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Mutations in the B30.2 domain of pyrin and the risk of ankylosing spondylitis in the Chinese Han population: A case–control study
Immunology Letters 162 (2014) 49–52
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Mutations in the B30.2 domain of pyrin and the risk of ankylosing spondylitis in the Chinese Han population: A case–control study Chongru He 1 , Jia Li 1 , Weidong Xu ∗ Department of Orthopedics, Changhai Hospital Affiliated to the Second Military Medical University, 168 Changhai Road, Shanghai 200433, People’s Republic of China
a r t i c l e
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Article history: Received 17 December 2013 Received in revised form 18 June 2014 Accepted 7 July 2014 Available online 15 July 2014 Keywords: Ankylosing spondylitis Familial Mediterranean fever MEFV Pyrin Mutation
a b s t r a c t Ankylosing spondylitis (AS) and familial Mediterranean fever (FMF) are a common autoimmune disease and a classic autoinflammatory disease, respectively. Mediterranean fever (MEFV) encodes the pyrin protein and is the causal disease gene in FMF. This protein is an important regulator of innate immunity and may play a key role in the development of AS. To identify the mutations in the B30.2 domain of pyrin and to uncover the relationships between these mutations and AS risk in the Chinese Han population, we extracted genomic DNA from the peripheral blood of 200 AS patients and 200 matched controls and performed polymerase chain reactions (PCRs) and direct sequencing on those samples. Statistical analysis indicated that only Met694Val (rs61752717) in the B30.2 domain of pyrin could affect the risk of AS (P = 0.042; odds ratio [OR] = 5.103; 95% confidence interval [CI] = 1.111–23.437 for the model of Met (M) vs. Val (V), P = 0.040; OR = 5.211; 95% CI = 1.127–24.091 for the model of MM vs. MV + VV). Moreover, M694V is significantly associated with a higher level of erythrocyte sedimentation rate (ESR) and Creactive protein (CRP) in AS patients. Our results are the first to suggest that the M694V allele of the pyrin was associated with AS risk in the Chinese Han population and that this mutation may be associated with the inflammatory response in the development of AS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Ankylosing spondylitis (AS) is a chronic inflammatory disease that may ultimately lead to physical disability [1–4]. After New York criteria and modified New York criteria for AS was established, researchers were seeking a deeper understanding of this disease [5–7]. According to current knowledge, chronic inflammation and pathological ossification typically manifest in AS patients [3]. However, the molecular pathogenesis of this disease remains unclear. Recent insights into the genetic factors of AS have led to the development of disease diagnosis and therapy. The expression of a major histocompatibility complex (MHC) class I gene, human leukocyte antigen B27 (HLA-B27), is considered the major cause of
Abbreviations: AS, ankylosing spondylitis; MHC, major histocompatibility complex; HLA, human leukocyte antigen; TNF, tumor necrosis factor; TAP, ATP-binding cassette transporter; ERAP, endoplasmic reticulum aminopeptidase; IL, interleukin; FMF, familial Mediterranean fever; MEFV, Mediterranean fever; SD, standard deviation; OR, odds ratios; CI, confidence interval; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; NF-B, nuclear factor-B. ∗ Corresponding author. Tel.: +86 021 81873393; fax: +86 021 65492727. E-mail address: [email protected]. 1 These authors contributed equally to this paper. http://dx.doi.org/10.1016/j.imlet.2014.07.002 0165-2478/© 2014 Elsevier B.V. All rights reserved.
