Deletion of Complement Factor H–Related Genes CFHR1 and CFHR3 Is Associated with Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. Disease-associated mutations have been described in the genes encoding the complement regulators complement factor H, membrane cofactor protein, factor B, and factor I. In this study, we show in two independent cohorts of aHUS patients that deletion of two closely related genes, complement factor H–related 1 (CFHR1) and complement factor H–related 3 (CFHR3), increases the risk of aHUS. Amplification analysis and sequencing of genomic DNA of three affected individuals revealed a chromosomal deletion of ∼84 kb in the RCA gene cluster, resulting in loss of the genes coding for CFHR1 and CFHR3, but leaving the genomic structure of factor H intact. The CFHR1 and CFHR3 genes are flanked by long homologous repeats with long interspersed nuclear elements (retrotransposons) and we suggest that nonallelic homologous recombination between these repeats results in the loss of the two genes. Impaired protection of erythrocytes from complement activation is observed in the serum of aHUS patients deficient in CFHR1 and CFHR3, thus suggesting a regulatory role for CFHR1 and CFHR3 in complement activation. The identification of CFHR1/CFHR3 deficiency in aHUS patients may lead to the design of new diagnostic approaches, such as enhanced testing for these genes

Atypical Haemolytic Uraemic Syndrome Associated with a Hybrid Complement Gene

BACKGROUND: Sequence analysis of the regulators of complement activation (RCA) cluster of genes at chromosome position 1q32 shows evidence of several large genomic duplications. These duplications have resulted in a high degree of sequence identity between the gene for factor H (CFH) and the genes for the five factor H-related proteins (CFHL1–5; aliases CFHR1–5). CFH mutations have been described in association with atypical haemolytic uraemic syndrome (aHUS). The majority of the mutations are missense changes that cluster in the C-terminal region and impair the ability of factor H to regulate surface-bound C3b. Some have arisen as a result of gene conversion between CFH and CFHL1. In this study we tested the hypothesis that nonallelic homologous recombination between low-copy repeats in the RCA cluster could result in the formation of a hybrid CFH/CFHL1 gene that predisposes to the development of aHUS. METHODS AND FINDINGS: In a family with many cases of aHUS that segregate with the RCA cluster we used cDNA analysis, gene sequencing, and Southern blotting to show that affected individuals carry a heterozygous CFH/CFHL1 hybrid gene in which exons 1–21 are derived from CFH and exons 22/23 from CFHL1. This hybrid encodes a protein product identical to a functionally significant CFH mutant (c.3572C>T, S1191L and c.3590T>C, V1197A) that has been previously described in association with aHUS. CONCLUSIONS: CFH mutation screening is recommended in all aHUS patients prior to renal transplantation because of the high risk of disease recurrence post-transplant in those known to have a CFH mutation. Because of our finding it will be necessary to implement additional screening strategies that will detect a hybrid CFH/CFHL1 gene

MLPA Binding Sites Used to Identify Deletions of <i>CFH</i> Exons 22 and 23

<p>The hybridisation sequence for the 5′ and 3′ probes are shown by red and blue, respectively. The genomic sequence of <i>CFH</i> is shown aligned above <i>CFHL1</i>.</p

cDNA Evidence of a Hybrid Gene

<div><p>(A) Inverted <i>CFH</i> exons 20–23 cDNA sequence showing the site of the first-round forward primers (blue), the second round forward primers (green), and the reverse primers for both rounds (red). The nucleotides at which the <i>CFH</i> and <i>CFHL1</i> sequences differ are shown in bold and highlighted (excluding exon 20).</p> <p>(B) Inverted cDNA sequence of <i>CFH</i> exons 20–23 from III:6 (affected family member) showing evidence for a hybrid <i>CFH/CFHL1</i> gene. Positions at which the <i>CFH</i> and <i>CFHL1</i> sequences differ are indicated by arrows. At the three differences in exons 22 and 23 (numbered 1–3) there is a heterozygous base change, one allele being wild-type <i>CFH</i> and the other the equivalent base from <i>CFHL1</i>.</p> <p>(C) Inverted cDNA sequence showing hybrid <i>CFH/CFHL1</i> sequence (c.3590T>C, V1197A, and c.3572C>T, S1191L) in III:6 (affected) and III:3 (unaffected carrier) compared to normal <i>CFH</i> sequence in III:1 (unaffected), III:7 (unaffected), and a normal unrelated control.</p></div

