MEFV gene polymorphisms and TNFRSF1A mutation in patients with inflammatory myopathy with abundant macrophages

Abstract

Inflammatory myopathy with abundant macrophages (IMAM) has recently been proposed as a new clinical condition. Although IMAM shares certain similarities with other inflammatory myopathies, the mechanisms responsible for this condition remain unknown. Patients with familial Mediterranean fever (FMF) and tumour necrosis factor receptor-associated periodic syndrome (TRAPS) also often develop myalgia. We therefore investigated the polymorphisms or mutations of MEFV and TNFRSF1A genes in patients with IMAM to identify their potential role in this condition. We analysed the clinical features of nine patients with IMAM and sequenced exons of the MEFV and TNFRSF1A genes. The patients with IMAM had clinical symptoms such as myalgia, muscle weakness, erythema, fever and arthralgia. Although none of the patients were diagnosed with FMF or TRAPS, seven demonstrated MEFV polymorphisms (G304R, R202R, E148Q, E148Q-L110P and P369S-R408Q), and one demonstrated a TNFRSF1A mutation (C43R). These results suggest that MEFV gene polymorphisms and TNFRSF1A mutation are susceptibility and modifier genes in IMAM.

Introduction

Inflammatory myopathy with abundant macrophages (IMAM), a rare and new clinical condition, has been proposed as one aspect of inflammatory myopathy [1]. IMAM is characterized by diffuse infiltration of macrophages in muscle and fascia with muscle fibre damage. The clinical condition is expressed as dermatomyositis (DM)-like disease, but distinct from DM in pathological findings [2]. Additionally, IMAM shares similarities with cytophagic histiocytic panniculitis (CHP) [1], which is characterized by diffuse subcutaneous panniculitis composed of macrophages showing haemophagocytic activity. The underlying mechanisms responsible for IMAM remain unknown.

In autoinflammatory syndrome causing genetic variants of the innate immune system, macrophages play a crucial role by mediating inflammation through cytokines, such as interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF) [3]. Familial Mediterranean fever (FMF) and TNF receptor-associated periodic syndrome (TRAPS) are more common disorders among autoinflammatory syndrome. FMF is an autosomal-recessive inherited disorder characterized by self-limiting attacks of fever and serositis causing MEFV gene mutations or polymorphisms [3]. IL-1β, its activation regulated by pyrin protein encoded by the MEFV gene, is considered to play a major role in FMF. Conversely, TRAPS is an autosomal-dominant inherited disorder characterized by periodic fever, abdominal pain, myalgia, exanthema, arthralgia and ocular involvement [3]. One of the mechanisms is misfolding of the extracellular receptor domain and retention of the mutated TNFRSF1A protein in the endoplasmic reticulum caused by TNFRSF1A gene mutations [4]. Myalgia is often observed in these conditions. Protracted febrile myalgia syndrome (PFMS) in FMF patients [5] and monocytic fasciitis in TRAPS patients [6] are known as rare conditions with severe myalgia and fever, which resemble IMAM in clinical findings. In this study, we investigated the MEFV gene polymorphisms and TNFRSF1A mutations in patients with IMAM.

Patients and methods

Patients

Nine patients, who had been diagnosed with IMAM between 2004 and 2013 in the hospitals affiliated with Nagasaki University, were enrolled into this study. Their mean age was 56 years (median: 54, min–max: 40–71). The diagnosis of IMAM was based on muscle symptoms such as myalgia and/or muscle weakness with infiltration of CD68⁺ macrophages into the muscle and fascia that were biopsied. We excluded the patients who had a DM-specific skin manifestation, including heliotrope erythema, Gottron's papules or Gottron's signs. None of the individuals had a family history of FMF or TRAPS. The definition of haemophagocytic syndrome (HPS) was made based on criteria including phagocytosis by macrophages in histopathological findings, which has been described by Tsuda [7]. In case 6, because of obtaining no biopsied specimen, a clinical diagnosis of IMAM was made by muscle symptoms, fever, erythema and elevated ferritin levels along with the thickening of fascia in magnetic resonance imaging (MRI). Information on demographics (sex, age), clinical symptoms, the presence of HPS, treatment and outcome was collected for all patients.