this disease [1,2]. Other AS susceptibility genes include HLA-B60, HLA-B61 [8], HLA-DR [9], tumor necrosis factor (TNF)-␣ [10], ATPbinding cassette transporter (TAP)-1/2 [11], endoplasmic reticulum aminopeptidase (ERAP)-1, interleukin (IL)-23R [12], IL-6 [13] and IL-10 [14]. Familial Mediterranean fever (FMF) is a classic autoinflammatory disorder that may ultimately lead to amyloidosis [15,16]. The Jewish, Turkish, Arabian, and Armenian populations are the most susceptible [17–22]. Since 1992, several independent groups identified the Mediterranean fever (MEFV) gene as the causal gene of FMF [23–26]. Pyrin is encoded by MEFV and is considered a key modulator of innate immunity and autoimmune disease [27–29]. Because AS is a systemic autoimmune disease, we aimed to test the hypothesis that pyrin may play an important role in AS development. According to the data from two online databases, the Human gene mutation database [30] and Infevers [31], over 200 mutations in MEFV have been identified in over 600 publications. Approximately 20 high frequency mutations, including E148Q, M680I, M694V, M694I and V726A, were identified in the FMF population [30,31]. Interestingly, many high frequency mutations are located in the B30.2 domain of pyrin. This domain participates in protein interactions and is an indispensable for its normal biochemical function. Therefore, these mutations represent disease-causing
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C. He et al. / Immunology Letters 162 (2014) 49–52
candidates of AS. The relationships between mutations in the B30.2 domain of pyrin and the risk of AS in a Han population were tested in this study.
2. Materials and methods 2.1. Collecting sample AS patients (305) and healthy controls (400), who visited the Department of Orthopedics in our hospital from 2009 to 2012, were enrolled in this experiment. Face-to-face questionnaire surveys were used to collect informed consent and detailed information, including the name, sex, age, weight, family history, disease stage and blood routine parameters. To reduce interference from different genetic backgrounds and some medications, 200 samples with detailed information who met the modified diagnostic criteria of AS, were from the Chinese Han population and did not use the medications for AS in the past three months were chosen from 305 patients for further experiments. Two hundred controls without common orthopedic disorders and autoimmune diseases, including AS, osteoproliferation, rheumatoid arthritis, aseptic necrosis of head of femur, systemic lupus erythematosus, and other related diseases, were carefully selected from 400 persons. Peripheral blood was collected from each person and stored at −20 ◦ C. This study was approved by the committee on ethics of biomedicine research in Changhai Hospital of Second Military Medical University. 2.2. DNA extraction, PCR amplification and DNA sequencing Following the protocol provided with the BloodGen Mini Kit (CWBIO, China), 200 l of peripheral blood was used for DNA extraction. The quality of the DNA extraction was assayed by 1% agarose electrophoresis. Primer Premier (Version 6.0, Premier Biosoft, USA) was used to design the primers (5 CCAACCTCATCTTCTCTGAT-3 , 5 -GTTCCTAACTTACCTCTGCTA-3 ). The following polymerase chain reaction (PCR) program conditions were used: 1 cycle of 94 ◦ C for 10 min, followed by 30 cycles of 94 ◦ C for 30 s, 50 ◦ C for 30 s, and 72 ◦ C for 1 min, and a final step at 72 ◦ C for 10 min. Before DNA sequencing, the PCR product was purified using the DNA purification kit (CWBIO, China) to remove interference segments. The DNA Sequencer 3730xl was used to analyze the target sequence (690 bp, NG007871:g.17967 to 18656). Eight reported MEFV missense variations (M680I [rs28940580; c.2040G>C or c.2040G>A; p.Met680Ile], M694V [rs61752717; c.2080A>G; p.Met694Val], M694I [rs28940578; c.2082G>A; p.Met694Ile], V722M [rs104895201; c.2164G>A; p.Val722Met], V726A [rs28940579; c.2177T>C; p.Val726Ala], I729V [rs140434276; c.2185A>G; p.Ile729Val], N733S [rs146720938; c.2198A>G; p.Asn733Ser] and S749C [rs104895171; c.2246C>G; p.Ser729Cys]) were tested in this study. 2.3. Statistical analysis The data are presented as the mean ± standard deviation (SD). The mean comparisons and proportions were based on an unpaired t test and chi-square test, respectively. Yates’ correction was used for tables containing cells where values are less than five individuals. Unconditional logistic regression was used to compute the odds ratio (OR) with 95% confidence intervals (CIs). Moreover, P values were corrected using Bonferroni correction for multiple comparisons. P < 0.05 was considered statistically significant. All statistical analyses were performed using the computer program SPSS software (Version 17.0, San Rafael, USA).
