Blau syndrome, clinical and genetic aspects

Autoimmunity Reviews 12 (2012) 44–51

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Review

Blau syndrome, clinical and genetic aspects Paolo Sfriso a,⁎, Francesco Caso a, Sofia Tognon b, Paola Galozzi a, Alessandra Gava a, Leonardo Punzi a a b

Rheumatology Unit, Department of Medicine, University of Padova, Padova, Italy Ophthalmology Unit, Department of Neurosciences, University of Padova, Padova, Italy

a r t i c l e

i n f o

a b s t r a c t Blau syndrome (BS) is a rare autosomal dominant, autoinflammatory syndrome characterized by the clinical triad of granulomatous recurrent uveitis, dermatitis and symmetric arthritis. The gene responsible for BS has been identified in the caspase recruitment domain gene CARD15/NOD2. In the majority of patients, the disease is characterized by early onset, usually before 3–4 years of age. The manifestations at disease onset are usually represented by articular and cutaneous involvement signs, generally followed later by ocular manifestations which are often the most relevant morbidity of BS. In some cases the presence of fever is also observed; atypical cases of BS have been reported with cardiovascular, neurological, renal, intestinal and other organ involvement. The rarity and the variations in the severity and evolution of its expressions do not permit sufficient data about optimal treatment for patients with BS. The first step of therapy is represented by the use of corticosteroids and successively, in case of unsatisfactory response, by additional treatment with immunosuppressive agents. The results with biologic anti-cytokine agents, such as anti-TNFα and anti-IL1β, are different, particularly with regard to ocular morbidity. Clinical and genetic aspects of the familial and the sporadic form of BS will be discussed and focused on. A description of a case study of an Italian family is also included. © 2012 Elsevier B.V. All rights reserved.

Available online 2 August 2012 Keywords: Blau syndrome Genetic mutation NOD2 Uveitis Camptodactyly Autoinflammatory disease

Contents 1. Introduction . . . . . . . . 2. Clinical aspects . . . . . . . 3. Genetic and functional aspects 4. An Italian family with BS . . . References . . . . . . . . . . . .

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1. Introduction Blau syndrome is a rare autoinflammatory granulomatous disorder [1]. In 1985, the pediatrician Edward Blau described for the first time the disease as a dominantly inherited, chronic inflammatory syndrome characterized by the clinical triad of granulomatous dermatitis, symmetric arthritis and recurrent uveitis with onset below 4 years of age. The original description of Blau about the disorder, reported four generations with 11 members of the same family affected. Ten showed arthritis; two showed skin, eye and joint involvement; one showed skin and joint disease; and one manifested isolated ocular involvement with iritis [2]. Tromp et al. mapped the ⁎ Corresponding author at: Rheumatology Unit, Department of Medicine, University of Padova, via Giustiniani 2, 35128 Padova, Italy. Tel.: +39 0498212190; fax: +39 0498214382. E-mail address: [email protected] (P. Sfriso). 1568-9972/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.autrev.2012.07.028

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44 46 47 49 50

BS locus in 1996 by genotyping 72 of the 74-member pedigree with dinucleotide-repeat markers to the chromosomal region 16q12.1–13, which contain one of the susceptibility genes for Crohn's disease [3]. In 2001, Miceli-Richard et al. identified the gene that confers susceptibility for BS, discovering three missense mutations (R334Q, R334W and L469F) in the region encoding the nucleotide-binding domain (NBD) of the caspase recruitment domain gene (CARD15/NOD2) in four French and German BS affected families [4]. Hence, CARD15/NOD2 is involved in susceptibility to a second granulomatous disorder [5] in addition to Crohn's disease. Further reports of CARD15/NOD2 mutations were published in other families with BS, successively. Originally BS was considered a distinct entity from early onset sarcoidosis (EOS), in spite of the striking clinical similarities between them. Genetic analyses subsequently have shown that many patients with EOS have mutations in CARD15/NOD2. Thereafter, some authors proposed that BS and EOS are the familial and sporadic forms, respectively, of the same disease [6,7]. Some others proposed the term ‘pediatric granulomatous arthritis’

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Table 1 (a) Reported families with Blau syndrome (April 2012); (b) reported BS-associated mutations in CARD15/NOD2. a. Authors

