Association of IRF5 in UK SLE families identifies a variant involved in polyadenylation

Abstract

Results from two studies have implicated the interferon regulatory gene IRF5 as a susceptibility gene in systemic lupus erythematosus (SLE). In this study, we conducted a family-based association analysis in 380 UK SLE nuclear families. Using a higher density of markers than has hitherto been screened, we show that there is association with two SNPs in the first intron, rs2004640 (P = 3.4 x 10(-4)) and rs3807306 (P = 4.9 x 10(-4)), and the association extends into the 3'-untranslated region (UTR). There is a single haplotype block encompassing IRF5 and we show for the first time that the gene comprises two over-transmitted haplotypes and a single under-transmitted haplotype. The strongest association is with a TCTAACT haplotype (T:U = 1.92, P = 5.8 x 10(-5)), which carries all the over-transmitted alleles from this study. Haplotypes carrying the T alleles of rs2004640 and rs2280714 and the A allele of rs10954213 are over-transmitted in SLE families. The TAT haplotype shows a dose-dependent relationship with mRNA expression. A differential expression pattern was seen between two expression probes located each side of rs10954213 in the 3'-UTR. rs10954213 shows the strongest association with RNA expression levels (P = 1 x 10(-14)). The A allele of rs10954213 creates a functional polyadenylation site and the A genotype correlates with increased expression of a transcript variant containing a shorter 3'-UTR. Expression levels of transcript variants with the shorter or longer 3'-UTRs are inversely correlated. Our data support a new mechanism by which an IRF5 polymorphism controls the expression of alternate transcript variants which may have different effects on interferon signalling.

Figure 1

Genomic organization, haplotype architecture and haplotype-TDT across the human IRF5 gene. (A) The exons are marked with black boxes and numbered above. The untranslated alternative 5′-exons are labelled 1a–c and the 3′-UTR is shown as a white box. The polymorphisms selected for the study are numbered 1–8 below the gene. (B) The haplotype block structure across IRF5, as constructed using Haploview, from 357 EC parent-proband trios and 22 IP trios. There is a single haplotype block across the gene, consisting of seven haplotypes, numbered 1–7 down the left-hand side. The haplotype frequency is shown to the right of each haplotype. The marker numbers are shown above the haplotypes and correspond to the SNPs illustrated in (A). The haplotype-tagging SNPs are indicated by asterisks under the marker numbers. The over-transmitted haplotype 3 and haplotype 7 are outlined by solid boxes. The under-transmitted haplotype 2 is outlined by a dashed box; the box incorporates IRF5 SNP 1, to distinguish it from haplotype 4. The over-transmitted rare allele of IRF5 SNP 7 which is unique to over-transmitted haplotype 3 is indicated in bold type. (C) The pair-wise pattern of LD across IRF5 is shown in a matrix. The left-hand diagram shows the pair-wise LD quantified by correlation, r² and the right-hand diagram the D′ plot. Stronger LD is depicted graphically by denser shading.

Figure 2

Correlation of RNA expression level with genotype for variants across IRF5 in CEU CEPH individuals. (A) The gene diagram of IRF5 shows the location of the SNPs genotyped in the UK SLE families (IRF5 SNPs 1–8) as in Figure 1A. The expression variants (E1–8) identified by sequencing in the CEPH individuals and correlate with IRF5 mRNA levels are marked by an asterisk. They are shown directly underneath the SNPs used in the association analysis. The location of the expression probe sets, Probe 1 (205469_s_at) and Probe 2 (239412_at) are shown above exon 9. (B) This panel illustrates the correlation of the level of IRF5 mRNA with the TAT haplotype (defined by IRF5 SNP 2, IRF5 SNP 6 and IRF5 SNP 8) in 91 CEPH family samples, with Probe 1 (205469_s_at) and Probe 2 (239412_at) RNA expression probes.

Figure 3

A novel polyadenylation site in IRF5. (A) Diagrammatic representation of the 3′-UTR of IRF5, showing the location of the Affymetrix expression probes, designated Probe 1 and Probe 2 in greater detail than Figure 2. The position of the RT-PCR amplicon relative to IRF5 SNP 6 (rs10954213) is also shown. (B) Alternatively polyadenylated transcripts from IRF5 are illustrated, the size being dependent on the allele at IRF5 SNP 6. (C) Strength of association between RNA expression levels across for each expression SNP across IRF5 for two expression probes 205469_s_at and 239412_at. For each probe, the −log₁₀ P-value of the association is plotted against marker number (E1–8). (D) Quantification of expression differences for IRF5 SNP 6 for the two Affymetrix expression probes in 30 CEPH parental samples. The samples were grouped according to genotype at IRF5 SNP 6 and the mean expression of each sample group was calculated separately for each probe. The fractional change (as a percentage) in the signal from each probe is shown between the A/A and G/G homozygotes.

Figure 4

Graphical model representing relationships between SNP genotypes and mRNA expression. Open circles correspond to three probe sets from Affymetrix HGU-133 Plus 2.0 GeneChip. Filled circles correspond to genotypes at five SNPs [SNP E1 (rs3757385), SNP E3 (Ins GGGGC), SNP E4 (rs2004640/IRF5 SNP 2), SNP E5 (30 bp del), SNP E6 (rs10954213/IRF5 SNP 6), SNP E8 (rs2280714)] and sex. A line connecting two circles represents direct association between the two corresponding variables.

Figure 5

Polyadenylation polymorphism and the expression of the long transcript. The relative units of expression of the longer IRF5 transcript are represented on a log₂ scale in a box and whisker plot, showing 95%CI. The expression levels are normalized with respect to HPRT1. The IRF5 genotype, either A/A or G/G represents the allelic composition at IRF5 SNP 6, which influences polyadenylation.