Fig 1.
Relationships among and characteristics of patients in this study.
The three patients had multifocal micronodular pneumocyte hyperplasia. Patients 1 and 3 (daughter and son) were diagnosed with tuberous sclerosis complex in accordance with the 2012 diagnostic criteria. The father of patients 1 and 3 did not display any clinical indications of a tuberous sclerosis complex. *Major features included in the 2012 diagnostic criteria.
Fig 2.
Radiological and histopathological findings of multifocal micronodular pneumocyte hyperplasia.
(A) Chest high-resolution computed tomography (HRCT) image of patient 1 (daughter) showed several ground glass nodules measuring 1–7 mm in size. Cystic changes suggestive of lymphangioleiomyomatosis (LAM) were not observed. (B–D) Histopathological images of multifocal micronodular pneumocyte hyperplasia (MMPH) of patient 1. (B) Low-powered view (×20), haematoxylin and eosin (HE) staining. There were well-demarcated, nodular lesions ranging from 2 to 5 mm in the pulmonary parenchyma. (C) High-powered view (×200), HE staining. Alveolar structure was maintained. Enlarged type II pneumocytes lining the thickened alveolar septa had abundant eosinophilic cytoplasm and spherical nuclei with mild nuclear atypia. (D) High-powered view (×200), Elastica-Masson staining. The alveolar septa were thickened by proliferation of elastic fibres. (E) Chest HRCT image of patient 2 (mother) showed several micro ground glass nodules. (F) Histopathological image of lung specimen of patient 2. High-powered view (×200), HE staining. Multifocal well-demarcated nodular lesions were observed. Enlarged cuboidal type II pneumocytes had swollen nuclei. The alveolar septa were thickened by fibrous proliferation. (G) Chest HRCT image and (H) histopathological image, high-powered view (×200), HE staining, of the lung lesion of patient 3 (son) showed findings consistent with MMPH.
Fig 3.
Axial magnetic resonance imaging (MRI; T2 fluid-attenuated inversion recovery) (A) and computed tomography (CT) imaging (B) of patient 3. The high-intensity area near the left lateral ventricle represents cortical dysplasia (tubers and radial migration lines; white arrow). The high-intensity area, which was observed in MRI (black arrow) with calcification in CT (white arrowhead) near the left Monro foramen, represents a subependymal nodule. (C) MRI (T2 fluid-attenuated inversion recovery) of patient 1. The high-intensity area in the left frontal lobe represents a cortical tuber (black arrowhead).
Fig 4.
Analysis of RNA and introns of TSC1 and TSC2.
Three patients (patients 1, 2, and 3), their healthy father (husband of patient 2), and two healthy controls were assessed. Total RNA was extracted from peripheral blood lymphocytes and template DNA was synthesised. (A, B) Reverse transcription-PCR (RT-PCR) with the template DNA and primer pair covering exons 5–7 of TSC1. Full-length agarose gel electrophoresis of RT-PCR products is shown in S1 Fig. In lanes for all three patients, two additional, longer amplification product bands were observed (mutations 1 and 2). (C) Direct DNA sequencing with products amplified using RT-PCR revealed an insertion of 68 base pairs (bp) (mutation 1) or 92 bp (mutation 2) of a part of the intron 5 sequence between exons 5 and 6. (D) The sequences of a part of intron 5 for the three patients and their father were detected via direct sequencing of DNA extracted from peripheral blood. The three patients harboured point mutations (c.363+668G>A) in the heterojunction. (E) A section of intron 5. Intron 5 consists of 2096 bp in total. A segment of the sequence of intron 5 of TSC1 including the point mutation was detected by direct DNA sequencing. Sixty-eight base pairs (yellow) and 92 bp (yellow and green) were inserted between exons 5 and 6, as shown in (C). The 6-bp sequence including the point mutation (light blue) may serve as a new branch point. AG (red) immediately upstream of the inserted sequence may function as the splice acceptor site, and 2 GT (brown) after the inserted sequence may function as the splice donor sites. Therefore, the point mutation may cause splicing anomalies. Because 68 and 92 are not multiples of 3, the splicing anomaly may cause a frame shift and loss of function of TSC1 in the three patients.
Fig 5.
Loss of heterozygosity (LOH) analysis of lung tissue from patient 2 (mother).
Polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) revealed that mutant alleles increased due to LOH in all lung lesions of patient 2 (mother). (A) DNA was extracted from four lesions in paraffin-embedded lung tissue obtained via video-assisted thoracic surgery lung biopsy. Lesions 1–3 included numerous multifocal micronodular pneumocyte hyperplasia (MMPH) cells, and lesion 4 included scant MMPH cells. (B) PCR-RFLP analysis of lung lesions. Full-length agarose gel electrophoresis of RT-PCR products is shown in S2 Fig. Lane numbers match the lesion numbers in (A). In all four lanes, there were bands indicating 30- and 91-bp fragments cleaved by SspI. (C) PCR-RFLP analysis of peripheral blood DNA from the three patients, the father, and one healthy control. Full-length agarose gel electrophoresis of RT-PCR products is shown in S3 Fig. Lanes 1–5 indicate the father, patient 2, patient 1, patient 3, and the healthy control, respectively. In the three lanes for the patients, there were 30- and 91-bp bands indicating the mutant alleles. (D, E) Densitometry analysis of bands in lung lesion DNA (D) and peripheral blood DNA (E). Letters indicating the area match those in (B, C). The area of each band determined via densitometric analysis is shown in Tables 1 and 2.
Table 1.
Densitometric analysis of lung lesions in patient 2 (mother).
Table 2.
Densitometric analysis of DNA from peripheral blood of each patient.
Fig 6.
Immunohistochemical staining of lung lesions in patient 2.
Multifocal micronodular pneumocyte hyperplasia (MMPH) lesions of patient 2 were assessed immunohistochemically. High-powered views (×200). Phospho-p70S6K (A) and phospho-4E-BP1 (B) were expressed in the cytoplasm of MMPH cells. Phospho-AKT was not expressed in MMPH cells (C).