Table 1 Selected characteristics of AS patients. Age (mean ± SD), range Sex Male/female Marital status No/yes Race Clinical scoring system (mean ± SD) BASDAI BASFI HLA-B27 Positive/negative Family history Medicationb NSAID Disease-modifying anti-rheumatic drugs TNF blockers Traditional Chinese medicine Biochemical index (mean ± SD) ESR (mm/h) CRP (mg/L) Age of onset (years) <30, 30–40, >40 Disease duration (years) <10, 10–30, >30 Clinical symptoms Articular pain Hip joint involvement Pulmonitis Dorsal kyphosis Colonitis Oral aphthous ulcers Iridic inflammation Tympanitis
33.95 ± 10.55,a 19–76 81%/19% 33%/67% Han 4.68 ± 2.92 3.39 ± 2.19 94%/6% 10% 98% 40% 10% 60% 55.05 ± 39.10c 41.12 ± 52.57d 74%, 24%, 2% 79%, 18%, 3% 80% 35% 25% 20% 18% 15% 12% 3%
BASDAI: Bath ankylosing spondylitis disease activity index; BASFI: Bath ankylosing spondylitis functional index; NSAID: non-steroidal anti-inflammatory drugs; TNF: tumor necrosis factor; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein. a The average age of the controls was 32.02 ± 11.20, and the range was from 20 to 68. t-Test demonstrated P > 0.05 in the age difference between patients and controls. b From our investigation, many patients used more than one type of drug. c Recommended value of ESR was less than 20 mm/h. d Recommended value of CRP was less than 10 mg/L.
3. Results 3.1. Characteristics of study subjects The distributions of selected characteristics among subjects are summarized in Table 1. Our analysis included 200 cases and 200 controls. There were no significant differences in the age, sex ratio and marital status between the patients and controls. 3.2. Relationships between common mutations and AS risk We searched the sequencing results for 8 missense variations (M680I, M694V, M694I, V722M, V726A, I729V, N733S and S749C). In total, we identified 20 mutations in 17 AS patients at M680I, M694V, I729V and S749C sites (Tables 2 and 3). However, the mutations M694I, V722M, V726A and N733S were not observed in either the AS patients or the controls. Moreover, all of the samples lacked homozygous mutations. The relationships between the four mutations (M680I, M694V, I729V and S749C) in the B30.2 domain of pyrin and AS risk are represented in Table 2. M694V (rs61752717) was the most frequently mutated site and was identified in ten AS patients. The frequency of the minor G allele of M694V (A2120G) in AS patients (2.5%) was significantly higher than in the controls (0.5%). In the model of the major allele (A) vs. the minor allele (G), P = 0.042 (OR, 95% CI: 5.103, 1.111–23.437). In the model of the homozygous frequent allele vs. homozygous rare allele + heterozygous allele, P = 0.040 (OR, 95% CI: 5.211,
C. He et al. / Immunology Letters 162 (2014) 49–52 Table 2 Distribution of MEFV variants in patients and controls. Variants M680I M/I MM MI II M694V M/V MM MV VV I729V I/V II IV VV S749C S/C SS SC CC
AS patients n = 200
Controls n = 200
P value
398/2 198 2 0
399/1 199 1 0
NSa NSb
390/10 190 10 0
398/2 198 2 0
0.042a 0.040b
395/5 195 5 0
394/6 194 6 0
NSa NSb
397/3 197 3 0
399/1 199 1 0
NSa NSb
OR (95% CI)
5.103 (1.111, 23.437) 5.211 (1.127, 24.091)
OR: odds ratio; 95% CI: 95% confidence interval; NS: not significant. a Wild-type allele vs. minor allele. b Homozygous frequent allele vs. homozygous rare + heterozygous.