Families

Members affected

Generations

Involvement Joint

Skin

Eye

Visceral

Blau [2] Jabs [23] Pastores [64] De Chadarévian [65] Hafner [24] Moraillon [66] Saini [28] Tromp [3] Scerri [67] Manouvier-Hanu [18] Ting [29] Miceli-Richard [4] Wang [16]

1 1 1 1 1 1 1 1 1 1 1 4 10

11 4 3 2 2 2 3 16 2 4 3 11 50

4 3 2 2 2 2 2 4 2 2 2 2 2–4

10 4 3 2 2 2 3 13 2 4 2 9 50

3 No 2 2 1 1 1 8 2 2 3 7 40

3 3 3 2 1 No 1 6 2 2 No 5 33

Latkany [25] Ewida [68] Alonso [22] Kurokawa [26] Masel [20] Van Duist [10] Snyers [27] Schaffer [38] Becker [34] Emaminia [30] Aróstegui [14]

8 1 1 1 1 1 1 1 1 1 9

16 4 4 3 1 2 3 4 4 3 12

3 2 2 2 1 2 2 3 2 2 1–2

15 4 4 3 1 1 3 No 4 3 11

9 4 4 2 1 1 2 2 No 1 10

16 2 2 3 1 1 2 3 3 1 7

Dhondt [31] Okafuji [15] Milman [9]

1 2 1

1 4 4

1 2 2

1 4 4

1 4 3

1 4 4

Villanueva-Mendoza [69] Akil [33] Stoevesandt [70] Son [71] Martin [72] Raiji [73] Jimenez-Martinez [74]

1 1 1 1 1 1 1

3 3 1 2 1 1 1

2 1 1 2 1 1 1

3 3 1 2 1 1 1

2 3 1 2 1 1 1

1 3 1 No 1 1 1

Xiang [75]

1

3

2

3

2

3

None None None None None None Hepatic Not evaluated None/neurological None Renal None Large vessel arteritis and cranial neuropathy None None None None Ichthyosis None None None Pneumonitis Neurological Renal, neurological, pericarditis, hepatic, adenopathies Leg ulcers None Neurological, parotid gland, intestinal None Renal None Renal None None Interstitial pneumopathy, hepatic, renal None

b. Mutation

Sequence variant

Functional study a

Family (number of affected)

Country of origin

Reference

R334W (p.Arg334Trp)

c.1000C>T

Yes

R334Q (p.Arg334Gln)

c.1001G>A

Yes

E383K (p.Glu383Lys)

c.1147G>A

Yes

E383G (p.Glu383Gly) G464W (p.Gly464Trp) L469F (p.Leu469Phe) W490L (p.Trp490Leu) E498_L500delinsV (p.Glu498_Leu500delinsVal) M513R (p.Met513Arg)

c.1148A>G

Yes

1 (5) 2 (15) 1 (2) 1 (3) 1 (3) 3 (9) 1 (3) 2 (4) 3 (13) 1 (4) 1 (2) 2 (6) 1 (2) 1 (2) 1 (2 + 4 asympt.) 1 (2)

Germany Japan Japan Unknown USA Spain China France Japan Unknown Spain Spain Italy Mexico Spain Japan

[4] [16] [26] [27] [38] [39] [75] [4] [16] [34] [14] [39] [10] [69] [76] [15]

c.1390G>T

No

Unknown

India

c.1405C>T

Yes

1 (2)

France

Khubchandani RP et al., personal communicationa [4]

c.1469G>T

Unknown

Unknown

Italy

c.1493_1498del6

Unknown

1 (1)

Japan

Van Duist M et al., personal communicationa [77]

c.1538T>G

No

1 (1)

Mexico

[74] (continued on next page)

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P. Sfriso et al. / Autoimmunity Reviews 12 (2012) 44–51

Table 1 (continued) b. Mutation

Sequence variant

Functional study a

Family (number of affected)

Country of origin

Reference

R587C (p.Arg587Cys) T605N (p.Thr605Asn)

c.1759C>T

No

1 (3)

Spain

[14]

c.1814C>A

No

1 (4)

Norway

[9]

a

These data were obtained from http://fmf.igh.cnrs.fr/ISSAID/infevers/.

