SCIENCE CHINA Life Sciences doi: 10.1007/s11427-017-9090-6
THEMATIC ISSUE: Frontiers in rare diseases
• RESEARCH PAPER •
Gene mutations and clinical phenotypes in Chinese children with Blau syndrome Caifeng Li*, Junmei Zhang, Shipeng Li, Tongxin Han, Weiying Kuang, Yifang Zhou, Jianghong Deng & Xiaohua Tan Department of Rheumatology and Immunology, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing 100045, China Received April 4, 2017; accepted May 22, 2017; published online June 16, 2017
The mutations of CARD15 gene and clinical features of Chinese patients with Blau syndrome were analyzed. We identified 10 missense mutations, out of which five were new: R334L, E383D, R471C, C495R and D512F. The rest of them, R334W, R334Q, G481D, M513T and R587C, have been reported previously. Among all the mutations, R334W, R334Q and C495R had the highest frequency. Blau syndrome was found at early age after birth. It began with lepidic rash and symmetric polyarthritis and was phenotypically characterized by typical rash, arthritis, iridocyclitis and arteritis. Cardiac involvement was also found in Blau syndrome. In addition to nerve deafness, renal involvement, osteochondroma and central nervous system involvement were also found in our patients. Therefore, Chinese children with Blau syndrome have unique gene mutations and complicated clinical phenotypes. Pathologic examination and CARD15 mutation testing should be considered for diagnosis as early as possible for suspected patients. Blau syndrome, genetic mutation, clinical phenotype Citation:
Li, C., Zhang, J., Li, S., Han, T., Kuang, W., Zhou, Y., Deng, J., and Tan, X. (2017). Gene mutations and clinical phenotypes in Chinese children with Blau syndrome. Sci China Life Sci 60. doi: 10.1007/s11427-017-9090-6
INTRODUCTION In 1985, Edward Blau described for the first time the granulomatous disease of the skin, joints, and eyes of 11 family members in four generations (Blau, 1985). Later, these similar symptoms could be found in some families. They were also observed sporadically in population. Now, this syndrome is uniformly named Blau syndrome (Sfriso et al., 2012; Wouters et al., 2014). Blau syndrome is an autoimmune disease characterized by early onset of granulomatous inflammation of the skin, eye, and joints. There are more than 200 cases of Blau syndrome reported worldwide with no significant ethnic and gender predisposition. *Corresponding author (email:
[email protected]) © Science China Press and Springer-Verlag Berlin Heidelberg 2017
Blau syndrome is a chronic granulomatous inflammatory disease with an autosomal dominant inheritance pattern, characterized by granulomatous arthritis, uveitis, and typical skin rash with an onset age before five years. The morbidity of Blau syndrome is not so clear. Two major cases of the disease are familial forms and sporadic forms. Previous studies found that mutations in the caspase recruitment domain gene 15 (Caspase Recruitment Domain 15, CARD15) may lead to this disease. Clinical manifestations of Blau syndrome are complex and it involves numerous organs. In this study, we analyzed mutations in CARD15 gene collected from 30 children with Blau syndrome in our hospital. The clinical manifestations and organ involvement were described. The relationship between the phenotype and genotype was summarized. life.scichina.com link.springer.com
2
Sci China Life Sci
Li, C., et al.
RESULTS CARD15 gene mutation After gene sequencing, 10 mutation sites were found, they were 1000C/T(R334W)(7/30), 1001G/A(R334Q)(4/30), 1001G/T(R334L)(1/30), 1149G/T(E383D)(2/30), 1411C/T (R471C)(1/30), 1443G/A(G481D)(1/30), 1483T/C(C495R) (3/30), 1538C/T(M513T)(2/30), 1759C/T(R587C)(2/30) and 1534-5AG/TT(D512F)(2/30). The sequencing peak graph is shown in Figure 1. 1411C/T(R471C) was homozygous mutation while the rest were heterozygous mutation. Among the 10 mutations, R334W, R334Q, G481D, M513T and R587C have been reported already, while R334L, E383D, R471C, C495R and D512F were newly found in Chinese pediatric patients with Blau syndrome. Seven patients had mutation in R334W, four had mutation in R334Q and three had mutation in C495R. We found that R334W, R334Q and C495R are the most common mutation sites. Clinical phenotype Initial phenotype All patients were early onset after birth. The earliest onset age was after birth, the latest was at the age of three years and
CARD15 Figure 1 gene mutation sites in Chinese children with Blau syndrome.
