A patient with TSC1 germline mutation whose ... - Wiley Online Library

Journal of Internal Medicine 2004; 256: 166–173

CASE REPORT

A patient with TSC1 germline mutation whose clinical phenotype was limited to lymphangioleiomyomatosis T. SATO1,2, K. SEYAMA1,2, T. KUMASAKA3, H. FUJII3, Y. SETOGUCHI1, T. SHIRAI3, Y. TOMINO4, O. HINO2,3 & Y. FUKUCHI1 From the 1Department of Respiratory Medicine, Juntendo University School of Medicine, Hongo Bunkyo-Ku, Tokyo; 2Department of Experimental Pathology, Cancer Institute, Kami-Ikebukuro Toshima-Ku, Tokyo; Departments of 3Pathology and 4Nephrology, Juntendo University School of Medicine, Hongo Bunkyo-Ku, Tokyo; Japan

Abstract. Sato T, Seyama K, Kumasaka T, Fujii H, Setoguchi Y, Shirai T, Tomino Y, Hino O, Fukuchi Y (Juntendo University School of Medicine and Cancer Institute, Tokyo, Japan). A patient with TSC1 germline mutation whose clinical phenotype was limited to lymphangioleiomyomatosis (Case report). J Intern Med 2004; 256: 166–173. Background: Lymphangioleiomyomatosis (LAM) can occur as in isolated form (sporadic LAM) or as a pulmonary manifestation of tuberous sclerosis complex (TSC) (TSC-associated LAM). Recent studies, however, revealed that both forms of LAM are genetically related but that sporadic LAM is a distinct clinical entity caused by somatic mutations of TSC2 (not TSC1) rather than a forme fruste of TSC carrying either of the TSC1 or TSC2 germline mutations. Method: Case presentation and in-depth molecular and histopathological examinations. A 34-year-old Japanese woman was diagnosed as having pulmonary lymphangioleiomyomatosis (LAM) when bilateral pneumothoraces were surgically treated in 1992. Although slowly progressive renal disfunction was observed due to bilateral multiple renal cysts during the past 4 years, she had no other

Introduction Lymphangioleiomyomatosis (LAM) is a rare disorder that occurs almost exclusively in women of reproductive age. LAM is characterized by hamartomatous proliferation of smooth muscle cells (LAM cells) 166

clinical features of TSC and was diagnosed as having sporadic LAM with multiple renal cysts of undetermined aetiology. Her subsequent clinical course was complicated by an endobrochial carcinoid tumour, which eventually resulted in her death in June 1999 due to massive haemoptysis. Results: Postmortem examination revealed the presence of LAM lesions in the lungs, mediastinal lymph nodes, kidneys and uterus. Diffuse renal LAM lesions are presumed to generate multiple renal cysts by constricting the nephron rather than epithelial hyperplasia obstructing lumina, which is analysis of the TSC genes demonstrated that she did not have TSC2/PKD1 contiguous gene syndrome but had a TSC1 germline mutation (Sato T et al. J Hum Genet 2002; 47: 20–8) that had occured de novo. Conclusion: This patient therefore illustrates that clinical manifestations of TSC are sufficiently diverse as to allow a forme fruste of TSC that mimics sporadic LAM and that TSC1 mutation can cause multiple renal cysts resulting in renal failure. Keywords: contiguous gene syndrome, forme fruste, germline mutation, lymphangioleiomyomatosis, polycystic kidney disease, tuberous sclerosis complex.

in the lungs and frequently involves lymphatic structures such as mediastinal and retroperitoneal lymph nodes [1]. This condition can occur as an isolated form (sporadic LAM) or as a pulmonary manifestation of tuberous sclerosis complex (TSC) (TSC-associated LAM), an autosomal dominant 2004 Blackwell Publishing Ltd