due to the prevalence of the disease among infants. However this was inadequate to represent the systemic nature of this disease [8]. Thus, N. Milman, et al. proposed to classify these patients as sporadic BS due to de novo mutations, restricting the term EOS to patients with features of sarcoidosis and without mutations in CARD15/NOD2 [9]. This review will discuss the clinical and genetic aspects of BS, both in its familial and sporadic forms. In addition, we will describe an Italian family affected by BS who has been under our observation over the past 25 years [10]. We hope that this description of the characteristics and the evolution process of the disease over such a long time period may contribute to a better understanding of BS. 2. Clinical aspects To our knowledge, BS has been observed until now in 193 persons belonging to 63 families (Table 1a). The occurrence of BS is reported primarily among Caucasians although it has been also reported in Asians [6] as well as AfroAmericans [11]. No information is available regarding the extent to which the disease is spread. For most patients, the disease is characterized by early onset, typically at ages before 3–4 years [12]. Sometimes, however, symptoms appear only after 10 years of age [9]. Onset occurs mostly with articular and cutaneous symptoms [13–15] while eye symptoms typically appear later, between 7 and 12 years of age [2,16]. The most common manifestation of the disease is arthritis, and in general it appears within the first 10 years of life [17]. Often the initial manifestation of arthritis in the absence of ocular or skin findings is mistaken for juvenile rheumatoid arthritis (JRA). Joint manifestations usually appear such as symmetric polyarthritis, involving wrists, metacarpophalangeal (MCP), 1st metatarsophalangeal (MTP) and proximal interphalangeal (PIP) joints of hands and feet, ankles and occasionally elbows (Fig. 1a). Joint swelling, with moderate redness, warmth and tenderness, often accompanies arthritis due to synovial thickening and effusion [13,14]. Additionally, frequent granulomatous inflammation in the periarticular structures is present leading to marked periarticular swelling and tenosynovial cysts particularly affecting the wrists and dorsa of the hands. These patients exhibit a chronically evolving arthritis which may cause deformity of fingers and wrist ankylosis. Joint involvement generally has a juvenile onset, appearing as painless cysts on the back of feet and wrists which may evolve into mild ‘boutonnière finger deformities’ [18,19]. Traditional X-rays reveal deformities typical of periarticular swelling and, at times, joint space narrowing [18,19]. Radiographic signs of erosive joint changes are sometimes absent in the earlier stage of the disease process. Nonetheless, progression to deforming arthritis may occur leading to severe handicap caused by flexion contractures of fingers and toes (camptodactyly) and decreased motion of large joints. BS has been described with various skin manifestations but such descriptions have not been uniformly consistent. These usually report two main types of asymptomatic eruptions: a papulonodular tender brownish rash and multiple, firm, subcutaneous nodules (Fig. 1b). The exanthema usually appears as an erythema with maculopapular configuration. Pinhead-sized, lichenoid, yellow to brown papules typically appear in clusters and may become confluent. The appearance

is variable, often symmetric, located on the trunk and/or extremities [16,12,20,21]. During a period of years, intermittent episodes of generalized exanthema with spontaneous resolution can happen, eventually leaving pitted scars at sites of previous papules. The exanthema can even go unnoticed when it is mild. The eruptions are described in some cases as papular or plaque-like, maculopapular and tiny red dots; they have also frequently been described as erythematous, intermittent and generalized [22]. Histology of the lesions consistently demonstrates non-caseating granulomas with multinucleated giant cells [2,3,16,23,24] (Fig. 1c). ‘Comma-shaped bodies’ in epithelioid cells, which seem to be a marker for BS, may be revealed by electron microscopy [16,18]. The most relevant morbidity of BS is eye involvement [2,25–27]. The ocular symptoms of BS require the closest attention, from the perspective of quality of life. The most common manifestations are recurrent anterior uveitis or panuveitis together with eye pain, photophobia and blurred vision. Granulomatous uveitis, often bilateral, can evolve into a cataract and band keratopathy, often requiring surgery (Fig. 1d). Most ocular structures such as conjunctiva, lachrymal gland, retina and optic nerve may be afflicted by inflammation. Vitritis, granulomatous disks and bilateral disseminated chorioretinal lesions that are surrounded by retinal hemorrhages and progressive subretinal fibrosis are also included among the other findings observed [2,3,26] At times fundus modifications resemble those in sarcoid uveitis [26]. Most patients typically require continuing follow-up and treatment for eye symptoms all through their life. Often chorioretinitis, cataract, glaucoma and retinal detachment complicate eye involvement, leading to significant visual impairment and even blindness. Atypical cases of BS have been reported which involve organs other than the skin, joints or eyes. BS has been reported together with hepatic and renal granulomatous involvement [28,29], cerebral infarction [30] and malignant hypertension [16]. In another case a family with BS was observed to have ichthyosis vulgaris [20]. Persistent or intermittent fever, granulomatous arteritis with central nervous system involvement including cranial neuropathies [23] and corticosteroid-responsive hearing loss [23,30] have also been reported. Recently, a BS patient with very large and painful ulcerations on both legs was described, where the biopsy of the ulcers suggested that these are part of a granulomatous disease [31]. Patients with BS have also been reported to have granulomas in the lymph nodes, parotid glands and intestines [9]. With regards to the cardiovascular manifestations of BS, very little information exists. A case of sinus of Valsalva aneurysm has been recently described, suggesting some form of auto-immune aortitis [32]. A case associating BS with chronic tubulointerstitial nephritis and renal clear cell carcinoma has been reported [33]. Interestingly, histopathological examination revealed giant cell granulomas in both the tumor and non-neoplastic renal tissue. The authors suggest, given the relation between chronic inflammation and cancer, that patients with BS should be monitored for malignancies. An unusual case of BS with an R334Q mutation associated with granulomatous lymphadenitis and interstitial pneumonitis has been