six months. Twenty-three cases were started with recurrent scaly rash (76.7%). The other seven cases began with cystic swelling arthritis (23.3%). The symptoms showed repeatedly and accompanied by gradual appearance of other clinical manifestation. Clinical manifestation and organ involvement The clinical manifestations and organ involvement of the 30 Chinese pediatric patients with Blau syndrome are shown in Table 1. All patients had arthritis. The majority of joint symptoms appeared within three years of age, except only one at the age of eight. The symptom of arthritis was symmetrical multi-joint inflammation. Joints with all size could be affected. There were multiple cystic masses with different sizes around the joint, no obvious pain and limited activity in the affected joints (Figure 2). The most frequently affected joints were ankle (90%), wrist (76.7%), knee (66.7%) and interphalangeal joint (50%). Twenty-seven patients had typical rash, the majority of which were scaly rash, or pink or brown popular skin rash, which recurrently occurred on face, trunk and limbs (Figure 3). Nineteen patients had iridocyclitis. The eye syndrome began with nebula, corneal congestion or albugo, and grad-
Sci China Life Sci
Li, C., et al.
Table 1 Clinical phenotype of children with Blau syndrome Percentage
Phonotype
Case
Arthritis
30/30
100%
Rash
27/30
90.0%
Iridocyclitis
19/30
63.3%
Liver and spleen lymph node enlargement
15/30
50.0%
Fever
13/30
43.4%
Arteritis
8/30
26.7%
Cardiac involvements
5/30
16.7%
Nerve deafness
3/30
10.0%
Renal involvement
2/30
6.7%
Chondroma
2/30
6.7%
Central nervous system involvement
1/30
3.3%
Lung
0/30
0.0%
3
Moreover, this research discovered that 15 patients had painlessly enlarged liver, spleen and lymph node. Thirteen patients had different degrees of fever. Most of them had low to moderate fever with ineffective anti-infection treatment. Eight patients had aorto-arteritis. Four of them had hypertension and two of them had severe renal artery stenosis. There were five patients with cardiac involvement with main manifestations of pericardial effusion and increased left ventricular diameter. Three patients had nerve deafness. Two patients had renal lesions, with manifestation of microscopic hematuria. Two patients complicated with cartilaginous tumor. Only one patient had demyelinating lesion in the central nervous system, but no obvious neurological symptom observed. Routine MRI examination revealed that the lesions disappeared after treatment. None of the 30 patients had lung lesion. Accessory examination During the onset of the disease, white blood cell (WBC) would be raised (10.13−15.1×109 L−1) along with the rise of platelet (304−571×109 L−1). They could reduce to normal during the remission period. Mild to moderate anemia could be found, the range of haemoglobin was between 80 and 120 g L−1. The inflammatory indexes like ESR, CRP and fibrous protein could also increase in different degrees. After treatment these indexes could reduce to normal. ANA, ds-DNA, RF and ANCA of all pediatric patients were negative. No bone destruction was found in their joint imaging examination.
DISCUSSION Joint Figure 2 performance of Blau syndrome patients.
Rash Figure 3 of Blau syndrome patients.
ually deteriorated with bilateral iridocyclitis, glaucoma and cataract. Two patients had severe monocular lesions, and were left with little light sensation.
Blau syndrome is an autosomal dominant condition with the mutations in NOD2 (nucleotide-binding oligomerization domain protein 2, also called caspase recruitment domain-containing protein 15 (CARD15)) which was located in 16p12.1-13 (Tromp et al., 1996). So far, more than 10 mutations in CARD15 which are closely associated with the disease have been reported in different races and regions. R334Q and L469F were the earliest discovered mutations (Miceli-Richard et al., 2001), and R334W and R334Q were the most frequently found mutations, which were observed in more than 50% of patients (Milhavet et al., 2008; Rodriguez-Perez et al., 2009). van Duist et al. found a new mutation site, E383K, in a study of Italian families with Blau syndrome (van Duist et al., 2005). Milman et al. reported a family of Blau syndrome with the mutation of T605N (Milman et al., 2009). Further studies of the sporadic cases of Blau syndrome revealed more mutations, such as D382E, E383G, G464W, G481D, W490L, C495Y, H496L, M513T, R587C, T605P, A612T, and N670K. In this study, a total of 10 mutations were detected. R334W, R334Q, G481D, M513T and R587C have also been reported
4
Sci China Life Sci
Li, C., et al.