Southern Blot Evidence of Genomic Rearrangement

<div><p>(A) A Southern blot using a 1.1 kb probe overlying <i>CFH</i> exon 21 and <i>CFHL1</i> exon 4 hybridised to HindIII-digested DNA (sites shown as arrows) will result in fragments of 11.2 kb from <i>CFH,</i> 1.4 kb from <i>CFHL1,</i> and 8.6 kb from a <i>CFH/CFHL1</i> hybrid gene. The site of the 1.1 kb probe is indicated above.</p> <p>(B) Southern blot showing an additional 8.6 kb band (indicated by B) in lanes 2 and 3, which represent III:3 (unaffected carrier) and III:6 (affected) compared to lanes 1, 4, and 5, which represent III:1 (unaffected) and individuals with homozygous deletion of <i>CFHL1</i>. Bands at A and C represent fragments of 11.2 kb from <i>CFH</i> and 1.4 kb from <i>CFHL1,</i> respectively. A size ladder is shown to the right with heavy arrows indicating the expected sizes.</p></div

Genomic Structure of the Region of the RCA Cluster Containing the Genes Encoding Factor H and the Five factor H-Related Proteins

<div><p>Genomic duplications including the different exons of the six genes were originally determined by Male et al. [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030431#pmed-0030431-b004" target="_blank">4</a>] and are colour-coded. Exons are indicated as vertical lines.</p> <p>(A) Position of the genes encoding factor H and the factor H-related proteins in a centromeric segment of the RCA cluster at 1q32.</p> <p>(B and C) Nonhomologous recombination occurring at X would result in a hybrid gene consisting of the first 21 exons of <i>CFH</i> (encoding SCRs 1–18 of the hybrid gene) and the last 2 exons of <i>CFHL1</i> (encoding SCRs 19 and 20 of the hybrid gene). If the recombination occurred at Y this would result in deletion of <i>CFHL3</i> and <i>CFHL1</i> but <i>CFH</i> would remain intact.</p> <p>(D) The recombination event would also potentially lead to a duplication of <i>CFHL1</i> and <i>CFHL3</i>.</p> <p>(Figure adapted from <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030431#pmed-0030431-g001" target="_blank">Figure 1</a> of [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030431#pmed-0030431-b015" target="_blank">15</a>] with kind permission of Human Mutation C 2006, Wiley Liss Inc., A Wiley Company.)</p></div

Identifying and Screening the Breakpoint Region

<div><p>(A) Sequence of unique PCR product generated with specific <i>CFH</i> (forward) and <i>CFHL1</i> (reverse) primers from III:6 (affected) demonstrating the hybrid product. Unique <i>CFH</i> positions are indicated with black arrows, and unique <i>CFHL1</i> nucleotides are indicated with red arrows.</p> <p>(B) The genomic sequence of <i>CFH</i> is shown aligned above <i>CFHL1</i>. Exons 21 and 4 of the two genes respectively are highlighted in grey. The primer-binding sites for the PCR are shown in red. The differences visible in intron 21/4 from sequencing the product are highlighted in the standard base colours A (green), C (blue), G (black), and T (red). The breakpoint is within the region underlined.</p> <p>(C) Sequence of the intron between exons 21 and 22 shows a switch from heterozygosity at <i>CFH/CFHL1</i> unique bases to a <i>CFHL1</i> sequence in III:3 (unaffected carrier) and III:6 (affected) compared with III:1 (unaffected), III:7 (unaffected), and a normal unrelated control.</p></div