Jessica Caprioli

Relative Role of Genetic Complement Abnormalities in Sporadic and Familial aHUS and Their Impact on Clinical Phenotype

BACKGROUND AND OBJECTIVES Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Most childhood cases are caused by Shiga toxin-producing bacteria. The other form, atypical HUS (aHUS), accounts for 10% of cases and has a poor prognosis. Genetic complement abnormalities have been found in aHUS. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS We screened 273 consecutive patients with aHUS for complement abnormalities and studied their role in predicting clinical phenotype and response to treatment. We compared mutation frequencies and localization and clinical outcome in familial (82) and sporadic (191) cases. RESULTS In >70% of sporadic and familial cases, gene mutations, disease-associated factor H (CFH) polymorphisms, or anti-CFH autoantibodies were found. Either mutations or CFH polymorphisms were also found in the majority of patients with secondary aHUS, suggesting a genetic predisposition. Familial cases showed a higher prevalence of mutations in SCR20 of CFH and more severe disease than sporadic cases. Patients with CFH or THBD (thrombomodulin) mutations had the earliest onset and highest mortality. Membrane-cofactor protein (MCP) mutations were associated with the best prognosis. Plasma therapy induced remission in 55 to 80% of episodes in patients with CFH, C3, or THBD mutations or autoantibodies, whereas patients with CFI (factor I) mutations were poor responders. aHUS recurred frequently after kidney transplantation except for patients with MCP mutations. CONCLUSIONS Results underline the need of genetic screening for all susceptibility factors as part of clinical management of aHUS and for identification of patients who could safely benefit from kidney transplant.

Mutations in factor H reduce binding affinity to C3b and heparin and surface attachment to endothelial cells in hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Recent studies have identified a factor H-associated form of HUS, caused by gene mutations that cluster in the C-terminal region of the complement regulator factor H. Here we report how three mutations (E1172Stop, R1210C, and R1215G; each of the latter two identified in three independent cases from different, unrelated families) affect protein function. All three mutations cause reduced binding to the central complement component C3b/C3d to heparin, as well as to endothelial cells. These defective features of the mutant factor H proteins explain progression of endothelial cell and microvascular damage in factor H-associated genetic HUS and indicate a protective role of factor H for tissue integrity during thrombus formation.

Familial haemolytic uraemic syndrome and an MCP mutation

BACKGROUND Mutations in factor H (HF1) have been reported in a consistent number of diarrhoea-negative, non-Shiga toxin-associated cases of haemolytic uraemic syndrome (D-HUS). However, most patients with D-HUS have no HF1 mutations, despite decreased serum concentrations of C3. Our aim, therefore, was to assess whether genetic abnormalities in other complement regulatory proteins are involved. METHODS We screened genes that encode the complement regulatory proteins-ie, factor H related 5, complement receptor 1, and membrane cofactor protein (MCP)-by PCR-single-strand conformation polymorphism (PCR-SSCP) and by direct sequencing, in 25 consecutive patients with D-HUS, an abnormal complement profile, and no HF1 mutation, from our International Registry of Recurrent and Familial HUS/TTP (HUS/thrombotic thrombocytopenic purpura). FINDINGS We identified a heterozygous mutation in MCP, a surface-bound complement regulator, in two patients with a familial history of HUS. The mutation causes a change in three aminoacids at position 233-35 and insertion of a premature stop-codon, which results in loss of the transmembrane domain of the protein and severely reduced cell-surface expression of MCP. INTERPRETATION Results of previous studies on HF1 indicate an association between HF1 deficiency and D-HUS. Our findings of an MCP mutation in two related patients suggest that impaired regulation of complement activation might be a factor in the pathogenesis of genetic forms of HUS. MCP could be a second putative candidate gene for D-HUS. The protein is highly expressed in the kidney and plays a major part in regulation of glomerular C3 activation. We propose, therefore, that reduced expression of MCP in response to complement-activating stimuli could prevent restriction of complement deposition on glomerular endothelial cells, leading to microvascular cell damage and tissue injury.

