Reactive arthritis (ReA) is a postinfectious, nonseptic arthritis included within the broad spectrum of spondyloarthritis (SpA). The prevalence of ReA following infectious outbreaks estimated by telephonic survey-based follow-up studies is between 6% and 13%.[1,2] Incidence rates within the last two decades have varied between 13 and 90/100,000 patients.[3,4]
The increasingly understood role of inflammasome pathway in the pathogenesis of ankylosing spondylitis (AS) has positioned SpA at the crossroads of autoimmunity and autoinflammation. Upregulation of NLR family pyrin domain containing 3 (NLRP3), NLR family caspase recruitment domain family member (CARD) domain-containing protein 4 (NLRC4), and interferon-inducible protein (AIM2) pathways was demonstrated in the gut of human leukocyte antigen B27 (HLA-B27) transgenic rats, SKG mice as well as gut tissue and peripheral blood mononuclear cells of patients of AS. Genes pertaining to inflammasome pathway such as CARD9, interleukin 1 receptor Type I, Mediterranean fever (MEFV), and immunity-related GTPase family M have been demonstrated to be upregulated in SpA.
The prevalence of AS, defined by the radiographic arm of the assessment of SpA international society criteria, was found to be 7.5% in a cohort of 157 patients of familial Mediterranean fever (FMF). Five sequence alterations (M694V, V726A, M680I, M694I, and E148Q), in the MEFV gene, account for the majority of FMF chromosomes. The wide clinical variability of the disease has been related to MEFV allelic heterogeneity. M694V homozygotes have a severe form of the disease. The frequency of the M694V allele was around four times higher among patients with FMF with sacroiliitis compared to those without. In addition, none of these patients were positive for HLA-B27. Conversely, in a cohort of 193 patients of AS, 43 (22.3%) were positive for heterozygous MEFV variants. Recently, a meta-analysis concluded that the odds of developing AS are three times higher in patients who are heterozygous for M694V. Genome-wide association studies have identified a strong association between a novel coding MEFV variant and AS patients in a cohort of Turkish and Iranian patients. This association was observed in patients with HLA-B27 positive and negative. Patients harboring polymorphisms also had higher interleukin (IL-23) and IL-1 production. In addition to association, the severity of autoimmune disease may also be more in the setting of MEFV mutations.[13,14,15]
FMF is classically described in ethnic groups such as North African Jews, Arabs, Turks, Greeks, and Italians. Musculoskeletal symptoms, reported in 63%–72% of patients, are the second most common type of acute attack in FMF. Acute, recurrent monoarthritis is the most common form of musculoskeletal involvement in FMF. In this regard, the clinical presentation of FMF is similar to ReA, as fever and acute monoarthritis with lower limb predominance, though less common than oligoarthritis, is characteristic of the ReA. Although the prevalence of FMF mutations has not been studied in India, MEFV gene variants and mevalonate kinase mutations have been reported. Considering the wide ethnic diversity in India, the occurrence of these genetic mutations cannot be ruled out. Such associations may carry important therapeutic implications, including the utility of colchicine, an affordable and easily available drug for low-middle income countries. In this context, we conducted a pilot study with the aim to investigate the presence of M694V gene polymorphisms in the MEFV gene in patients with ReA.
Patients with ReA fulfilling the modified Braun’s criteria, or diagnosed as per the consensus of at least two rheumatologists, and visiting the Department of Clinical Immunology and Rheumatology of a Tertiary Care Teaching Hospital between January 2019 and March 2020 with active symptoms, were enrolled in this observational cross-sectional study. Approval from the Institutional Review Board was obtained before recruiting patients (IEC 2018-124-IMP-105). Clinical details and laboratory parameters were recorded on a prespecified form. Patients with age <16 years were classified as juvenile ReA. Sacroiliitis was defined according to the modified New York criteria. FMF was diagnosed in those meeting the Tel Hashomer criteria. A control group comprising patients without arthritis or any medical disease was included for comparison.
Deoxyribonucleic acid isolation and quantification
The ethylenediaminetetraacetic acid (EDTA) blood samples were collected from each patient and stored at −80°C. The frozen EDTA sample was thawed at room temperature for further deoxyribonucleic acid (DNA) isolation. DNA samples were isolated for all samples using the QIAamp® DNA Blood mini kit (Lot 16301933). Both qualitative and quantitative analyses of DNA samples were done using the 1% agarose electrophoresis and NanoDrop.
Mediterranean fever mutation detection using restricted fragment length polymorphism
The isolated DNA samples were amplified at concentration of 200–250 pg/mL with the following steps: 1 (Initial denaturation): 94°C 60s; 2 (Denaturation): 94°C 45s; 3(Annealing): 55°C 30s; 4 (Extension): 72°C 60s for 40 cycles; and 5 (Final extension) 72°C 600 s, at 50 mL final reaction volume, with specific sets of primer against the exon 2 5’-GAATGGCTACTGGGTGGAGAT-3’ and 3’- GCTGTCACATTGTAAAAGGAG-5’. The polymerase chain reaction (PCR) product was run on the 2% agarose gel. The further amplified product was digested with Restriction enzyme Hph I (Haemophilus parahaemolyticus) for the 1 h at 37°C at 25 mL volume as prescribed format. The restricted PCR product was run on 2% agarose gel to see the band size 154 bp (Normal) and 118 bp and 36 bp (Mutant) [Figure 1].