Histology and immunohistochemistry

A full-thickness wedge biopsy of abnormal lesion detected by MRI was performed following written informed consent from each individual. The biopsied specimens were fixed in formalin followed by paraffin-embedding and processed for light microscopy. Microscopic evaluation was demonstrated in haematoxylin and eosin (HE)-stained sections. Immunohistochemistry was performed to identify macrophages (CD68), T lymphocytes (CD4 and CD8) and B lymphocytes (CD20).

MEFV and TNFRSF1A gene analysis

MEFV gene analysis was carried out at the Clinical Research Center of National Hospital Organization (NHO) Nagasaki Medical Center. TNFRSF1A gene analysis was carried out at the Department of Immunogenetics, Institute of Tropical Medicine, Nagasaki University. The genetic analysis was approved by the Ethics Committee of Nagasaki Medical Center, and written informed consent was obtained from each individual. Exons 1, 2, 3, 4 and 10 of the MEFV gene (RefSeq ID: NM_000243) and all 10 exons of the TNFRS1A gene (RefSeq ID: NM_001065) were analysed for all patients by direct sequencing. Two millilitres of blood samples were collected from all subjects. Genomic DNA was extracted from whole blood by means of the Promega Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). MEFV and TNFRSF1A gene analysis was performed by genomic sequencing, as described previously [8].

Results

Patients

Table 1 summarizes the demographics of the nine individual cases. IMAM patients had myalgia, muscle weakness, non-specific erythema, fever and arthralgia. Muscle symptoms such as myalgia and/or muscle weakness were observed. Skin erythema in the trunk and/or extremities was observed in seven patients. Seven patients showed fever, but only one patient (case 3) had a periodic fever. Five patients had arthralgia, but no patients had abdominal pain suggesting serositis. In two cases, phagocytosis by macrophages was revealed by aspiration biopsy of bone marrow, and diagnosed in both patients as haemophagocytic syndrome. None of the patients were diagnosed with FMF or TRAPS on the basis of their clinical findings. In laboratory findings, creatine phosphokinase (CPK) levels were elevated in six patients (mean: 1116, median: 497, min–max: 16–3717 U/ml). The levels of C-reactive protein (mean: 8·3, median: 2·1, min–max 0·4–19·3 mg/dl), serum ferritin (mean: 1116, median: 1096, min–max 159–24 337 ng/ml) and aldolase (mean: 21·7, median: 18·8, min–max 7·9–63·2 IU/l) were elevated in all patients. In short-term inversion recovery (STIR) images from a plain MRI, thickening of fascia was revealed in all patients. In four cases, high-intensity lesions of STIR images were observed not only in fascia but also in muscle fibres. All patients were treated with prednisolone, and five patients were also treated with an immunosuppressant such as tacrolimus, cyclosporin, methotrexate or cyclophosphamide. In case 3, colchicine was partially effective, but treatment with steroid and tacrolimus was needed for improvement of the symptoms. In all patients, the symptoms improved during the clinical course.

Table 1.

Demographic features of nine patients with inflammatory myopathy with abundant macrophages (IMAM)