Combined Complement Gene Mutations in Atypical Hemolytic Uremic Syndrome Influence Clinical Phenotype

Several abnormalities in complement genes reportedly contribute to atypical hemolytic uremic syndrome (aHUS), but incomplete penetrance suggests that additional factors are necessary for the disease to manifest. Here, we sought to describe genotype-phenotype correlations among patients with combined mutations, defined as mutations in more than one complement gene. We screened 795 patients with aHUS and identified single mutations in 41% and combined mutations in 3%. Only 8%-10% of patients with mutations in CFH, C3, or CFB had combined mutations, whereas approximately 25% of patients with mutations in MCP or CFI had combined mutations. The concomitant presence of CFH and MCP risk haplotypes significantly increased disease penetrance in combined mutated carriers, with 73% penetrance among carriers with two risk haplotypes compared with 36% penetrance among carriers with zero or one risk haplotype. Among patients with CFH or CFI mutations, the presence of mutations in other genes did not modify prognosis; in contrast, 50% of patients with combined MCP mutation developed end stage renal failure within 3 years from onset compared with 19% of patients with an isolated MCP mutation. Patients with combined mutations achieved remission with plasma treatment similar to patients with single mutations. Kidney transplant outcomes were worse, however, for patients with combined MCP mutation compared with an isolated MCP mutation. In summary, these data suggest that genotyping for the risk haplotypes in CFH and MCP may help predict the risk of developing aHUS in unaffected carriers of mutations. Furthermore, screening patients with aHUS for all known disease-associated genes may inform decisions about kidney transplantation.

Combined kidney and liver transplantation for familial haemolytic uraemic syndrome.

Recurrent haemolytic uraemic syndrome (HUS) is a genetic form of thrombotic microangiopathy that is mostly associated with low activity of complement factor H. The disorder usually develops in families, leads to end stage renal disease, and invariably recurs after kidney transplantation. We did a simultaneous kidney and liver transplantation in a 2-year-old child with HUS and a mutation in complement factor H to restore the defective factor H, with no recurrence of the disease. The operation was successful, and at discharge, the child had healthy kidney and liver function, with no sign of haemolysis.

Complement Factor H Mutation in Familial Thrombotic Thrombocytopenic Purpura with ADAMTS13 Deficiency and Renal Involvement

Thrombotic thrombocytopenic purpura is a rare disorder of small vessels that is associated with deficiency of the von Willebrand factor-cleaving protease ADAMTS13, which favors platelet adhesion and aggregation in the microcirculation. The disease manifests mainly with central nervous system symptoms, but cases of renal insufficiency have been reported. Presented are findings of the genetic basis of phenotype heterogeneity in thrombotic thrombocytopenic purpura in two sisters within one family. The patients had ADAMTS13 deficiency as a result of two heterozygous mutations (causing V88M and G1239V changes). In addition, a heterozygous mutation (causing an S890I change) in factor H of complement was found in the patient who developed chronic renal failure but not in her sister, who presented with exclusive neurologic symptoms.

Mutations in FN1 cause glomerulopathy with fibronectin deposits

Glomerulopathy with fibronectin (FN) deposits (GFND) is an autosomal dominant disease with age-related penetrance, characterized by proteinuria, microscopic hematuria, hypertension, and massive glomerular deposits of FN that lead to end-stage renal failure. The genetic abnormality underlying GFND was still unknown. We hypothesized that mutations in FN1, which encodes FN, were the cause of GFND. In a large Italian pedigree with eight affected subjects, we found linkage with GFND at the FN1 locus at 2q32. We sequenced the FN1 in 15 unrelated pedigrees and found three heterozygous missense mutations, the W1925R, L1974R, and Y973C, that cosegregated with the disease in six pedigrees. The mutations affected two domains of FN (Hep-II domain for the W1925R and the L1974R, and Hep-III domain for the Y973C) that play key roles in FN–cell interaction and in FN fibrillogenesis. Mutant recombinant Hep-II fragments were expressed, and functional studies revealed a lower binding to heparin and to endothelial cells and podocytes compared with wild-type Hep-II and an impaired capability to induce endothelial cell spreading and cytoskeletal reorganization. Overall dominant mutations in FN1 accounted for 40% of cases of GFND in our study group. These findings may help understanding the pathogenesis of proteinuria and glomerular FN deposits in GFND and possibly in more common renal diseases such as diabetic nephropathy, IgA nephropathy, and lupus nephritis. To our knowledge no FN1 mutation causing a human disease was previously reported.

The hemolytic uremic syndromes.

