Somatic mosaicism of CIAS1 in a patient with chronic infantile neurologic cutaneous articular syndrome.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 52, No. 11, November 2005, pp 3579–3585 DOI 10.1002/art.21404 © 2005, American College of Rheumatology Somatic Mosaicism of CIAS1 in a Patient With Chronic Infantile Neurologic, Cutaneous, Articular Syndrome Megumu Saito, Akihiro Fujisawa, Ryuta Nishikomori, Naotomo Kambe, Mami Nakata-Hizume, Momoko Yoshimoto, Katsuyuki Ohmori, Ikuo Okafuji, Takakazu Yoshioka, Takashi Kusunoki, Yoshiki Miyachi, Toshio Heike, and Tatsutoshi Nakahata Chronic infantile neurologic, cutaneous, articular syndrome (CINCA syndrome) is a severe inflammatory disease that was recently found to be associated with mutations in CIAS1. However, CIAS1 mutations have been detected in only half of CINCA syndrome patients, and it remains unclear which genes are responsible for the syndrome in the remaining patients. We describe here a patient with CINCA syndrome who exhibited CIAS1 somatic mosaicism. We genetically analyzed the CIAS1 gene in various blood cells and the buccal mucosa of the patient. The production of interleukin-1␤ (IL-1␤) by peripheral blood mononuclear cells (PBMCs) was measured by enzyme-linked immunosorbent assay, and the ability of the mutant CIAS1 gene to enhance ASC-dependent NF-B activation was assessed to confirm that the mutations of CIAS1 found were responsible for the patient’s clinical manifestations of the CINCA syndrome. The patient had 1 heterologous single-nucleotide polymorphism, 587G>A (S196N), and 1 heterologous mutation, 1709A>G (Y570C), in exon 3 of CIAS1. The latter mutation was found to occur as somatic mosaicism. The patient’s PBMCs produced a large amount of IL-1␤ in the absence of stimulation, unlike those from controls or from his mother, who also bore the S196N polymorphism. In addition, the Y570C mutation (with or without the S196N polymorphism) increased the ability of CIAS1 to induce ASC-dependent NF-B activation, unlike the wild-type gene or the gene bearing the S196N polymorphism alone. The findings in this patient indicate that somatic mosaicism is one reason CIAS1 mutations have not been detected in some patients with CINCA syndrome. Chronic infantile neurologic, cutaneous, articular syndrome (CINCA syndrome) (MIM no. #607115), also known as neonatal-onset multisystem inflammatory disease, is a recently recognized disorder characterized by recurrent episodes of inflammation (1–5). Patients with CINCA syndrome exhibit recurrent fever, an urticarial rash beginning in the neonatal period, arthropathy characterized particularly by epiphyseal patellar overgrowth, chronic meningitis, papilledema, hearing loss, and growth retardation. The gene responsible for the syndrome is reported to be CIAS1, which has also been associated with 2 less severe but phenotypically similar syndromes, familial cold autoinflammatory syndrome (FCAS) (MIM no. #120100) and Muckle-Wells syndrome (MWS) (MIM no. #191900) (6–10). CIAS1 (also designated PYPAF1 and NALP3) has been reported to be expressed in polymorphonuclear cells, monocytes, chondrocytes, and activated T cells (9,11,12). Its product is cryopyrin, which contains an amino-terminal pyrin domain, a centrally located NACHT domain, and carboxy-terminal leucine-rich repeats (LRRs). Cryopyrin localizes in the cytosol and is believed to function as a pattern recognition receptor. It can activate caspase 1 in the presence of an adaptor protein called ASC, and it converts the proform of interleukin-1␤ (IL-1␤) into the mature and biologically Supported in part by the Morinaga Hoshi-Kai, the Sapporo Bioscience Foundation, the Ministry of Education, Science, Sports, and Culture, and the Ministry of Health, Labor, and Welfare, Japan. Megumu Saito, MD, Akihiro Fujisawa, MD, Ryuta Nishikomori, MD, PhD, Naotomo Kambe, MD, PhD, Mami NakataHizume, MS, Momoko Yoshimoto, MD, PhD, Katsuyuki Ohmori, PhD, Ikuo Okafuji, MD, Takakazu Yoshioka, MD, Takashi Kusunoki, MD, PhD, Yoshiki Miyachi, MD, PhD, Toshio Heike, MD, PhD, Tatsutoshi Nakahata, MD, PhD: Kyoto University, Kyoto, Japan. Drs. Saito and Fujisawa contributed equally to this work. Address correspondence and reprint requests to Ryuta Nishikomori, MD, PhD, Department of Pediatrics, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: email@example.com. Submitted for publication May 10, 2005; accepted in revised form August 9, 2005. 