1.127–24.091). Only the M694V mutation displayed a significant association with AS risk. 3.3. Association between MEFV M694V and AS manifestations The analysis of the relationships between the MEFV mutations and AS manifestation is presented in Table 3. No significant results were observed for the age of onset and clinical scoring systems. Only the expression levels of ESR and CRP in AS patients with the M694V allele were significantly higher than those without any mutation or without the M694V mutation. The relationships between different medication history and the ESR and CRP levels of AS patients were evaluated in supplement. 4. Discussion AS and FMF are autoimmune and autoinflammatory diseases, respectively. They both elicit sacroiliitis in affected individuals [32,33]. However, whether they share the same molecular pathogenesis remains unclear. Recent studies suggested that MEFV was a key modulator of innate immunity [15,19,22,28]. Some experiments tried to uncover the relationship between MEFV and AS. Duman et al. described a patient with AS and FMF who harbored the MEFV M680I mutation [34]. Durmus et al.’s study suggested that the frequency of the MEFV mutation in AS patients was not
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significantly higher than that in healthy controls. However, the presence of the MEFV mutation seemed to aggravate the disease course of AS [35]. A separate analysis at each mutation site revealed that M694V was significantly associated with AS risk [36]. From 2010 to 2012, three independent groups demonstrated that MEFV mutations positively associated with the pathogenesis of AS [37–39]. In 2006, Han et al. detected 12 mutations in MEFV from 57 samples, 54% of which harbored at least one MEFV mutation. Contrary to the low disease incidence of FMF in the Chinese Han population, the mutation frequency of MEFV was considerably high [40]. Based on the evidence mentioned above, we hypothesized that mutations in FMF-related MEFV were associated with AS in the Chinese Han population. To prove this hypothesis, we performed a case–control study to discuss the relationships between common mutations in the B30.2 domain of pyrin and the risk of AS in the Han population. By sequencing PCR products from 400 blood samples, four mutations in the B30.2 domain of MEFV were identified. Association analysis suggested that M694V (rs61752717) was a risk factor for AS development. Additionally, M694V significantly correlated higher levels of ESR and CRP (Table 3). Because ESR and CRP are important inflammatory markers that are indicative of immune system activity, the M694V mutation may play a key role in the inflammatory response during AS development. Mutations in the B30.2 domain, which is located at the C terminus of pyrin, may affect protein stability, pyrin’s three-dimensional structure and its interactions [41,42]. Pyrin can directly interact with pro-caspase-1 and inhibitors of nuclear factor-B (NF-B) complexes, which are core inflammatory proteins [43–45]. Mutations in the C terminus of pyrin could affect its binding with a pro-apoptotic protein Siva. This protein interaction disturbance may lead to cell apoptosis and inflammation [46]. According to docking data, M680I and M694V are the binding sites between pyrin and caspase-1. In normal conditions, pyrin can bind to caspase-1 and limit its activity; therefore, the M694V mutation may suppress these protein interactions and enhance the capacity of caspase-1 to catalyze the 31-kDa IL-1  precursor to the 17-kDa activator. The latter is a main up-regulator of fever and inflammation [47]. This result may explain why the M694V mutation significantly correlated with higher levels of CRP and ESR in AS patients in this study. The M694V (rs61752717) mutation in the B30.2 domain of pyrin affected the risk of AS in the Chinese Han population. Moreover, M694V significantly correlated with higher levels of ESR and CRP. These results suggested that the M694V allele may play an important role in inflammatory processes of AS in the Chinese Han population. Conflict of interest The authors declare that they have no conflict of interest.
Table 3 Association between MEFV variations and the clinical features of AS.
The numbers of patients Age of onset, mean ± SD BASDAI, mean ± SD BASFI, mean ± SD ESR (mm/h) CRP (mg/L)
Without mutation
Without M694V mutation
With M694V mutation
183
190
10
21.87 ± 5.47
21.66 ± 4.67
19.24 ± 5.76
4.74 ± 0.76 3.41 ± 1.03 55.51 ± 37.32 41.21 ± 47.43
4.71 ± 1.34 3.4 ± 0.99 53.74 ± 39.57 39.6 ± 52.91
4.10 ± 1.25 3.20 ± 1.20 80.02 ± 30.10a 70.03 ± 40.10a
a Comparison with patients without any mutation or without the M694V mutation, where significantly different (P < 0.05, with Bonferroni correction) ESR and CRP levels were observed in patients with the M694V mutation.
