TREX1 acts in concert with the SET complex in granzyme A-mediated apoptosis, and mutations in TREX1 cause Aicardi-Goutières syndrome and familial chilblain lupus. Here, we report monoallelic frameshift or missense mutations and one 3' UTR variant of TREX1 present in 9/417 individuals with systemic lupus erythematosus but absent in 1,712 controls (P = 4.1 x 10(-7)). We demonstrate that two mutant TREX1 alleles alter subcellular targeting. Our findings implicate TREX1 in the pathogenesis of SLE.

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TREX1 constitutes the major 3'-->5' DNA exonuclease activity measured in mammalian cells. Recently, biallelic mutations in TREX1 have been shown to cause Aicardi-Goutieres syndrome at the AGS1 locus. Interestingly, Aicardi-Goutieres syndrome shows overlap with systemic lupus erythematosus at both clinical and pathological levels. Here, we report a heterozygous TREX1 mutation causing familial chilblain lupus. Additionally, we describe a de novo heterozygous mutation, affecting a critical catalytic residue in TREX1, that results in typical Aicardi-Goutieres syndrome.

Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological features of apoptosis. Single-stranded DNA damage is initiated when the endonuclease NM23-H1 becomes activated to nick DNA after granzyme A cleaves its inhibitor, SET. SET and NM23-H1 reside in an endoplasmic reticulum-associated complex (the SET complex) that translocates to the nucleus in response to superoxide generation by granzyme A. We now find the 3'-to-5' exonuclease TREX1, but not its close homolog TREX2, in the SET complex. TREX1 binds to SET and colocalizes and translocates with the SET complex. NM23-H1 and TREX1 work in concert to degrade DNA. Silencing NM23-H1 or TREX1 inhibits DNA damage and death of cells treated with perforin (PFN) and granzyme A, but not of cells treated with perforin and granzyme B (GzmB). After granzyme A activates NM23-H1 to make single-stranded nicks, TREX1 removes nucleotides from the nicked 3' end to reduce the possibility of repair by rejoining the nicked ends.

The 335 exonucleases catalyze the excision of nucleoside monophosphates from the 3 termini of DNA. We have identified the cDNA sequences encoding two 335 exonucleases (TREX1 and TREX2) from mammalian cells. The TREX1 and TREX2 proteins are 304 and 236 amino acids in length, respectively. Analysis of the TREX1 and TREX2 sequences identifies three conserved motifs that likely generate the exonuclease active site in these enzymes. The specific amino acids in these three conserved motifs suggest that these mammalian exonucleases are most closely related to the proofreading exonucleases of the bacterial replicative DNA polymerases and the RNase T enzymes. Expression of TREX1 and TREX2 in Escherichia coli demonstrates that these recombinant proteins are active 335 exonucleases. The recombinant TREX1 protein was purified, and exonuclease activity was measured using single-stranded, partial duplex, and mispaired oligonucleotide DNA substrates. The greatest activity of the TREX1 protein was detected using a partial duplex DNA containing five mispaired nucleotides at the 3 terminus. No activity was detected using single-stranded RNA or an RNA-DNA partial duplex. Identification of the TREX1 and TREX2 cDNA sequences provides the genetic tools to investigate the physiological roles of these exonucleases in mammalian DNA replication, repair, and recombination pathways.The multistep processes of DNA replication, repair, and recombination in human cells often require the excision of nucleotides from the DNA 3Ј termini. For each cell division 4 billion nucleotides must be correctly replicated. The polymerization of incorrect or structurally modified nucleotides into DNA generates the 3Ј termini that block chain elongation by the DNA polymerases. Oxidative damage to DNA can result in fragmented nucleotides at the 3Ј termini that can not be elongated by DNA polymerases. Genetic recombination and mismatch repair pathways can require the removal of normal nucleotides from the 3Ј termini of DNA chains. Enzymes containing 3Ј35Ј exonuclease activity remove these mismatched, modified, fragmented, and normal nucleotides to generate the appropriate 3Ј termini for subsequent steps in the DNA metabolic pathways.Several 3Ј35Ј exonucleases have been described from a variety of animal cells (1-5). These exonucleases demonstrate similar biochemical properties, but the relationships between these enzymes are not known. Also, there are 3Ј35Ј exonucleases contained in the structural domains of mammalian DNA pols 1 ␦ (6), ⑀ (7), and ␥ (8). These proofreading 3Ј35Ј exonucleases excise incorrectly polymerized nucleotides during DNA synthesis. Thus, a variety of 3Ј35Ј exonucleases are present in mammalian cells. These exonucleases might function in multiple pathways to generate 3Ј termini that support further steps such as polymerization or ligation.Excision of incorrectly polymerized nucleotides by proofreading 3Ј35Ј exonucleases is an important mechanism to minimize errors during DNA synthesis. The polymerase-associated proofreading exonuclease was ...