	Muscle symptoms		Skin lesions	Other manifestations			Laboratory findings		MRI findings (STIR high lesion)
Case (age/sex)	Myalgia	Weakness	Erythema	Fever	Arthralgia	HPS	CPK (U/l)	Ferritin (ng/ml)	Fascia	Muscle	Treatment	Outcome
1 (58/M)	+	Moderate	Extremities	+	−	−	1927	316	+	−	PSL, mPSL	Remission
2 (54/F)	+	Mild	–	−	−	−	278	159	+	−	PSL, TAC, MTX	Improve
3 (40/M)	+	Mild	–	+	+	−	497	843	+	+	PSL, TAC, Colchitine	Improve
4 (68/M)	+	Moderate	Trunk	+	+	−	16	2600	+	+	PSL, TAC, mPSL	Improve
5 (66/M)	+	–	Extremities/trunk	+	−	−	1431	1702	+	+	PSL, mPSL	Remission
6 (53/F)	+	Mild	Extremities	+	+	−	77	1096	+	−	PSL, TAC	Remission
7 (71/F)	+	Mild	Trunk	+	+	+	32	24 337	+	+	PSL, mPSL	Remission
8 (53/F)	+	Moderate	Extremities	−	+	−	2066	241	+	−	PSL	Remission
9 (45/F)	+	Mild	Extremities	+	−	+	3717	12 984	+	−	PSL, CsA, CTX, mPSL	Remission

CsA, cyclosporin A; CPK, creatine phosphokinase; CTX, cyclophosphamide; HPS, haemophagocytic syndrome; mPSL, methyl prednisolone pulse therapy; MRI, magnetic resonance imaging; MTX, methotrexate; PSL, prednisolone; TAC, tacrolimus; M, male; F, female.

Histopathology

In biopsied specimens, thickening of the fascia was identified. Infiltration of CD68-positive macrophages was abundant. CD4⁺ and CD8⁺ T lymphocytes were scattered in the fascia, but few CD20⁺ B lymphocytes were identified. Few inflammatory cells were detected in the muscle fibres (Fig. 1).

Fig. 1.

Pathological findings in inflammatory myopathy with abundant macrophages (IMAM) (case 5). (a) Thickening of fascia and cellular inflammation. (b) Macrophage infiltration in the fascia. (c–d) Infitration of CD68-positive macrophages was abundant. (e–g) CD4⁺ and CD8⁺ T lymphocytes were scattered in the fascia, but few CD20⁺ B lymphocytes were identified. (a,b) Haematoxylin and eosin staining.; (c,d) CD68 immunohistochemistry; (e) CD4; (f) CD8; (g) CD20. Magnification: (a,c,e,f,g) ×100; (b,d) ×400.

MEFV and TNFRSF1A gene analysis

In the gene analysis, MEFV polymorphisms were observed in seven of the nine patients with IMAM: heterozygous G304R (n = 2), R202Q (n = 1) and E148Q (n = 1); compound heterozygous E148Q-L110P (n = 2) and P369S-R408Q (n = 1); and a TNFRSF1A mutation was observed in one patient: heterozygous C43R (T214C) (Table 2).

Table 2.

Mediterranean fever (MEFV) polymorphisms and tumour necrosis factor receptor superfamily member 1A (TNFRSF1A) mutation in the patients with inflammatory myopathy with abundant macrophages (IMAM)

Case	MEFV	TNFRSF1A
1	G304R	–
2	G304R	–
3	R202Q	–
4	E148Q	–
5	E148Q/L110P	–
6	E148Q/L110P	–
7	P369S/R408Q	–
8	–	C43R
9	–	–

Discussion

IMAM is a rare inflammatory myopathy which was described initially in 2009 by Brunn et al. [1]. The pathological finding of IMAM is characterized by diffuse destructive infiltration of macrophages into the fascia, rather than the muscle. Although IMAM shares certain similarities with DM in clinical findings, it is distinct from DM in pathological findings [2]. Inflammatory cells infiltrate predominantly CD68⁺ macrophages in IMAM, as opposed to predominantly CD3⁺ CD4⁺ T cells and CD20⁺ B cells in DM. In idiopathic inflammatory myopathies, recent data suggest that not only adaptive immune but also innate immune activation, through Toll-like receptors (TLRs), nuclear factor kappa B (NF-κB) and nucleotide-binding oligomerization domain-like receptor (NLR)-inflammasome, contribute to disease pathogenesis [9]. In IMAM, macrophage infiltration also indicates that innate immune activation occurs in the fascia.

