Clustering of missense mutations in the C-terminal region of factor H in atypical hemolytic uremic syndrome

Abstract

Hemolytic-uremic syndrome (HUS) is a microvasculature disorder leading to microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Most cases of HUS are associated with epidemics of diarrhea caused by verocytotoxin-producing bacteria, but atypical cases of HUS not associated with diarrhea (aHUS) also occur. Early studies describing the association of aHUS with deficiencies of factor H suggested a role for this complement regulator in aHUS. Molecular evidence of factor H involvement in aHUS was first provided by Warwicker et al., who demonstrated that aHUS segregated with the chromosome 1q region containing the factor H gene (HF1) and who identified a mutation in HF1 in a case of familial aHUS with normal levels of factor H. We have performed the mutational screening of the HF1 gene in a novel series of 13 Spanish patients with aHUS who present normal complement profiles and whose plasma levels of factor H are, with one exception, within the normal range. These studies have resulted in the identification of five novel HF1 mutations in four of the patients. Allele HF1 Delta exon2, a genomic deletion of exon 2, produces a null HF1 allele and results in plasma levels of factor H that are 50% of normal. T956M, W1183L, L1189R, and V1197A are missense mutations that alter amino acid residues in the C-terminal portion of factor H, within a region--SCR16-SCR20--that is involved in the binding to solid-phase C3b and to negatively charged cellular structures. This remarkable clustering of mutations in HF1 suggests that a specific dysfunction in the protection of cellular surfaces by factor H is a major pathogenic condition underlying aHUS.

Figure 1

Mutations in factor H in patients with aHUS. a, Organization of the 1q32 genomic region containing the human HF1 gene. Colored arrows indicate the location and transcriptional orientation of the HF1 gene and the FHR1–FHR5 genes. For each gene, the exon/intron organization is indicated by vertical bars. Genomic duplications within the region are indicated by colored boxes. These duplications are 1.2–38 kb in size and present a pairwise nucleotide identity of 85%–97%. The exon/intron organization of the HF1 gene, as well as the nucleotide sequences of the intronic flanking regions, was determined on the basis of data published by Male et al. (2000) and of data generated in our laboratory by PCR analysis and sequencing of PAC clones RPCIP704A14355, RPCIP704L20665, RPCIP704M03650, RPCIP704O14608 (authors' unpublished data). These data and sequence data generated by the Human Genome Project (clones AL049741, AL049744, AL139418, and AL353809) provided the genomic sequence for the HF1 gene and the FHR1–FHR5 genes. HF1 comprises 23 exons and spans >94 kb of genomic DNA. Exon 10 does not contribute to the factor H transcript; it is utilized only in the alternative HF1 transcript that codes for the factor H–like 1 molecule (also known as “FHL-1”) (Estaller et al. 1991). To amplify each of the HF1 exons present in the factor H transcript, we designed PCR primers to the intronic flanking sequences (table 1). Primers for exons 8/9 and 21–23 were designed to avoid amplification of homologous exons in the FHR3 gene and the FHR1 gene, respectively. b, Identification of mutations in the factor H gene in four patients with aHUS. The pedigrees of patients HUS2, HUS3, HUS11, and HUS12 are shown, as are the chromatograms corresponding to the DNA sequence surrounding the mutated nucleotide for each patient and family members studied. The positions of nucleotide changes are according to the cDNA sequence reported by Ripoche et al. (1988). The ATG initiation codon is located at nucleotide position c.74. Amino acid numbering includes the signal peptide. c, Functional domains and mutations in the factor H molecule. A diagram of the structure of human factor H with the 20 SCR repeats is shown. Functional domains are indicated schematically. The location of the six missense mutations thus far characterized in patients with aHUS (four of whom reported here) is indicated to illustrate that they are clustered within a specific region of factor H, a region that has been reported to be important for the control of C3b deposited on surfaces.