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Systemic lupus erythematosus (SLE) is a prototypic autoimmune disorder with a complex pathogenesis in which genetic, hormonal and environmental factors have a role. Rare mutations in the TREX1 gene, the major mammalian 3 0 -5 0 exonuclease, have been reported in sporadic SLE cases. Some of these mutations have also been identified in a rare pediatric neurological condition featuring an inflammatory encephalopathy known as Aicardi-Goutiè res syndrome (AGS). We sought to investigate the frequency of these mutations in a large multi-ancestral cohort of SLE cases and controls. A total of 40 single-nucleotide polymorphisms (SNPs), including both common and rare variants, across the TREX1 gene, were evaluated in B8370 patients with SLE and B7490 control subjects. Stringent quality control procedures were applied, and principal components and admixture proportions were calculated to identify outliers for removal from analysis. Population-based case-control association analyses were performed. P-values, false-discovery rate q values, and odds ratios (OR) with 95% confidence intervals (CI) were calculated. The estimated frequency of TREX1 mutations in our lupus cohort was 0.5%. Five heterozygous mutations were detected at the Y305C polymorphism in European lupus cases but none were observed in European controls. Five African cases incurred heterozygous mutations at the E266G polymorphism and, again, none were observed in the African controls. A rare homozygous R114H mutation was identified in one Asian SLE patient, whereas all genotypes at this mutation in previous reports for SLE were heterozygous. Analysis of common TREX1 SNPs (minor allele frequency (MAF)410%) revealed a relatively common risk haplotype in European SLE patients with neurological manifestations, especially seizures, with a frequency of 58% in lupus cases compared with 45% in normal controls (P ¼ 0.0008, OR ¼ 1.73, 95% CI ¼ 1.25-2.39). Finally, the presence or absence of specific autoantibodies in certain populations produced significant genetic associations. For example, a strong association with anti-nRNP was observed in the European cohort at a coding synonymous variant rs56203834 (P ¼ 2.99EÀ13, OR ¼ 5.2, 95% CI ¼ 3.18-8.56). Our data confirm and expand previous reports and provide additional support for the involvement of TREX1 in lupus pathogenesis.

We recently described a novel autosomal-dominant genodermatosis, termed familial chilblain lupus, and mapped its genetic locus to chromosome 3p21. Familial chilblain lupus manifests in early childhood with ulcerating acral skin lesions and is associated with arthralgias and circulating antinuclear antibodies. In this study, we report the identification of a heterozygous missense mutation (D18N) in TREX1 encoding the 3'-5'repair exonuclease 1 in affected individuals of the family with chilblain lupus. The homodimeric TREX1 is the most abundant intracellular DNase in mammalian cells. We have recently shown that TREX1 plays a role in apoptotic single-stranded DNA damage induced by the killer lymphocyte protease granzyme A. D18N affects a highly conserved amino acid residue critical for catalytic activity. Recombinant mutant TREX1 homodimers are enzymatically inactive, while wild type/mutant heterodimers show residual exonucleolytic activity, suggesting a heterozygous loss of function. Lymphoblastoid cells carrying the D18N mutation are significantly less sensitive to granzyme A-mediated cell death, suggesting a novel role for this caspase-independent form of apoptosis in the pathogenesis of familial chilblain lupus. Our findings also warrant further investigation of TREX1 in common forms of lupus erythematosus.