Abstract
Autoinflammatory diseases include a large spectrum of monogenic diseases, e.g. familial Mediterranean fever (FMF), as well as complex genetic trait diseases, e.g. adult-onset Still's disease (AOSD). In populations where FMF is common, an increased MEFV mutation rate is found in patients with rheumatic diseases. The aim of this study was to examine MEFV mutations in Japanese patients with AOSD. Genomic DNA was isolated from 49 AOSD patients and 105 healthy controls, and exons 1, 2, 3 and 10 of the MEFV gene genotyped by direct sequencing. MEFV mutation frequencies in AOSD patients were compared with controls. We found no significant difference in overall allele frequencies of MEFV variants between AOSD patients and controls. However, MEFV exon 10 variants (M694I and G632S) were significantly higher in AOSD patients than controls (6·1 versus 0%). In addition, there was no significant difference between MEFV variant carriers and non-carriers with clinical manifestations, but the monocyclic clinical course of the AOSD disease phenotype was observed less frequently in patients without MEFV variants. AOSD patients had significantly higher frequencies of MEFV exon 10 mutations, suggesting that low-frequency variants of MEFV gene may be one of the susceptibility factors of AOSD.
Keywords: adult-onset Still's disease, autoinflammatory disease, MEFV
Introduction
Adult-onset Still's disease (AOSD) is a systemic inflammatory disease characterized by fever, skin rash, and joint pain 1. The pathophysiology of AOSD has remained unclear; however, it is presumed to be an autoinflammatory disease due to the absence of autoantibodies and autoantigen-specific T cells 2. Patients with autoinflammatory diseases, including classical hereditary periodic syndromes, share clinical features (e.g. spiking fever, skin rash, and arthritis) with the major symptoms of AOSD 3. Familial Mediterranean fever (FMF) is a common autoinflammatory disease, characterized by recurrent inflammatory attacks of fever, skin rash, synovitis, and serositis 4. FMF is thought to be caused by gain-of-function mutations in the MEFV gene 5. Although AOSD etiology and pathogenesis are largely unknown, a growing number of studies support the hypothesis that similar to other autoinflammatory diseases, dysregulation of inflammasome activation and the related overproduction of interleukin-1 (IL-1) β plays a pivotal role 6. Accordingly, IL-1 blockade shows efficacy in treating AOSD symptoms in refractory cases 7. Recent advances in sequencing technology are allowing investigators to sequence selected genes to discover low-frequency variants in patients with complex and genetically matched controls 8. Thus, we propose that MEFV mutations/polymorphisms may be one of the genetic factors associated with AOSD. Therefore, in this study we investigated MEFV gene variations in Japanese AOSD patients.
Materials and methods
Patients
Forty eight patients diagnosed with AOSD between 2012 and 2014, and according to the diagnostic criteria of Yamaguchi et al. 9, were enrolled in the study from hospitals affiliated with Nagasaki University. One hundred five healthy individuals were recruited as controls. In the patient group, medical histories and clinical findings were collected by reviewing electronic medical records. Clinical, demographic, and genetic features of the 49 AOSD patients and genetic features of the 105 healthy controls were analyzed. The study protocol was approved by the ethics committees of the Sasebo City Hospital institutional review board (No 2012-A-22). Written informed consent was obtained from each individual for their clinical records to be used in this study.
Mutational analysis
Blood samples (2 ml) were collected from all subjects. Genomic DNA was extracted from whole blood using the Promega Wizard® Genomic DNA Purification Kit (Madison, WI, USA). Mutational analysis was performed by direct DNA sequencing. Polymerase chain reaction (PCR) was performed using forward and reverse primers for each exon of the MEFV gene, as described previously 10. PCR products were purified using ExoSAP-IT (GE Healthcare, Tokyo, Japan) and directly sequenced using specific primers and BigDye Terminator v1·1 (Applied Biosystems, Tokyo, Japan). MEFV genetic analysis was approved by the Ethics Committee of Nagasaki Medical Center (No. 21003, 2009).
