CFH and CFHR structural variants in atypical Hemolytic Uremic Syndrome: Prevalence, genomic characterization and impact on outcome

Abstract

Introduction: Atypical hemolytic uremic syndrome (aHUS) is a rare disease that manifests with microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure, and is associated with dysregulation of the alternative complement pathway. The chromosomal region including CFH and CFHR1-5 is rich in repeated sequences, favoring genomic rearrangements that have been reported in several patients with aHUS. However, there are limited data on the prevalence of uncommon CFH-CFHR genomic rearrangements in aHUS and their impact on disease onset and outcomes.

Methods: In this study, we report the results of CFH-CFHR Copy Number Variation (CNV) analysis and the characterization of resulting structural variants (SVs) in a large cohort of patients, including 258 patients with primary aHUS and 92 with secondary forms.

Results: We found uncommon SVs in 8% of patients with primary aHUS: 70% carried rearrangements involving CFH alone or CFH and CFHR (group A; n=14), while 30% exhibited rearrangements including only CFHRs (group B; n=6). In group A, 6 patients presented CFH::CFHR1 hybrid genes, 7 patients carried duplications in the CFH-CFHR region that resulted either in the substitution of the last CFHR1 exon(s) with those of CFH (CFHR1::CFH reverse hybrid gene) or in an internal CFH duplication. In group A, the large majority of aHUS acute episodes not treated with eculizumab (12/13) resulted in chronic ESRD; in contrast, anti-complement therapy induced remission in 4/4 acute episodes. aHUS relapse occurred in 6/7 grafts without eculizumab prophylaxis and in 0/3 grafts with eculizumab prophylaxis. In group B, 5 subjects had the CFHR3_1-5::CFHR4₁₀ hybrid gene and one had 4 copies of CFHR1 and CFHR4. Compared with group A, patients in group B exhibited a higher prevalence of additional complement abnormalities and earlier disease onset. However, 4/6 patients in this group underwent complete remission without eculizumab treatment. In secondary forms we identified uncommon SVs in 2 out of 92 patients: the CFHR3_1-5::CFHR4₁₀ hybrid and a new internal duplication of CFH.

Discussion: In conclusion, these data highlight that uncommon CFH-CFHR SVs are frequent in primary aHUS and quite rare in secondary forms. Notably, genomic rearrangements involving the CFH are associated with a poor prognosis but carriers respond to anti-complement therapy.

Keywords: atypical hemolytic uremic syndrome (aHUS); complement; copy number variations (CNVs); eculizumab; factor H (FH); factor H-related proteins (FHRs); single molecule real-time (SMRT); structural variants (SVs).

Figure 1

Structure of Factor H family: genes and proteins. (A) The human complement factor H (CFH) gene family is located on chromosome 1q31.3 and includes six genes: CFH, CFHR3, CFHR1, CFHR4, CFHR2 and CFHR5. For each gene, the corresponding protein was represented. Each short consensus repeat (SCR) is composed of about 60 amino acids and is encoded by a single exon, with the exception of SCR2 of Factor H (FH), encoded by exon 3 and 4. Exon 1 of each gene encodes 18 amino acids of the signal peptide (SP). CFH gene is composed of 23 exons and, through two alternative splicing, produces FH, deriving from 22 exons, and Factor H-like protein 1 (FHL-1), deriving from 10 exons. Exon 10 is not included in the FH transcript and encodes the C-terminal four amino acids (Ser-Phe-Leu-Thr; indicated in the Figure with the green SFLT) and the 3’UTR of FHL-1. FHR-1 exists in two isoforms that differ in three amino acids in the SCR3: FHR-1*A is known as acidic isoform and has His at position 157 (H157), Leu at 159 (L159) and Glu at 175 (E175); FHR-1*B is the basic isoform with Tyr at position 157 (Y157), Val at 159 (V159) and Gln at 175 (Q175). (B) Factor H-related proteins (FHRs) share a high degree of conservation within the C-terminal domains of FH (SCR18-SCR19-SCR20), and FHR-1 is the most similar (the percentage of amino acids identity between each SCR of FHR and those of FH is indicated by blue numbers under the SCRs). As represented in the Figure, SCR3 of FHR-1*A differs from SCR18 of FH for 3 amino acids (H157, L159 and E175) while SCR5 differs from FH-SCR20 for 2 amino acids (L290 and A296). At variance, SCR3 of FHR-1*B has the same amino acids of SCR18 of FH. Of note, the N-terminal domains of FHR-1, FHR-2 and FHR-5 (SCR1 and SCR2) have a high sequence identity (indicated by black percentage numbers) and include a dimerization motif which explains their presence in plasma as either homo-or heterodimers. This image was inspired by Jozsi et al. (7) Trends immunology, 2015.

