Overall Cohort Characteristics
Over a period of 6 years, we tested a total of 2181 samples for an interferon signature. These samples were from 1565 individuals, comprising 75 persons considered as controls (96 samples); 1264 patients (1827 samples); and 209 parents (241 samples) and 17 siblings (17 samples) of affected patients (Fig. 1).
We excluded the following from this total of 2181 samples: 187 samples from controls, parents, and siblings where data on age, sex, or genotype were unavailable or where a proband was also excluded (see below); 42 samples from 12 AGS patients under treatment with reverse transcriptase inhibitors (https://clinicaltrials.gov/ct2/show/NCT02363452?term=Aicardi+Goutieres&rank=1); 51 samples from seven IFIH1- and TMEM173-mutated patients under treatment with the JAK1/2 inhibitor ruxolitinib ; 175, seven, and 13 results from 161, seven, and 10 individuals with, respectively, adult-onset SLE/mixed connective tissue disease, retinal vasculopathy and cerebral leukodystrophy (RVCL), and STAT1 gain-of-function mutations which were performed as part of separate studies. We also excluded 714 samples from 597 patients where there was no genetic diagnosis and/or where phenotypic data were limited/allowed for no definite clinical diagnosis, there was no family history of similarly affected relatives, and for whom we had tested less than three ISs.
Following the above exclusions, our cohort consisted of 992 samples from 630 individuals, specifically: 78 samples from 65 controls (including 31 parents and seven siblings to patients with a molecularly proven autosomal dominant type I interferonopathy, most particularly due to heterozygous mutations in TREX1, ADAR1, IFIH1, and TMEM173, where the mutation was shown to have occurred de novo) (Table S1); 84 samples from 71 parents and five samples from five siblings where the parent/sibling of a person with biallelic mutations in a known type I interferonopathy-causing gene was shown to be heterozygous for one familial mutation (Table S2); and 825 samples from 489 patients which we divided into three groups for ease of analysis (Table 1). Group 1 comprises patients with one or more interferon signature reading(s) and a confirmed molecular diagnosis in any of the following 13 genes considered to have a proven link to type I interferon production/signaling: TREX1 (Table S3), RNASEH2A (Table S4), RNASEH2B (Table S5), RNASEH2C (Table S6), SAMHD1 (Table S7), ADAR1 (Table S8), IFIH1 (Table S9), ACP5 (Table S10), TMEM173 (Table S11), C1Q (Table S12), C2 (Table S12), ISG15 (Table S13), and SKIV2L (Table S14). Of note, we only included patients with biallelic mutations in these genes, except for those individuals with recognized dominant mutations in TREX1 (at positions p.Asp18 and p.Asp200), ADAR1 (at position p.Gly1007), IFIH1, and TMEM173. In group 2, we included patients with a confirmed molecular diagnosis of any other genotype, where at least one patient from each of at least two different families tested positive for an interferon signature on at least one occasion. This group thus comprised patients with mutations in DNASE1L3 (Table S15), PRKDC (Table S16), CECR1 (Table S17), RNASET2 (Table S18), and TRNT1 (Table S19). Finally, in group 3, we included patients with a clinical diagnosis of juvenile SLE (JSLE) (Table S20); juvenile DM (JDM) (Table S21); systemic juvenile idiopathic arthritis (sJIA) (Table S24) and other, non-systemic, JIA (pJIA) (Table S25); a molecularly undetermined phenotype which was clinically labeled as autoinflammatory (defined here as unexplained recurrent fevers and/or organ-specific features with elevated markers of systemic inflammation in the absence of autoimmunity and underlying infection) (Table S23); and patients with a phenotype variably comprising neurological, dermatological, rheumatological, and immunological features reminiscent of the known type I interferonopathy spectrum (Table S22), who screened negative for mutations in relevant type I interferonopathy-associated genes and who demonstrated a positive IS on three or more occasions or had a similarly affected relative with a minimum of three positive ISs shared between affected family members.