Immunoblot analysis
Patient's sera (1·5 μl) were diluted 10-fold with phosphate-buffered saline (PBS). We separated these diluted serum sample plus 5 μl of protein loading buffer under reducing conditions by NuPAGE 3–8% Tris-acetate gel electrophoresis (Invitrogen Carlsbad, CA, USA). Proteins were electrophoretically transferred onto an Invitrogen polyvinylidene fluoride membrane and incubated overnight at 4°C with blocking solution [5% nonfat milk in Tris-buffered saline with 0·05% Tween 20 (TTBS)]. The blocked membrane was incubated with rabbit anti-human cleaved IL-1β polyclonal antibody (MyBioSource, San Diego, CA, USA; 1:200 dilution with 1% nonfat milk in TTBS) for 1 h at room temperature and then washed five times with TTBS buffer for 10 min each time at room temperature with constant shaking. Then, the membrane was incubated with horseradish peroxidase–conjugated second antibody (1:2000 dilution; Santa Cruz Biotechnology) for 1 h at room temperature and washed five times with TTBS buffer for 10 min each time at room temperature with constant shaking. Immunodetection analysis was performed using a ECL Western blotting kit (Amersham, Little Chalfont, UK). Images of the developed film were scanned using LAS-3000 image analyzer (FUJIFILM, Tokyo, Japan).
Statistical analyses
For continuous variables, results were expressed as mean ± standard deviation (SD). For quantitative data, analysis was performed using a Mann–Whitney U rank-sum test to compare two independent groups. For categorical variables, a chi-square test (or Fisher's exact test when appropriate) was used for comparisons. Two-sided P values less than 0·05 were considered statistically significant. Data were analyzed using SPSS software (SPSS Inc., Chicago, IL, USA).
Results
Demographic features
In total, 49 AOSD patients (8 males and 41 females) were included in the study. Table 1 shows the clinical and demographic features of the AOSD patients. The mean age of patients was 51·1 ± 19·4 years (min–max: 19–84, median: 53), mean age of disease onset was 45·9 ± 20·3 years (min–max: 17–83, median: 45).
Table 1.
Demographic and clinical features of adult-onset Still's disease (AOSD) patients with or without MEFV variants
| Demographic/clinical features | MEFV variants (+) (n = 31) | MEFV variants (−) (n = 18) | P-value |
|---|---|---|---|
| Age (years) | 50·4 ± 18·3 | 52·4 ± 21·7 | 0·510 |
| Onset age (years) | 45·0 ± 19·8 | 47·4 ± 21·5 | 0·572 |
| Duration (years) | 5·4 ± 5·6 | 5·0 ± 6·2 | |
| Gender (female/male) | 25/6 | 16/2 | 0·372 |
| Fever (%) | 31 (100) | 18 (100) | |
| Skin rash (%) | 30 (96·8) | 18 (100) | 0·633 |
| Sore throat (%) | 21 (67·7) | 14 (77·8) | 0·453 |
| Arthritis (%) | 26 (83·9) | 12 (66·7) | 0·150 |
| Myalgia (%) | 7 (23·3) | 9 (50·0) | 0·058 |
| Liver dysfunction (%) | 25 (80·6) | 16 (88·9) | 0·372 |
| Disseminated intravascular coagulation (%) | 3 (9·7) | 2 (11·1) | 0·614 |
| Macrophage activation syndrome (%) | 9 (29·0) | 3 (16·7) | 0·270 |
| Treatment | |||
| Steroid treatment (%) | 30 (96·8) | 18 (100) | 0·633 |
| Steroid pulse therapy (%) | 22 (71·0) | 12 (66·7) | 0·753 |
| Immunosuppressant (%) | 19 (61·3) | 11 (61·1) | 0·990 |
| Biologicals (%) | 6 (19·4) | 0 | 0·053 |
Mutational analysis
All individuals were successfully genotyped for the MEFV gene. MEFV variants were identified in 31 AOSD patients (63·3%), and the genotypes were shown in Table 2. Distributions of MEFV variants in patient and control groups are shown in Table 3. There was no statistical difference between AOSD patients and healthy subjects with regards allele frequencies of MEFV exon 1 (E84K), exon 2 (L110P, E148Q, R202Q, and G304R), and exon 3 (P369S and R408Q) variants. However, the carriage rates of exon 10 MEFV variants (M694I and G632S) were significantly higher in AOSD patients than those of healthy subjects (6·1% versus 0%, P = 0·031).
