposed to development of posttransplant aHUS recurrence.
We discuss the implications of these findings for pretransplant genetic counseling in patients who developed ESRD following STEC-HUS.
Material and Methods
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- Material and Methods
The two patients were selected among those with diagnosis of STEC-HUS referred to the International Registry of HUS/TTP.
The patients and healthy controls provided informed written consent. The study was approved by the Ethics Committee of the Azienda Sanitaria Locale, Bergamo, Italy.
Genomic DNA was extracted from peripheral blood leukocytes (Nucleon BACC2 kit, Amersham, UK). The coding sequences and the intronic flanking regions of complement genes were screened by direct sequencing (AB-3730 sequencer).
CFHR3-CFHR1 deletion was detected by Multiplex Ligation-dependent Probe Amplification, MLPA (SALSA MLPA P236-A1 kit, MRC Holland).
Complement C3 and C4 levels in serum were evaluated by kinetic nephelometry; CFH and CFI levels were measured by enzyme-linked immunosorbent assay (ELISA).
Paraffin-embedded sections of kidney biopsy of patient #1 were analyzed by light microscopy after staining with hematoxylin and eosin for histopathological evaluation. Additional sections were analyzed for deposition of C3 and C5b-9 using mAb anti-C3 (Quidel, San Diego, CA) and mAb WU 13–15 anti-C9 neoantigen (a gift from Prof. R. Wurzner, Insbruck, Austria) as primary antibodies and the streptavidin–biotin–peroxidase complex system.
The clinical course of the patient, at present 47 years old, was partially described in a previous report . She was first hospitalized at the age of 26 years because of watery diarrhea, profuse vomiting and syncope. Laboratory exams showed platelet count 27,000/µL, hemoglobin 8.1 gr/dL, hematocrit 26%, LDH 2.200 U/L and serum creatinine 2 mg/dL. The diagnosis of HUS was made. Search for E. coli infection showed positivity for anti-Stx and anti-E. coli O157:H7 lipopolysaccharide antibodies in serum. A renal biopsy demonstrated classic histological features of thrombotic microangiopathy (Figure 1A–D). A diffuse and intense reactivity for C3 and C9, the latter reflecting formation of the terminal complement complex (C5b-9), was evident in glomeruli with overt ischemic alterations (Figure 1E–F).
Figure 1. Renal biopsy of the native kidney from patient #1. Images show typical features of active microangiopathic process characterized by glomerular alterations of variable severity from slight ischemic changes (A) with capillary engulfment and mesangial proliferation (B) to infarction and mesangiolysis (C) (original magnification 400×). Small arteries and arterioles showed prominent intimal (black arrows) and subintimal proliferation leading to lumen occlusion (D) (original magnification 400×). Panels E and F: Immunohistochemistry showing C3 deposition on the endothelial layer of the glomerular tuft (E). C9 immunostaining demonstrates diffuse terminal complement activation (C5b-9) in glomeruli with overt ischemic alterations (F). Original magnification 400×, Streptavidin-biotin-peroxidase complex method.
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Biochemical analyses, performed during the acute phase, showed low C3 levels (65.6 mg/dL; normal range: 90–180 mg/dL) and normal C4 levels (14.6 mg/dL; normal range: 10–40 mg/dL), suggesting activation of the AP.
Despite intensive plasma exchange the disease progressed, renal function deteriorated and hemodialysis was started. Seizures and hypertensive encephalopathy developed and the patient underwent bilateral nephrectomy that was followed by complete neurological and hematological remission. At discharge, the patient was on chronic hemodialysis but her blood pressure was well-controlled .
At 33 years of age the patient received a cadaveric renal allograft under immunosuppressive therapy with cyclosporine, mycophenolate mofetil and methylprednisolone; however, one year after transplantation she developed clinical and laboratory signs of HUS recurrence (platelet count 37 000/µL, hemoglobin 9 gr/dL, hematocrit 29%), with severe renal and neurological involvement (serum creatinine 4 mg/dL, seizures). No improvement was achieved with plasma exchange and infusion, the patient developed ESRD and the graft was explanted. About 10 years later, before planning a second kidney transplantation, the patient was referred to our center to investigate her possible genetic predisposition to HUS and hence the risk of disease recurrence. No relevant clinical history was reported in her family.
