Resequencing the susceptibility gene, ITGAM, identifies two functionally deleterious rare variants in systemic lupus erythematosus cases

Abstract

Introduction: The majority of the genetic variance of systemic lupus erythematosus (SLE) remains unexplained by the common disease-common variant hypothesis. Rare variants, which are not detectable by genome-wide association studies because of their low frequencies, are predicted to explain part of this "missing heritability." However, recent studies identifying rare variants within known disease-susceptibility loci have failed to show genetic associations because of their extremely low frequencies, leading to the questioning of the contribution of rare variants to disease susceptibility. A common (minor allele frequency = 17.4% in cases) nonsynonymous coding variant rs1143679 (R77H) in ITGAM (CD11b), which forms half of the heterodimeric integrin receptor, complement receptor 3 (CR3), is robustly associated with SLE and has been shown to impair CR3-mediated phagocytosis.

Methods: We resequenced ITGAM in 73 SLE cases and identified two previously unidentified, case-specific nonsynonymous variants, F941V and G1145S. Both variants were genotyped in 2,107 and 949 additional SLE cases, respectively, to estimate their frequencies in a disease population. An in vitro model was used to assess the impact of F941V and G1145S, together with two nonsynonymous ITGAM polymorphisms, A858V (rs1143683) and M441T (rs11861251), on CR3-mediated phagocytosis. A paired two-tailed t test was used to compare the phagocytic capabilities of each variant with that of wild-type CR3.

Results: Both rare variants, F941V and G1145S, significantly impair CR3-mediated phagocytosis in an in vitro model (61% reduction, P = 0.006; 26% reduction, P = 0.0232). However, neither of the common variants, M441T and A858V, had an effect on phagocytosis. Neither rare variant was observed again in the genotyping of additional SLE cases, suggesting that their frequencies are extremely low.

Conclusions: Our results add further evidence to the functional importance of ITGAM in SLE pathogenesis through impaired phagocytosis. Additionally, this study provides a new example of the identification of rare variants in common-allele-associated loci, which, because of their extremely low frequencies, are not statistically associated. However, the demonstration of their functional effects adds support to their contribution to disease risk, and questions the current notion of dismissing the contribution of very rare variants on purely statistical analyses.

Figure 1

ITGAM transcript, CD11b primary protein domains, and CR3 protein. (A)ITGAM genomic organization showing relative positions of common polymorphisms (R77H, M441T, A858V) and novel rare variants (F941V, G1145S). (B) Primary protein domains of CD11b and the relative location of the missense variants. The β-propellor domain is shown in dark blue; the ligand-binding I-domain is shown in green; light blue represents the other extracellular domains; the transmembrane domain is shown in yellow, and the short cytoplasmic tail is represented in orange. (C) A cartoon diagram of the CR3 protein (Protein domain ratios = extracellular/transmembrane/cytoplasmic = 1:6:6). The same colors as (B) are used to represent the various domains; CD18 is shown in light purple. Asterisks are used to indicate the position of missense variants used in the functional studies: M441T, A858V, F941V, and G1145S.

Figure 2

Comparisons of CD11 amino acid sequences. CD18 pairs with CD11b, CD11a, CD11c, and CD11d, and together, these make up the β₂ integrin family. The four CD11 protein sequences (encoded by ITGAM, ITGAX, ITGAD, and ITGAL) were aligned by using MutAlin. The numbers indicate the CD11b codons, and all other sequences are aligned relative to this. F941 and G1145 of CD11b are indicated by arrows. The phenylalanine at codon 941 is present across all four CD11 polypeptides; the glycine at codon 1145 is not found in any other CD11 polypeptide.

Figure 3

Association of CR3-expressing COS-7 cells with sRBC_iC3b. Association Index = mean number of associated (internal and external) sRBC_iC3b/100 COS-7 cells. No significant difference was observed between WT and M441T (P = 0.79), A858V (P = 0.65), G1145S (P = 0.85), or F941V (P = 0.61).

Figure 4

Phagocytosis of sRBC_iC3b by CR3-expressing COS-7cells as measured with the Phagocytic Index. Phagocytic Index, mean number of internal sRBC_iC3b/100 COS-7 cells. F941V significantly impairs phagocytosis (P = 0.002). No significant difference observed between WT and either common polymorphism M441T (P = 0.74) and A858V (P = 0.92), or the rare variant G1145S (P = 0.37).

Figure 5

Phagocytosis of sRBC_iC3b by CR3-expressing COS-7cells as measured with percentage phagocytosis. Percentage phagocytosis, mean percentage phagocytosis/COS-7 cell. Both rare variants F941V and G1145S significantly impair phagocytosis (P = 0.006 and 0.0232, respectively). No significant difference was observed between WT and either common polymorphism M441T (P = 0.70) and A858V (P = 0.47).

Figure 6

Percentage reduction in phagocytosis of CD11b variants compared with WT. Comparison of the functional impact of the two rare variants with that of the SLE-associated R77H (previously published data [19]). Mean and SEM are shown.