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Fig. 1. Clinical and histological aspects of Blau syndrome. a) Asymptomatic diffuse brownish papulae of some millimeter in diameter, firm subcutaneous nodules, varying in size from 5 to 30 mm in diameter, on the extensor surfaces of the legs. b) Enlargement of interphalangeal joints. c) Skin biopsy from the right forearm. Non-caseating granulomas, containing several histiocytes and multinucleated giant cells with strong PAS positivity and rare lymphocytes and eosinophils. d) Granulomatous uveitis, evolved into cataract and band keratopathy.

reported [34]. However, unlike in the case of sarcoidosis [19], the involvement of the lungs has rarely been described in BS [12,21]. No studies on the optimal treatment for patients with BS have been made yet, owing to its rarity and the variations in the severity and evolution of its expressions. The major concern is with eye involvement, which may, at times, be severe [2,10,26]. Low-dose glucocorticoids are generally satisfactory at the quiescent stage [25]. In some acute stages, high doses of glucocorticoids are, however, necessary. Its long-term use, especially at high doses, may be problematic, as disease onset is often during childhood. Additional treatment with immunosuppressive agents should be tried if the therapeutic response to corticosteroids is unsatisfactory, or a maintenance dose of prednisolone ≥ 10 mg/day is needed for prolonged periods. What may represent promising therapeutic approaches in refractory cases include biologic anti-cytokine agents such as infliximab, a tumor necrosis factor (TNF)-α inhibitor and anakinra, the IL-1 receptor antagonist [14,21]. The results however are variable, particularly with regard to ocular morbidity. Moreover, a recent in vitro study suggests that BS is not mediated by excess IL-1 beta or other IL-1 activity, in contrast to related IL-1 beta-dependent auto-inflammatory cryopyrinopathies. Finally, a pilot study on two sporadic BS patients yielded results suggesting that thalidomide is a promising agent for the reduction of granulomatous inflammation by means of its suppression of nuclear factor-κB (NF-κB) activation and interference with the proliferation and differentiation of monocytes [35]. 3. Genetic and functional aspects Blau syndrome (MIM# 186580) is an autosomal dominantly inherited disorder, due to mutations in the caspase recruitment domain gene CARD15/NOD2.

