Mutations involved in Aicardi-Goutières syndrome implicate SAMHD1 as regulator of the innate immune response

Abstract

Aicardi-Goutières syndrome is a mendelian mimic of congenital infection and also shows overlap with systemic lupus erythematosus at both a clinical and biochemical level. The recent identification of mutations in TREX1 and genes encoding the RNASEH2 complex and studies of the function of TREX1 in DNA metabolism have defined a previously unknown mechanism for the initiation of autoimmunity by interferon-stimulatory nucleic acid. Here we describe mutations in SAMHD1 as the cause of AGS at the AGS5 locus and present data to show that SAMHD1 may act as a negative regulator of the cell-intrinsic antiviral response.

Figure 1

SAMHD1 is the AGS5 gene. Schematic of the AGS5 critical region and the SAMHD1 gene and protein. (a) Genetic map of chromosome 20q. The AGS5 critical interval is defined by the overlapping homozygous segment in AGS165 and the ancestral haplotype shared by two Maltese famillies (marker distances derived from the UCSC Browser, March 2006 assembly). (b) SAMHD1 spans 59,532 bp of genomic sequence (chromosome 20:34,954,059– 35,013,590) in 16 exons and encodes a 626 amino-acid (AA) protein with a molecular weight of 72.2 kDa. (c) Schematic of the SAMHD1 protein indicating position of identified substitutions and conserved domains.

Figure 2

Localization of SAMHD1. (a) Localization of SAMHD1 in MRC-5 control and Q149X fibroblast cells (AGS128). Cells immunolabeled with DAPI (DNA), α-tubulin (to show microtubules) and antibody to SAMHD1. Scale bar, 10 µm. Top, specific nuclear localization of SAMHD1 in MRC-5 control cells. Bottom, nuclear localization is maintained in subject fibroblasts expressing the Q149X nonsense substitution (nonsignificant difference from MRC-5 cells), although expression is reduced and localization to nuclear structures is less well defined. (b) Localization of wild-type (WT) GFP-SAMHD1 and GFP-SAMHD1 carrying a Q149X substitution in HeLa cells. Cells immunolabeled with DAPI (DNA), α-tubulin and GFP. Scale bar, 10 µm. Top, GFP is expressed throughout the cell. Middle, specific nuclear localization of SAMHD1-GFP was seen in >98% of cells. Bottom, nuclear localization but with reduced expression and an altered intranuclear distribution is maintained in cells expressing a SAMHD1-GFP carrying the Q149X nonsense substitution. (c) Immunoblot of cell lysates containing expressed GFP constructs. Lane 1, molecular weight marker; lane 2, Optimem; lane 3, GFP control; lane 4, wild-type SAMHD1-GFP; lane 5, Q149X SAMHD1-GFP. In lane 4, full-length wild-type SAMHD1-GFP is indicated by the band between 75 and 100 kDa, whereas the smaller bands suggest protein degradation. A truncated Q149X-GFP gene product is seen in lane 5. FL, full-length; TP, truncated protein; PD, protein degradation; GFP, GFP construct.

Figure 3

Fold induction of IFN-β, Samhd1 and Trex1 following transfection with interferon-stimulatory DNA. We transfected bone marrow–derived macrophages from MyD88^−/− and MyD88/Ifnar1 double knockout mice with calf-thymus interferon-stimulatory DNA and recorded fold induction of IFN-β, Samhd1 and Trex1 at the specified time points.