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Atypical hemolytic uremic syndrome: telling the difference between H and Y

2010; Elsevier BV; Volume: 78; Issue: 8 Linguagem: Inglês

10.1038/ki.2010.222

1523-1755

Elena Goicoechea de Jorge, Matthew C. Pickering,

Renal Diseases and Glomerulopathies

Mutations in the complement factor H (CFH) gene are frequently associated with atypical hemolytic uremic syndrome (aHUS). Hakobyan et al. have developed novel reagents that can rapidly determine the contribution of each CFH allele to the total plasma CFH pool, showing that low-expression CFH alleles are important risk factors for the development of aHUS. These reagents represent a significant contribution to the techniques used to determine susceptibility factors among individuals with aHUS. Mutations in the complement factor H (CFH) gene are frequently associated with atypical hemolytic uremic syndrome (aHUS). Hakobyan et al. have developed novel reagents that can rapidly determine the contribution of each CFH allele to the total plasma CFH pool, showing that low-expression CFH alleles are important risk factors for the development of aHUS. These reagents represent a significant contribution to the techniques used to determine susceptibility factors among individuals with aHUS. The hemolytic uremic syndrome (HUS, MIM 235,400) is a condition characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure due to glomerular thrombotic microangiopathy. The majority of HUS episodes are triggered by Escherichia coli 0157:H7 infection. However, a minority of cases are not associated with infection; this form, termed atypical HUS (aHUS), has the poorest long-term prognosis. During the last decade there has been dramatic progress in understanding the pathogenesis of aHUS, particularly through the study of familial forms. Approximately half of all aHUS cases have been associated with mutations and/or polymorphisms in genes encoding proteins of the complement system. Mutations in the regulatory proteins of this system resulted in ‘loss of function,’ whereas mutations in genes encoding complement activation proteins resulted in ‘gain of function.’1.Noris M. Remuzzi G. Atypical hemolytic-uremic syndrome.N Engl J Med. 2009; 361: 1676-1687Crossref PubMed Scopus (928) Google Scholar Mutations in thrombomodulin, an endothelial glycoprotein, have recently been associated with aHUS.2.Delvaeye M. Noris M. De Vriese A. et al.Thrombomodulin mutations in atypical hemolytic-uremic syndrome.N Engl J Med. 2009; 361: 345-357Crossref PubMed Scopus (419) Google Scholar These mutations were associated with impaired complement regulation in vitro. Together with other lines of evidence, aHUS is now viewed as a disorder in which there is defective complement regulation (Figure 1). In many individuals, both genetic susceptibility factors and an environmental insult are required for the syndrome to develop. For example, many environmental factors—such as infection, pregnancy, and drugs—have been reported to trigger episodes of aHUS.1.Noris M. Remuzzi G. Atypical hemolytic-uremic syndrome.N Engl J Med. 2009; 361: 1676-1687Crossref PubMed Scopus (928) Google Scholar Family studies have clearly shown that multiple genetic risk factors are generally required for the condition to become manifest. For example, in one pedigree in which there were three independently segregating aHUS-associated risk factors, the syndrome developed only among individuals possessing all three risk factors.3.Esparza-Gordillo J. Jorge E.G. Garrido C.A. et al.Insights into hemolytic uremic syndrome: segregation of three independent predisposition factors in a large, multiple affected pedigree.Mol Immunol. 2006; 43: 1769-1775Crossref PubMed Scopus (107) Google Scholar The investigation of individuals with aHUS for genetic susceptibility factors has become increasingly complex because both the number and nature of reported genetic defects has expanded. The diagnostic workup of affected individuals is summarized in Figure 2. Exon sequencing to screen for mutations in complement regulatory (CFH, CFI, and MCP (membrane cofactor protein, also known as CD46)) and activation (C3, CFB) genes and the thrombomodulin gene (THRB) is relatively straightforward. However, significant complexity arises within the CFH gene family. Complement factor H (CFH) is the major regulator of the complement alternative pathway, and mutations in the CFH gene are among the most frequent alterations detected in the majority of aHUS cohorts. The CFH gene is located in the ‘regulators of complement activation’ (RCA) gene cluster on chromosome 1q32.4.Rodriguez de Cordoba S. Esparza-Gordillo J. Goicoechea de Jorge E. et al.The human complement factor H: functional roles, genetic variations and disease associations.Mol Immunol. 