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832

CONCISE REPORT

Autoinflammatory gene mutations in Behçet’s disease


I Koné-Paut, E Sanchez, A Le Quellec, R Manna, I Touitou
...................................................................................................................................

Ann Rheum Dis 2007;66:832–834. doi: 10.1136/ard.2006.068841

deficiency (MKD), cryopyrin associated periodic syndromes


Background: Behçet’s disease (BD) shares clinical features with
(CAPS) and pyogenic sterile arthritis, pyoderma gangrenosum,
well-recognised autoinflammatory disorders. In addition, muta-
and acne (PAPA) syndrome, respectively, as possible suscept-
tions in genes for familial Mediterranean fever and tumour ibility factors for BD. We did not identify any relevant
necrosis factor receptor-associated periodic syndrome have association with the main mutations responsible for these
been reported to have increased in patients with BD. disorders. Interestingly, however, two patients were found to
Patients and methods: DNA samples from 97 patients with BD have genetic MKD.
and 51 matched healthy controls were analysed for the
mevalonate kinase (MVK), cold-induced autoinflammatory syn-
drome 1 (CIAS1) and proline/serine/threonine phosphatase-
PATIENTS AND METHODS
We analysed 97 DNA samples from patients with BD for MVK,
interacting protein 1 (PSTPIP1) genes, responsible for mevalonate
CIAS1 and PSTPIP1,responsible for MKD, CAPS and PAPA,
kinase deficiency (MKD), cryopyrin associated periodic syn-
respectively. Complete clinical information was obtained from
dromes (CAPS) and pyogenic sterile arthritis, pyoderma gang- their medical chart reviews. The DNA analysis was performed
renosum and acne (PAPA) syndrome, respectively. Over 90% of systematically in patients who fulfilled the international criteria
known mutations were screened using restriction fragment length for BD.4 None of their doctors had clinical suspicions of another
polymorphism analysis and/or sequencing. disease. We also analysed DNA from 51 ethnically matched
Results: Two patients had paired mutations in the MVK gene healthy controls. The mean ages of the patients and controls were
(genotypes V377I/V377I and V377I/S135L) and displayed comparable (35 vs 37 years). The male:female sex ratio was
typical features of BD and MKD. Another was heterozygotic for 0.83:1 among patients and 0.59:1 among controls. Over 90% of
the V377I genotype. The V198M mutation in the CIAS1 gene was the known mutations were screened using restriction fragment
identified in one patient with typical BD but no symptoms of length polymorphism analysis and/or sequencing. The MVK and
CAPS. No mutations were identified in the control group. PSTPIP1 CIAS1 mutational hot spots (ie, V377I, exon 2 and exon 9 for
analysis revealed a new exon 10 insertion variant MVK and exon 3 for CIAS1) were screened as described
(c.741+33_741+34insGT) in 2 of 97 patients and in 1 of 51 previously by Federici et al.5 (MVK exons 2 and 9, CIAS1 exon 3
controls (p.0.05), indicating that it is a polymorphism rather and the corresponding intronic boundaries of these genes were
than a true mutation. amplified using M13-tagged primers. A total of 50 ng of genomic
Discussion: This study could not demonstrate any significant DNA, 2.5 mm MgCl2, 0.25 mm each of forward and reverse
increases in MVK, CIAS1 or PSTPIP1 mutations in patients with primers, 0.025 U of Taq gold polymerase and 0.2 mm dNTP were
BD as compared with controls. used for amplification reactions as follows: an initial denatura-
tion at 95˚C for 9 min; 30 cycles of denaturing at 94˚C (30 s),
annealing at 58˚C and extension at 72˚C (1 min); and a final
9 min extension at 72˚C. PCR efficiencies were verified on a 5%