Table 2.
MEFV genotypes of adult-onset Still's disease (AOSD) patients
| MEFV genotypes | AOSD n = 49 (%) |
|---|---|
| M694I/normal | 2 (4·1) |
| G632S/E148Q | 1 (2·0) |
| P369S/R408Q | 3 (6·1) |
| E148Q/P369S | 1 (2·0) |
| E148Q/E148Q/P369S/R408Q | 1 (2·0) |
| L110P/E148Q/E148Q/P369S/R408Q | 1 (2·0) |
| E148Q/normal | 10 (20·4) |
| R202Q/normal | 3 (6·1) |
| E148Q/E148Q | 2 (4·1) |
| L110P/E148Q | 1 (2·0) |
| L110P/E148Q/E148Q | 2 (4·1) |
| L110P/E148Q/R202Q | 1 (2·0) |
| L110P/L110P/E148Q/E148Q | 1 (2·0) |
| E84K/normal | 1 (2·0) |
| E84K/L110P/E148Q | 1 (2·0) |
| Normal | 18 (36·7) |
| Total | 49 |
Table 3.
Allele frequencies and carriage rates of MEFV variants in patients with adult-onset Still's disease (AOSD)
| AOSD patients (n = 49) | Healthy subjects (n = 105) | P-value | ||
|---|---|---|---|---|
| Allele frequencies (%) | ||||
| Exon 10 | M694I | 2 (2·0) | 0 | 0·101 |
| G632S | 1 (1·0) | 0 | 0·318 | |
| Exon 3 | P369S | 6 (6·1) | 13 (6·2) | 0·982 |
| R408Q | 5 (5·1) | 12 (5·7) | 0·827 | |
| Exon 2 | L110P | 8 (8·2) | 15 (7·1) | 0·751 |
| E148Q | 29 (29·6) | 52 (24·8) | 0·370 | |
| R202Q | 4 (4·1) | 6 (2·9) | 0·399 | |
| G304R | 0 | 6 (2·9) | 0·098 | |
| Exon 1 | E84K | 2 (2·0) | 2 (1·0) | 0·380 |
| Carriage rate (%) | ||||
| Total exon 10 variants | 3 (6·1) | 0 | 0·031 | |
| Total exon 1,2,3 variants | 28 (57·1) | 61 (58·1) | 0·911 | |
| Total MEFV variants | 31 (63·3) | 61 (58·1) | 0·542 |
Clinical features in patients with or without MEFV variants
Clinical features of patients with or without MEFV variants were compared (Table 4). Although there was no statistically significant difference, AOSD patients with MEFV variants were more frequently treated with biologics (tocilizumab) compared with those without MEFV variants (19·4% versus 0%, P = 0·053). No other significant differences were found between patients with and without MEFV variants. However, there was a correlation between MEFV gene variants and AOSD disease phenotype (Table 5). Among clinical progression types 11, the monocyclic systemic phenotype was significantly lower in AOSD patients with MEFV variants. We also examined association of MEFV variants with AOSD treatments.
Table 4.
