Biomedical subjects

Stefan Heinen

Publications and source records attributed to Stefan Heinen.

7 recordsLinked to original sources

PubMed2007

Hemolytic uremic syndrome: a factor H mutation (E1172Stop) causes defective complement control at the surface of endothelial cells.

Defective complement regulation results in hemolytic uremic syndrome (HUS), a disease that is characterized by microangiopathy, thrombocytopenia, and acute renal failure and that causes endothelial cell damage. For characterization of how defective complement regulation relates to the pathophysiology, the role of the complement regulator factor H and also of a mutant factor H protein was studied on the surface of human umbilical vein endothelial cells. The mutant 145-kD factor H protein was purified to homogeneity, from plasma of a patient with HUS, who is heterozygous for a factor H gene mutation G3587T, which introduces a stop codon at position 1172. Functional analyses show that the lack of the most C-terminal domain short consensus repeats 20 severely affected recognition functions (i.e., binding to heparin, C3b, C3d, and the surface of endothelial cells). Wild-type factor H as well as the mutant protein formed dimers in solution as shown by cross-linking studies and mass spectroscopy. When assayed in fluid phase, the complement regulatory activity of the mutant protein was normal and comparable to wild-type factor H. However, on the surface of endothelial cells, the mutant factor H protein showed severely reduced regulatory activities and lacked protective functions. Similarly, with the use of sheep erythrocytes, the mutant protein lacked the protective activity and caused increased hemolysis when it was added to factor H-depleted plasma. This study shows how a mutation that affects the C-terminal region of the factor H protein leads to defective complement control on cell surfaces and damage to endothelial cells in patients with HUS. These effects explain how mutant factor H causes defective complement control and in HUS-particularly under condition of inflammation and complement activation-causes endothelial cell damage.

Cells, Cultured↗

PubMed2006

De novo gene conversion in the RCA gene cluster (1q32) causes mutations in complement factor H associated with atypical hemolytic uremic syndrome.

Many of the complement regulatory genes within the RCA cluster (1q32) have arisen through genomic duplication and the resulting high degree of sequence identity is likely to predispose to gene conversion events. The highest degree of identity is between the genes for factor H (CFH) and five factor H-related proteins--CFHL1, CFHL2, CFHL3, CFHL4, and CFHL5. CFH mutations are associated with atypical hemolytic uremic syndrome (aHUS). In the Newcastle cohort of 157 aHUS patients we have identified CFH mutations in 25 families or individuals. Eleven of these 25 independent mutations are either c.3226C>G,Q1076E; c.3572C>T,S1191L; c.3590T>C,V1197A or combined c.3572C>T,S1191L/c.3590T>C,V1197A. Sequence analysis shows that all four of these changes could have arisen as a result of gene conversion between CFH and CFHL1. Analysis of parental samples in two patients with S1191L/V1197A has shown that the changes are de novo thus providing conclusive evidence that gene conversion is the mutational mechanism in these two cases. To confirm that S1191L and V1197A are disease predisposing we examined their functional significance in three ways - analysis of the C3b/C3d binding characteristics of recombinant mutant S1191L/V1197A protein, heparin affinity chromatography and haemolytic assays of serum samples from aHUS patients carrying these changes. The results showed that these changes resulted in impaired C3b binding and a defective capacity to control complement activation on cellular surfaces. We, therefore, provide conclusive evidence that gene conversion is responsible for functionally significant CFH mutations in aHUS.

Animals↗

PubMed2006

Complement and diseases: defective alternative pathway control results in kidney and eye diseases.

The complement system is a central part of innate immunity and in its normal setting aimed to recognize and eliminate microbes. For elimination toxic activation products are generated locally and are reported directly of the surface of the invading microbe. A deregulation of the alternative pathway results in defective recognition and toxic activation products can be formed on the surface of host tissues and structures. Recent studies have shown that mutated or defective regulators of the alternative pathway of complement are associated with auto immune diseases of the kidney, including the atypical form of hemolytic uremic syndrome (HUS), membranoproliferative glomerulonephritis (MPGN) and also of the eye, such as age-related macular degeneration (ARMD). Current research provides clues how mutations occurring in genes coding for single complement components or the inactivation of single regulators lead to defective alternative pathway amplification, via the convertase C3bBb. These scenarios explain how defects of a single regulator lead to local, organ specific damage.

Animals↗

PubMed2005

Factor H and atypical hemolytic uremic syndrome: mutations in the C-terminus cause structural changes and defective recognition functions.

