Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis

Abstract

Lymphangioleiomyomatosis (LAM) is a progressive and often fatal interstitial lung disease characterized by a diffuse proliferation of abnormal smooth muscle cells in the lungs. LAM is of unusual interest biologically because it affects almost exclusively young women. LAM can occur as an isolated disorder (sporadic LAM) or in association with tuberous sclerosis complex. Renal angiomyolipomas, which are found in most tuberous sclerosis patients, also occur in 60% of sporadic LAM patients. We previously found TSC2 loss of heterozygosity in 7 of 13 (54%) of angiomyolipomas from sporadic LAM patients, suggesting that LAM and TSC could have a common genetic basis. In this study, we report the identification of somatic TSC2 mutations in five of seven angiomyolipomas from sporadic LAM patients. In all four patients from whom lung tissue was available, the same mutation found in the angiomyolipoma was present in the abnormal pulmonary smooth muscle cells. In no case was the mutation present in normal kidney, morphologically normal lung, or lymphoblastoid cells. Our data demonstrate that somatic mutations in the TSC2 gene occur in the angiomyolipomas and pulmonary LAM cells of women with sporadic LAM, strongly supporting a direct role of TSC2 in the pathogenesis of this disease.

Figures

Figure 1

Examples of SSCP analyses. The variant band is indicated with an arrow. (A) Patient 487. The variant band in the angiomyolipoma (A) was present in microdissected LAM cells from two separate paraffin blocks (L1 and L2), but not in normal kidney (NK) or in two regions of normal lung (NL1 and NL2). The variant band was more intense in specimen L2, which contained more actin-positive cells than specimen L1. (B) Patient 490. The variant band in the angiomyolipoma (A) and microdissected LAM cells (L) was not present in normal kidney (NK), or microdissected normal lung (NL). (C) Patient 491. The variant band in the angiomyolipoma (A) was present in microdissected LAM cells (L) and faintly present in the microdissected specimen that consisted primarily of smooth muscle actin-negative cells (NL). This band was absent from the lymphoblastoid cells (Fig. 2C) and from control lymphoblastoid cells, an example of which is shown in the lane labeled C. The four lanes are from the same gel. (D) Patients 621 and 651. The angiomyolipomas from patients 621 and 651 had the same exon 16 mutation. The variant bands in the angiomyolipomas (A) were not present in the lymphoblastoid (LB) cells of either patient. For patient 651, the variant bands were present in the microdissected LAM cells (L) but not in normal kidney or normal lung.

Figure 2

Examples of DNA sequencing results. (A) Patient 487. Wild-type sequence is present in normal kidney DNA. Overlapping peaks representing the wild-type (G) and mutant (T) bases are present in the angiomyolipoma (arrow). The variant band in DNA from the angiomyolipoma was cut from the SSCP gel, reamplified, and sequenced (angiomyolipoma variant band). Only the mutant sequence, containing the T, is present. Overlapping peaks are seen at the same position in the microdissected L2 LAM cells (arrow). The mutant peak is predominant in both the LAM and the angiomyolipoma, consistent with the LOH results. (B) Patient 490. A 13-bp deletion in one copy of TSC2 results in overlapping peaks beginning at position 2061 in the angiomyolipoma (arrow). DNA from the angiomyolipoma variant band that was cut from the gel shows the mutant sequence, with the arrow indicating the first affected base. (C) Patient 491. A 4-bp deletion in one copy of TSC2 results in overlapping peaks beginning at position 529 in the angiomyolipoma (arrow). The angiomyolipoma variant band shows the deletion, indicated by the open triangle. The deletion, indicated by the four outlined bases, is not present in lymphoblastoid cells. (D) Patient 621. Wild-type sequence is seen in lymphoblastoid cells and normal kidney. Overlapping peaks of wild-type sequence (G) and mutant (A) are present in the angiomyolipoma (arrow). (E) Patient 651. Wild-type sequence is seen in lymphoblastoid cells, normal kidney, and normal lung. Overlapping peaks of wild-type sequence (G) and mutant (A) are present in the angiomyolipoma and LAM cells (arrows).

Figure 3

Examples of chromosome 16p13 LOH analyses. The upper band of each allele is indicated with a line and the lost allele is indicated with an arrow. (A) Patient 487. Two alleles of the marker kg8 are present in normal kidney (NK) and normal lung (NL). Decreased intensity of the upper allele was found in the angiomyolipoma (A) and microdissected LAM specimen (L2) consistent with LOH. (B) Patient 490. Two alleles of the marker D16S525 are present in normal kidney and normal lung. Each allele is represented by a ladder of three or four bands, the highest of which is indicated with a line. Decreased intensity of the lower allele was found in the angiomyolipoma (A) and microdissected LAM specimen (L) consistent with LOH. (C) Patient 621. Two alleles of the marker D16S291 are present in normal kidney. Each allele is represented by two bands. Decreased intensity of the upper allele was found in the angiomyolipoma (A) consistent with LOH. (D) Patient 651. Two alleles of the marker D16S283 are seen in lymphoblastoid cells (LB), normal kidney, and normal lung. Each allele is represented by a ladder of three bands. Decreased intensity of the upper allele was seen in the angiomyolipoma (A) consistent with LOH. Decreased intensity of the upper allele was also seen in the LAM cells (L), suggestive of LOH.

Figure 4

Patient 487. Adjacent sections of the lung biopsy stained with hematoxylin and eosin (A) and immunostained with muscle-specific actin (B). Regions with primarily actin-positive cells are evident. Within these regions, actin-negative cells are also present. Microdissection from this region represented the L2 specimen in Fig. 1A.