C3 serum levels were persistently low (73 mg/dL) despite the patient was in remission without hematological signs of the disease, while C4 levels were normal (36 mg/dL). Serum levels of factor H (415 mg/L, normal range: 350–750 mg/L) and factor I (60.9 mg/L normal range: 38–65 mg/L) were normal. Genetic analyses of CFH, CFI and MCP were performed on leukocyte DNA and we found a new c.1234G > A heterozygous missense mutation in CFI, causing the aminoacidic substitution p.Val412Met (Figure 2A). This residue is localized in the serine protease domain SP (Figure 2B) that is responsible for C3b inactivation by CFI . The aminoacidic change Val412Met is predicted as damaging by PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/, score 0.997) and Sift (http://sift.jcvi.org, score 0.01) softwares. The variant is not reported in NCBI online database and was not found in any of 220 healthy controls. Homozygous deletion of CFHR3-CFHR1, previously associated with development of anti-CFH autoantibodies and aHUS , was excluded by MLPA .
Figure 2. Sequencing electropherograms showing the c.1234G > A heterozygous mutation in CFI of patient #1. A: upper chromatogram, forward; lower chromatogram, reverse. B: Schematic structure of CFI protein with the localization of the mutation.
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The clinical history of this patient, a 23-year-old woman, started at the age of 16 years when she presented with diarrhea, thrombocytopenia (platelet count 33 000/µL), microangiopathic hemolytic anemia (hemoglobin 6.1 gr/dL, hematocrit 17,3%), hypertension and very severe renal failure, quickly developing ESRD (serum creatinine 21 mg/dL). Infection by E. coli O157:H7 producing Stx1 and Stx2 was documented in stool cultures.
At 18 years of age living-related kidney transplantation from her mother was performed. Seven months after transplantation the patient manifested graft dysfunction. A graft biopsy revealed intraglomerular thrombi, ischemic alterations and arteriolar thrombosis leading to diagnosis of thrombotic microangiopathy. Despite intensive plasma-exchange, anemia (hemoglobin 8 gr/dL), thrombocytopenia (platelet count 80 000/µL) and severe graft dysfunction (serum creatinine 10 mg/dL) persisted and 2 months later the patient lost the graft.
Before planning kidney transplantation, DNA samples of the patient, of her parents and of the two siblings were sent to our center to investigate a possible genetic predisposition to HUS and to evaluate the further risk of disease recurrence. No relevant clinical history was reported in her family. Both the parents and the siblings were healthy (Figure 3).
Figure 3. Family pedigree of patient #2. The black circle represents the proband carrying the heterozygous MCP mutation c.286 + 2T > G; healthy carriers are indicated by a black dot. WT: Wild-type.
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C3 and C4 serum levels were normal in the patient. Serum levels of factor H (474 mg/L) were also normal. Genetic analyses of CFH, CFI, MCP, C3, CFB and THBD were performed on leukocyte DNA. The patient was found to be heterozygous for the c.286 + 2T > G (also known as IVS2 + 2T > G ) splice site mutation in MCP. This mutation has been previously described in aHUS patients [23, 24] and has been found to cause an abnormal splicing between exons 2 and 3, resulting in either protein truncation  or in the deletion of 48 aminoacids . The mutation was absent in 319 healthy controls. Analysis of relatives' DNA revealed the same mutation in the mother and in the brother, but not in the father and the sister (Figure 3 and 4).
Figure 4. Sequencing electropherograms of patient #2 and her parents showing the c.286 + 2T > G heterozygous mutation in MCP.
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Homozygous deletion of CFHR3-CFHR1 was excluded by MLPA .
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- Material and Methods
Here we describe two unrelated patients who debuted with STEC-HUS, developed ESRD soon after the onset of the disease and experienced HUS recurrence after kidney transplantation. This disease course is rather unusual. First, patients with STEC-HUS usually recover from acute episode, although on long term 25% of them develop some degree of renal impairment . Second, the disease rarely recurs in kidney transplants performed after STEC-HUS . STEC-released Stx is the causative agent of STEC-HUS, therefore a re-exposure to STEC would be required to trigger recurrence. In addition patients with STEC-HUS develop neutralizing anti-Stx antibodies that persist for long-time, thus protecting from recurrences .
