Abstract
Lymphangioleiomyomatosis (LAM) (MIM #606690) is a rare lung disorder leading to respiratory failure associated with progressive cystic destruction due to the proliferation and infiltration of abnormal smooth muscle-like cells (LAM cells). LAM can occur alone (sporadic LAM, S-LAM) or combined with tuberous sclerosis complex (TSC-LAM). TSC is caused by a germline heterozygous mutation in either TSC1 or TSC2, and TSC-LAM is thought to occur as a result of a somatic mutation (second hit) in addition to a germline mutation in TSC1 or TSC2 (first hit). S-LAM is also thought to occur under the two-hit model involving a somatic mutation and/or loss of heterozygosity in TSC2. To identify TSC1 or TSC2 changes in S-LAM patients, the two genes were analyzed by deep next-generation sequencing (NGS) using genomic DNA from blood leukocytes (n = 9), LAM tissue from lung (n = 7), LAM cultured cells (n = 4), or LAM cell clusters (n = 1). We identified nine somatic mutations in six of nine S-LAM patients (67 %) with mutant allele frequencies of 1.7–46.2 %. Three of these six patients (50 %) showed two different TSC2 mutations with allele frequencies of 1.7–28.7 %. Furthermore, at least five mutations with low prevalence (<20 % of allele frequency) were confirmed by droplet digital PCR. As LAM tissues are likely to be composed of heterogeneous cell populations, mutant allele frequencies can be low. Our results confirm the consistent finding of TSC2 mutations in LAM samples, and highlight the benefit of laser capture microdissection and in-depth allele analyses for detection, such as NGS.
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Acknowledgments
We thank LAM patients and transplantation centers (Tohoku University, Okayama University, and Fukuoka University) for participating in this work. We also thank Carl Zeiss Microscopy Co., Japan for the opportunity to use the PALM MicroBeam for LAM cell microdissection, Dr. T. Kumasaka (Japan Red Cross Medical Center) for histopathologic investigations, and Ms. S. Sugimoto and K. Takabe for their technical assistance.
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This work was supported in part by a grant for Research on Measures for Intractable Diseases, a grant for Comprehensive Research on Disability Health and Welfare, the Strategic Research Program for Brain Science (SRPBS) from Japan Agency for Medical Research and Development (AMED); a Grant-in-Aid for Scientific Research on Innovative Areas (Transcription Cycle) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT); Grants-in-Aid for Scientific Research (A, B, and C), and challenging Exploratory Research from the Japan Society for the Promotion of Science (JSPS); the fund for Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation Systems from the Japan Science and Technology Agency (JST); the Takeda Science Foundation; the Yokohama Foundation for Advancement of Medical Science; and the Hayashi Memorial Foundation for Female Natural Scientists.
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.
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Fujita, A., Ando, K., Kobayashi, E. et al. Detection of low-prevalence somatic TSC2 mutations in sporadic pulmonary lymphangioleiomyomatosis tissues by deep sequencing. Hum Genet 135, 61–68 (2016). https://doi.org/10.1007/s00439-015-1611-0
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DOI: https://doi.org/10.1007/s00439-015-1611-0