2004; 41: 355-367Crossref PubMed Scopus (487) Google Scholar The gene encodes the CFH protein, an abundant plasma protein comprising 20 globular domains termed short consensus repeat (SCR) domains. In addition, through alternative splicing, the CFH gene also encodes a smaller protein consisting of only seven SCR domains, termed factor H–like 1 (FHL-1). With the exception of the C-terminal four amino acids, the FHL-1 protein sequence is identical to the first seven SCR domains of CFH. In close proximity to the CFH locus are five genes encoding proteins that are structurally related to CFH. These five proteins are termed CFH-related proteins. Their respective genes are arranged in the genomic sequence CFHR3, CFHR1, CFHR2, CFHR4, and CFHR5. The CFH–CFHR1–5 gene region contains multiple genomic duplications that render this area susceptible to genomic rearrangements through mechanisms such as gene conversion or nonhomologous recombination. Some of these rearrangements have been associated with aHUS. Examples include a rearrangement that results in the formation of a CFH–CFHR1 hybrid gene.5.Venables J.P. Strain L. Routledge D. et al.Atypical haemolytic uraemic syndrome associated with a hybrid complement gene.PLoS Med. 2006; 3: e431Crossref PubMed Scopus (166) Google Scholar Furthermore, a common polymorphic rearrangement that results in the deletion of CFHR1 and CFHR3 genes is associated with anti-CFH autoantibodies and aHUS.6.Jozsi M. Licht C. Strobel S. et al.Factor H autoantibodies in atypical hemolytic uremic syndrome correlate with CFHR1/CFHR3 deficiency.Blood. 2008; 111: 1512-1514Crossref PubMed Scopus (287) Google Scholar, 7.Zipfel P.F. Edey M. Heinen S. et al.Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome.PLoS Genet. 2007; 3: e41Crossref PubMed Scopus (260) Google Scholar Identification of these rearrangements requires the use of techniques (such as multiplex ligation-dependent probe amplification analysis) that assess copy-number variation across the CFH–CFHR1–5 gene region. In addition to genetic analysis, important investigations in aHUS patients include assays to measure plasma complement components (e.g., CFH), measurement of CD46 expression on peripheral blood mononuclear cells, and techniques to detect anti-CFH autoantibodies. Again, the situation for CFH is not straightforward. The normal range for plasma CFH is quite wide and influenced by both genetic and environmental (e.g., smoking) factors.8.Esparza-Gordillo J. Soria J.M. Buil A. et al.Genetic and environmental factors influencing the human factor H plasma levels.Immunogenetics. 2004; 56: 77-82Crossref PubMed Scopus (135) Google Scholar Consequently, heterozygous CFH deficiency states may not be readily detectable by measurement of plasma CFH levels. The difficulty is how to distinguish the contribution of each CFH allele to the plasma CFH pool. In this issue, Hakobyan et al.9.Hakobyan S. Tortajada A. Harris C.L. et al.Variant-specific quantification of factor H in plasma identifies null alleles associated with atypical hemolytic uremic syndrome.Kidney Int. 2010; 78: 782-788Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar report having addressed this problem by developing a novel method to rapidly measure allele-specific CFH protein products (Figure 3). They used the common nonsynonymous CFH polymorphism termed Y402H (rs1061170), located within the SCR7 domain of both CFH and FHL-1, to generate monoclonal antibodies specific for either the Y402 or H402 protein allotypes. These reagents allowed the authors to determine, among individuals who are heterozygous for Y402H, the contribution of each CFH allele to the total plasma CFH and FHL-1 protein pool. With these new tools, they assessed plasma levels of the Y402 and H402 protein allotypes among individuals with aHUS in their Spanish cohort. Among 48 cases who were heterozygous for Y402H, 3 unrelated individuals with low expression of the H402 allele were identified. In 2 of these (H29 and H90), total plasma CFH levels were in the lower part of the normal range (124.4–402 mg/liter) when a non-allele-specific CFH ELISA was used. In each family, the inheritance of low- or null-expression alleles was an independent risk factor for aHUS. Several important messages emerged from the analysis of these individuals. First, using an ELISA assay, these antibodies provide a rapid and simple tool for the identification of CFH null alleles. In addition, these novel reagents may circumvent the need to perform cloning and expression studies to determine whether a particular mutant affects protein expression. Two