TSC1 GERMLINE MUTATION

inheritance disorder characterized by hamartomatous lesions in the brain, retina, skin, heart, kidney and lungs. TSC is a tumour-suppressor syndrome caused by mutations in either of the two genes, TSC1 or TSC2 [2, 3]. As both forms of LAM are histopathologically identical and are often complicated with renal angiomyolipoma (AML), both could be aetiologically related and sporadic LAM would represent a forme fruste of TSC [4]. Recent studies, however, revealed that both forms of LAM are genetically related but that sporadic LAM is a distinct clinical entity caused by somatic mutations of TSC2 (not TSC1) rather than a forme fruste of TSC carrying either of the TSC1 or TSC2 germline mutations. Complete inactivation of TSC2 function was demonstrated in microdissected LAM cells from renal AMLs and the lungs [5], but patients with sporadic LAM do not carry the TSC2 germline mutation [6]. The most common sites of extrapulmonary LAM are along the axial lymphatics including mediastinal, retroperitoneal and pelvic lymph nodes [7], and patients with LAM frequently develop renal AMLs [8]. The kidneys are commonly affected in TSC, and cysts and AMLs are representative renal lesions of TSC. According to a cross-sectional study of 139 patients with TSC, the prevalence of renal involvement was reported to be 61% [9]. However, TSC patients with renal failure appear to be rare, and its prevalence is estimated to be approximately 1% of TSC patients [10]. Amongst underlying renal diseases of 65 patients with TSC and end-stage renal failure requiring haemodialysis, AMLs alone were present in 23.1%, renal cysts alone in 18.5% and both in 53.8% of cases [10]. Regarding the development of polycystic kidney diseases in patients with TSC, the role of PKD1, a causative gene for autosomal dominant polycystic kidney disease (ADPKD) [11], has been reported to be important [12]. As TSC2 and PKD1 lie adjacent to each other in chromosomal region 16p13.3, a contiguous gene syndrome with deletions disrupting both TSC2 and PKD1 simultaneously has been described in TSC patients with severe polycystic kidney diseases [13]. Accordingly, the majority of TSC patients with significant renal cystic diseases are considered to reflect the involvement of PKD1 in addition to TSC2 and the role of TSC1 in this condition seems very small [12]. Here we describe the histopathological findings of an adult female who can be diagnosed as having

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sporadic pulmonary LAM with renal failure due to multiple renal cysts rather than TSC-associated LAM. Autopsy and genetic analysis disclosed that she had a TSC1 germline mutation and its phenotypic expression was limited to LAM lesions in the lungs, mediastinal lymph nodes, kidneys and uterus.

Case report A 34-year-old Japanese female was admitted to the hospital because of sudden onset of chest pains and shortness of breath in August 1992. Chest X-rays revealed bilateral pneumothoraces. She had undergone a supravaginal hysterectomy because of adenomyosis and leiomyoma of the uterus at the age of 25 years. She had a 4-year history of proteinuria and chronic renal dysfunction, and her serum creatinine level at admission was 8.8 mg dL)1 (reference value 0.5–0.8 mg dL)1). Although the size of both kidneys was almost within normal limits, multiple bilateral renal cysts were detected by ultrasonography and computed tomography (CT) (Fig. 1a). Neither AMLs nor cysts in other organs were detected. Accordingly, a renal biopsy had never been performed and the precise aetiology of renal dysfunction remains unclarified. There was no history of mental retardation or seizures and no family history of TSC or polycystic kidney disease. Chest tube drainage failed to adequately control the pneumothoraces, therefore bilateral bullectomy with concurrent lung biopsy was performed with median sternotomy in September 1992. Histopathological examination of the resected lung tissue established the diagnosis of pulmonary LAM. Chest CT images taken after surgery for the treatment of pneumothoraces revealed characteristic thin-walled cysts in the lungs (Fig. 1b). She had no skin lesions such as facial angiofibromas and periungual fibromas. No

Fig. 1 CT findings of the abdomen and chest. (a) Multiple cysts in both kidneys without angiomyolipoma (AML) were identified. (b) Multiple thin-walled cysts were revealed in both lungs.

2004 Blackwell Publishing Ltd Journal of Internal Medicine 256: 166–173

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abnormality of the brain such as cortical tubers, intracerebral calcifications and subependymal nodules was identified with cranial CT. Ophthalmological examinations revealed no bilateral hamartomatous lesions in the optic fundi. According to the clinical diagnostic criteria for TSC [14], she was diagnosed as having sporadic LAM from the absence of clinical signs and features of TSC. She was discharged in November 1992 and managed with haemodialysis. Two years later, she occasionally expectorated bloody sputum and had haemoptysis in February 1995. Fibreoptic bronchoscopy revealed a tumour obstructing the right intermediate trunk and transbronchial biopsy disclosed a bronchial carcinoid tumour. Because of severely impaired renal functions, surgical resection of the carcinoid tumour could not be performed and she was treated with supportive care. Thereafter, she was repeatedly hospitalized due to obstructive pneumonia and gradual progression of the endobronchial carcinoid tumour, which eventually led to massive haemoptysis and death in early June 1999. A postmortem examination was performed, but examination of the brain was not permitted. A part of the clinical characteristics and serial changes of pulmonary function tests have been previously presented (case 9 in ref. [15]).

