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.