Atypical hemolytic uremic syndrome is a disease that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Mutations in the complement regulator factor H are associated with the inherited form of the disease, and >60% of the mutations are located within the C terminus of factor H. The C-terminus of factor H, represented by short consensus repeat 19 (SCR19) and SCR20, harbors multiple functions; consequently, this study aimed to examine the functional effects of clinically reported mutations in these SCR. Mutant factor H proteins (W1157R, W1183L, V1197A, R1210C, R1215G, and P1226S) were recombinantly expressed and functionally characterized. All six mutant proteins showed severely reduced heparin, C3b, C3d, and endothelial cell binding. By peptide spot analyses, four linear regions that are involved in heparin, C3b, and C3d binding were localized in SCR19 and SCR20. A three-dimensional homology model of the two domains suggests that these four regions form a common binding site across both domains. In addition, this structural model identifies two types of residues: Type A residues are positioned on the SCR surface and are represented by mutants W1157R, W1183L, R1210C, and R1215G; and type B residues are buried within the SCR structure and affect mutations V1197A and P1226S. Mutations of both types of residue result in the same functional defects, namely the reduced binding of factor H to surface-attached C3b molecules and reduced complement regulatory activity at the cell surfaces. The buried type B mutations seem to affect ligand interaction of factor H more severely than the surface-exposed mutations.

Amino Acid Sequence↗

PubMed2005

Binding of complement factor H to endothelial cells is mediated by the carboxy-terminal glycosaminoglycan binding site.

Factor H (FH), the major fluid phase regulator of the alternative complement pathway, mediates protection of plasma-exposed host structures. It has recently been shown that short consensus repeats 19 to 20 of FH are mutational hot spots associated with atypical hemolytic uremic syndrome (aHUS), a disease with endothelial cell damage. Domain 20 of FH contains binding sites for heparin, C3b, and the cleavage product C3d. To study the role of these binding sites in target recognition, we performed site-directed mutagenesis in domain 20 and assayed the resulting recombinant proteins. The mutant FH15-20A (substitutions R1203E, R1206E, and R1210S) bound neither heparin nor endothelial cells. Similarly, an aHUS-derived mutant FH protein (E1172Stop, lacking domain 20) failed to bind endothelial cells and showed impaired binding to heparin. Binding of FH to endothelial cells was inhibited by heparin and a specific monoclonal antibody that inhibited heparin but not C3d binding, demonstrating that the heparin site on domains 19 to 20 mediates interaction of FH to endothelial cells. Binding of FH15-20 to heparin was inhibited by several cell surface- and basement membrane-associated glycosaminoglycans, suggesting that binding site specificity is not restricted to heparin. Thus, defective heparin/glycosaminoglycan-binding site on domains 19 to 20 of FH most probably mediates complement-induced endothelial cell damage in aHUS.

Amino Acid Substitution↗

PubMed2005

Successful plasma therapy for atypical hemolytic uremic syndrome caused by factor H deficiency owing to a novel mutation in the complement cofactor protein domain 15.

Quantitative or functional deficiency of complement factor H results in uncontrolled complement activation. This leads to thrombotic microangiopathy and finally causes renal failure (atypical hemolytic uremic syndrome [aHUS]). By regular analysis of factor H in patients with aHUS, the authors found a complete factor H deficiency in an infant in whom aHUS developed at 8 months of age. Factor H was quantified by enzyme-linked immunosorbent assay and further analyzed by Western blot using a factor H-specific antibody. Complement activation was determined by measuring total hemolytic activity of the classical (CH50) and alternative (APH50) pathways, C3 and C3d. The sequence of factor H gene was determined. Serial factor H measurements after fresh frozen plasma infusion allowed calculation of a factor H half-life. Factor H was absent in plasma (<1 mug/mL), and the complement system was highly activated (CH50, APH50, C3 decreased; C3d increased). Genetic analysis identified a novel homozygous factor H mutation (T2770A; Y899Stop) in CCP domain 15, most likely causing defective protein secretion. Time course measurements of factor H after plasma infusion established a factor H half-life of about 6 days. By repetitive plasma infusions (20 mL/kg over about 2 to 3 hours) the authors were able to interrupt the vicious circle of thrombotic microangiopathy in a factor H-deficient patient with aHUS. Based on the measured factor H half-life of about 6 days, regular plasma infusions in 2-week intervals were given, which prevented further aHUS episodes and stopped the decline of kidney function.

Complement Factor H↗

PubMed2003

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.

Animals↗