Materials and methods The lungs, kidneys and other tissues obtained at autopsy were fixed in 10% buffered neutral formaldehyde solution for 24–48 h, embedded in paraffin after routine processing, sectioned and stained with haematoxylin and eosin (H & E). The archival paraffin-embedded blocks of the uterus obtained during supravaginal hysterectomy were also available. Immunohistochemical stainings were performed on paraffin-embedded sections using monoclonal antibodies to a-smooth muscle actin (a-SMA) (dilution 1 : 200; Dako corporation, Tokyo, Japan), oestrogen receptor-a (ER) (dilution 1 : 50; Novocastra, Newcastle, UK), cytokeratin (dilution 1 : 100; Immunotech, Cedex, France), HMB-45 (dilution 1 : 30; Dako) and microphthalmia transcription factor (MIFT) (dilution 1 : 50; Novocastra). An LSAB kit for a-SMA and cytokeratin immunostaining and an EnVision kit for HMB-45 and MITF immunostaining were utilized to detect antibody

binding and 3, 3¢-diaminobenzidine tetrahydrochloride was used as the chromogen for all markers except for ER. For ER, biotinylated anti-mouse rabbit antibody (Dako) and alkaline phosphatase-conjugated streptoavidin (Dako) were utilized to detect antibody binding and fast red was used as the chromogen. Microdissection from paraffin-embedded tissues and sequencing of the TSC1 gene were performed as described previously [16]. Microsatellite markers of TSC1 loci (D9S149, D9S1198 and D9S1199) were amplified by PCR using fluorescence-labelled primers (Applied Biosystems, Foster City, CA, USA) and DNA fragments was separated by ABI model 377 sequencer equipped with GeneScan software (Applied Biosystems).

Results Postmortem findings supported the clinical diagnosis of sporadic LAM, although an examination of the brain was not allowed. Pathological findings were noted exclusively in the lungs, mediastinal lymph nodes and kidneys. Macroscopically, the left lung and the right upper lobe showed diffuse emphysematous changes and the right middle and lower lobes had obstructive pneumonia with a bronchial carcinoid tumour (3.0 · 3.0 · 2.5 cm) extruding from the right B6 segmental bronchus to the right main bronchus. Several moderately swollen lymph nodes were observed in the mediastinum. Both kidneys were slightly atrophic and the majority of renal parenchyma was occupied by multiple cysts. No nodules were observed on the surface or sections of the kidneys. Histopathological examination of all organs except the brain was carried out and revealed that LAM cells proliferated not only in the lungs and mediastinal lymph nodes, but also in the kidneys. The architecture of the lung was destroyed and replaced by numerous cysts. Spindle-shaped LAM cells with clear cytoplasm proliferated along the cyst wall (Fig. 2a), lymphatic vessels and bronchioles. Accumulation of haemosiderin-laden macrophages in the air spaces was noted. The majority of the LAM cells showed immunoreactivity to anti-a-SMA antibody and many of them were positively immunostained with both anti-ER and anti-progesterone receptor antibodies (data not shown). Reactivity to anti-HMB45 antibody was detected in a small