Acknowledgments This work was supported by a grant from the Natural Science Foundation of Shanghai (no. 10JC1417900). References [1] Brewerton DA, Hart FD, Nicholls A, Caffrey M, James DC, Sturrock RD. Ankylosing spondylitis and HL-A 27. Lancet 1973;1:904–7. [2] Schlosstein L, Terasaki PI, Bluestone R, Pearson CM. High association of an HL-A antigen, W27, with ankylosing spondylitis. N Engl J Med 1973;288:704–6. [3] Braun J, Sieper J. Ankylosing spondylitis. Lancet 2007;369:1379–90. [4] Joshi AB, Markovic L, Hardinge K, Murphy JC. Total hip arthroplasty in ankylosing spondylitis: an analysis of 181 hips. J Arthroplast 2002;17:427–33.
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Mutations in the B30.2 domain of pyrin and the risk of ankylosing spondylitis in the Chinese Han population: A case–control study Chongru He 1 , Jia Li 1 , Weidong Xu ∗ Department of Orthopedics, Changhai Hospital Affiliated to the Second Military Medical University, 168 Changhai Road, Shanghai 200433, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 17 December 2013 Received in revised form 18 June 2014 Accepted 7 July 2014 Available online 15 July 2014 Keywords: Ankylosing spondylitis Familial Mediterranean fever MEFV Pyrin Mutation
a b s t r a c t Ankylosing spondylitis (AS) and familial Mediterranean fever (FMF) are a common autoimmune disease and a classic autoinflammatory disease, respectively. Mediterranean fever (MEFV) encodes the pyrin protein and is the causal disease gene in FMF. This protein is an important regulator of innate immunity and may play a key role in the development of AS. To identify the mutations in the B30.2 domain of pyrin and to uncover the relationships between these mutations and AS risk in the Chinese Han population, we extracted genomic DNA from the peripheral blood of 200 AS patients and 200 matched controls and performed polymerase chain reactions (PCRs) and direct sequencing on those samples. Statistical analysis indicated that only Met694Val (rs61752717) in the B30.2 domain of pyrin could affect the risk of AS (P = 0.042; odds ratio [OR] = 5.103; 95% confidence interval [CI] = 1.111–23.437 for the model of Met (M) vs. Val (V), P = 0.040; OR = 5.211; 95% CI = 1.127–24.091 for the model of MM vs. MV + VV). Moreover, M694V is significantly associated with a higher level of erythrocyte sedimentation rate (ESR) and Creactive protein (CRP) in AS patients. Our results are the first to suggest that the M694V allele of the pyrin was associated with AS risk in the Chinese Han population and that this mutation may be associated with the inflammatory response in the development of AS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Ankylosing spondylitis (AS) is a chronic inflammatory disease that may ultimately lead to physical disability [1–4]. After New York criteria and modified New York criteria for AS was established, researchers were seeking a deeper understanding of this disease [5–7]. According to current knowledge, chronic inflammation and pathological ossification typically manifest in AS patients [3]. However, the molecular pathogenesis of this disease remains unclear. Recent insights into the genetic factors of AS have led to the development of disease diagnosis and therapy. The expression of a major histocompatibility complex (MHC) class I gene, human leukocyte antigen B27 (HLA-B27), is considered the major cause of
Abbreviations: AS, ankylosing spondylitis; MHC, major histocompatibility complex; HLA, human leukocyte antigen; TNF, tumor necrosis factor; TAP, ATP-binding cassette transporter; ERAP, endoplasmic reticulum aminopeptidase; IL, interleukin; FMF, familial Mediterranean fever; MEFV, Mediterranean fever; SD, standard deviation; OR, odds ratios; CI, confidence interval; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; NF-B, nuclear factor-B. ∗ Corresponding author. Tel.: +86 021 81873393; fax: +86 021 65492727. E-mail address: [email protected]. 1 These authors contributed equally to this paper. http://dx.doi.org/10.1016/j.imlet.2014.07.002 0165-2478/© 2014 Elsevier B.V. All rights reserved.
