Kuniaki Seyama

Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis

AbstractPulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM.

Lymphangiogenesis-mediated shedding of LAM cell clusters as a mechanism for dissemination in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) affects exclusively women of reproductive age, involves the lungs and axial lymphatic system, and is frequently complicated with renal angiomyolipomas. LAM lesions are generated by the proliferation of LAM cells with mutations of one of the tuberous sclerosis complex (TSC) genes. Recent studies indicate that LAM cells can migrate or metastasize to form new lesions in multiple organs, although they show a morphologically benign appearance. In the previous study, we reported LAM-associated lymphangiogenesis and implicated its role in the progression of LAM. In this study, we further focused on the lymphatic abnormalities in LAM: LAM-associated chylous fluid (5 pleural effusion and 2 ascites), surgically resected diaphragm (1 patient), and axial lymphatic system including the thoracic duct, lymph nodes at various regions, and diaphragmatic lymphatic system (5 autopsy cases). We demonstrated that LAM cell clusters enveloped by lymphatic endothelial cells (LCC) in all chylous fluid examined. We identified LAM lesion in the diaphragm (2 of 5 autopy cases and one surgical specimen), thoracic duct (5 of 5), and lymph nodes (retroperitoneal (5 of 5), mediastinal (4 of 5), left venous angle (5 of 5) with total positive rate of 68% to 88% at each region of the lymph node, but less frequent or none at remote lymph nodes located away from the axial lymph trunk (cervical [1 of 5] and axillary [0 of 5]). LCCs were identified in intra-LAM lesional lymphatic channels where LAM cells proliferate along lymphatic system. In in vitro culture system, LCC can fragment into each proliferating LAM cell. These findings suggest that LAM-associated lymphangiogenesis demarcates LAM lesion into bundle- or fascicle-like structure and eventually shed LCC into the lymphatic circulation and that LCCs play a central role in the dissemination of LAM lesion.

Lymphangiogenesis in Lymphangioleiomyomatosis Its Implication in the Progression of Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is characterized by the proliferation of abnormal smooth muscle cells (LAM cells) in the lungs, lymph nodes, and/or other organs. We examined lymphangiogenesis using immunohistochemistry for Flt-4 (VEGFR-3), a new specific marker for lymphatic endothelial cells, as well as the expression of vascular endothelial growth factor (VEGF)-C in LAM. Specimens were obtained from 6 autopsy cases, a single lung transplant case, and 8 surgical cases for analyses. We demonstrated that lymphatics were extremely abundant in both pulmonary and extrapulmonary LAM and that lymphatic endothelial cells not only proliferated encompassing LAM foci but also infiltrated the intra-LAM foci, and that in advanced LAM, lymphangiogenesis involved vascular walls and interstitium surrounding the area where LAM cells proliferate. In contrast, angiogenesis, confirmed with CD31 immunostaining, was observed less in the LAM foci. LAM cells demonstrated positive reactivity against anti-VEGF-C antibody at varying intensities. Significant correlation (P < 0.001) was noted between the degree of lymphangiogenesis in LAM or VEGF-C expression on LAM cells and lymphagioleiomyomatosis histologic score (LHS), which represents the histologic severity of pulmonary LAM and has been reported to have prognostic significance. Our study is likely to provide a novel point of view on the pathophysiologic significance of lymphangiogenesis in LAM.

Mutations of the Birt-Hogg-Dube gene in patients with multiple lung cysts and recurrent pneumothorax

Rationale: Birt–Hogg–Dubé (BHD) syndrome, a rare inherited autosomal genodermatosis first recognised in 1977, is characterised by fibrofolliculomas of the skin, an increased risk of renal tumours and multiple lung cysts with spontaneous pneumothorax. The BHD gene, a tumour suppressor gene located at chromosome 17p11.2, has recently been shown to be defective. Recent genetic studies revealed that clinical pictures of the disease may be variable and may not always present the full expression of the phenotypes. Objectives: We hypothesised that mutations of the BHD gene are responsible for patients who have multiple lung cysts of which the underlying causes have not yet been elucidated. Methods: We studied eight patients with lung cysts, without skin and renal disease; seven of these patients have a history of spontaneous pneumothorax and five have a family history of pneumothorax. The BHD gene was examined using PCR, denaturing high-performance liquid chromatography and direct sequencing. Main results: We found that five of the eight patients had a BHD germline mutation. All mutations were unique and four of them were novel, including three different deletions or insertions detected in exons 6, 12 and 13, respectively and one splice acceptor site mutation in intron 5 resulting in an in-frame deletion of exon 6. Conclusions: We found that germline mutations of the BHD gene are involved in some patients with multiple lung cysts and pneumothorax. Pulmonologists should be aware that BHD syndrome can occur as an isolated phenotype with pulmonary involvement.