B
ehçet’s disease (BD) is a complex chronic and relapsing acrylamide gel, and 1–4 ml of amplified product was directly
inflammatory disorder that has been extensively described submitted to sequencing in both directions with M13 universal
in young adults from Eastern Asia and Mediterranean primers using the Big Dye Terminator V.3.1 kit (Applied
countries. Its essential manifestations are oral and genital Biosystems, Foster City, California, USA), following the manu-
ulcerations, folliculitis, erythema nodosum and uveitis. The facturer’s suggestions. Sequencing products were run on an ABI
presence of vasculitis worsens the prognosis, introducing the Prism 3100 Genetic Analyzer (Applied Biosystems). MVK exon 11
potential for life-threatening complications such as throm- was amplified using 1176 and 1381 primers6 and the PCR
bophlebitis, arterial aneurysms and occlusion. The aetiology of
fragments were digested with BsmA1 to examine V377I, the most
BD is unknown. Because BD shares clinical similarities with frequent hyperimmunoglobulinemia D and periodic fever syn-
certain well-recognised autoinflammatory disorders, we won-
drome (HIDS) mutation.)
dered whether the gene responsible for familial Mediterranean
The two known PAPA mutations were analysed by PCR
fever (MEFV) could be involved. We have previously demon-
amplification using primers PST10FM13 (tgtaaaacgacggccag-
strated a higher frequency of MEFV mutations in patients with
tactgggcttccagcagagag) and PST10RM13 (caggaaacagctatgacct-
BD, with respect to their ethnicity.1 Other groups have
gagctgctgaggcctgaga), followed by sequencing in both directions
confirmed these data and found an association between the
for mutation A230T, and primers PST11FM13 (tgtaaaacgacggc-
presence of MEFV mutations and the severity of vasculitis.2
cagtcacaatggcctgtgaggag) and PST11RM13 (caggaaacagctatgac-
Recently, the R92Q mutation in the TRAPS (tumour necrosis
caagggagctgtgagctac), followed by BstN1 digestion for mutation
factor receptor-associated periodic syndrome) gene, which is
E250Q.
responsible for another defect of innate immunity, was reported
to have increased in patients with BD.3 In view of these
findings, and because BD is a multifactorial disease, we decided Abbreviations: BD, Behçet’s disease; CAPS, cryopyrin-associated periodic
syndromes; CIAS1, cold-induced autoinflammatory syndrome 1; HIDS,
to test additional autoinflammatory genes—namely mevalo- hyper IgD syndrome; FMF/MEFV, familial Mediterranean fever MKD,
nate kinase (MVK), cold-induced autoinflammatory syndrome mevalonate kinase deficiency; MVK, mevalonate kinase; PAPA, pyogenic
1 (CIAS1) and proline/serine/threonine phosphatase-interact- sterile arthritis, pyoderma gangrenosum and acne; PSTPIP1, proline/
ing protein 1 (PSTPIP1)—responsible for mevalonate kinase serine/threonine phosphatase interacting protein 1

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Gene mutations in Behçet’s disease 833

RESULTS producing the pro-inflammatory cytokines interleukin (IL) 2, IL6,


Mutations were identified in 4 (4%) of 97 patients (table 1). Two IL8, IL12, IL18, tumour necrosis factor -a and interferon c. On the
of the four had paired mutations in the MVK gene (genotypes basis of certain clinical similarities, its episodic nature and the
V377I/S135L and V377I/V377I) and displayed features typical of importance of granulocyte activation in its pathogenesis, BD has
both BD and MKD (table 2). The first was a 16-year-old French been suggested to be another autoinflammatory disease. The
girl with BD who had bipolar aphtosis, erythema nodosum, severe participation of MEFV and tumour necrosis factor a receptor 1
acne, and transient knee and ankle arthritis. She also had mutations in the course and pathogenesis of the disease was
recurrent fevers that appeared monthly, accompanied by chills, described previously. As autoinflammatory diseases share many
headaches, abdominal pain, diarrhoea and conjunctivitis. She clinical features, and as the proteins involved have molecular and
experienced adverse reactions to immunisation, which induced functional similarities, we wondered whether non-FMF gene
flares of erythema nodosum. She had marked hyper-IgA (5 g/l; mutations could be detected in patients with BD. We analysed
IgD and mevalonic aciduria not performed) and was negative for DNA samples from 97 patients with BD who fulfilled the
HLAB51. The second patient was a 30-year-old French woman international criteria. Our results did not identify any relevant
with recurrent fevers, bipolar aphtosis, erythema nodosum, skin association between BD and the genes responsible for MKD,
reactions after immunisation, polyarthralgia and keratitis. A third CAPS or PAPA.
patient was heterozygotic for V377I and displayed a typical BD Interestingly, two patients with typical features of BD were
phenotype without other MKD-related symptoms. The fourth discovered to have genetic MKD (two mutations). MKD (also
patient had the V198M mutation in the CIAS1 gene and showed called, in milder forms, hyper-IgD syndrome or HIDS) is much
typical BD but no features of CAPS. Clinical phenotypes and DNA less frequent than BD. The main presentation is recurrent fever
analysis results of other family members of this last patient were lasting 5–7 days, accompanied by headaches, lethargy, cervical
not available. Considering the low penetrance of the V198M adenitis, abdominal distress, diarrhoea, urticarial rash and
mutation, there was only a low probability of a diagnosis of CAPS transient arthritis. The first manifestations of HIDS start during
in this patient. the first year of life and are triggered by infections and
No mutations were identified in the control group. We did immunisations.9 A high level of serum IgD was previously
not find any patient or control with either of the two known considered to be a hallmark of HIDS, but an increasing number
mutations (A230T and E250Q) in the PSTPIP1 gene. We did, of case reports have shown that this finding is neither constant
however, identify a new exon 10-intron insertion variant nor specific to the disease. The detection of slight mevalonic
(c.741+33_741+34insGT) in two non-paediatric patients. This aciduria in a febrile patient allows a diagnosis, as HIDS is caused
insGT was identified in 2 of 97 patients and in 1 of 51 controls by a partial mevalonate kinase deficiency. Genetic confirmation is
(p.0.05), indicating that it is a polymorphism and not a true performed by identifying mutations in the MVK gene on
mutation. RNA from these patients was unavailable to test chromosome 12p13.10 11 Our two patients showed symptoms of
whether this insertion affected gene expression. These results both diseases, specifically bipolar aphtosis and headaches. In both
do not support a strong role for non-familial Mediterranean patients, skin hypersensitivity was not strictly a pathergic
fever (FMF) hereditary periodic fever genes in BD, although we phenomenon, but was rather erythema nodosum and urticarial
cannot rule out the possibility that rare mutations are involved. rash triggered by antigen stimulation (especially immunisations).
Although recurrent fevers and eye symptoms were present in both
DISCUSSION patients, the reported symptoms (keratitis and conjunctivitis)
Autoinflammatory disorders represent a new category of heritable were distinct from typical BD uveitis. In patient 1, the fact that
diseases, characterised by recurrent self-limited inflammatory chronic diarrhoea was present could have shifted the diagnosis
attacks occurring without evident precipitating events. They towards MKD.12 Conversely, the presence of erythema nodosum
include FMF (the prototypic disease), MKD (complete and hyper- in our two genetically proven patients with MKD was initially a
IgD syndrome (HIDS)), TRAPS, CAPS and, more recently, PAPA strong argument for a diagnosis of BD. To our knowledge, we are
syndrome.7 These diseases result from mutations in five distinct the first to report erythema nodosum, a key feature of BD, as part
genes that are listed on the regularly updated central INFEVERS of the clinical spectrum of MKD. Another patient with only one
website.8 BD, by contrast, is considered a systemic inflammatory MKD mutation presented with a typical BD phenotype, but in this
disease, most likely involving adaptive immunity and particularly case bipolar aphtosis could have been part of the two diseases.
T lymphocytes skewed towards the T helper 1 phenotype and Thus, patients with BD with early-onset recurrent fevers, bipolar

