We initially ascertained three patients demonstrating a wide range of autoimmunity in association with intracranial calcification and spasticity. We considered the immuno-osseous dysplasia spondyloenchondrodysplasia (MIM 271550) as a differential diagnosis in these cases. Although regarded primarily as a skeletal dysplasia1
, SPENCD patients have been reported to show immune dysfunction and neurological involvement2,3
. Radiological investigations in our patients revealed the characteristic metaphyseal and vertebral bone lesions described in SPENCD. We therefore sought to determine the genetic basis of this phenotype.
The demographic characteristics and clinical features of a total of ten patients with SPENCD are summarised in , , and Supplementary Table 1
. Four patients fulfilled American College of Rheumatology classification criteria for a diagnosis of SLE4
. These included elevated anti-nuclear antibodies, anti-dsDNA antibodies, thrombocytopenia, and nephritis or non-erosive arthritis. Additionally, patient 1 demonstrated overlapping features of systemic sclerosis, Sjögren's syndrome and an inflammatory myositis. Three further patients exhibited significantly elevated anti-dsDNA and anti-nuclear antibody titers. Possibly related to immune activation, four of six patients assessed showed intracranial calcification, a recognized feature of neuro-lupus5
Demographic, molecular and immunological characteristics of SPENCD patients.
Bone, brain and skin involvement in patients with mutations in ACP5
Whole-genome genotyping analysis in three unrelated patients born to consanguineous parents determined an overlapping region of homozygosity on chromosome 19p13 (). Linkage analysis gave a maximum multipoint lod score of 3.6 between base-pair positions 10,527,380-13,214,722. This region contained a total of 95 RefSeq annotated genes. Genotyping of patient 1 demonstrated two contiguous markers, one copy number probe (CN_784690) and one SNP (rs2071484), which failed to hybridize, indicating a possible homozygous deletion within the ACP5
gene. DNA from this patient was refractory to PCR amplification of all exons of ACP5
, despite good amplification of non-ACP5
PCR products, and good amplification of DNA from the single available parent and from control DNA. Further evidence of a homozygous deletion in this patient came from quantitative multiplex PCR of short fluorescent fragments (QMPSF) of DNA from the patient and her mother, and reverse transcription PCR analysis (Supplementary Fig. 1
). We were not able to define the precise breakpoints of the deletion by PCR. Although the parents of patient 1 were not knowingly consanguineous, genotype data demonstrated a run of 35 homozygous SNPs in a 270kb segment between rs4804628 and rs318699 encompassing ACP5
, possibly suggesting distant shared ancestry.
Sequencing of the complete coding region of ACP5
revealed mutations in the further nine patients tested (). All mutations segregated with the disease in the families investigated, and all parents tested were heterozygous for a relevant familial mutation. All missense mutations were in highly conserved residues from a representative sample of eukaryotic species containing TRAP (Supplementary Fig. 2
). None of the mutations identified were present on 210 alleles from control samples of mixed ethnicity.
The observation of biallelic null mutations in four of eight families indicated that the SPENCD phenotype results from a loss of TRAP activity. To explore this possibility further, we measured levels of total TRAP protein and its 5a isoform in plasma from six patients. Compared to five age-matched controls, and an unaffected sibling to patients 2 and 3 who was homozygous for the wild-type allele on gene sequencing, levels of total TRAP protein were negligible, and TRAP 5a protein undetectable, indicating an almost complete lack of TRAP synthesis or secretion in the affected patients tested (). It is of note that patients 4 and 9 were homozygous for missense mutations, and that patients 2 and 3 carried a missense alteration on one allele, suggesting that these missense changes also act as null mutations, perhaps through protein misfolding and the induction of protein degradation. This hypothesis is supported by bioinformatic assessment of the protein structure, in which all four missense changes are predicted to result in significant protein destabilisation ().
Levels of TRAP protein and interferon alpha activity in patients with mutations in ACP5
Computational analysis of missense mutations in the context of protein structure
null mice demonstrate a skeletal dysplasia highly reminiscent of the human disease SPENCD6
, they do not develop an overt autoimmune phenotype. However, these mice do show disordered macrophage pro-inflammatory cytokine production7
. Considering both the importance of the cytokine interferon alpha in the pathogenesis of the prototypic autoimmune disorder SLE8
, and the clinical overlap with the Mendelian interferon-opathy Aicardi-Goutières syndrome3,9
, we sought to assess type I interferon activation in patients affected with SPENCD. Remarkably, type I interferon activity was elevated in serum from all eight patients sampled (), with a persistent elevation demonstrated in five patients where two or more measurements were recorded. This activity was completely abolished with anti-sera against interferon alpha, but not with anti-sera against interferon beta (Supplementary Table 2
SLE patients frequently demonstrate an increased expression of type I interferon stimulated genes, a so-called interferon signature10
. To determine if a similar signature was present in SPENCD patients, we undertook whole-genome microarray analysis in three affected individuals. This analysis identified 18 genes which were greater than four-fold over-expressed in patient whole blood compared to age-matched controls (, Supplementary Fig. 3 and Supplementary Table 3
), 15 of which are recognized as interferon stimulated genes. These data were confirmed by qPCR analysis in five patients. We did not find evidence of a circulating inducer of interferon activity in patient sera (Supplementary Table 4
), possibly suggesting an up-regulation of type I interferon via a cell-intrinsic mechanism.
Gene expression analysis in patients with TRAP deficiency
The above data demonstrate that loss of TRAP protein results in a dramatic up-regulation of interferon alpha and type I interferon stimulated genes in patients affected with SPENCD. In marked contrast, we found normal levels of interferon gamma protein (Supplementary Table 5
), and IL-10
RNA in patient whole blood (). Moreover, we saw normal numbers of T and B cells, including T regulatory cell subsets, in two patients, 4 and 6 (data not shown), suggesting that the autoimmune predisposition in TRAP deficiency is not related to a quantitative defect of regulatory T cells. These data indicate a primary role for TRAP in innate immunity, a contention further supported by the observation that TRAP is induced by lipopolysaccharide11
and by nucleic acid ligands in a sequence non-specific and non-TLR9 dependent manner12
Osteopontin, encoded by the OPN
gene, is a recognized substrate for osteoclast-derived TRAP in vivo13
. Interestingly, polymorphisms in OPN
and serum osteopontin levels show an association with interferon alpha levels in lupus patients14
, possibly through an involvement of osteopontin in the regulation of interferon alpha production by plasmacytoid dendritic cells (pDCs)15
, a major source of type I interferon. For these reasons, we assayed total osteopontin in patient serum, but did not see a statistically significant difference between patients and controls (Supplementary Fig. 4
). Furthermore, although we observed fold-levels of ACP5
expression in pDCs equivalent to peripheral blood mononuclear cells, much higher levels were seen in macrophages and non-plasmacytoid dendritic cells (Supplementary Table 6
). Of note, ACP5
mRNA expression by human pDCs (Supplementary Table 7
), as well as mouse macrophage and mouse dendritic cells (data not shown), was not increased following interferon alpha stimulation, perhaps indicating that TRAP does not act in a simple feedback regulatory loop within these cells. However, the possibility remains that loss of TRAP activity causes an increase in phosphorylated intracellular osteopontin16
in pDCs or other relevant cell types, leading to an increase in circulating interferon alpha through an as yet undefined pathway.
SPENCD patients demonstrate a truly remarkable spectrum of autoimmunity. In relation to SLE, together with deficiency of early components of the classical complement pathway17
and mutations in TREX118
, TRAP deficiency now represents a third monogenic disorder associated with the development of lupus also showing an up-regulation of type I interferon activity19,20,21
. Our findings reveal a previously unrecognised link between TRAP activity and interferon metabolism, and further emphasise the importance of the type I interferon response in autoimmunity.