This gene, mapped on the chromosomal region 16q12.1–13 [3], has been firstly associated with Crohn's disease (MIM# 266600), a granulomatous inflammatory bowel disease [36,37]. The identification of CARD15/NOD2 as the gene responsible for BS marked the major turning-point for BS studies [4]. Since 2001, several cohorts have been genotyped and different CARD15/NOD2 mutations have been identified in BS patients from distinct ethnic origins (Table 1b; Fig. 2a). The most commonly observed mutations are missense substitutions affecting the highly evolutionary conserved arginine residue at position 334 (R334W or R334Q) [4,14,16,26,27,34,38,39]. These mutated alleles make codon 334 a genetic hot spot for mutations. Most of the other mutations are found only once or few times. There are no known mutations involving the untranslated and the intronic regions of the gene, even though this has not been extensively studied. Mutations in CARD15/NOD2 are also found in children with early-onset sarcoidosis (EOS, MIM# 609464), a sporadically occurring condition that shares a common genetic etiology with Blau syndrome [7,40]. CARD15/NOD2 encodes a multi-domain protein of 1040 amino acids, namely nucleotide oligomerization domain 2 (NOD2). NOD2 is a member of the NOD-like receptor family (NLR) that plays an important role in innate immune defense. NOD2 expression has mainly occurred in the cytosol of myelomonocytic cells [41], dendritic cells [42] and Paneth cells [43], which are major component of non-caseating granulomas. More recently NOD2 was shown to be associated with the plasma membrane of intestinal epithelial cells [44]. The baseline expression of NOD2 by epithelial cells is low, and TNF can induce upregulation of expression and this positive effect is augmented by IFNγ [45]. NF-κB-binding sites in the CARD15/NOD2 promoter are involved in the response to TNF, implying that, when NOD2 activates NF-κB following activation by its ligand, NOD2 can upregulate itself [46].

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Fig. 2. Genetical and biochemical aspects of Blau syndrome. a) Schematic presentation of CARD15/NOD2 gene and the nucleotide positions of BS-associated mutations (NM_022162. 1). Exons are represented by boxes whereas horizontal lines stand for introns. b) Detailed representation of NOD2 displaying amino acid positions of BS-associated mutations. The numbers under the diagram are the first and last amino acids of each structural domain (data obtained from http://www.uniprot.org/uniprot/Q9HC29). c) Schematic representation of the NOD2 signaling pathway. MDP activates cytosolic NOD2 through direct interaction with the leucine-rich repeat (LRR) region. Binding leads to the oligomerization of NOD2 and recruitment of the RICK (RIP-like interacting CLARP kinase) through CARD–CARD interactions. RICK then activates the IKK complex through IKK/NEMO. This results in phosphorylation, ubiquitination and degradation of IκB, and downstream activation of associated NF-κB (nuclear factor-kappaB). The IKK complex is also activated by TGF-β activated kinase 1 (TAK1), which is downstream of TLR activation.

As shown in Fig. 2b, NOD2 has a typical three-domain structure composed of two N-terminal effector regions consisting of a caspase recruitment domain (CARD), a centrally located NACHT that is critical for activation, and nine C-terminal leucine-rich repeats (LRRs) that sense the pathogen-associated molecular patterns (PAMPs) [47].

The N-terminal domain mediated signaling through its homophilic interactions with other CARD containing proteins. The centrally located NACHT domain displays ATPase activity and is necessary for activation and oligomerization leading to inflammatory signaling responses [48].