Clinical and genetic spectrum of Birt–Hogg–Dubé syndrome patients in whom pneumothorax and/or multiple lung cysts are the presenting feature

Background Birt–Hogg–Dubé syndrome (BHDS) is an inherited autosomal genodermatosis characterised by fibrofolliculomas of the skin, renal tumours and multiple lung cysts. Genetic studies have disclosed that the clinical picture as well as responsible germline FLCN mutations are diverse. Objectives BHDS may be caused by a germline deletion which cannot be detected by a conventional genetic approach. Real-time quantitative polymerase chain reaction (qPCR) may be able to identify such a mutation and thus provide us with a more accurate clinical picture of BHDS. Methods This study analysed 36 patients with multiple lung cysts of undetermined causes. Denaturing high performance liquid chromatography (DHPLC) was applied for mutation screening. If no abnormality was detected by DHPLC, the amount of each FLCN exon in genome was quantified by qPCR. Results An FLCN germline mutation was found in 23 (63.9%) of the 36 patients by DHPLC and direct sequencing (13 unique small nucleotide alterations which included 11 novel mutations). A large genomic deletion was identified in two of the remaining 13 patients by qPCR (one patient with exon 14 deletion and one patient with a deletion encompassing exons 9 to 14). Mutations including genomic deletions were most frequently identified in the 3′-end of the FLCN gene including exons 12 and 13 (13/25=52.0%). The BHDS patients whose multiple cysts prompted the diagnosis in this study showed a very low incidence of skin and renal involvement. Conclusions BHDS is due to large deletions as well as small nucleotide alterations. Racial differences may occur between Japanese and patients of European decent in terms of FLCN mutations and clinical manifestations.

Characteristics of pulmonary cysts in Birt–Hogg–Dubé syndrome: Thin-section CT findings of the chest in 12 patients

PURPOSE To describe in detail the characteristic chest computed tomography (CT) findings of Birt-Hogg-Dubé (BHD) syndrome. MATERIALS AND METHODS Thin-section chest CT scans of consecutive 12 patients with genetically diagnosed BHD syndrome were retrospectively evaluated by two observers, especially about the characteristics (distribution, number, size, shape and relation to pleura) of pulmonary cysts. Interobserver agreement in the identification of abnormalities on the CT images was achieved using the κ statistic, and the degree of interobserver correlation for the characterization of pulmonary cysts was assessed using the Spearman rank correlation coefficient. RESULTS Multiple pulmonary cysts were seen in all patients. The number of cysts in each patient was various (range, 29-407), and cysts of various sizes (from a few mm to 2 cm or more) were seen in all patient. 76.6% (mean) of cysts were irregular-shaped, and 40.5% (mean) of cysts were located along the pleura. The mean extent score of cysts was 13% of the whole lung, and the distribution of cysts was predominantly in the lower medial zone. Finally, cysts abutting or including the proximal portions of lower pulmonary arteries or veins were also seen in all patients. CONCLUSION Multiple, irregular-shaped cysts of various sizes with lower medial lung zone predominance are characteristic CT findings of BHD syndrome. Cysts abutting or including the proximal portions of lower pulmonary arteries or veins may also exist in this syndrome in a high probability.