Figure 2

Graphic representation of MLPA results from aHUS patients carrying structural variants (SVs) in the CFH and CFHR genes. (A) MLPA patterns consistent with CFH::CFHR1 hybrid genes. In patient #4, MLPA pattern shows one copy of exons 22 and 23 of CFH, one copy of CFHR3, one copy of CFHR1 until intron 3, 2 copies of exons 5 and 6 of CFHR1 and two normal copies of CFHR4, CFHR2 and CFHR5, consistent with CFH_1-21::CFHR1_5-6 hybrid gene (29). In patient #5 MLPA results evidence 1 copy of exon 23 of CFH, one copy of CFHR3, one copy of CFHR1 until exon 5, 2 copies of exons 6 of CFHR1 and two normal copies of CFHR4, CFHR2 and CFHR5, consistent with CFH_1-22::CFHR1₆ hybrid gene (30). In patient #6 MLPA analysis shows one copy of exons 22 and 23 of CFH, one copy of CFHR3, normal copy number from the CFHR1-intron 1 to CFHR1-intron 3, 3 copies of exons 5 and 6 of CFHR1, and two normal copies of CFHR4, CFHR2 and CFHR5. This abnormal MLPA pattern was further characterized through long PCR, Sanger sequencing and SMRT which, as reported in Figure 3, led to the identification of the CFH_1-20::CFHR1_4-6 hybrid gene. (B) Representation of FH::FHR-1 hybrid proteins resulting from SVs identified in patients #1, #2, #3, #4, #5 and #6. SCRs translated from CFH are indicated in blue while SCRs deriving from CFHR1 are indicated in brown. The number “100” indicates that SCR4 of FHR-1 is identical to SCR19 of FH; similarly, SCR3 of FHR-1*B is identical to SCR18 of FH. The total identity between FH and FHR-1 indicates that the translated FH::FHR-1 protein is the same in all the above described cases. (C) MLPA pattern consistent with reverse CFHR1::CFH hybrid genes. MLPA results in patient #9 show 3 copies of CFH-exon 23, normal CFHR3 copies, two CFHR1 copies until exon 5, one copy of CFHR1-exon 6, two normal copies of CFHR4, CFHR2 and CFHR5, consistent with the reverse CFHR1_1-5::CFH₂₃ hybrid gene. In patient #10, #11 and #12 MLPA analysis shows a gain starting from CFH-exon 22 until CFHR1-intron 3 and a loss of CFHR1-exon 5-6 consistent with a reverse CFHR1_1-4::CFH_22-23 hybrid gene. In addition, patient #10 carries 3 copies of CFHR3 and 2 copies of normal CFHR1. In patient #11 MLPA provides a copy of normal CFHR1 and 2 copies of CFHR3, consistent with the presence of CFHR1_1-4-CFH_22-23 on one allele and CFHR3-CFHR1 del on the other allele (see Supplementary Figure 1). Unlike patient #11, the abnormal MLPA pattern of patient #12 does not involve the probe located downstream of CFH. (D) Representation of reverse FHR-1::FH hybrid proteins resulting from SVs identified in patients #7, #8, #9, #10, #11 and #12. The translated fusion protein is the same in all cases due to the 100% of identity between SCR19-FH and SCR4-FHR-1.

Figure 3

Patient #1 with a hybrid CFH_1-21::CFHR1 _5-6 gene and a de novo CFHR3-CFHR1 duplication. (A) The proband (black arrow) is patient II:1, his father is I:1 (samples are not available, n.a.), his mother is I:2 and his brother (unaffected carrier of hybrid CFH_1-21::CFHR1 _5-6 gene) is II:2. Genotype of CFH single nucleotide polymorphisms (snps) targeting the CFH-H3 risk (TGTGT) haplotype (c.1–331C>T, rs3753394; c.184G>A, p.V62I, rs800292; c.1204T>C, p.Y402H, rs1061170; c.2016A>G, p.Q672Q, rs3753396; c.2808G>T, p.E936D, rs1065489) and the CD46 snp (rs7144, c.*897 T>C) targeting the CD46 _GGAAC risk haplotype are reported with a yellow square and in red, respectively. (B) MLPA analysis over the CFH-CFHR region in proband’s relatives shows three different patterns. Patient (II:1) exhibits one copy of CFH exons 22 and 23 and 3 copies of CFHR1 exons 5 and 6. His brother (II:2) exhibits a large heterozygous deletion from CFH exon 22 to CFHR1 intron 3, consistent with the presence of hybrid CFH_1-21-CFHR1 _5-6 gene. The mother (I:2) has a normal copy number. These results suggest that the patient and his brother inherited the hybrid CFH_1-21::CFHR1_5-6 gene from their father and evidenced the presence of a de novo CFHR3-CFHR1 duplication only in the patient.