in other populations. R334W and R334Q mutation were the most common mutations, which were consistent with the results of foreign studies. R334L, E383D, R471C, C495R and D512F were newly discovered mutations in Chinese patients of Blau syndrome. We suspect that the differences in genetic background among various populations may account for this phenomenon. More research with larger sample size is required to confirm these results. The age of onset of Blau syndrome is usually very young, and in our study the number was as young as three years and six months. Onset occurs mostly with repeated scaly rash, while arthritis, uveitis and involvement of other organs appear later. However, not all patients have the typical triad of granulomatous uveitis, dermatitis and symmetric arthritis. In our study, arthritis was the dominant symptom and appeared in all patients, while most patients had typical rash, and more than 50% of patients had iridocyclitis. Since the 1980s, patients of familial granulomatous arteritis with polyarthritis were reported, with some cases complicating with large aneurysms (Gedalia et al., 1996; Mourad and Tang, 2010; Rotenstein et al., 1982). In recent years, Khubchandani et al. reported a case of Blau syndrome with Takayasu’s disease (Khubchandani et al., 2012). Moreover, he found that the clinical manifestations of Takayasu’s disease may be related to mutation of G464W. In our study, eight patients had Takayasu’s arteritis, suggesting that inflammation of the large artery is also a common manifestation of Blau syndrome. Large vessels and medium vessels could be involved. Severe renal artery stenosis was observed in two cases in our cohort of patients. Cardiac involvements including pericardial effusion, increased left ventricular diameter, and cardiac dysfunction were found in five patients. In addition, two patients had microscopic hematuria, suggesting renal involvement, which is required to be confirmed by renal biopsy. There has been no report of Blau syndrome complicating with chondroma in foreign studies. However, chondromas were observed in two patients in our study. One patient in our cohort had demyelinating disease of the central nervous system. Also there are reports of Blau syndrome complicating with the clinical manifestations caused by involvement of the central nervous system (Emaminia et al., 2007). Recently, Kamio et al. reported a case of Blau syndrome with refractory skin ulcers in the leg (Kamio et al., 2016). There has been a report of R334Q mutation in one patient with pulmonary interstitial change (Becker et al., 2007). However, in this study, no patient had clinical manifestations or imaging abnormalities suggesting pulmonary involvement. Laboratory tests of patients of Blau syndrome usually showed mild anemia, elevated levels of white blood cells, platelets, ESR, and CRP. In addition to the typical clinical manifestations, genetic testing and biopsy of skin or synovium should be performed to confirm the diagnosis of Blau
syndrome. The clinical manifestations of Blau syndrome are complex and various, and the degree of organ involvement is different. Misdiagnosis and missed diagnosis of Blau syndrome may occur when the triad is atypical. Pathological biopsy or genetic examination should be carried out as soon as possible to confirm the diagnosis in patients who had lepidic rash or symmetrical cystoid arthritis. Early diagnosis and treatment can significantly improve symptoms, reduce the degree of important organs’ damage and improve the quality of life.
MATERIALS AND METHODS Patients We collected 30 patients with Blau syndrome from the Department of Rheumatology and Immunology in Beijing Children’s Hospital from November 2006 to November 2016. The patients got at least one of the three typical characteristics, including cystic swelling of arthritis, uveitis and rash. Also, their rash or synovial tissue may have the pathologic results of noncaseating granulomas lesions. The patients included 16 males and 14 females with the sex ratio of 1.14:1. Their age ranged from one year old to 13 years old, with an average age of four years. The course of disease ranged from 10 months to 12 years. Detection of mutation in CARD15 Informed consent was obtained from all the patients’ parents. The peripheral blood was collected from patients with EDTA solution and the genomic DNA were extracted using the conventional salting out method. Primer design Primer 3 software by the Whitehead Institute for Biomedical research was used to design primers. The primers were designed according to the sequences of exon 4 in CARD15 and synthesized by Beijing SaiBaiSheng Gene Technology Company with Tm value ranged between 57.3 and 59.4 (Table S1 in Supporting Information). PCR amplification Each 50 µL PCR amplification reaction was performed with 5 μL genomic DNA, 2 μL of each of the forward and reverse primers, 31.6 μL deionized water, 5 μL 10× buffer, 4 μL dNTP mixture and 0.4 μL DNA polymerase (Takara Biotechnology Co., Dalian). The modified thermal profile consisted of an initial denaturation step at 95°C for 2 min, followed by a 35 cycles of denaturation at 94°C for 1 min, then annealing at 60°C for 1 min and extension at 72°C for 1 min, and a final extension at 72°C for 5 min. Products were electrophoresed by agarose gel, followed by EB staining and identified under
Sci China Life Sci
Li, C., et al.
UV light. Sequencing Each 10 µL sequencing reaction was performed with 2 μL PCR products as templates and sequenced by the forward primers after initial purification using Multiscreen PCR plates. The PCR products were labeled by Big-Dye labeling kit (ABI PRIMERTM). Sequencing was completed by ABI 3730 automatic sequencer. Chromas software was used to analyze the sequence, diagram and sequence data were compared by DNAMAN software. For uncertain sequencing results, such as suspicious mutation sites, the reverse primer sequencing would be taken. Clinical phenotype The clinical data of 30 patients with Blau syndrome were collected, and the age of onset, initial manifestation, clinical manifestation and organ involvement were summarized (Table S2 in Supporting Information). Data availability: All the clinical data and identified genetic variations have been deposited into the rare disease database, eRAM, at http://www.pediascape.org/eram/. The Compliance and ethics author(s) declare that they have no conflict of interest and followed out policy concerning informed consent.