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Fig. 2 Histopathological findings of the lungs and kidneys. (a) Destructed lung parenchyma showed thickened alveolar walls where spindleshaped LAM cells proliferated with hyalinous change. Haemosiderin-laden macrophages accumulated in the air spaces (HE stain, ·100). Some of the spindle-shaped LAM cells showed characteristic immunoreactivity for HMB-45 (inset, ·400). (b) Bronchial carcinoid tumour originated from the right lower bronchus. Polygonal tumour cells with round nuclei were arranged in cords or ribbons separated by a fibrovascular stroma (HE stain, ·100). (c) At lower magnification, multiple cysts in various sizes and a few small nodules were observed in the kidneys (HE stain, ·1.25). (d) The small nodular lesions were composed of a-SMA-positive spindle-shaped cells proliferating around the renal tubuli (immunohistochemistry for ma-SMA, ·100). (e) The cyst wall was lined by a monolayer of flattened epithelium which showed immunoreactivity to cytokeratin (immunohistochemistry for cytokeratin, ·100; inset, ·400). (f) ER-positive epithelioid cells (red colour) proliferated amongst the renal tubuli (brown colour) in the renal interstitium [double immunohistochemistry for ER (red) and cytokeratin (brown), ·200]. Some of the ER-positive epithelioid cells showed immunoreactivity to anti-MITF antibody (inset: immunohistochemistry for MITF, ·200).

fraction of LAM cells (Fig. 2a, inset). The bronchial carcinoid tumour was well demarcated and consisted of uniform tumour cells of polygonal shape arranged in cords or ribbons (Fig. 2b). The tumour cells were separated by a fibrovascular stroma and few mitotic figures were observed. Microscopical examination of the kidneys revealed multiple cysts and microscopical nodules with concomitant atrophy of the glomerulo-tubular units (Fig. 2c). Nodular lesions were composed of a-SMA-positive spindle-shaped cells with clear cytoplasm (data not

shown). Some of the cysts and renal tubuli were surrounded with proliferating spindle-shaped cells with positive reactivity to anti-a-SMA antibody (Fig. 2d). The cysts were lined with flattened epithelial cells, based on their positive reactivity to anticytokeratin antibody (Fig. 2e), rather than the endothelial cells characteristic of lymphangiomatous cysts [17]. Despite extensive analysis, there was neither micropapillary epithelial hyperplasia in the cyst wall nor AMLs, which are characteristic pathological findings of the kidneys in TSC [18]. In


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hamartomatous lesion was identified in the heart, liver, pancreas, spleen or gastrointestinal tract. Mutation analysis of the TSC genes revealed that she had a germline nonsense mutation C165X resulting from a base substitution (TGCﬁTGA) in exon 6 of TSC1 that was previously reported (patient LNK8 in ref. [16]). Sequencing of the same exon in her parents and brother revealed the wildtype unmutated sequence only (Fig. 3a), suggesting that she could carry a de novo mutation in the TSC1 gene. Further analysis of microsatellite markers at the TSC1 locus demonstrated Mendelian inheritance of D9S149 in the family (Fig. 3b). Identical findings were observed in the analysis of the D9S1199 marker (data not shown). In addition, LAM cells microdissected from the lung (Fig. 3b), lymph nodes, kidneys and uterus were previously demonstrated to have identical LOH at D9S149 [16]. Assuming that the LOH serves to delete the normal TSC1 allele, it is likely that the patient TSC1 mutation is linked to the F2 allele of D9S149. As illustrated in Fig. 3, the same F2 allele was inherited from the father to the patient and his brother. Hence, the patient carried a germline TSC1 mutation that had occurred de novo on the TSC1 allele inherited from his father. Furthermore, TSC1 LOH was not demonstrated in

some of the renal interstitium, however, ER-positive epithelioid cells proliferated amongst the renal tubuli and cysts (Fig. 2f). The immunoreactivity to HMB45 was difficult to demonstrate in these ER-positive epithelioid cells, but instead MITF, another melanoma-related antigen [19], was detected in the nuclei of some ER-positive cells (Fig. 2f, inset). Archival pathological specimen of the uterus revealed nodular lesions consisting of immature smooth muscle cells with clear cytoplasm in the hypertrophic myometrium layer (data not shown). No