Table 1 Mutations and sequence variants detected in our study population


BD Controls

In FEVERS11
Diseases Paediatric BD, Non paediatric BD, In this study Frequency of data
(transmission) Genes n = 32 n = 65 n = 51 mutations in controls

MKD (recessive) MVK V377I/V377I 1 V377I/S135L 0 V377I


1 0
V377I/- S135L
1 0
CAPS (dominant) CIAS1 0 V198M 0 V198M
1 0
PAPA (dominant) PSTPIP1 0 Mutation Mutation 0
0 0
Variant* Variant*
2 1

BD, Behçet’s disease; CAPS, cryopyrin-associated periodic syndromes; CIAS1, cold-induced autoinflammatory
syndrome 1; MKD, mevalonate kinase deficiency; MVK, mevalonate kinase; PAPA, pyogenic sterile arthritis, pyoderma
gangrenosum and acne; PSTPIP1, proline/serine/threonine phosphatase interacting protein 1.
*c.741+33_741+34insertGT.

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834 Koné -Paut, Sanchez, Quellec, et al

Table 2 Clinical features of patients with Behçet’s disease carrying autoinflammatory gene mutations
Age at onset Age at diagnosis
Patient number Genes Mutations Gender Country (years) of BD (years) Clinical features

1 MVK V377I/S135L F France ,1 year 16 years Fever: 40˚C, chills/monthly;


headaches; bipolar aphthosis;
erythema nodosum; severe acne;
conjunctivitis; abdominal pain;
diarrhoea; transient arthralgia;
arthritis; febrile and skin reaction
after immunisations

2 MVK V377I/V377I F France 30 years Fever; bipolar aphthosis; macular


rash; skin hypersensitivity;
erythema nodosum; keratitis;
transient arthralgia

3 MVK V377I/- M Italian 24 years 30 years bipolar aphthosis; erythema


nodosum; folliculitis; uveitis;
transient arthralgia

4 CIAS1 V198M M France 44 years buccal aphthosis; skin aphthosis;


erythema nodosum; uveitis;
venous thrombosis; ulcerative
colitis; transient arthralgia

BD, Behçet’s disease; CIAS1, cold-induced autoinflammatory syndrome 1; F, female; MKD, mevalonate kinase deficiency; M, male.

aphtosis, abdominal complaints and atypical eye involvement REFERENCES


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Competing interests: None declared. autoinflammatory syndromes associated with the CIAS1/PYPAF1/NALP3 gene.
Arthritis Rheum 2004;50:4045–50.
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Pediatric Rheumatology, Hôpital de Bicêtre, 78 rue du Général Leclerc, autosomal dominant disorder of pyogenic sterile arthritis, pyoderma
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16 Wise CA, Gillum JD, Seidman CE, Lindor NM, Bashiardes S, Lovett M. Mutations
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Published Online First 9 January 2007 an autoinflammatory disorder. Hum Mol Genet 2002;11:961–9.

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