of the index cases identified by Hakobyan et al.9.Hakobyan S. Tortajada A. Harris C.L. et al.Variant-specific quantification of factor H in plasma identifies null alleles associated with atypical hemolytic uremic syndrome.Kidney Int. 2010; 78: 782-788Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar were found to have mutations affecting cysteine residues within the CFH protein (C853R in index case H90 and C1218R in index case H169). Both index cases were heterozygous for the Y402H polymorphism. With the allotype-specific reagents developed by Hakobyan et al.,9.Hakobyan S. Tortajada A. Harris C.L. et al.Variant-specific quantification of factor H in plasma identifies null alleles associated with atypical hemolytic uremic syndrome.Kidney Int. 2010; 78: 782-788Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar it was straightforward to demonstrate that these cysteine mutations are associated with no expression of the CFH protein. Using western blotting, it was also possible to show that FHL-1 expression from the affected alleles was normal. This would be expected, since these cysteine mutations affected exons encoding for SCR domains that are not present in the FHL-1 protein. In another report, these reagents were used to demonstrate that the CFH Y899D mutation was associated with minimal protein expression.10.Johnson S.A. Williams J.M. Hakobyan S. et al.Impact of compound heterozygous complement factor H mutations on development of atypical hemolytic uremic syndrome—a pedigree revisited.Mol Immunol. 2010; 47: 1585-1591Crossref PubMed Scopus (9) Google Scholar Second, these techniques allowed Hakobyan et al.9.Hakobyan S. Tortajada A. Harris C.L. et al.Variant-specific quantification of factor H in plasma identifies null alleles associated with atypical hemolytic uremic syndrome.Kidney Int. 2010; 78: 782-788Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar to track two abnormal CFH alleles in another aHUS-affected pedigree. The index case (H29), a Y402H heterozygote, had low levels of the plasma H402 protein allotype. In contrast, plasma levels of the Y402 protein allotype were normal. However, this individual carried a common aHUS-associated CFH gene mutation (R1210C) on the CFH Y402 allele. The R1210C mutation was present in heterozygosity in five other members of this pedigree, none of whom had developed aHUS. Two of these individuals, one the sister of the index case, were Y402H heterozygotes. Hakobyan et al.9.Hakobyan S. Tortajada A. Harris C.L. et al.Variant-specific quantification of factor H in plasma identifies null alleles associated with atypical hemolytic uremic syndrome.Kidney Int. 2010; 78: 782-788Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar were able to show that these individuals had not inherited the abnormal low-expressing CFH H402 allele present in the H29 index case. Therefore, in this pedigree, aHUS had developed only when both abnormal CFH alleles were present. Only through the use of these novel reagents was it possible to conclude that the sister of the index case is not at high risk of developing aHUS. This illustrates an important clinical application for these antibodies. Third, interesting insights into the differential regulation of CFH and FHL-1 emerged from the study of the H29 pedigree. The genetic basis of the low-expression CFH H402 allele was not found, even though an extensive search for the causative mutation was performed. However, although the plasma levels of the CFH 402H allotype were markedly reduced, western blot analysis surprisingly showed that the FHL-1-H402 allotype was increased fivefold compared with controls. This suggests that the defect has disrupted differential CFH gene splicing. The significance of this is not clear but warrants further study. In summary, the investigation of patients with aHUS has become increasingly complex, and in many cases no susceptibility factors have been identified. This indicates that our understanding of the condition remains incomplete. The work of Hakobyan et al.9.Hakobyan S. Tortajada A. Harris C.L. et al.Variant-specific quantification of factor H in plasma identifies null alleles associated with atypical hemolytic uremic syndrome.Kidney Int. 2010; 78: 782-788Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar provides valuable additional tools for the investigation of patients with aHUS. With the novel reagents developed by these investigators, it is now possible to rapidly identify low-expressing or null CFH alleles. These are independent risk factors for aHUS that should be looked for in other cohorts. MCP is a Wellcome Trust Senior Fellow in Clinical Science (WT082291MA). EGdJ is funded through this fellowship.