Fig. 3 Genetic analysis of the TSC1 gene and microsatellite marker of the TSC1 loci in peripheral blood and LAM cells. (a) Demonstration of a TSC1 germline mutation, C165X, in the patient but not in the parents and the brother. The nucleotide sequence of exon 6 in the TSC1 gene was analysed using genomic DNA obtained from peripheral blood leucocytes. A part of the sequence ladder of the parents, brother and patient was shown in the direction from 3¢ to 5¢ (i.e. nucleotide sequence is complementary to the normal nucleotide sequence). Note that two bands [wild-type (G) and mutated (T) nucleotides] were observed in the same position (indicated with an arrowhead) in the patient but not in the parents and the brother. Through this nucleotide shift (TGCﬁTGA) a premature stop codon is introduced instead of a cysteine at amino acid position 165. (b) Comparison of microsatellite marker D9S149 amongst the parents, brother and patient. D9S149 was amplified by PCR and analysed by ABI 377 Sequencer equipped with GeneScan software. Parents have four different alleles, arbitrarily designated as F1 and F2 from the father and as M1 and M2 from the mother. The brother inherited F2 and M1 whilst F2 and M2 were transmitted to the patient. LAM cells microdissected from the lung specimen of the patient showed LOH at D9S149 in which the M2 allele disappeared, indicating that the F2 allele is linked with the TSC1 allele carrying the nonsense mutation. In contrast, the brother who inherited the same F2 allele as the patient, has no TSC1 germline mutation (see Fig. 2a). No LOH was detected in carcinoid tumour cells for D9S149 or other microsatellite markers examined.



microdissected tumour cells from the bronchial carcinoid tissue (Fig. 3b).

Discussion Pulmonary LAM can occur either as a pulmonary manifestation of TSC (TSC-associated LAM) or as an isolated form (sporadic LAM). The clinical features of our patient were insufficient to establish a diagnosis of TSC-associated LAM according to the revised criteria for clinical diagnosis of TSC [14]; she has LAM and polycystic kidney disease, but has no other major features of TSC such as cortical tubers, retinal hamartomas, or skin manifestations including facial angiofibromas, subungual fibromas and hypopigmented macules. It is well known that the clinical phenotype of TSC vary widely not only amongst patients but also within families [20, 21] and even between identical twins affected with TSC [22]. As the histopathological findings of TSC-associated and sporadic LAM are identical and TSC is often variably expressed, sporadic LAM has been postulated as a forme fruste of TSC from the 1970s [4]. On the contrary, a series of studies revealed that both TSC-associated LAM and sporadic LAM are genetically related diseases, but that sporadic LAM is a distinct clinical entity caused by somatic mutations of TSC2 (not TSC1) rather than a form fruste of TSC carrying either of TSC1 or TSC2 germline mutations [5, 6]. Here we have described a patient for the first time who is clinically a sporadic LAM but genetically a TSC-associated LAM due to TSC1 mutation rather than TSC2 mutation. Our patient illustrates how the diverse clinical expressivity of TSC can generate a forme fruste of TSC that mimics sporadic LAM. It is unclear whether this particular TSC1 mutation, C165X, has some role in developing her unique phenotype when compared with other patients with TSC1 mutations. There are many studies reporting the lack of genotype-phenotype correlation in TSC [23, 24]. It is postulated therefore that the severity and phenotype of TSC would be determined by stochastic events rather than the type and location of the mutation, which is in accordance with the pathogenic mechanism of tumour suppressor genes; lesions will occur as a second hit, inactivating the wild allele, has occurred. However, TSC1 diseases seems to be milder than TSC2 diseases [25–27] and therefore a limited phenotypic expression like in our patient is expected to occur in TSC1 disease.

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As bronchial carcinoid tumour is derived from pulmonary neuroendocrine cells of which embryological origin is postulated to be a neural crest ancestor, there might be a disorder in relation to TSC [28]. However, there is no report regarding the association between TSC and carcinoid tumour in the literature. Interestingly, inactivation of the TSC1 gene was not demonstrated in carcinoid cells whilst demonstrated in LAM cells. This may be in accordance with the histopathological examinations that pulmonary neuroendocrine cells were probably derived from the endoderm [29] and makes it undetermined whether bronchial carcinoid tumours in our patient is a spectrum of TSC-related tumours. The kidneys are frequently affected in patients with TSC and approximately 50–80% of patients are found to have renal abnormalities such as AMLs and cysts [9, 17, 30]. These lesions are usually multiple and bilateral, and may enlarge to replace or compress the renal parenchyma resulting in renal failure. In TSC patients with severe renal cystic disease, cystic formation often begins during childhood, sometimes in early infancy and histopathological appearances seem to be distinctive. The cysts are lined with hypertrophic and hyperplastic tubular epithelial cells, often producing a ‘piled up’ appearance with nodular extension of the epithelium into the cyst lumen [30]. The epithelial hyperplasia is considered to be responsible for cystogenesis by obstructing the tubular or ductal lumina [30–32]. In contrast, diffuse proliferation of LAM cells and multiple cysts were prominent findings in the kidneys of our patient. Neither epithelial hyperplasia on the cyst wall nor AML was identified. To the best of our knowledge, this is the first report describing a case with diffuse LAM lesions causing polycystic kidney disease and renal failure. We postulate that proliferating LAM cells play a role in cystogenesis by constricting the renal tubules externally rather than internal obstruction of the lumina by epithelial hyperplasia – the mechanism commonly observed in TSC with polycystic kidney disease. Regarding the molecular basis of polycystic kidney disease in patients with TSC, the significant role of PDK1 has been reported [33]. In a study of unrelated TSC patients with polycystic kidney disease, 22 of 27 patients had contiguous deletions of TSC2 and PKD1. Inactivation of both copies of PKD1 in the tubular epithelium is postulated to cause hyperplasia resulting in the obstruction of lumina and renal