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NOD2 is a general detector of intracellular invasive bacterial infections, sensing small peptides such as muramyl dipeptide (MDP), a degradation product from both Gram-positive and Gram-negative bacterial peptidoglycan [49]. Thus far, a crucial role for NOD2 in bacterial clearance has been demonstrated in vitro for infection with a variety of pathogens including Streptococcus pneumoniae [50], Mycobacterium tuberculosis [51] and Staphylococcus aureus [52]. In vivo evidence of NOD2 during host defense has been clearly demonstrated for Listeria monocytogenes [53] and M. tuberculosis [54]. NOD2 was shown to mediate additional cellular responses to the host defense system. Recent findings have provided evidence that NOD2 is important in the induction of type I IFN in response to viral ssRNA [55] and is involved in the production of the microbicidal ROS [56]. Moreover, it initiates autophagy by recruiting one of the key components for autophagosome formation, ATG16L1 [57]. Basically, NOD2 activation leads to the activation of NF-κB which is present in the cytoplasm as an inactive form bound to inhibitory proteins known as inhibitor of κBs (IκB) (Fig. 2c). NOD2 resides in the cytoplasm of resting cells in an autoinhibited conformation due to the intramolecular interactions of its LRR and CARD domains: the response to bacterial components (MDP) results in an oligomerization via the NACHT domain, thus exposing the CARD-containing effector domain and inducing the recruitment of the serine–threonine kinase RICK (also known as RIP2) through a CARD–CARD interaction. Following its activation by NOD2, RICK activates the inhibitor of NF-κB (IκB)-kinase-γ (IKKγ, also known as NEMO), the regulatory subunit of the IKK complex, and promotes the phosphorylation of the catalytic subunit IKKβ. The IKK complex is also activated by TGF-β activated kinase 1 (TAK1), which is downstream of TLR activation. Subsequently the activated IKK complex phosphorylates the inhibitor IκB, thus releasing the active NF-κB which translocates to the nucleus where it initiates the transcriptional activation of inflammatory genes. It has been shown recently that NOD2 might also need to interact with another intracellular molecule, GRIM19 (gene-associated with retinoid-IFN-induced mortality 19), for optimal activation of NF-κB [58]. Recent results showed the essential role of RICK in the modulation of innate and adaptive immunity triggered by NOD2 ligands [59] and established that in RICK-deficient mice this protein played a critical role not only in NF-κB, but also in mitogen-activated protein kinase signaling (MAPK), in macrophages stimulated with NOD ligands [60]. However, NOD-dependent but RICK-independent signaling also exists: Travassos and colleagues demonstrated that in RICK-deficient cells a NOD-dependent induction of autophagy occurs [57]. Two distinct groups of functional alterations of CARD15/NOD2 are involved in the two granulomatous diseases (Crohn's disease and Blau syndrome): Crohn's disease-associated variants predominantly reside in the LRRs, while Blau syndrome-associated mutations occur in the NACHT domain. Because activation of NOD2 involves oligomerization initiated by the NACHT-domain, mutations in this domain might decrease the threshold for spontaneous oligomerization of NOD2. This might explain the finding that epithelial cells expressing BS-mutated forms of NOD2 show increased NF-κB activation, even in the absence of stimulation with MDP [61]. Since BS is due to an increased activity of NF-κB signaling, it is generally classified as an autoinflammatory disease. However, it remains unknown how these gain-of-function mutations in CARD15/NOD2 affect the pathogenesis of Blau syndrome and whether a genotype–phenotype correlation exists between the clinical manifestations and CARD15/NOD2 mutations. Recent studies [62] suggest the possibility that an elevated basal NF-κB activity may affect disease progression rather than disease onset since they found that patients with mutated CARD15/NOD2 and low NF-κB activity tended to experience complications (i.e. arthritis and uveitis) at a later age. A multiple sequence alignment of NOD2 with homologous proteins has shown that some residues affected by BS mutations correspond to

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the position of pathogenic mutations in the closely related protein PYPAF1 (also known as NLRP3 or CIAS1), linked to CAPS, cryopyrinassociated periodic syndrome [63]. Identical mutations at analogous sequence positions seem to be extremely improbable to occur by chance; this observation suggests a close connection between the two diseases.

4. An Italian family with BS The proband, a 31-year-old Caucasian woman, was referred to our Unit in 1984, having arthritis of hands and feet and a papulonodular skin eruption. The patient reported that when she was 20 years old she had developed chronic, bilateral uveitis and glaucoma and then also cataracts a few years later. She underwent an iridectomy when she was 24 years old and 7 years later a cataract operation. Symmetrical arthritis involving fingers, wrists and feet, and skin manifestations, consisting of widespread papules and firm subcutaneous nodules on the extremities appeared during adolescence and thereafter intermittently although kept under control by non-steroidal anti-inflammatory drugs (NSAIDs). The patient showed asymptomatic, diffuse, miliary brownish papule and firm subcutaneous nodules, varying in size from 5 to 30 mm in diameter, on the dorsa of hands and feet and on the extensor leg surfaces. Arthritis was evident on the bilateral MCP and PIP joints. The joints were only slightly tender and swollen, and they were neither red nor hot. Similarly, the bilateral 1st and the 2nd MTPs of the feet were involved. Camptodactyly (flexion contractures of the fingers and toes) was absent. Radiographs of hands and feet showed only a slight periosteal enlargement of the bone and a chest X-ray was negative. All laboratory test results including inflammatory indices were normal. Human leucocyte antigen (HLA) typing showed A2 and A3; B8 and B21; DR3 and DR7. Forearm skin biopsy specimens were prepared for light and electron microscopy examination. Non-caseating granulomas, containing several histiocytes and multinucleated giant cells with strong PAS positivity and rare lymphocytes and eosinophils were showed by histology. Polyploid histiocytes with abundant cytoplasm containing a welldeveloped Golgi complex and endoplasmic reticulum, profuse mitochondria, lysosomes and glycogen granules, probably determining PAS positivity, were revealed by transmission electron microscopy. In a few cells pleomorphic cytoplasmatic inclusions were found. Some collagen fibers were in close apposition to cellular membranes, usually surrounded by a cytoplasmatic process of histiocytes, but without clear evidence of phagocytosis. Capillary vessels with swollen endothelial cells due to cellular infiltrates were found. Low doses of steroids (methylprednisolone 4 mg/day) were introduced due to the limited efficacy of NSAIDs, and these are continued even now with satisfactory control of all disease manifestations. The proband consulted us one month after her first examination in our Division, this time about her 5-year-old daughter who had recently developed skin manifestations similar, although less severe, to her own. No therapy was prescribed at that time, as the entity of the disturbances was negligible. However, four years later she developed arthritis with slight swelling of all fingers of both hands and moderate pain. Because there was a simultaneous worsening of dermatitis, she was admitted to the Dermatological Department of the University of Padova. All routine laboratory investigations, including serum lipids, rheumatoid factor and antinuclear antibodies (ANAs), were within normal limits. A right forearm skin biopsy revealed some granulomas, prevalently composed of PAS-positive histiocytes and multinucleated giant cells without intracellular lipids, similar to those observed in the proband. BS was diagnosed, but no specific treatment was prescribed except for NSAIDs, which were to be taken when necessary for a few days' time for pain control.

