Type I interferon (IFN) plays an important role in the immune defence against viral infections [1]. Excessive activation of type I IFN can lead to disturbances in immune function. Type I interferonopathies are autoinflammatory and autoimmune disorders [1, 2] characterised by the upregulation of type I IFN. Notwithstanding type I IFN upregulation, each disease in the category of type I interferonopathies has its own clinical manifestations. The main types of diseases and their causative genes are shown in Table 1 [1, 3].

Table 1 The main types of type I interferonopathies and their causative genes

Three prime repair exonuclease 1 (TREX1) plays an important role in DNA damage repair [1, 4]. A growing number of studies have shown that TREX1 mutations are associated with multiple type I interferonopathies [1, 5]. Aicardi-Goutières syndrome (AGS) is a subgroup of type I interferonopathies [1, 3, 6]. Familial chilblain lupus (FCL) is a subtype of AGS characterised by papular skin lesions, which occur as a result of the cold [1]. FCL affects the tip of the finger or toe and the folds of the nail, and there is usually no associated neurological condition [1, 7]. The pathogenic genes of FCL include TREX1, SAMHD1 and STING. Tofacitinib, a Janus kinase (JAK) inhibitor, has been described to improve type I interferonopathies by suppressing the overactivated JAK / signal transducing activator of transcription (STAT) pathway.

In this article, we describe the clinical, pathological and genetic characteristics of two cases with complex heterozygous variants in the TREX1 gene. These two variants have not been reported in previous studies and have not been reported as a polymorphic change in public databases; the frequency of the variants in the population is extremely low. Tofacitinib treatment led to an improvement in the patient.

Materials and methods


A 2-year-old female was admitted to our outpatient clinic in the Department of Dermatology due to painful bluish-red papular lesions of the skin in acral locations. The erythematous infiltrates first occurred when she was 8 months old, affecting mainly the interphalangeal joints of her fingers, toes and ears. Ulcerations were commonly noted in distal portions of her toes and fingers, causing nail changes. Scars were left without blisters or necrotic areas (Figs. 1a and 2a). Erythematous to violaceous indurated infiltrative plaques appeared on the face (Fig. 3a).

Fig. 1
figure 1

Clinical manifestations on the foot of the proband. a Chilblain lesions on the toes and the right dorsal feet. b After treatment, the skin lesions were controlled

Fig. 2
figure 2

Clinical manifestations on the hands of the proband. a Chilblain lesions on the fingers. b After treatment, the skin lesions were controlled

Fig. 3
figure 3

Clinical manifestations on the face of the proband. a Chilblain lesions on the face. b After treatment, the skin lesions were controlled

She is the 3rd child of a healthy non-consanguineous couple with no family history of haemophilia or skin diseases (Fig. 4a). Her eldest brother was in good health, while her elder brother presented similar skin lesions, albeit much more severe, at the same age, with necrotic destruction leading to the falling off of his fingers. In addition to the skin disorder, he also had cerebral-palsy-like abnormality in motor development and posture.

Fig. 4
figure 4

Pedigree of the family and Sanger sequencing results and identified mutations. a Pedigree of the family with FCL. The arrow indicates the proband, and the asterisks indicate family members who had genetic testing in this study. b, c Sanger sequencing results and identified mutations in pedigree. (b: TREX1 NM_016381; c.C227T; p.Ala76Val. c: TREX1 NM_016381; c.458dupA; p.Gln153fs3)

A skin biopsy taken from the borders of an erythematous lesion upon her right foot showed significant hyperkeratosis and acanthosis. There was lymphocyte-dominated inflammatory infiltration at the dermal junction and around the dermal vessels and adnexa, and neovascularisation in the dermal papilla (Fig. 5).

Fig. 5
figure 5

Histopathological findings of a skin lesion of the proband. Histopathological examination showed significant hyperkeratosis and acanthosis. There were lymphocyte-dominated inflammatory infiltration at the dermal junction and around the dermal vessels and adnexa, and neovascularisation in the dermal papilla (H&E, × 100)

Participants and patients in this study have given written informed consent to be in the study and to allow the publication of their case details. The ethics review board of the institution approved the study.

DNA sample preparation

Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany), according to the standard protocol, and quantified using a NanoDROP 2000 spectrophotometer (Thermo Scientific; Waltham, MA, USA).

Mutational analysis of the TREX1 gene

Whole exome sequencing (WES) was conducted in the proband by the Novogene company on the Illumina NovaSeq platform, with an average sequencing depth of 100X. Raw sequence results were aligned to the human reference genome (GRCh37/hg19) and annotated to obtain the candidate variants. Then the candidate variants were validated using Sanger sequencing to confirm the WES results. Primers were designed using Primer3web, version 4.1.0 for the suspected disease-causing genes. Variants were interpreted using VarCards ( and PolyPhen2 ( And we also used SWISS-MODEL ( to predict the effect of the two mutations on protein structure.

Measuring the expression levels of IFN-stimulated genes in the patient

Real-time quantitative PCR was performed to measure the expression levels of five IFN-stimulated genes (ISGs) (IFI27, IFI44L, IFIT1, ISG15, RSAD2) in the patient after treatment. The results were compared with the expression levels of these genes in normal controls.