this disease [1,2]. Other AS susceptibility genes include HLA-B60, HLA-B61 [8], HLA-DR [9], tumor necrosis factor (TNF)-␣ [10], ATPbinding cassette transporter (TAP)-1/2 [11], endoplasmic reticulum aminopeptidase (ERAP)-1, interleukin (IL)-23R [12], IL-6 [13] and IL-10 [14]. Familial Mediterranean fever (FMF) is a classic autoinflammatory disorder that may ultimately lead to amyloidosis [15,16]. The Jewish, Turkish, Arabian, and Armenian populations are the most susceptible [17–22]. Since 1992, several independent groups identified the Mediterranean fever (MEFV) gene as the causal gene of FMF [23–26]. Pyrin is encoded by MEFV and is considered a key modulator of innate immunity and autoimmune disease [27–29]. Because AS is a systemic autoimmune disease, we aimed to test the hypothesis that pyrin may play an important role in AS development. According to the data from two online databases, the Human gene mutation database [30] and Infevers [31], over 200 mutations in MEFV have been identified in over 600 publications. Approximately 20 high frequency mutations, including E148Q, M680I, M694V, M694I and V726A, were identified in the FMF population [30,31]. Interestingly, many high frequency mutations are located in the B30.2 domain of pyrin. This domain participates in protein interactions and is an indispensable for its normal biochemical function. Therefore, these mutations represent disease-causing
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candidates of AS. The relationships between mutations in the B30.2 domain of pyrin and the risk of AS in a Han population were tested in this study.
2. Materials and methods 2.1. Collecting sample AS patients (305) and healthy controls (400), who visited the Department of Orthopedics in our hospital from 2009 to 2012, were enrolled in this experiment. Face-to-face questionnaire surveys were used to collect informed consent and detailed information, including the name, sex, age, weight, family history, disease stage and blood routine parameters. To reduce interference from different genetic backgrounds and some medications, 200 samples with detailed information who met the modified diagnostic criteria of AS, were from the Chinese Han population and did not use the medications for AS in the past three months were chosen from 305 patients for further experiments. Two hundred controls without common orthopedic disorders and autoimmune diseases, including AS, osteoproliferation, rheumatoid arthritis, aseptic necrosis of head of femur, systemic lupus erythematosus, and other related diseases, were carefully selected from 400 persons. Peripheral blood was collected from each person and stored at −20 ◦ C. This study was approved by the committee on ethics of biomedicine research in Changhai Hospital of Second Military Medical University. 2.2. DNA extraction, PCR amplification and DNA sequencing Following the protocol provided with the BloodGen Mini Kit (CWBIO, China), 200 l of peripheral blood was used for DNA extraction. The quality of the DNA extraction was assayed by 1% agarose electrophoresis. Primer Premier (Version 6.0, Premier Biosoft, USA) was used to design the primers (5 CCAACCTCATCTTCTCTGAT-3 , 5 -GTTCCTAACTTACCTCTGCTA-3 ). The following polymerase chain reaction (PCR) program conditions were used: 1 cycle of 94 ◦ C for 10 min, followed by 30 cycles of 94 ◦ C for 30 s, 50 ◦ C for 30 s, and 72 ◦ C for 1 min, and a final step at 72 ◦ C for 10 min. Before DNA sequencing, the PCR product was purified using the DNA purification kit (CWBIO, China) to remove interference segments. The DNA Sequencer 3730xl was used to analyze the target sequence (690 bp, NG007871:g.17967 to 18656). Eight reported MEFV missense variations (M680I [rs28940580; c.2040G>C or c.2040G>A; p.Met680Ile], M694V [rs61752717; c.2080A>G; p.Met694Val], M694I [rs28940578; c.2082G>A; p.Met694Ile], V722M [rs104895201; c.2164G>A; p.Val722Met], V726A [rs28940579; c.2177T>C; p.Val726Ala], I729V [rs140434276; c.2185A>G; p.Ile729Val], N733S [rs146720938; c.2198A>G; p.Asn733Ser] and S749C [rs104895171; c.2246C>G; p.Ser729Cys]) were tested in this study. 2.3. Statistical analysis The data are presented as the mean ± standard deviation (SD). The mean comparisons and proportions were based on an unpaired t test and chi-square test, respectively. Yates’ correction was used for tables containing cells where values are less than five individuals. Unconditional logistic regression was used to compute the odds ratio (OR) with 95% confidence intervals (CIs). Moreover, P values were corrected using Bonferroni correction for multiple comparisons. P < 0.05 was considered statistically significant. All statistical analyses were performed using the computer program SPSS software (Version 17.0, San Rafael, USA).