Demographic and clinical features of adult-onset Still's disease (AOSD) patients with or without MEFV variants
| Demographic/clinical features | MEFV variants (+) (n = 31) | MEFV variants (−) (n = 18) | P-value |
|---|---|---|---|
| Age (years) | 50·4 ± 18·3 | 52·4 ± 21·7 | 0·510 |
| Onset age (years) | 45·0 ± 19·8 | 47·4 ± 21·5 | 0·572 |
| Duration (years) | 5·4 ± 5·6 | 5·0 ± 6·2 | |
| Gender (female/male) | 25/6 | 16/2 | 0·372 |
| Fever (%) | 31 (100) | 18 (100) | |
| Skin rash (%) | 30 (96·8) | 18 (100) | 0·633 |
| Sore throat (%) | 21 (67·7) | 14 (77·8) | 0·453 |
| Arthritis (%) | 26 (83·9) | 12 (66·7) | 0·150 |
| Myalgia (%) | 7 (23·3) | 9 (50·0) | 0·058 |
| Liver dysfunction (%) | 25 (80·6) | 16 (88·9) | 0·372 |
| Disseminated intravascular coagulation (%) | 3 (9·7) | 2 (11·1) | 0·614 |
| Macrophage activation syndrome (%) | 9 (29·0) | 3 (16·7) | 0·270 |
| Treatment | |||
| Steroid treatment (%) | 30 (96·8) | 18 (100) | 0·633 |
| Steroid pulse therapy (%) | 22 (71·0) | 12 (66·7) | 0·753 |
| Immunosuppressant (%) | 19 (61·3) | 11 (61·1) | 0·990 |
| Biologicals (%) | 6 (19·4) | 0 | 0·053 |
Table 5.
Clinical progression type of adult-onset Still's disease (AOSD) patients with or without MEFV variants
| Clinical progression type | Total (n = 49) | MEFV variants (+) (n = 31) | MEFV variants (−) (n = 18) | P-value | (Pc-value) |
|---|---|---|---|---|---|
| Monocyclic systemic (%) | 15 (30·6) | 5 (16·1) | 10 (55·6) | 0·004 | (0·016) |
| Polycyclic systemic (%) | 24 (49·0) | 17 (54·8) | 7 (38·9) | 0·282 | (n.s.) |
| Chronic articular monocyclic systemic (%) | 6 (12·2) | 5 (16·1) | 1 (5·6) | 0·271 | (n.s.) |
| Chronic articular polycyclic systemic (%) | 4 (8·2) | 4 (12·9) | 0 | 0·149 | (n.s.) |
Pc= corrected P-value; n.s. = not significant.
Clinical features of AOSD patients with MEFV exon 10 mutations
Among 49 patients with AOSD, 3 patients had exon10 mutations of MEFV gene. They had episodes of high fever, sore throat and arthritis. Two of them had fever-associated skin eruption colored salmon pink, and all of them were diagnosed with AOSD. In addition to oral prednisolone (12·5–40 mg/day), they were treated with methylprednisolone pulse therapy (case1), methylprednisolone pulse therapy and methotrexate (case2), and methotrexate and IL-6 inhibitor (tocilizumab) (case 3) respectively. Eventually all patients achieved remission. They were not complicated with disseminated intravascular coagulation (DIC) or macrophage activation syndrome (MAS). We previously demonstrated that the cleaved form of IL-1β, p17, was detectable in the sera from FMF patients with MEFV mutations 12. We examined the circulating cleaved IL-1β in AOSD patients with MEFV mutations. As shown in Fig. 1, serum cleaved IL-1β bands were detectable in all AOSD patients with MEFV exon 10 mutations and in one of three patients with MEFV exon 1, 2 mutations during the febrile periods.
Fig 1.
Cleaved interleukin (IL)-1β (p17) in sera from adult-onset Still's disease (AOSD) patients with MEFV exon 10 variants or exon 1, 2 variants. Sera from AOSD patients with MEFV exon 10 variants (n = 3) or exon 1, 2 variants (n = 3) were analysed by anti-cleaved IL-1β immunoblot analysis. The concentrations of C-reactive protein (CRP) were shown in the lower panel of each sample. Additional brood bands observed under reducing conditions are light chains generated from cleavage of thiol-disulphide bridge of immunoglobulins.