3579 3580 SAITO ET AL active IL-1␤ molecule (13,14). Cryopyrin also participates in regulating NF-B (14,15). All CIAS1 mutations found to date in patients with CINCA syndrome, FCAS, and MWS are located in exon 3, which encodes the NACHT domain (7,9,16–19). It is speculated that these CIAS1 mutations may lead to an aberrantly functioning cryopyrin protein, which then leads to dysregulated production of inflammatory cytokines such as IL-1␤ (7,13,14,20). However, although patients with FCAS and MWS tend to show familial inheritance patterns, CINCA syndrome usually occurs de novo, and approximately half the patients with CINCA syndrome lack detectable mutations in the CIAS1 coding region (7,9,16,17). It has been postulated that different genes or a modifier gene could be involved in these latter cases, although these patients do not differ clinically from those with CIAS1 mutation(s). Herein we describe a patient with typical symptoms of CINCA syndrome who was found to have somatic mosaicism of CIAS1. Such somatic mosaicism could be easily missed with the sequencing method usually used to identify CIAS1 mutations. This case may explain why CIAS1 mutations have not been detected in some patients with CINCA syndrome. CASE REPORT The patient was a 15-year-old boy born by normal vaginal delivery with no consanguinity. During the neonatal period, he had developed an urticarial rash. At the age of 22 months, his abnormal gait led to the diagnosis of arthritis of the knee. At age 10 years, an ophthalmologic evaluation revealed papilledema, and cerebrospinal fluid examination showed chronic meningitis. At the age of 11 years, he underwent bilateral knee joint tendonectomy. When he was 12 years old, audiography showed mild sensorineural deafness. He had been treated with glucocorticoids since the age of 2 years and with methotrexate mini-pulse therapy since the age of 4 years. Laboratory examinations revealed persistent elevations of the C-reactive protein level, erythrocyte sedimentation rate, and white blood cell count, although eosinophilia was not detected. On the basis of these characteristic signs and symptoms, the clinical course, and the persistent inflammation, the patient was diagnosed, at the age of 12 years, as having CINCA syndrome. Physical examination of the patient at the age of 15 years showed marked growth retardation (height –8.5 SD, weight –8.2 SD), joint contracture of the knees bilaterally, an urticarial rash, and a saddlenose deform- ity. However, no mental retardation (IQ 82 by Wechsler Intelligence Scale for Children III) or epilepsy was detected. Magnetic resonance imaging and computed tomography of the head also did not show any abnormalities. The patient’s parents and sister did not have any of the symptoms noted above. METHODS Genetic analysis. Informed consent was obtained from the patient and his family according to a protocol approved by the Institutional Review Board of Kyoto University Hospital. Peripheral blood mononuclear cells (PBMCs) and neutrophils were obtained by density-gradient centrifugation over Ficoll (Amersham Biosciences, Piscataway, NJ) and with monopoly reagents (Dainippon, Osaka, Japan), respectively, and phytohemagglutinin (PHA) blasts were produced by culturing PBMCs with PHA (Invitrogen, Carlsbad, CA) as previously described (21). Genomic DNA from whole blood, PBMCs, neutrophils, and PHA blasts was then obtained using a Puregene DNA isolation kit according to the protocol of the manufacturer (Gentra Systems, Minneapolis, MN), and genomic DNA from a buccal mucosa sample was obtained using a DNA isolation kit from Qiagen (Valencia, CA). For each genomic DNA sample, each CIAS1 exon, including the exon–intron boundaries, was amplified by polymerase chain reaction (PCR) and sequenced by the dideoxynucleotide termination method with ABI 3100 (Applied Biosystems, Foster City, CA). To analyze the frequency with which the CIAS1 mutation occurred, the amplified exon 3 was subcloned using a TOPO TA cloning kit (Invitrogen) and sequenced. Reverse transcriptase–PCR (RT-PCR) for CIAS1 was performed as previously described (21). Enzyme-linked immunosorbent assay for IL-1␤. For cytokine analysis, PBMCs were cultured with or without lipopolysaccharide (LPS; 10 ng/ml) (Sigma, St. Louis, MO) and supernatants were collected. Enzyme-linked immunosorbent assay for IL-1␤ was performed according to