By analysing the WES results, we found two variants of the TREX1 gene. One was a missense variant (NM_016381; c.C227T; p.Ala76Vla), and the other was a frameshift variant (NM_016381; c.458dupA; p.Gln153Glnfs*3). The two variants were verified using Sanger sequencing (Fig. 4b, c). To verify whether the two variants were de novo or not, the patients’ parents were tested using Sanger sequencing. As shown in Fig. 4b, the father of the proband was a heterozygote of the variant c.C227T, and the mother was a heterozygote of the variant c.458dupA (Fig. 4c). Both mutations were novel. The proband’s affected brother carried the same variants as the proband, so the inheritance was in accordance with an autosomal recessive pattern. The damaging score of the missense mutation was 0.83 (damaging score of loss-of-function variant is deemed to be 1) by using VarCards and this mutation was predicted to be probably damaging with a score of 1.000 by using PolyPhen2. According to the SWISS-MODEL prediction, the overall structure of the protein with either missense mutation or frameshift mutation of TREX1 had undergone tremendous changes (Fig. 6), which indicated that these two mutations would affect the structure and function of the protein. Real-time quantitative PCR revealed significantly elevated expression levels of five ISGs (IFI27, IFI44L, IFIT1, ISG15, RSAD2) in the patient than in the normal controls (Fig. 7). Accordingly, the patient and her affected brother were diagnosed with FCL.

Fig. 6
figure 6

Effects of the mutations in TREX1 in the patient were simulated by means of SWISS-MODEL. a Predicted normal TREX1 protein structure. b Predicted TREX1 protein structure with a specific missense mutation. c Predicted TREX1 protein structure with a specific frameshift mutation

Fig. 7
figure 7

Targeted IFN-stimulated gene RNA expression in total blood in patient compared with normal control. The ratio of the gene of interest to GAPDH in the normal control was set to 1. Data shown were mean ± SD of 3 three experiments, and each experiment was performed in duplicate. P: Patient, NC: normal control

After the diagnosis was made, the proband was given tofacitinib 2.5 mg bid, which led to marked improvement (Figs. 1b, 2b and 3b). During the following 2 years, the skin lesions and fever could be controlled with tofacitinib alone during the summer and autumn, but got worse during the winter with arthralgia, which could be controlled with tofacitinib 2.5 mg bid and prednisone 5 mg qd. No mild or severe adverse events were observed. She currently lives a normal kindergarten life.


In this study, we found two unreported mutations in TREX1 in a Chinese family. One mutation was a missense mutation (NM_016381; c.C227T; p.Ala76Val), and the other was a frameshift mutation (NM_016381; c.458dupA; p. Gln153Glnfs*3). Previously, researchers had reported on the mechanism of TREX1 causing type I interferonopathies [8]. Diseases associated with TREX1 are shown in Table 2.

Table 2 Diseases associated with TREX1

The correlation of gene mutations and clinical manifestations of the patients was consistent with the diagnosis of FCL, a subtype of AGS and one of the type I interferonopathies. The proband and her brother carried the same mutations, but her brother had neurological symptoms in addition to the symptoms on the skin. This was also in consistence with the fact that a deficient activity of TREX1 could affect both the nervous system and the skin, which led to corresponding manifestations in AGS patients.

The proband was given tofacitinib to treat her FCL symptoms and showed improvement. This was consistent with recent studies revealing that JAK or reverse transcriptase inhibitors could benefit patients with type I interferonopathies [9,10,11,12,13]. König et al. reported a family with five members affected by FCL. Two of the members were treated with tofacitinib 5 mg twice daily for 17 days. On the 14th day of treatment, the IFN signature was significantly suppressed, and the pain and discomfort of the fingers were relieved [11]. The mechanism of the therapeutic effects of tofacitinib in interferonopathies might be that the tofacitinib inhibits the abnormally activated IFN-α/β receptor, the JAK/STAT pathway, and the subsequent autoinflammation [11, 14]. In the CD4 T cells and CD19 B cells from 2 patients with mutation of stimulator of interferon genes (STING) treated with tofacitinib, the phosphorylation of STAT1, STAT2 and STAT3 was blocked [14]. Tofacitinib opened up a new choice of treatment for one of the affected members of the family in this study.

The limitation of this study is that we did not measure the changes in type I IFN in the patient before the treatment by JAK inhibitor. We sampled the peripheral blood of the patient at his first visit but didn’t extract the RNA immediately so that RNA was all gone after it had been stored for too long. Due to the extremely low concentration of type I interferon in blood, it can’t be detected directly by using conventional ELISA. Currently, the expression levels of five IFN-stimulated genes (ISGs) (IFI27, IFI44L, IFIT1, ISG15, RSAD2) are generally used to assess the activity of type I interferon signaling pathway [15,16,17] by real-time quantitative PCR. Here, to provide the supportive evidence for the pathogenicity of the novel compound heterozygous TREX1 mutations, we measured the differences of five ISGs between the patient after treatment and normal controls (Fig. 7). The expression level of the five ISGs of the patient was higher than that of the normal control. Studies have confirmed that the expression of ISGs in patients with type I interferonopathies would have a significant decline before and after treatment by JAK inhibitor [18]. In our study, as shown in Fig. 7, although we only measured the changes of five ISGs in this patient after treatment, we reasonably speculated that the expression level of ISGs in this patient before treatment was also elevated and had be higher than that after treatment. Therefore, this result can be an indirect line of evidence to support the genetic diagnosis of this patient.

This study expanded the mutation spectrum of the TREX1 gene and provided an accurate genetic diagnosis for the affected members of the family. The accuracy of the diagnosis has greatly helped the dermatologists to make decisions on the patient’s follow-up treatment, especially for the skin symptoms. In clinical practice, the same or similar symptoms can occur in a variety of skin diseases, which greatly adds to the difficulty in making the correct skin disease diagnosis. The emergence of WES has greatly helped physicians in the diagnosis and treatment of skin diseases. WES has proved to be a valuable tool in the diagnosis and research of hereditary skin diseases such as interferonopathies and is worthwhile performing.


In summary, we identified two novel complex heterozygous variants in the TREX1 gene, which may participate in the pathogenesis of the type I interferonopathies observed in patients in this family. Tofacitinib could be an alternative treatment for this disease.