Induction of human neutrophil chemotaxis by Candida albicans-derived β-1,6-long glycoside side-chain-branched β-glucan

Polysaccharide β‐1,3‐D‐glucans (β‐glucans) are components of the cell wall of various fungi and show immunomodulatory activities. β‐Glucans have been reported to enhance neutrophil accumulation during pathogenic fungi‐induced lung inflammation. Therefore, we examined whether β‐glucans themselves possess chemotactic activities for human neutrophils. Among several kinds of β‐glucans, β‐1,6‐long glucosyl side‐chain‐branched β‐glucan, isolated from Candida albicans [Candida soluble β‐D‐glucan (CSBG)], dose‐dependently induced neutrophil migration in a Boyden chamber system. In contrast, 1,6‐monoglucosyl‐branched β‐glucans, such as Sparassis crispa‐derived β‐glucan (SCG) and grifolan (GRN), which were derived from nonpathogenic fungi, hardly induced neutrophil migration. Moreover, CSBG‐induced neutrophil migration was inhibited completely by liposomes containing neutral glycosphingolipid lactosylceramide (LacCer; Galβ1‐4Glc‐ceramide) but not NeuAcα2‐3Galβ1‐4Glcβ1‐1′‐Cer ganglioside. Furthermore, binding experiments demonstrated that CSBG bound to glycosphingolipids (such as LacCer) with a terminal galactose residue; however, SCG and GRN (1,6‐monoglucosyl‐branched β‐glucans) did not bind to LacCer. It is important that a Src kinase inhibitor protein phosphatase 1, a phosphatidylinositol‐3 kinase (PI‐3K) inhibitor wortmannin, and a Gαi/o inhibitor pertussis toxin inhibited neutrophil migration toward CSBG. Taken together, our results suggest that β‐1,6‐long glucosyl side‐chain‐branched β‐glucan CSBG binds to LacCer and induces neutrophil migration through the activation of Src family kinase/PI‐3K/heterotrimeric G‐protein signal transduction pathways.

The epidemiology of lymphangioleiomyomatosis in Japan: a nationwide cross-sectional study of presenting features and prognostic factors.

Background and objective:  To evaluate the characteristics and prognostic factors of Japanese patients with lymphangioleiomyomatosis (LAM).

Senescence marker protein-30 knockout mouse as a novel murine model of senile lung

Senescence marker protein‐30 (SMP30) was originally identified as a novel protein of which expression decreases in an androgen‐independent manner with aging in the rat liver and functions to protect cells from apoptosis. By reverse transcription–polymerase chain reaction analysis, SMP30 mRNA transcripts were found in the mouse lung, liver, kidney, testis and cerebrum. We examined SMP30 expression in the mouse liver, kidney and lung during aging and a distinct temporal profile of SMP30 expression was found in each tissue; the SMP30 mRNA level peaked at 1–3 months of age and decreased thereafter in the liver (the highest at 1 month of age followed by a rapid decline and consistently low thereafter in the kidney), and peaked at 12 months of age in the lung. To investigate the physiological role of SMP30 in the lung, immunohistochemical studies of wild‐type (SMP30Y/+) mice and histopathological examinations of SMP30 knockout (SMP30Y/–) mice were performed. Immunoreactivity against anti‐SMP30 antibody was mainly detected in bronchial epithelial cells and strongly detected at 6–12 months of age. Morphometric analysis was performed to measure the mean linear intercept and destructive index, and found peripheral airspace enlargement without alveolar destruction in SMP30Y/– mice at 1, 3 and 6 months of age compared with the SMP30Y/+ mice. Our results strongly suggest that SMP30Y/– mice could be a novel model for a senile lung and further examinations of SMP30Y/– mice may offer clues to elucidate the mechanisms of the development of pulmonary diseases in the elderly.

Official American Thoracic Society/Japanese Respiratory Society Clinical Practice Guidelines: Lymphangioleiomyomatosis Diagnosis and Management

BACKGROUND Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease that primarily affects women. The purpose of these guidelines is to provide recommendations for the diagnosis and treatment of LAM. METHODS Systematic reviews were performed to summarize evidence pertinent to our questions. The evidence was summarized and discussed by a multidisciplinary panel. Evidence-based recommendations were then formulated, written, and graded using the Grading of Recommendations, Assessment, Development, and Evaluation approach. RESULTS After considering the panels confidence in the estimated effects, the balance of desirable (i.e., benefits) and undesirable (i.e., harms and burdens) consequences of treatment, patient values and preferences, cost, and feasibility, recommendations were formulated for or against specific interventions. These included recommendations for sirolimus treatment and vascular endothelial growth factor D testing and recommendations against doxycycline and hormonal therapy. CONCLUSIONS Evidence-based recommendations for the diagnosis and treatment of patients with LAM are provided. Frequent reassessment and updating will be needed.