the protocol recommended by the manufacturer (eBioscience, San Diego, CA). NF-B luciferase assay. Wild-type CIAS1 and its LRRs-lacking mutant (⌬LRRs; amino acids 1–742) in pEFBOS vector (22) were kindly provided by Dr. T. Suda (Kanazawa University, Ishikawa, Japan). Other mutants were generated using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). ASC complementary DNA was kindly provided by Dr. J. Sagara (Shinshu University, Nagano, Japan). HEK293FT cells (105) were transfected using TransIT293 Transfection Reagent (Milus Bio, Madison, WI) with the NF-B reporter construct (pNF-B-luc; 20 ng) (BD Biosciences Clontech, Palo Alto, CA), an internal control construct (pRL-TK; 5 ng) (Toyo Ink, Tokyo, Japan), and wild-type or mutant CIAS1 in the presence or absence of ASC (16 ng). The total plasmid contents were adjusted by adding empty pEF-BOS vector. Twenty-four hours later, the transfected cells were harvested and subjected to dual luciferase assay as previously described (23). CIAS1 SOMATIC MOSAICISM IN CINCA SYNDROME 3581 Figure 1. Top, Pedigree of the patient’s family. The patient (family member 3) is indicated by the arrow. Bottom, Chromatograms of the CIAS1 gene at positions 587 and 1709, from CIAS1 gene analysis of the patient and his parents (family members 1 and 2). indicates position 1709 on the patient’s chromatogram. RESULTS Unexpectedly, 2 heterologous point mutations, 587G⬎A (S196N) and 1709A⬎G (Y570C), were detected in exon 3 of CIAS1 in the patient, as shown in Figure 1. Even though the chromatographic intensities of G and A at position 587 were almost identical, the intensity of the mutated G at position 1709 was, reproducibly, considerably lower than that of the A at this position (Figure 1). This suggests that the 1709A⬎G (Y570C) mutation may be a case of somatic mosaicism. To test this hypothesis, exon 3 of CIAS1 from the patient’s whole blood, amplified by PCR, was subcloned to measure the frequency of the mutant allele. This revealed that the 1709A⬎G mutation occurred with a frequency of 16.7% in whole blood (Figure 2). Thus, somatic mosaicism is the cause of the less frequent occurrence of the 1709G variant compared with 1709A in the direct sequencing analysis. When we mixed equal amounts of 2 subcloned plasmids that expressed G or A at position 1709, respectively, we confirmed that the signal for G was stronger than that for A (Figure 2). This would help to easily identify this mutation, even when it occurs at a frequency of ⬃10–20%. We also identified the mosaicism of CIAS1 at the messenger RNA level by RT-PCR (Figure 2). By sequencing exon 3 in the subcloned plasmid expressing G at position 1709, we found that both the 587G⬎A (S196N) and 1709A⬎G (Y570C) mutations were located on the same allele (data not shown). To further characterize the mosaicism in this 3582 SAITO ET AL allele; 1%), while no 1709A⬎G (Y570C) mutations were detected. CIAS1 mutations detected previously in patients with CINCA syndrome have been reported to activate caspase 1 via ASC, and as a result, monocytes from the patients produce IL-1␤ even in the absence of stimuli such as LPS (7,13,14,20). Consequently, we cultured PBMCs from the patient, his mother, and normal controls. The patient’s PBMCs produced large amounts of IL-1␤ in the absence of LPS stimulation, unlike those from his mother and the controls, whereas the LPS response of the patient’s PBMCs was comparable with that of PBMCs from his mother and controls after 24 hours of LPS stimulation (Figure 3). This indicates that the Y570C mutation, and not the S196N polymorphism found in the patient’s mother as well, was likely to be the CIAS1 mutation responsible for the clinical manifestations of CINCA syndrome in this patient. To address this question more directly, we measured the effect of various mutations in CIAS1 on ASC-dependent NF-B activity in NF-B reporter experiments. HEK293FT cells were cotransfected with wild-type or mutant CIAS1, ASC, and the NF-B reporter construct. We confirmed that the mutant lacking the LRRs (⌬LRRs) or bearing other reported point mutations, namely, R260W or D303N (14), activated Figure 2. Analysis of the frequency with which the 1709G⬎A mutation occurred in various cells from the patient. The chromatograms resulting from direct sequencing of isolated DNA and the results of frequency analysis performed by cloning and sequencing are shown. A:G ⫽ 1:1 indicates