This work was supported by Special Fund for Clinical Acknowledgements Medicine of Chinese Medical Association (12040690369). Becker, M.L., Martin, T.M., Doyle, T.M., and Rosé, C.D. (2007). Interstitial pneumonitis in Blau syndrome with documented mutation in CARD15. Arthritis Rheum 56, 1292–1294. Blau, E.B. (1985). Familial granulomatous arthritis, iritis, and rash. J Pediatrics 107, 689–693. Emaminia, A., Nabavi, M., Mousavi Nasab, M., and Kashef, S. (2007). Central nervous system involvement in Blau syndrome: a new feature of the syndrome? J Rheumatol 34, 2504–2505. Gedalia, A., Shetty, A.K., Ward, K., Correa, H., Venters, C.L., and Loe, W.A.
5
(1996). Abdominal aortic aneurysm associated with childhood sarcoidosis. J Rheumatol 23, 757–759. Kamio, Y., Kanazawa, N., Mine, Y., and Utani, A. (2016). Intractable leg ulcers in Blau syndrome. J Dermatol 43, 1096–1097. Khubchandani, R.P., Hasija, R., Touitou, I., Khemani, C., Wouters, C.H., and Rose, C.D. (2012). Blau arteritis resembling Takayasu disease with a novel NOD2 mutation. J Rheumatology 39, 1888–1892. Miceli-Richard, C., Lesage, S., Rybojad, M., Prieur, A.M., Manouvrier-Hanu, S., Häfner, R., Chamaillard, M., Zouali, H., Thomas, G., and Hugot, J.P. (2001). CARD15 mutations in Blau syndrome. Nat Genet 29, 19–20. Milhavet, F., Cuisset, L., Hoffman, H.M., Slim, R., El-Shanti, H., Aksentijevich, I., Lesage, S., Waterham, H., Wise, C., Sarrauste de Menthiere, C., and Touitou, I. (2008). The infevers autoinflammatory mutation online registry: update with new genes and functions. Hum Mutat 29, 803–808. Milman, N., Ursin, K., Rødevand, E., Nielsen, F.C., and Hansen, T.V.O. (2009). A novel mutation in theNOD2 gene associated with Blau syndrome: a Norwegian family with four affected members. Scandinavian J Rheumatology 38, 190–197. Mourad, F., and Tang, A. (2010). Sinus of valsalva aneurysm in Blau’s syndrome. J Cardiothorac Surg 5, 16. Rodriguez-Perez, N., Aguinaga-Barrilero, A., Gorrono-Echebarria, M.B., Perez-Blas, M., and Martin-Villa, J.M. (2009). Blau syndrome-related CARD15/NOD2 mutations are not linked to idiopathic uveitis in Spanish patients. Dis Markers 27, 1–5. Rotenstein, D., Gibbas, D.L., Majmudar, B., and Chastain, E.A. (1982). Familial granulomatous arteritis with polyarthritis of juvenile onset. N Engl J Med 306, 86–90. Sfriso, P., Caso, F., Tognon, S., Galozzi, P., Gava, A., and Punzi, L. (2012). Blau syndrome, clinical and genetic aspects. Autoimmunity Rev 12, 44–51. Tromp, G., Kuivaniemi, H., Raphael, S., Ala-Kokko, L., Christiano, A., Considine, E., Dhulipala, R., Hyland, J., Jokinen, A., Kivirikko, S., Korn, R., Madhatheri, S., McCarron, S., Pulkkinen, L., Punnett, H., Shimoya, K., Spotila, L., Tate, A., and Williams, C.J. (1996). Genetic linkage of familial granulomatous inflammatory arthritis, skin rash, and uveitis to chromosome 16. Am J Hum Genet 59, 1097–1107. van Duist, M.M., Albrecht, M., Podswiadek, M., Giachino, D., Lengauer, T., Punzi, L., and De Marchi, M. (2005). A new CARD15 mutation in Blau syndrome. Eur J Hum Genet 13, 742–747. Wouters, C.H., Maes, A., Foley, K.P., Bertin, J., and Rose, C.D. (2014). Blau Syndrome, the prototypic auto-inflammatory granulomatous disease. Pediatr Rheumatol 12, 33.
SUPPORTING INFORMATION Table S1 The primer details Table S2 Clinical manifestations and gene mutations of children with Blau syndrome
The supporting information is available online at life.scichina.com and link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.
