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

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

Pedigree of the Family

<p>Pedigree of the Family</p

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

Dosage Evidence of a Hybrid Gene

<p>(A) QF-PCR and (B) MLPA dosage quotients showed an apparent heterozygous “deletion” of <i>CFH</i> exons 22 and 23 in II:8 (unaffected carrier), III:3 (unaffected carrier), III:6 (affected), and IV:1 (affected). There was no evidence of a “deletion” in III:1 and III:7 (both unaffected).</p