Conversely, macrophagic myofasciitis (MMF) is considered persistent immune activation caused by injection of aluminium hydroxide-adjuvanted vaccines, described initially in 1998 [10]. In MMF, macrophage infiltrates are focal and form with crystalline aluminium hydroxide particles, distinct from IMAM. One of the mechanisms of MMF was expected to be autoinflammation, such as activating the intracellular NLRP3 inflammasome system caused by an injection of aluminium salts [11]. On this account, MMF has been classified as ‘auto-immune/inflammatory syndrome by adjuvants’ (ASIA).

Severe debilitating myalgia can occur in 25% of patients with FMF [12]. Usually, exercise-induced myalgia is well known in FMF, but protracted febrile myalgia syndrome (PFMS) is described in rare cases. PFMS is characterized by severe debilitating myalgia of extremities and high fever, occasionally accompanied by abdominal pain, diarrhoea, arthritis/arthralgia and transient vasculitic purpura. The response to corticosteroid therapy was prompt. The thickening of endomysial and perimysial spaces and the infiltration of fibroblasts, macrophages and a few mast cells has been reported in patients with PFMS [5]. IMAM has a great deal in common with PFMS with regard to clinical and pathological findings. In this study, IMAM patients demonstrated MEFV polymorphisms such as R202Q, G304R, R408Q, P369S, L110P and E148Q. The frequencies of these polymorphisms were, respectively, 0·2, 2·7, 4·4, 5·0, 8·7 and 23·3% in Japanese healthy controls [13,14]. In general, these polymorphisms, with the exception of E148Q, are not disease-associated polymorphisms. However, these are often observed in patients with atypical FMF in Japan [14]. This study is a small case-series, and we were unable to infer the statistical difference from healthy controls; however, the frequencies of these polymorphisms (R202Q: one from nine, G304R: two from nine, R408Q: one from nine, P369S: one from nine, L110P: two from nine and E148Q: three from nine) were expected to increase in patients with IMAM.

Monocytic fasciitis in patients with TRAPS was described initially as infiltration of monocytes/macrophages in fascia [6]. The patient had myalgia, fever, abdominal pain and rash, and demonstrated a high intensity of the muscles and dermis in MRI. These findings resemble IMAM. Additionally, in a group of multiple sclerosis patients carrying the TNFRSF1A R92Q mutation, additional TRAPS symptoms, such as myalgias, arthralgias/arthritis and rash, were observed [15]. In this study, one patient with IMAM demonstrated the TNFRSF1A mutation C43R, which is a disease-associated mutation, and has not been reported in Japanese TRAPS patients. These results suggest that MEFV and TNFRSF1A mutations are associated with the development of myalgia and fasciitis observed in IMAM.

Severe forms of IMAM may be associated with macrophage activation syndromes (MAS) [1]. MAS, such as familial haemophagocytic lymphohistiocytosis (HLH) and secondary HLH associated with systemic-onset juvenile idiopathic arthritis (JIA), have been classified as autoinflammatory diseases by Masters et al. [3]. Furthermore, association between MAS and autoinflammatory syndrome has been described in several case reports, such as FMF [16], TRAPS [17], chronic infantile neurological, cutaneous, articular (CINCA) syndrome [18] and hyperimmunoglobulinaemia D with periodic fever syndrome (HIDS) [19]. The MEFV polymorphisms and TNFRSF1A mutations observed in patients with IMAM might play important roles in the pathogenesis of MAS.

Our study showed MEFV polymorphisms and TNFRSF1A mutation in patients with IMAM. Although none of these patients were diagnosed with FMF or TRAPS by clinical findings, the results suggest that MEFV gene polymorphisms and TNFRSF1A mutations are susceptibility and modifier genes in IMAM. This possibility needs to be explored in a larger series of patients.

Disclosure

The authors declare that they have no competing interests.