Purpose of reviewRecent studies have provided a better understanding of the molecular mechanisms responsible for hemolytic uremic syndromes. In this review, we summarize biochemical and genetic data that may lead to new clinical approaches. Recent findingsThe structures and modes of action of Shiga toxins have been deciphered. Patients with non-Shiga-like toxin hemolytic uremic syndrome have been found to carry mutations in three genes that encode for regulators of the complement system (factor H, membrane cofactor protein, and factor I). SummaryShiga-like toxin-1 and Shiga-like toxin-2 regulate genes that encode for chemokines, cytokines, cell adhesion molecules, and transcription factors involved in immune response and apoptosis. Mutations in factor H, membrane cofactor protein and factor I have recently been identified. Reduced expression of compliment regulators might prevent restriction of complement deposition on glomerular endothelial cells, leading to microvascular cell damage and tissue injury. Shiga-like toxin hemolytic uremic syndrome in children has a favorable prognosis in 90% of cases; kidney transplantation shows a good graft survival rate (80%) in children who progress to end stage renal disease. As for non-Shiga-like toxin hemolytic uremic syndrome, treatment with plasma infusion or exchange has been used with controversial effects. Kidney transplantation is not recommended in those patients with mutations in factor H and factor I; however, a kidney transplant corrects membrane cofactor protein dysfunction. These findings vividly underscore the clinical heterogeneity of outcomes depending upon the nature of the underlying cause of the disease.

Complement factor H and hemolytic uremic syndrome.

Factor H is a 150 kDa single chain plasma glycoprotein that plays a pivotal role in the regulation of the alternative pathway of complement. Primary sequence analysis reveals a structural organization of this plasma protein, in 20 homologous units, called Short Consensus Repeats (SCRs), each about 60 amino acids long. Biochemical and genetic studies show an association between factor H deficiency and human diseases, including Systemic Lupus Erythematosus, susceptibility to pyogenic infection and a form of membranoproliferative glomerulonephropathy. More recently, factor H deficiency has also been associated with susceptibility to Hemolytic Uremic Syndrome (HUS), a disease consisting of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure, caused by platelet thrombi which mainly, but not exclusively, form in the microcirculation of the kidney. In this review, we summarize recent genetic and biochemical data, which indicate a critical role for factor H in the pathogenesis of HUS and suggest an important role of the most C-terminal domain, i.e. SCR 20, in the disease. In addition, we discuss the physiological consequences of these findings, as novel functional data show a particular essential role of SCR 20 of factor H as the central discriminatory and regulatory site of this multidomain, multifunctional plasma protein.

Single Strand Conformation Polymorphism (SSCP) as a quick and reliable method to genotype M235T polymorphism of angiotensinogen gene.

OBJECTIVES Conflicting results on the relationship between M235T polymorphism of angiotensinogen (AGT) gene and diabetic nephropathy are reported in the literature, probably due to the small number of subjects, to different inclusion criteria and the different genotype analysis methods used. The aim of the present study was to set up a fast, cheap and reliable method to allow the genotyping of M235T polymorphism in a large number of subjects. DESIGN AND METHODS We developed in our laboratory a new specifically designed PCR-SSCP method for M235T genotyping whose specificity was compared with that of Allele Specific PCR (ASPCR) and Mutagenically Separated PCR (MS-PCR). The exact M235T genotype was estabilished by direct sequencing. The new PCR-SSCP method was then used to genotype a population of 1171 hypertensive, normoalbuminuric type II diabetes mellitus patients. The patients were also genotyped for ACE I/D polymorphism. For comparison a group of hypertensive non diabetic patients (n = 88) were also screened. RESULTS The PCR-SSCP method identified the M235T polymorphism with no misinterpretation at variance with ASPCR and MS-PCR methods that showed a preferential amplification of the T allele. The rare Y248C polymorphism of the AGT gene was also detected by PCR-SSCP. In diabetic hypertensive patients the prevalence of TT genotype was higher than in normotensive healthy controls and equivalent to that found in hypertensive non diabetic patients. CONCLUSIONS The PCR-SSCP method for detection of M235T polymorphism is a powerful and sensitive tool for rapid, cheap and efficient screening of a large number of samples. The results obtained with this method demonstrate an association of the TT genotype of AGT gene with hypertension, both in diabetic and non diabetic patients.