title: Detection of low-prevalence somatic TSC2 mutations in sporadic pulmonary lymphangioleiomyomatosis tissues by deep sequencing.
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.
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Fujita
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan.
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Ando
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan.
authors.forename: Etsuko
authors.initials: E
authors.lastname: Kobayashi
authors.affiliation: Division of Human Pathology, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Mitani
authors.affiliation: Department of Pathology, Yokohama City University Graduate School of Medicine Yokohama, Yokohama, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Okudera
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan.
authors.forename: Mitsuko
authors.initials: M
authors.lastname: Nakashima
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Miyatake
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Tsurusaki
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan.
authors.forename: Hirotomo
authors.initials: H
authors.lastname: Saitsu
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan.
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Seyama
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan. nmiyake@yokohama-cu.ac.jp.
authors.forename: Noriko
authors.initials: N
authors.lastname: Miyake
authors.affiliation: Department of Human Genetics, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan. naomat@yokohama-cu.ac.jp.
authors.forename: Naomichi
authors.initials: N
authors.lastname: Matsumoto
mesh_terms: D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D019656:Loss of Heterozygosity; D008175:Lung Neoplasms / Q000235:genetics*; D018192:Lymphangioleiomyomatosis / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins