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S.D., J.U. and T.A.B. performed SNP genotyping. T.A.B. performed sequencing with contributions from S.D., H.G., G.I.R. and M.S. QMPSF analysis was undertaken by J.U. and M.S. IFN assays were conducted by P.L. L.A.H.Z. performed microarray analysis. RT and qPCR was conducted by G.I.R., T.A.B., H.G., E.H. and E.S.F. Protein assays were preformed by A.J. for TRAP and N.S. for OPN. ACP5 expression in different human cell types was assessed by K.B.E. and A.W. Lymphocyte subset analysis was conducted by C.P., A.P. and F.R-L. Bioinformatics analysis was conducted by S.C.L. All other co-authors identified subjects with SPENCD and performed related clinical and laboratory studies (B.B-M., K.B., S.B., V.B., M.W.B., F.dZ., C.F., C.J-d., M.L.K., M.LM., A.L., A.L., K.M., L.M., V.N., C.M.R., S.S-B., C.W). G.I.R., J-L.C., A.R.H., R.J.D., and G.C.M.B. provided critical input into project direction and manuscript preparation. Y.J.C. designed and supervised the project and wrote the manuscript.
Written informed consent was obtained for data collection, blood sampling and genetic testing from patients, family members and healthy controls. The study was approved by the Leeds (East) Multi-centre Research Ethics Committee (Reference number 07/Q1206/7).
We studied ten individuals from eight families showing features consistent with the immuno-osseus dysplasia spondyloenchondrodysplasia (SPENCD). Of particular note was the diverse spectrum of autoimmune phenotypes observed in these patients, including systemic lupus erythematosus (SLE), Sjögren's syndrome, haemolytic anemia, thrombocytopenia, hypothyroidism, inflammatory myositis, Raynaud's disease, and vitiligo. Haplotype data indicated the disease gene to be on chromosome 19p13 and linkage analysis yielded a combined multipoint lod score of 3.6. Sequencing of the ACP5 gene, encoding tartrate resistant acid phosphatase (TRAP), identified biallelic mutations in each of the patients studied, and in vivo testing confirmed a loss of expressed protein. All eight patients assayed demonstrated elevated serum interferon alpha activity, and gene expression profiling in whole blood defined a type I interferon signature. Our findings reveal a previously unrecognised link between TRAP activity and interferon metabolism, and highlight the importance of type I interferon in the genesis of autoimmunity.
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 Table 1, Fig. 1, 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.
Whole-genome genotyping analysis in three unrelated patients born to consanguineous parents determined an overlapping region of homozygosity on chromosome 19p13 (Fig. 2). 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 (Table 1). 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 (Fig. 3). 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 (Fig. 4).
Although ACP5 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 (Fig. 3), 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 (Fig. 5a, 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.
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 and IL-12 RNA in patient whole blood (Fig. 5b). 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.
Genome-wide scans were performed in three unrelated patients (4, 6 and 7) born to consanguineous parents, and two unrelated affected individuals (patients 1 and 2) of non-consanguineous parents. Genotypes were generated using the Affymetrix GeneChip® single nucleotide polymorphism (SNP) Human Mapping 6.0 array. Datasets were collated and standardized to a single recent genome build (NCBI36/hg18) using the SNPsetter program (http://dna.leeds.ac.uk/snpsetter/), and the combined data were analyzed using AutoSNPa (http://dna.leeds.ac.uk/autosnpa/). Subsequently, siblings to patients 2 (patient 3) and 4 (patient 5), and a further 3 unrelated children (patients 8, 9 and 10), 2 born to consanguineous parents (paitents 8 and 10), were ascertained and DNA collected.
Linkage analysis was performed assuming autosomal recessive inheritance with a disease allele frequency of 0.001, complete penetrance, and an equal frequency for marker alleles. Lod scores were calculated using the Merlin linkage package (http:www.sph.umich.edu/csg/abecasis/Merlin).
Mutation screening of ACP5, encoding the enzyme tartrate resistant acid phosphatase (TRAP), was performed by polymerase chain reaction (PCR) amplification of genomic DNA segments and direct sequencing of the products using BigDye terminator chemistry and a 3130 DNA sequencer (Applied Biosystems). Mutation description is based on the reference complementary DNA (cDNA) sequence NM_001111035, with the ATG initiation site situated at the beginning of exon 4 and the termination codon in exon 7.
In order to confirm the genomic deletion of ACP5 in patient 1, we used a method of quantitative multiplex PCR of short fluorescent fragments (QMPSF) in which multiple short genomic sequences are amplified simultaneously with dye-labelled primers under quantitative conditions23.
TRAP isoform 5a protein levels in plasma were measured using a monoclonal antibody (mab220) raised against a unique epitope associated with the uncleaved loop peptide, which specifically captures serum TRAP 5a without cross-reaction to other acid phosphatases24. TRAP 5a protein bound by immobilized mab220 was detected with a second anti-TRAP monoclonal antibody (mab162) conjugated to horseradish peroxidase using a validated assay25. Total TRAP protein levels were assayed with a unique monoclonal antibody, mab14G6, to immobilize both isoforms 5a and 5b, which was then detected with the same peroxidase conjugate used for the 5a protein assay.
Type I interferon activity was measured by determining the cytopathic reduction of Madin-Darby bovine kidney (MDBK) cells infected with vesicular stomatitis virus26. A reference of human interferon alpha, standardized against the National Institutes of Heath reference Ga 023-902-530, was included with each titration. Titers were expressed as International Units (IU) per millilitre (ml). Interferon alpha activity in normal serum is <2 IU/ml27,28.
Total RNA was extracted from whole blood using PAXgene (PreAnalytix) or Tempus Spin (Applied Biosystems) RNA isolation kits. RNA concentration and integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies).
Whole transcriptome microarray expression analysis was performed in patients 1, 4, and 6 plus three age-matched controls. Amplified sense-strand cDNA generated from 100ng of total RNA was fragmented, labeled and hybridized to the Affymetrix Genechip Human Exon 1.0 ST ArrayR, and data analysed using Genomics Solution software (Partek Inc., St. Charles, MO, USA). Differential expression between patients and controls was assessed using t-test/ANOVA analysis. A q-value score was generated as a correction factor for false discovery29. Microarray data has been submitted in a MIAME compliant standard to the Array Express database (Experiment E-MEXP-2699; http://www.ebi.ac.uk/arrayexpress).
Quantitative PCR (qPCR) analysis was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems), and cDNA derived from 40ng total RNA. The relative abundance of target transcripts, measured using TaqMan probes for Ly6E (Hs00158942_m1), Mx1 (Hs00895598_m1), USP18 (Hs00276441_m1), RSAD2 (Hs01057264_m1), OAS1 (Hs00973637_m1), IFI44L (Hs00199115_m1), IL-10 (Hs99999035_m1) and IL-12 (Hs01073447_m1) was normalised to the expression level of HPRT1 (Hs03929096_g1) and 18s (Hs999999001_s1) and assessed with the Applied Biosystems StepOne Software v2.1. Statistical significance between groups was determined by t-tests using DataAssist v2.0 (Applied Biosystems). Patient data is expressed relative to the average of 4 age-matched normal controls.
Structural analysis was based on the crystal structure of human purple acid phosphatase (PDB code 2bq8)22. Hydrogen atoms were added using “Reduce”30, and interactions between residues of interest calculated with “Probe”31. To model missense mutations, the KiNG software32 was used to computationally substitute mutated residues. For each residue all low-energy side chain conformations (“rotamers”)33 were tested, and the interactions between the modelled mutation and the rest of the protein calculated with Probe. The conformation displayed in Fig. 4 is that with fewest van der Waals overlaps, i.e. the ‘best’ side chain conformation.
We would like to thank the families for their cooperation in the research presented here; Dr Belen Eguia, AP-HP, Department of Dermatology and Allergy, Hôpital Tenon, Paris, France for providing medical images; Mr Bob Smith for technical support; and Professor David Bonthron for insightful comments on the manuscript. We wish to acknowledge Leanne Wardleworth and Andy Hayes from the Microarray Facility, Faculty of Life Sciences, University of Manchester. T.A.B. is supported as a Wellcome Trust Clinical Fellow. Y.J.C., J.U. and S.D. acknowledge the Manchester NIHR Biomedical Research Centre. The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement number 241779.
ACP5 Ensembl transcript ID: ENSG00000102575; protein ID: ENSP00000218758. The microarray data is publicly available from ArrayExpress accession E-MEXP-2699.