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When the daughter was 12 years old she was admitted to our Rheumatological Unit because of painful arthritis. At the physical examination, she exhibited asymptomatic erythematous papules on the extremities, in particular on the extensor leg surfaces, symmetric arthritis involving PIP and MCP of the hands and 1st MTP of the feet, which were swollen, tender and slightly warm. Radiographs showed periarticular swelling and slight space narrowing of the PIP and of the first right MTP of the hands. Laboratory tests revealed a slightly elevated erythrocyte sedimentation rate (ESR) of 26 mm/1h and polyclonal hypergammaglobulinemia of 22.5% (total proteins 7.7 mg/dl). Rheumatoid factor and ANAs were absent. HLA typing revealed A23, A24, B18, B51, Bw4, DR11, DRw52 and DQw7. An electrocardiogram was normal and chest X-ray as well as abdominal ultrasound was negative. Ophthalmologic examination was negative. Synovial biopsy specimens, of the third left PIP, were similar to the skin biopsies previously taken, in particular with regard to the non-caseous granulomas. The diagnosis of BS was confirmed but because of the patient's age steroids were not prescribed and instead NSAIDs were begun. One year later, when the patient was 13 years old, she exhibited bilateral anterior uveitis, which was successfully treated with topical drugs. However, some weeks later, as arthritis and skin lesions worsened, low doses of oral steroids were introduced for 1–2 week cycles, which yielded a good response. References [1] Punzi L, Gava A, Galozzi P, Sfriso P. Miscellaneous non-inflammatory musculoskeletal conditions. Blau syndrome. Best Pract Res Clin Rheumatol 2011;25: 703-14. [2] Blau EB. Familial granulomatous arthritis, iritis, and rash. J Pediatr 1985;107: 689-93. [3] Tromp G, Kuivaniemi H, Raphael S, Ala-Kokko L, Christiano A, Considine E, et al. Genetic linkage of familial granulomatous inflammatory arthritis, skin rash, and uveitis to chromosome 16. Am J Hum Genet 1996;59:1097-107. [4] Miceli-Richard C, Lesage S, Rybojad M, Prieur AM, Manouvrier-Hanu S, Häfner R, et al. CARD15 mutations in Blau syndrome. Nat Genet 2001;29:19-20. [5] Cavanaugh J. NOD2: ethnic and geographic differences. World J Gastroenterol 2006 Jun 21;12:3673-7. [6] Kanazawa N, Okafuji I, Kambe N, Nishikomori R, Nakata-Hizume M, Nagai S, et al. Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factorkappaB activation: common genetic etiology with Blau syndrome. Blood 2005;105: 1195-7. [7] Rosé CD, Doyle TM, McIlvain-Simpson G, Coffman JE, Rosenbaum JT, Davey MP, et al. Blau syndrome mutation of CARD15/NOD2 in sporadic early onset granulomatous arthritis. J Rheumatol 2005;32:373-5. [8] Rosé CD, Martin TM, Wouters CH. Blau syndrome revisited. Curr Opin Rheumatol 2011;23:411-8. [9] Milman N, Ursin K, Rødevand E, Nielsen FC, Hansen TV. 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