Data are presented as numbers (percentage) for qualitative variables and as median (and interquartile range [IOR]) for quantitative variables. Statistical analysis was done using IBM SPSS 26.0 version (Chicago, Illinois, USA). Qualitative variables were compared by the Fischer’s test and quantitative variables by Mann–Whitney U nonparametric test, with P > 0.05 as the threshold of significance.
Forty-nine patients (male:female – 37:12), including 5 juvenile ReA were included during the study. The median age was 25 (±11) years and disease duration 0.76 (±1.33) months standard deviation. Twenty-six cases were triggered by preceding enteritis and 23 by urethritis. Ten healthy controls of age 27 (male:female – 7:3, IOR ± 1.5) were included for comparison.
Clinical characteristics of the cohort
Arthritis was present in 42 patients (86%), knee in 40 (82%), ankle in 17 (35%), shoulder in 1 (2%), elbow in 3 (6%), wrist in 4 (8%), and metacarpophalangeal joint in 3 (6%). Enthesitis was present in 13 (26%) and dactylitis was present in 6 (12%). Inflammatory back pain was present in 9 (18%) and buttock pain in 8 (16%).
Extra-articular features were present in 12 (24%) patients. History of conjunctivitis was present in 4 patients (8%) and uveitis in 1 patient (2%). Circinate balanitis was present in 4 (8%), oral ulcers in 3 (6%), psoriasiform skin lesions in 2 (4%), and keratoderma blennorrhagica and genital ulcers in 1 patient, each (2%). Fever was reported by 9 patients (18%).
Among those with fever, one patient had psoriasiform skin lesion and conjunctivitis, one had circinate balanitis and uveitis, and one each had circinate balanitis and oral ulcer. Family history of psoriasis and undifferentiated SpA was present in 2 (4%). None of the patients met the classification criteria for FMF as the typical recurrent short-lasting monoarthritis was not noted in any.
The hemoglobin was 12 g/dL (11–13), total leukocyte counts were 11080 cells/mm3 (8925–14775), platelet count was 3 lakhs/mm3 (2.1–5). The erythrocyte sedimentation rate was 50 mm/1st h (27–80) and C-reactive protein 5.5 mg/dL (1–11.25). Radiographic sacroiliitis was present in 7 of 22 (22%) patients.
All 49 patients of adult and juvenile ReA were negative for the M694V mutation, comparable to the healthy controls [Figure 2].
This is the first study in India assessing the prevalence of MEFV mutation in patients with SpA, to specifically explore if ReA, characterized by short-lasting fever and transient arthritis, could represent a forme fruste of FMF. Although monoarthritis was present in 34%, fever which was present in 18% was only associated with oligoarthritis, unlike the classical transient monoarthritis of FMF. Serositis and erythematous rashes were not noted in any patient. M694V mutation, which is the most common allele variant described in FMF, was not detected in any patient of ReA.
None of the patients in our study met the description of “typical” attack of arthritis in FMF where the patient experiences “recurrent (at least 3) short lasting (12 h to few days) episodes.” However, musculoskeletal symptoms not meeting the classical description is described. Chronic arthritis of hip and knee has been reported in up to 5% of patients. Clinical features of SpA such as sacroiliitis and enthesitis, which are not part of classification criteria, are well described in patients with FMF.[9,10,24] Other presentations include myalgia, protracted febrile myalgia, and arthralgia. In a cohort of 136 children with FMF, 4%–17% had miscellaneous musculoskeletal symptoms.
We evaluated for the presence of M694V mutant allele only as this mutation has the highest penetrance among FMF variant alleles, is the most common allelic variant and is most strongly associated with arthritis. Other exon 10 variants of MEFV gene, namely M680I, V726A, E148Q, and M694I allele variants, have been associated with arthritis with the former three significantly being associated with AS.
Our study has few limitations. First, apart from a few case reports, the prevalence of FMF allele mutations in the Indian population is not known. The questionnaire was not targeted for capturing specific clinical features of FMF but rather sought to screen a homogenous cohort of patients with ReA for M694V gene mutations. In addition, only a single gene mutation, selected by virtue of its prevalence in most populations was assessed.
We fully acknowledge the limitation of the study including an ethnically diverse Indian population wherein the exact genetic/ethnic origins of the patients enrolled were not studied. Moreover, the small sample size limits firm conclusions and calls for confirmation of these observations in a larger cohort with multiethnic stratified subsets.
The overall result suggests that MEFV polymorphisms seem to be absent in adult and childhood ReA in North Indian patients. Because this is the first study studying these in ReA and is limited by a small number of cases, larger studies will be required to confirm the absence of these mutations in Indian patients with ReA.
Financial support and sponsorship
This study was funded by an Indian Rheumatology Association research grant (2019).
Conflicts of interest
There are no conflicts of interest.
AKA acknowledges receipt of the DBT-JRF/SRF -program fellowship from the Department of Biotechnology, Ministry of Science and Technology, Government of India. KN acknowledges the IRA Research Grant fund received from the Indian Rheumatology Association, India. MKS acknowledges the CSIR-JRF/SRF Fellowship program from the Ministry of Human Resource Development, India. LG acknowledges Intramural Grant from SGPGIMS, Lucknow.
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