the chromatogram resulting from sequencing analysis of a mixture of equal amounts of plasmids containing CIAS1 exon 3 with either 1709A or 1709G. indicates position 1709. PBMC ⫽ peripheral blood mononuclear cell. patient, the frequency with which the 1709A⬎G mutation occurred in PBMCs, neutrophils, PHA blasts, and buccal mucosa was analyzed. While the frequency varied depending on the cell type (Figure 2), the mutation was identified in the various hematopoietic and somatic cells examined, which indicates that the somatic mosaicism occurred at a very early time point during embryogenesis. We next analyzed samples from the patient’s parents and found that his mother also had the 587G⬎A (S196N) mutation. However, neither of his parents bore the 1709A⬎G (Y570C) mutation. We also performed single-nucleotide polymorphism (SNP) analysis of the 2 mutated sites in 100 normal Japanese controls and found that 587G⬎A (S196N) is a rare SNP (2/200 Figure 3. Spontaneous production of interleukin-1␤ (IL-1␤) by peripheral blood mononuclear cells (PBMCs) from the patient. PBMCs from the patient (who had the Y570C and S196N mutations), his mother (who had the S196N mutation), and 2 normal controls were cultured with and without lipopolysaccharide (LPS) (10 ng/ml). The concentrations of IL-1␤ in the harvested supernatants were then measured by enzyme-linked immunosorbent assay. Representative results from 1 of 2 independent experiments are shown. CIAS1 SOMATIC MOSAICISM IN CINCA SYNDROME 3583 Figure 4. Effect of the CIAS1 mutations in the patient on ASC-dependent NF-B activation. A, HEK293FT cells were cotransfected with 16 ng of CIAS1, wild-type (WT) or its mutants (carboxy-terminal leucine-rich repeat–lacking mutant [⌬LRR], S196N, Y570C, Y570C with S196N, R260W, D303N), in the presence or absence of 16 ng ASC. B, HEK293FT cells were cotransfected with the indicated amounts of CIAS1, wild-type or its mutants (S196N, Y570C, Y570C with S196N) in the presence or absence of ASC. Results are shown as fold induction compared with vector without ASC (set at 1); values are the mean and SD of triplicate determinations. Representative results from 1 of 3 independent experiments are shown. NF-B more vigorously than did wild-type CIAS1 (Figure 4A). We also showed that the mutant bearing the Y570C point mutation, with or without the polymorphism of S196N, activated NF-B (in the presence of ASC) more strongly than did the wild-type gene, whereas the S196N polymorphism alone did not (Figure 4A). These data indicate that of the CIAS1 mutation and the polymorphism found in the patient, the Y570C mutation was critical for NF-B activation and is likely to be the major cause of his CINCA syndrome. 3584 SAITO ET AL To further confirm the effect of the individual mutation and polymorphism detected in this patient on ASC-dependent NF-B activation, we transfected HEK293FT cells with different amounts of wild-type, S196N single-mutant, Y570C single-mutant, and S196N/ Y570C double-mutant CIAS1 in the presence of ASC. The Y570C single mutant and the double mutant induced more NF-B activation than did the wild-type CIAS1 at each of the 3 concentrations tested. Moreover, we found that the double mutant consistently induced lower levels of NF-B activation relative to that generated by the Y570C single mutant with all concentrations of ASC tested, although the difference was minimal (Figure 4B). DISCUSSION This is the first report of somatic mosaicism of CIAS1 in a patient with CINCA syndrome. Initially, we failed to detect the small 1709G peak and only identified the S196N polymorphism. Since we did not find the latter polymorphism in our database search of Japanese SNPs, we therefore thought that it was responsible for the clinical manifestations of CINCA syndrome in this patient. However, our subsequent analysis of the patient’s family and our own SNP analysis of 100 normal Japanese controls revealed that S196N is simply a rare SNP and not the disease-causing mutation. This led us to examine the patient’s sequence chromatography results more carefully. The Y570C mutation, which in this patient occurred as somatic mosaicism, is one of the most common CIAS1 mutations, and all 4 reported CINCA syndrome patients with this mutation have a very severe phenotype that includes mental retardation or epilepsy (7,17,18). However, our patient did not exhibit such central nervous system