Variation in CFHR3 determines susceptibility to meningococcal disease by controlling factor H concentrations

Abstract

Neisseria meningitidis protects itself from complement-mediated killing by binding complement factor H (FH). Previous studies associated susceptibility to meningococcal disease (MD) with variation in CFH, but the causal variants and underlying mechanism remained unknown. Here we attempted to define the association more accurately by sequencing the CFH-CFHR locus and imputing missing genotypes in previously obtained GWAS datasets of MD-affected individuals of European ancestry and matched controls. We identified a CFHR3 SNP that provides protection from MD (rs75703017, p value = 1.1 × 10^-16) by decreasing the concentration of FH in the blood (p value = 1.4 × 10^-11). We subsequently used dual-luciferase studies and CRISPR gene editing to establish that deletion of rs75703017 increased FH expression in hepatocyte by preventing promotor inhibition. Our data suggest that reduced concentrations of FH in the blood confer protection from MD; with reduced access to FH, N. meningitidis is less able to shield itself from complement-mediated killing.

Keywords: CFH; CFHR3; Complement; Factor H-related protein; Meningococcal disease; Neisseria meningitidis; factor H; genotyping; in-depth sequencing; infectious disease.

Figure 1

Fine mapping by sequencing of the CFH-CFHR locus Plot showing association results of all the SNPs (arranged according to their GRCh37/hg19 build chromosomal position on the x axis) from deep sequencing (circle) and from meta-analysis (square) with combined stage 1 and stage 2 cohorts. The top SNP from the analysis is labeled (rs75703017). The color intensity of each symbol reflects the extent of LD with the top GWAS SNP.

Figure 2

Forest plot of the top SNP, rs75703017 Plot showing odds ratios (ORs) and confidence intervals in each of the four cohorts; the summary estimate is shown below. The dotted vertical line indicates no effect. Cohort names with numbers of MD individuals and healthy controls are on the left, ORs and 95% CI are on the right.

Figure 3

Fine mapping of the CFH-CFHR locus by GWAS (A) Known variants reported in the NHGRI-EBI catalog of human genome-wide association studies. An asterisk represents the location of the rs426736 SNP within the CNVs and annotated as associated with MD. (B) The plot represents the genes located in the captured region (ranging from CFH to CFHR5) of the sequencing approach and shows association results of all variants (SNPs and InDels arranged according to their GRCh37/hg19 build chromosomal position on the x axis) from GWAS meta-analysis with the lead SNP, rs1065489, set as a reference variant (purple diamond). The color intensity of each symbol reflects the extent of LD with the top GWAS SNP. (C) dbVar (https://www.ncbi.nlm.nih.gov/dbvar/)-annotated common CNVs with partial (nsv3888824 results in a CFH/CFHR1 hybrid gene) or complete (nsv4649133) deletion of CFHR3 and CFHR1. (D) Plot showing association results of all variants (SNPs and InDels arranged according to their GRCh37/hg19 build chromosomal position on the x axis) from a GWAS meta-analysis with the lead SNP, rs75703017, from stages 1+2 set as a reference variant (purple diamond) mapping to a smaller genetic area focused on the start of the CNVs. Variants, which were either previously reported^, or notable findings from this study (stages 1–3) are annotated within the plot. ^#Annotated variants represent small InDels within the CNVs. Violet vertical and green lines represent the start of the CNVs nsv3888824 and nsv4649133, respectively.

Figure 4

FH and FHR-3 concentrations in MD survivors (A) Box plots with 95% range of FH serum concentrations (left panel), determined by ELISA. FH serum concentrations are increased (p < 0.0001, Mann-Whitney test) in MD survivors (n = 367) compared to healthy controls (n = 124), whereas FHR-3 serum concentrations are not significantly different between the two groups. (B) Violin plotsof FH serum concentrations delineated by genotype for the SNP (rs75703017) most associated with FH concentrations; p = 1.41 × 10⁻¹¹. (C) Violin plot of FHR-3 serum concentrations delineated by genotype for the SNP (rs75703017), p value < 2 × 10⁻¹⁶. For both (B) and (C), the x and y axes indicate genotypes and protein concentration (µg/mL), repsectively, with D = CFHR3/CFHR1 deletion, A = minor allele, and C = major allele for rs75703017. The white dots indicate median concentrations, the thick black bars indicate the interquartile range, and the thin black bars represent total range. p values were estimated by ANCOVA.

Figure 5

Functional validation of the top associated variant (A) Effect of 2.8 kb regulatory sequence and the lead SNP, rs75703017, on reporter (firefly luciferase) activity in the HepG2 human liver cell line. pGL3-empty is an empty construct only containing a promoter, whereas pGL3-C contains the 2.8 kb regulatory sequence with major allele C at rs75703017, pGL3-A contains minor allele A at rs75703017, and pGL3-AAC contains minor allele A at rs75703017 and minor allele C at rs446868 and rs385390. Firefly luciferase concentrations were normalized to renilla luciferase activity for each sample, and all values were plotted relative to the pGL3-empty construct. The graph is representative of six independent experiments, and error bars represent means with standard deviation. Level of significance, calculated by t test, is indicated. (B) Schematic depiction of CRISPR/Cas9 targeting of the CFHR3 liver-specific regulatory region comprising 2,844 base pairs (chr1: 196,743,825–196,746,668). Excision sites of each guide RNA, located around the SNP of interest (rs75703017), are indicated by scissors. The position of screening primers designed for selection of positive clones with excision of the targeted region are indicated by red arrows. (C) n-fold change, relative to the wild type, of CFH transcript expression levels of CRISPR-edited CFHR3 (CRISPR-edited H1; genotype DD) carrying one copy of CFHR3 with allele C (WT-H1; genotype DC) in liver-differentiated H1 human embryonic stem cells. Expression was measured by qRT-PCR. The graph represents three independent experiments with two biological replicates (different sets of gRNA were used for targeting; KO1 WT-Cas9 gRNA 1 and 3 and KO2 nickase-Cas9 gRNA 1, 2, 3, and 4; see Table S2) and one technical replicate of KO1. Error bars represent means with standard deviation. Level of significance, calculated by t test, is indicated. (D = CFHR3/CFHR1 deletion, and C = major allele rs75703017).

Figure 6

Schematic model of the effect of CFHR3/CFHR1 deletion and SNP (rs75703017) on FH concentrations, interactions with N. meningitidis, and susceptibility to MD The schematic diagram shows the structure of the gene region containing CFH and five CFHR genes. Carriers of the minor allele, A, on rs75703017 show the lowest FH concentrations. Increased concentrations of FH are found with the major allele, C, on rs7570317, whereas deletion of CFHR3/CFHR1 is associated with the highest concentrations of FH. Susceptibility to MD is driven by FH serum availability, which increases binding to the meningococcal surface protein fHbp. This binding results in FH’s impairing complement-mediated killing and allowing survival and growth of N. meningitidis in blood.