Figure 4

Identification of CFH_1-20::CFHR1_4-6 hybrid gene in patient #6. (A) The abnormal MLPA pattern found in patient #6 involves CFH, CFHR3 and CFHR1 genes. It results in: 1) loss of one copy of exons 22-23 of CFH; 2) loss of 1 copy of the entire CFHR3 gene; 3) 2 copies of intron 1-2 and 3 of CFHR1; 4) 3 copies of exon 5 and 6 of CFHR1. (B) Electropherogram including the sequence of the genomic breakpoint. Arrows indicate the nucleotide differences between CFH and CFHR1. The first part of the sequence corresponded to CFH; the red asterisk indicates the genomic position where the intron 4 CFHR1 sequence started. Four nucleotide differences were found in exon 4 CFHR1: c.469, c.475, c.523 (indicated in brown) and c.588 (not reported). Three of them led to three FHR-1 amino acid changes (p.Tyr157-Val159-Gln175, respectively) and are characteristic of the basic isoform of CFHR1 (CFHR1*B). The green bar highlights the target sequence of the “CFHR1-intron 3” probe, located in intron 3 of CFHR1 (194 nucleotides before exon 4), upstream of the breakpoint region, explaining the 2 copies identified by this probe. (C) Representation of ~84 kb deletion involving CFH, CFHR3 and CFHR1, resulting in the generation of CFH_1-20::CFHR1_4-6 hybrid gene that encodes the FH_1-17::FHR1_3-5 fusion protein. (D) Screenshot from IGV (Integrative Genomics Viewer) showing reads from SMRT sequencing. SMRT sequencing identified both CFH_1-20::CFHR1_4-6 hybrid gene and the CFHR1 duplication. Misaligned reads in the CFHR1-CFHR4 intragenic region and the CFHR2 are also shown.

Figure 5

The CFH _1-18 duplication identified in patient #13. (A) MLPA pattern showing 3 copies of CFH until exon 18 and 2 normal copies in the remaining CFH, CFHR exons. (B) Screenshot from IGV (Integrative Genomics Viewer) showing reads from SMRT sequencing of patient #13, carrying a tandem CFH _1-18 duplication. Reads originating from across the breakpoint were mapped as chimeric alignments (split-reads) with the second part of the read mapped upstream of the first part (and vice versa for the reverse reads). (C) Pedigree of patient #13 and FH levels: the CFH_1-18 was inherited from the unaffected father and was also found in both his healthy brother (III-5) and in unaffected paternal uncle (II-1). FH levels resulted in the normal range in all tested samples (n.r.: ≥193 mg/L) although in the proband’s sample were lower (210 mg/L) than in the other relatives. (D) Western Blot (WB) to detect FH was performed using a monoclonal anti-human FH antibody (OX-23, LSBio), under non-reducing conditions, using sample from the proband (III-4), his available relatives, a patient with FH deficiency (negative control) and a healthy control with normal FH (positive control). The presence of a band with a MW (around 100 kDa) lower than normal FH in all carriers of the CFH _1-18 duplication indicates that a short FH, likely missing the C-terminal domains [FH_1-15; (E)], is secreted. n.a., not available.

Figure 6

Identification of the reverse CFHR1_1-3::CFH_21-23 hybrid gene in patient #14. (A) The results of MLPA show in patient #14 three copies of both CFH exons 21-22-23 and CFHR3 exons 1-2-3, 2 copies of CFHR1, one of them lacking exons 4-5-6. These data suggest the presence of a reverse CFHR1_1-3::CFH_21-23 hybrid with a partial duplication of CFHR3 _1-3. The analysis has been performed with the SALSA MLPA P236-CFH-region Kit (MRC Holland) implemented with homemade probes (indicated by asterisks) analyzed in a separate assay and covering the last exons and introns of the CFH gene (27). (B) Sequence of the genomic breakpoint of the CFHR1_1-3::CFH_21-23 hybrid gene, mapped between chr1:196796490 (intron 3 of CFHR1) and chr1:196711901 (intron 20 of CFH). Arrows indicate the nucleotide differences between CFH and CFHR1. The green bars highlight the target sequence of “CFH-intron 20” probe (located 747 nucleotides before exon 21) and the “CFHR1-intron 3” probe (located 396 nucleotides after exon 3). (C) Representation of reverse CFHR1_1-3::CFH_21-23 and the corresponding FHR-1_1-2::FH_18-19-20 fusion protein. (D, E) To investigate the effect of CFHR3_1-3 duplication at protein level, we performed a WB analysis, under non-reducing conditions, using both an anti-FHR-1-2-5 monoclonal antibody and a FHR-3 polyclonal antiserum. FHR-1 staining showed FHR-1 bands at the same MW of the normal healthy control (D). Staining of FHR-3 showed both the bands corresponding to the three normal glycosylated isoforms of FHR-3 and a faint band at low MW consistent with a short FHR-3_1-2 (E).

Figure 7

Identification of CFH_2-9 duplication in patient #22. (A) MLPA pattern in patient #22 shows a high signal on the exon 3, exon 4 and exon 6-CFH probes consistent with a heterozygous duplication. (B) Screenshot from IGV (Integrative Genomics Viewer) showing reads from SMRT sequencing. SMRT sequencing identified CFH_2-9 duplication. (C) Electropherogram of the genomic breakpoint. The first part of the sequence corresponds to intron 9 of the CFH and the second part is the sequence of CFH exon 2. (D) Representation of 19,6 kb internal duplication of the CFH and the predicted resulting FH protein consisting of 26 SCRs.