Results in Controls
Of 78 ISs from 65 controls, seven (9.0%) were abnormal (median IS 0.688, IQR 0.427–1.196) (Table 1, Table S1). In only two of these was the IS above five. The IS was measured on more than one occasion in 12 control individuals, with three persons being sampled three times. On 26 of 27 occasions, the IS was normal (Figure S1), the one positive sample returning an IS of 23.4 which was normal on repeat sampling. Controls ranged from 1 year to 93 years of age and there was no correlation between age and IS (p = NS). Thirty-five of 65 controls were female.
Results in Group 1
In this group, we measured 455 interferon signatures from 265 mutation-positive patients belonging to 222 families. All of these genotypes were associated with a significant upregulation of type I interferon signaling (median IS 10.73, IQR 5.90–18.41) (Table 1, Fig. 2a, Figure S2) compared to controls. Thirty-three of 148 samples (22.30%) from patients with mutations in RNASEH2B demonstrated a normal IS, and a lower median IS (6.10, IQR 2.74–10.84) was observed in this group compared to all other genotypes (median IS 13.14, IQR 8.12–21.54), with the next lowest median ISs associated with mutations in the two other proteins comprising the RNase H2 complex. A comparison of the median RQ value for each of the six individual ISGs across the genotypes comprising group 1 revealed higher fold induction of IFI27 and SIGLEC1 in patients mutated in ACP5, TMEM173, C1Q, and ISG15 compared to all other group 1 genotypes and to patients in group 3 (Fig. 3).
Ten samples from patients mutated in TREX1 (two of 40 patients; three of 77 samples), ADAR1 (four of 34 individuals; four of 56 samples), and ACP5 (two of 12 patients; three of 17 samples) also returned normal results (Fig. 2a, Table S26), versus none of 95 patients (none of 157 samples) with the nine other genotypes. We note that some, but not all, of these interferon signature-negative patients demonstrated a milder clinical phenotype in comparison with other cases mutated in these same genes (data not shown). Of further note, one of the two patients with ACP5-related disease with a normal IS was on high-dose immunosuppressants and was reported to have responded well clinically at the time of sampling.
Results in Group 2
In this group, we measured 30 interferon signatures from 17 mutation-positive patients belonging to 14 families (Fig. 2b, Figure S3). For each of the five genotypes, CECR1 (11 samples, five patients), RNASET2 (six samples, five patients), PRKDC (six samples, two patients), TRNT1 (four samples, three patients), and DNASE1L3 (three samples, two patients), we recorded positive ISs in at least one patient from each of at least two different families (Fig. 2b).
Results in Group 3
Group 3 includes patients with molecularly undefined phenotypes, where we measured 340 interferon signatures from 207 patients belonging to 187 families (Fig. 2c, Figure S4). Our data mirror the results from multiple studies demonstrating an upregulation of interferon signaling in a significant proportion of individuals with JSLE and JDM, where 82% (64 of 78) and 75% (76 of 101) of samples were abnormal in these two phenotypic groupings, respectively (JSLE: median IS 10.60, IQR 3.99–17.27; JDM: median IS 9.02, IQR 2.51–21.73) (Table 1). We note that a number of these patients were under treatment, and some had clinically quiescent disease at the time of sampling. In contrast, a lower proportion (14 of 41) of patients with a clinical diagnosis of a non-molecularly determined autoinflammatory phenotype returned a positive IS (median 1.25, IQR 0.59–4.06). Considering another complex disease, sJIA, we identified five of 19 patients with a positive IS, 11 of whom were being treated with interleukin 1 (IL1) blockade. Finally, we also included in this group 72 samples from 24 patients belonging to 17 families who did not carry a mutation in known clinically relevant type I interferonopathy-associated genes and where we recorded an upregulation of type I interferon signaling measured on at least three occasions—thus, likely indicative of a true association with enhanced type I interferon signaling (median IS 9.38, IQR 6.67–13.98).
Results in Parents and Siblings of Patients in Group 1
The large majority of samples from proven heterozygous carrier parents (66 of 84 samples) and siblings (four of five samples) to patients with biallelic mutations in AGS1-7 and ACP5 did not demonstrate an interferon signature (median IS 0.862, IQR 0.493–1.942) (Figure S5). Ten of the 18 abnormal parental signatures were recorded in individuals heterozygous for a mutation in ADAR1.