Table 1 Selected characteristics of AS patients. Age (mean ± SD), range Sex Male/female Marital status No/yes Race Clinical scoring system (mean ± SD) BASDAI BASFI HLA-B27 Positive/negative Family history Medicationb NSAID Disease-modifying anti-rheumatic drugs TNF blockers Traditional Chinese medicine Biochemical index (mean ± SD) ESR (mm/h) CRP (mg/L) Age of onset (years) <30, 30–40, >40 Disease duration (years) <10, 10–30, >30 Clinical symptoms Articular pain Hip joint involvement Pulmonitis Dorsal kyphosis Colonitis Oral aphthous ulcers Iridic inflammation Tympanitis
33.95 ± 10.55,a 19–76 81%/19% 33%/67% Han 4.68 ± 2.92 3.39 ± 2.19 94%/6% 10% 98% 40% 10% 60% 55.05 ± 39.10c 41.12 ± 52.57d 74%, 24%, 2% 79%, 18%, 3% 80% 35% 25% 20% 18% 15% 12% 3%
BASDAI: Bath ankylosing spondylitis disease activity index; BASFI: Bath ankylosing spondylitis functional index; NSAID: non-steroidal anti-inflammatory drugs; TNF: tumor necrosis factor; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein. a The average age of the controls was 32.02 ± 11.20, and the range was from 20 to 68. t-Test demonstrated P > 0.05 in the age difference between patients and controls. b From our investigation, many patients used more than one type of drug. c Recommended value of ESR was less than 20 mm/h. d Recommended value of CRP was less than 10 mg/L.
3. Results 3.1. Characteristics of study subjects The distributions of selected characteristics among subjects are summarized in Table 1. Our analysis included 200 cases and 200 controls. There were no significant differences in the age, sex ratio and marital status between the patients and controls. 3.2. Relationships between common mutations and AS risk We searched the sequencing results for 8 missense variations (M680I, M694V, M694I, V722M, V726A, I729V, N733S and S749C). In total, we identified 20 mutations in 17 AS patients at M680I, M694V, I729V and S749C sites (Tables 2 and 3). However, the mutations M694I, V722M, V726A and N733S were not observed in either the AS patients or the controls. Moreover, all of the samples lacked homozygous mutations. The relationships between the four mutations (M680I, M694V, I729V and S749C) in the B30.2 domain of pyrin and AS risk are represented in Table 2. M694V (rs61752717) was the most frequently mutated site and was identified in ten AS patients. The frequency of the minor G allele of M694V (A2120G) in AS patients (2.5%) was significantly higher than in the controls (0.5%). In the model of the major allele (A) vs. the minor allele (G), P = 0.042 (OR, 95% CI: 5.103, 1.111–23.437). In the model of the homozygous frequent allele vs. homozygous rare allele + heterozygous allele, P = 0.040 (OR, 95% CI: 5.211,
C. He et al. / Immunology Letters 162 (2014) 49–52 Table 2 Distribution of MEFV variants in patients and controls. Variants M680I M/I MM MI II M694V M/V MM MV VV I729V I/V II IV VV S749C S/C SS SC CC
AS patients n = 200
Controls n = 200
P value
398/2 198 2 0
399/1 199 1 0
NSa NSb
390/10 190 10 0
398/2 198 2 0
0.042a 0.040b
395/5 195 5 0
394/6 194 6 0
NSa NSb
397/3 197 3 0
399/1 199 1 0
NSa NSb
OR (95% CI)
5.103 (1.111, 23.437) 5.211 (1.127, 24.091)
OR: odds ratio; 95% CI: 95% confidence interval; NS: not significant. a Wild-type allele vs. minor allele. b Homozygous frequent allele vs. homozygous rare + heterozygous.