Such severe clinical features instead closely resemble those reported in patients with aHUS and mutations in complement regulatory proteins. Consistently, genetic screening revealed the presence of a heterozygous CFI mutation associated with persistent low C3 levels in patient #1, whereas a heterozygous MCP splicing mutation was found in patient #2, who showed normal complement serum levels, and also in her mother, who donated the kidney.
CFI encodes a plasma serine protease that regulates the activation of complement system by inactivating C3b. Mutations in CFI have been reported in 4–10% of aHUS cases [1, 11] but had never been previously described in association with STEC-HUS. MCP is a transmembrane protein that serves as cofactor for CFI to cleave C3b and C4b deposited on host surfaces . MCP mutations have been previously reported in 10–15% of aHUS patients , and also in a severe fatal case of STEC-HUS .
Evidence is emerging that STEC triggers complement activation, as documented by finding low C3 serum levels, and transient plasma elevations of C3 (C3b, C3c and C3d) and CFB (Ba, Bb) breakdown products and of sC5b9, during the acute phase of STEC-HUS . In addition, Stx was found to cause complement activation and C3 deposition on endothelial cells in vitro . Stx binding to endothelial cells upregulates mRNA expression and protein levels of cell adhesion molecules, including P-selectin , which has been shown to bind C3b with high affinity . Finally, mice deficient in CFB treated with Stx and LPS exhibited less thrombocytopenia and were protected against glomerular abnormalities and renal function impairment as compared with wild-type mice, providing in vivo evidence of the role of the AP of complement in STEC-HUS . The evidence that complement activation may have a pathogenic role in the microangiopathic lesions of STEC-HUS  provided the rationale for complement inhibitor therapy in three children with severe STEC-HUS who fully recovered with Eculizumab , an anti-C5 antibody that prevents the formation of the anaphylotoxin C5a and of the C5b-9 lytic complex.
The above published data and present findings of intense deposition of C3 and C5b-9 in renal biopsy of patient #1 taken at the time of STEC infection indicate that exposure to STEC may cause complement activation and trigger severe HUS in subjects with genetic alterations of complement regulatory proteins. In patient #1, the kidney transplant did not correct the CFI genetic defect, since the altered protein is produced by the liver and persists in the circulation after kidney transplantation . In such conditions, the kidney graft was not protected from complement activation triggered by ischemia and reperfusion injury posttransplantation, alloimmune response and immunosuppressive drugs, resulting in aHUS. Hence, the recurrence was due to the CFI mutation and had nothing to do with the patient having had STEC exposure in the past.
The above interpretation is consistent with published data that in patients with aHUS carrying genetic abnormalities in circulating regulatory proteins like CFI and CFH, the risk of recurrence after renal transplantation is very high .
Eculizumab has been shown to be beneficial to treat acute episodes of aHUS and also as prophylaxis of recurrences in the kidney graft [29, 30] in patients with aHUS and complement gene mutations. The presence in patient #1 of a CFI mutation, along with the finding of intense deposition of C3 and C5b-9 in renal biopsy, would provide the rationale for a preemptive use of Eculizumab as part of a new renal transplant plan for this patient deemed at high risk for recurrent disease.
MCP is a transmembrane protein widely expressed in the kidney, thus transplantation of a kidney expressing normal MCP corrects the local genetic defect in aHUS-patient with MCP mutations. Consequently, these patients usually have a good graft outcome with a low incidence of posttransplant recurrence . Such observation in aHUS patients does not fit with the finding here that patient #2 carrying an MCP mutation experienced HUS recurrence in the graft. Such discrepancy can be explained by the fact that patient #2 received the graft from her mother who carries the same MCP mutation. Thus in this case the kidney graft produced the same dysfunctional MCP protein as the native kidney and it was not protected enough from complement hyperactivation leading to aHUS recurrence.
In conclusion, the cases here reported indicate that screening of HUS-associated complement genes should be performed in all patients on dialysis following severe forms of STEC-HUS, since they may carry complement gene mutations and be undiagnosed cases of aHUS precipitated by STEC infections.
A correct diagnosis is critical to predict the risk of recurrence after transplantation and accordingly to adopt suitable prophylactic measures. Furthermore, genetic analysis of potential donors is mandatory before living related transplantation.