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cystogenesis in patients with ADPKD, as LOH and loss of the wild-type PKD1 allele was demonstrated in the epithelial cells of renal cyst [34]. A recent cohort study of 224 TSC patients revealed that the diseases associated with TSC1 were, on the average, milder than those associated with TSC2 [26]. Patients with TSC1 mutations demonstrated a lower frequency of seizures and severity of mental retardation, less severe renal involvement and facial angiofibromas and no retinal hamartomas. Renal cystic disease occurred at similar rates in patients with sporadic TSC1 and sporadic TSC2 (16% vs. 25%), but the frequency of multiple renal cysts, cysts >2 cm in size, or classic polycystic disease with renal enlargement was very low in sporadic TSC1 disease when compared with sporadic TSC2 disease (0% vs. 19%). Although the precise mechanism for LAM lesions in the development of renal cysts remains unclarified, our patient clearly indicates that a TSC1 mutation can predispose to multiple and bilateral renal cysts and renal failure. The most common sites of extrapulmonary LAM are the mediastinal and retroperitoneal lymph nodes [7], but other structures such as the uterus [35, 36], liver [35], ovary [35], ureter [37] and adrenal gland [35] are occasionally affected. Whilst the kidneys in our patient may be added to the list of sites where LAM can occur, LAM lesions in the kidney could be conceived as a wide histopathological spectrum of AMLs in which smooth muscle cells became the principal constituent. AMLs are composed of abnormal blood vessels, smooth muscle and adipose tissue in which the proportion of each tissue component varies widely. For instance, a case with sporadic pulmonary LAM who had a well-defined renal tumour composed of LAM cells [38], and a case with a clearly demarcated hepatic AML composed exclusively or predominantly of smooth muscle cells [39], have been reported. It is proposed that perivascular epithelioid cells can differentiate into adipose tissue and smooth muscle cells [40]. This is supported by the fact that AML is a neoplasm of clonal origin [41] and three cellular components have the same LOH pattern around TSC1 or TSC2 loci [5, 16]. As AMLs have been found in over 50% of patients with sporadic LAM [8] and it is one of the most common renal findings in TSC [9, 17, 30], we can postulate renal LAM in our patient as an extremely skewed AML where perivascular epithelioid cells preferentially differentiate into LAM cells

and that LAM cells have expanded throughout the renal parenchyma. The demonstration of ER-positive and MITF-positive epithelioid cells proliferating in some areas of the renal interstitium support the speculation about monotypic cell differentiation towards LAM cells in AML. However, we could not detect in our patient the area where LAM cells form tumour clearly demarcated from the renal interstitium. Further studies on LAM, AMLs and renal diseases are required to explore the potential clinical correlation between the affected TSC genes, types of mutation and diverse clinical course of perivascular epithelioid cell-related lesions.

Conflict of interest No conflict of interest was declared.

Acknowledgements The authors thank Ms Keiko Mitani and Ms Sanae Souma for their technical assistance in the works of immunohistochemistry and genetic analysis, respectively. Part of this study is supported by Grant-in-Aid for Scientific Research No. 14570563 from the Ministry of Education, Culture, Sports, Science and Technology.

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Correspondence: Kuniaki Seyama MD, PhD, Department of Respiratory Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo, Japan. (fax: +81 3-5802-1617; e-mail: [email protected]).