1.127–24.091). Only the M694V mutation displayed a significant association with AS risk. 3.3. Association between MEFV M694V and AS manifestations The analysis of the relationships between the MEFV mutations and AS manifestation is presented in Table 3. No significant results were observed for the age of onset and clinical scoring systems. Only the expression levels of ESR and CRP in AS patients with the M694V allele were significantly higher than those without any mutation or without the M694V mutation. The relationships between different medication history and the ESR and CRP levels of AS patients were evaluated in supplement. 4. Discussion AS and FMF are autoimmune and autoinflammatory diseases, respectively. They both elicit sacroiliitis in affected individuals [32,33]. However, whether they share the same molecular pathogenesis remains unclear. Recent studies suggested that MEFV was a key modulator of innate immunity [15,19,22,28]. Some experiments tried to uncover the relationship between MEFV and AS. Duman et al. described a patient with AS and FMF who harbored the MEFV M680I mutation [34]. Durmus et al.’s study suggested that the frequency of the MEFV mutation in AS patients was not
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significantly higher than that in healthy controls. However, the presence of the MEFV mutation seemed to aggravate the disease course of AS [35]. A separate analysis at each mutation site revealed that M694V was significantly associated with AS risk [36]. From 2010 to 2012, three independent groups demonstrated that MEFV mutations positively associated with the pathogenesis of AS [37–39]. In 2006, Han et al. detected 12 mutations in MEFV from 57 samples, 54% of which harbored at least one MEFV mutation. Contrary to the low disease incidence of FMF in the Chinese Han population, the mutation frequency of MEFV was considerably high [40]. Based on the evidence mentioned above, we hypothesized that mutations in FMF-related MEFV were associated with AS in the Chinese Han population. To prove this hypothesis, we performed a case–control study to discuss the relationships between common mutations in the B30.2 domain of pyrin and the risk of AS in the Han population. By sequencing PCR products from 400 blood samples, four mutations in the B30.2 domain of MEFV were identified. Association analysis suggested that M694V (rs61752717) was a risk factor for AS development. Additionally, M694V significantly correlated higher levels of ESR and CRP (Table 3). Because ESR and CRP are important inflammatory markers that are indicative of immune system activity, the M694V mutation may play a key role in the inflammatory response during AS development. Mutations in the B30.2 domain, which is located at the C terminus of pyrin, may affect protein stability, pyrin’s three-dimensional structure and its interactions [41,42]. Pyrin can directly interact with pro-caspase-1 and inhibitors of nuclear factor-B (NF-B) complexes, which are core inflammatory proteins [43–45]. Mutations in the C terminus of pyrin could affect its binding with a pro-apoptotic protein Siva. This protein interaction disturbance may lead to cell apoptosis and inflammation [46]. According to docking data, M680I and M694V are the binding sites between pyrin and caspase-1. In normal conditions, pyrin can bind to caspase-1 and limit its activity; therefore, the M694V mutation may suppress these protein interactions and enhance the capacity of caspase-1 to catalyze the 31-kDa IL-1  precursor to the 17-kDa activator. The latter is a main up-regulator of fever and inflammation [47]. This result may explain why the M694V mutation significantly correlated with higher levels of CRP and ESR in AS patients in this study. The M694V (rs61752717) mutation in the B30.2 domain of pyrin affected the risk of AS in the Chinese Han population. Moreover, M694V significantly correlated with higher levels of ESR and CRP. These results suggested that the M694V allele may play an important role in inflammatory processes of AS in the Chinese Han population. Conflict of interest The authors declare that they have no conflict of interest.
Table 3 Association between MEFV variations and the clinical features of AS.
The numbers of patients Age of onset, mean ± SD BASDAI, mean ± SD BASFI, mean ± SD ESR (mm/h) CRP (mg/L)
Without mutation
Without M694V mutation
With M694V mutation
183
190
10
21.87 ± 5.47
21.66 ± 4.67
19.24 ± 5.76
4.74 ± 0.76 3.41 ± 1.03 55.51 ± 37.32 41.21 ± 47.43
4.71 ± 1.34 3.4 ± 0.99 53.74 ± 39.57 39.6 ± 52.91
4.10 ± 1.25 3.20 ± 1.20 80.02 ± 30.10a 70.03 ± 40.10a
a Comparison with patients without any mutation or without the M694V mutation, where significantly different (P < 0.05, with Bonferroni correction) ESR and CRP levels were observed in patients with the M694V mutation.
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