Discussion
AOSD etiopathogenesis has not been fully elucidated but genetic and environmental factors may play critical roles 13. Because of similarities in clinical findings between FMF and AOSD, MEFV gene mutations may be factors in AOSD etiology. The MEFV gene encodes a protein named pyrin, which is expressed in neutrophils and monocytes 14,15. Consequently, MEFV gene mutations may alter pyrin function, leading to so-called ‘autoinflammation’ 16. Among hereditary autoinflammatory diseases, FMF is most frequently observed. Although FMF is rare in Japan, MEFV mutation frequencies are relatively high 17. In this study, we show that Japanese AOSD patients have a significantly higher frequency of disease-causing MEFV mutations, especially exon 10 mutations, compared with healthy subjects. FMF is characterized by recurrent fever, skin rashes, synovitis, and serositis, resembling clinical presentation of AOSD. Although the function of pyrin is still unknown, it appears to inhibit processing of IL-1β to its active form 18. With MEFV mutations, this action of pyrin is deficient, resulting in restricted production of active IL-1β. In this study we found that rare and low-frequency variants in an innate immunity gene, MEFV, were more frequently demonstrated in patients with AOSD compared to healthy subjects. The role of MEFV in human disease is not fully understood, however, current evidence suggests that FMF-associated MEFV variants are gain-of-function mutations leading to increased responsiveness to bacterial products 5. Taken together, innate immune responses to bacterial components could be contributed to the pathogenesis of AOSD.
To date, higher frequencies of MEFV gene mutations have been identified in inflammatory conditions 19. In accordance with our findings, significantly higher prevalence of the MEFV exon 10 variant, M694V, was found in patients with ankylosing spondylitis (AS), while no significant difference in the frequency of E148Q was demonstrated, suggesting a link between MEFV exon 10 rare variants and AS 20,21, a form of non-hereditary arthritis. Our data suggest that MEFV exon 10 variants may be one of the susceptibility factors for a complex disease, AOSD. G632S is a new mutations identified in Iranian Jews with mild phenotype of FMF 22. G632S mutation is placed in a loop at the surface of B30·2 domain of pyrin, and this site is presumed to be functionally important 23,24.
Studies examining MEFV mutations in AOSD patients have previously been published in Korean and Turkish populations 25,26, both populations with high MEFV mutation rates. In the Turkish population, Cosan et al. found that although it was not significantly different, the MEFV gene mutation rate increased in the AOSD group 25. However, a Korean study did not find more frequent MEFV mutations in AOSD patients than the general population. In this study, Kim et al. demonstrated higher incidence of the E148Q mutation in AOSD patients, but it was not different to healthy controls 26. Our results on MEFV exon 2 and exon 3 variants are consistent with these findings. We found overall frequencies of MEFV variants were comparable between healthy subjects and AOSD patients. However, clinical patterns of AOSD between patients with or without MEFV mutations were different. MEFV gene polymorphisms may modify the clinical phenotype or course of inflammatory disorders 27. We found the monocyclic systemic phenotype was significantly less common in AOSD patients with MEFV variants. Additionally, biologics were used more frequently in AOSD patients with MEFV variants. Based on these results, we suggest that being MEFV variants carrier may be related to repeated or sustained inflammatory processes in AOSD. Our data are inconsistent with those of Kim et al., who found no association between MEFV exon 2 variants and AOSD clinical course 26. These discrepancies may be owing to the gene analysis system used: we screened all exon 1, 2, 3, and 10 mutations by direct sequencing.
Our study lacked sufficient statistical power for subgroup analysis of clinical findings. Although In this study, the monocyclic systemic disease phenotype was significantly less in AOSD patients with MEFV variants than those without MEFV variants, the findings should be replicated in a large group of patients.
In conclusion, our study provides preliminary evidence suggesting that FMF-related MEFV variants are implicated with the disease phenotype of AOSD. Specifically, our findings suggest that MEFV exon 10 mutations may be one of the susceptibility factors for AOSD in the Japanese population. Replication studies are required to confirm association between these rare FMF-related MEFV variants with AOSD and its clinical manifestations in a larger group of patients.
Acknowledgments
This work was supported by a Grant-in-Aid for Research on intractable diseases from the Ministry of Health, Labor, and Welfare of Japan.
Disclosures
The authors declare that they have no competing interests.
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