dysfunctions, although he did have severe arthropathy and epiphyseal overgrowth. The relatively mild phenotype in our patient could be explained by the lower dose of the active mutant due to the somatic mosaicism of CIAS1. Alternatively, the milder phenotype may be due to occurrence of the mutation with differing frequencies in various cell types, i.e., fewer cells in the central nervous system may express the active mutation compared with those in articular or cutaneous regions. Yet another possibility relates to the fact that the patient not only had the Y570C mutation, but also had the maternally inherited S196N mutation on the same allele. This latter mutation could lessen the severity of the phenotype. In support of this are the results of our in vitro analysis of the ability of wild-type and mutant CIAS1 to activate NF-B: the S196N/Y570C double mutant activated NF-B slightly, but consistently, less potently than did the Y570C single mutant. Further analysis of more patients with CINCA syndrome and how their disease phenotypes relate to their genotypes, especially in patients with somatic mosaicism or double mutation, will be needed before we can determine why our patient has a less severe phenotype than other CINCA syndrome patients bearing the Y570C mutation. To date, patients with somatic mosaicism of CIAS1 have not been reported, even though CINCA syndrome largely occurs de novo. In the cohort of 233 type 2 neurofibromatosis founders with bilateral vestibular schwannomas reported by Kluwe et al, the rate of mosaicism was 24.9% (58 of 233) (24). This suggests the probable existence of other cases of CINCA syndrome with somatic mosaicism. Identification of these patients by whole blood gene sequencing analysis is likely to be problematic. Indeed, we were able to identify only the 1709A⬎G mutation that encodes the Y570C amino acid change, because the chromatographic intensity of 1709G in the sequencing analysis was 2-fold stronger than that of 1709A; this made it easier for us to detect the small peak representing the extra G. In order for direct sequencing methods to be useful in detecting cases of CIAS1 somatic mosaicism, baseline “noise” on chromatography will have to be minimized. Since somatic mosaicism of CIAS1 may be difficult to detect by the usual direct sequencing method, we would like to emphasize the importance of functional studies such as the IL-1␤ secretion assay in diagnosing the CINCA syndrome. Once the patient is confirmed to have dysregulated IL-1␤ secretion, the investigation for CIAS1 mutations can proceed. In cases in which CIAS1 mosaicism is suspected, subcloning-based sequencing of CIAS1 would be useful. However, this procedure is very labor-intensive. To overcome this, we are currently seeking to establish a method to enrich CIAS1-mutated cells, which would enhance the probability of finding the relevant mutations. In the whole blood of the patient described herein, the frequency of the Y570C mutation was found to be only 16.7%. This indicates that ⬃33% of the whole blood cells bore a heterozygous mutation of Y570C. Thus, the presence of an active CIAS1 mutation in only a portion of the cells appears to be sufficient to cause the clinical manifestations of CINCA syndrome. We speculate that one reason our patient developed CINCA syndrome despite the somatic mosaicism of the CIAS1 mutation is that the Y570C mutation is particularly CIAS1 SOMATIC MOSAICISM IN CINCA SYNDROME potent in inducing ASC-dependent NF-B activation. In this sense, it may be useful to catalog the CIAS1 mutations that cause CINCA syndrome according to their relative ability to induce ASC-dependent NF-B activation; this may help in focusing on certain regions of the CIAS1 gene in the search for possible CIAS1 mosaicism. Furthermore, somatic mosaicism mutation analysis will help to identify the most suitable patients in whom to investigate for new candidate genes whose mutation(s) can cause CINCA syndrome. In conclusion, we have identified a patient with CINCA syndrome who had CIAS1 somatic mosaicism. This case reveals that CIAS1 somatic mosaicism is one reason CIAS1 mutations have not been detected in some cases of CINCA syndrome. ACKNOWLEDGMENTS We would like to thank Dr. T. Suda (Cancer Research Institute, Kanazawa University, Ishikawa, Japan) for providing the CIAS1 complementary DNA and Dr. J. 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