Abstract
Lymphangiomyomatosis (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 (TSC). Because only a minority of women with TSC develops symptomatic LAM, we hypothesized that a relationship might exist between the type of germline TSC1 or TSC2 gene mutation and the risk of developing LAM. We examined all 41 exons of the TSC2 gene and 21 coding exons of the TSC1 gene for mutations in a group of 14 women with both TSC and LAM using single-strand conformation polymorphism analysis. Seven mutations were found in TSC2 and one in TSC1. Of the seven patients with TSC2 mutations, two had the same in-frame exon 40 deletion and one had an exon 41 missense change. We conclude that germline mutations in the extreme carboxy-terminus of tuberin can result in LAM. Further studies will be required to determine whether mutations in exons 40 and 41 are associated with an increased incidence and/or severity of LAM in women with TSC.
Lymphangiomyomatosis (LAM), which affects almost exclusively women, was first described 60 yr ago (1). Microscopically, LAM is characterized by a cystic distortion of the pulmonary architecture by smooth muscle cells (2). The symptoms of LAM include dyspnea, cough, and pneumothorax, with an average age at diagnosis of 33 yr (3, 4). Chest radiographs typically reveal a diffuse interstitial infiltrate with no zonal predominance, and computed tomography (CT) scans show homogeneously distributed thin-walled cysts. Although most LAM is pulmonary, retroperitoneal and pelvic lymph node involvement can also occur (5). Many patients have a slowly declining clinical course (3). Lung transplantation appears to be a valuable option for patients with end-stage disease (6, 7).
LAM can occur as an isolated disorder or in association with tuberous sclerosis complex (TSC). TSC is an autosomal dominant disorder characterized by seizures, mental retardation, autism, and hamartomatous tumors of the brain, heart, kidney, lung, and skin. These tumors include cerebral cortical tubers, subependymal giant cell astrocytomas, retinal hamartomas, cardiac rhabdomyomas, renal angiomyolipomas, and facial angiofibromas. LAM affects at least 4.6% of women with TSC (8). Among patients with TSC, LAM is the third most frequent cause of TSC-related death, after renal disease and brain tumors (8).
Germline mutations in two genes, TSC1 on chromosome 9q34 and TSC2 on chromosome 16p13, cause TSC. The TSC2 gene, cloned in 1993 (9), encodes a 1807 amino acid protein, tuberin, that has homology to Rap1 GTPase-activating protein (GAP) near its carboxy-terminus. The TSC1 gene, cloned in 1997 (10), encodes a 1164 amino acid protein, hamartin. TSC1 has no homology to known vertebrate genes.
In this study, we screened all 41 exons of TSC2 and the 21 coding exons of TSC1 for germline mutations in 14 women with TSC and symptomatic LAM. Our goal was to determine whether there is evidence of a relationship between LAM and the type or location of germline mutations in the TSC1 or TSC2 gene.
This study was approved by the Institutional Review Board of Fox Chase Cancer Center and was performed with the informed consent of the participants. All 14 patients had a clinical diagnosis of TSC, as defined by the 1998 Tuberous Sclerosis Complex Consensus Conference (11), and all had symptomatic LAM. The diagnosis of LAM was made by high-resolution CT scan in all cases. Six patients had lung biopsies that confirmed the diagnosis.
DNA was prepared from lymphoblastoid cell lines. Single-strand conformation analysis (SSCP) was used to search for mutations in each of the 41 exons of the TSC2 gene and 21 coding exons of the TSC1 gene, using 43 primer pairs for TSC2 and 26 for TSC1. The primers and polymerase chain reaction (PCR) conditions have been previously reported for TSC2 (12) and TSC1 (13). A single round of amplification using 35 cycles was performed. PCR was performed with [32P]dGTP in the reaction mix. The PCR products were run on MDE gels (FMC Bioproducts, Rockland, ME). To maximize the detection of variant bands, each PCR product was run on two gels: one without glycerol and one with 5% glycerol. All reactions were repeated at least twice for confirmation.
Samples in which variant bands were detected (Figure 1) were reamplified and sequenced in both the forward and reverse directions (Figure 2). In some cases, the variant band was also excised from the gel, reamplified, and sequenced. Sequence variations were compared with those in the on-line TSC Variation Database (http://expmed.bwh. harvard.edu/ts/), which maintains an up-to-date listing of all reported germline mutations and polymorphisms in the TSC1 and TSC2 genes. All sequencing reactions in which mutations were identified were repeated at least once for confirmation. For patients 562 and 624, in whom the same exon 40 mutation was identified, the reaction was repeated on a separately collected DNA sample.
Fig. 1. Examples of SSCP analyses. (A) Exon 9 of the TSC1 gene. The variant bands in DNA from patient 622 (lane 6) are indicated with arrows. The normal pattern of bands, indicated with lines, is seen in DNA from six other patients, in lanes 1–5 and lane 7. (B) Exon 41 of the TSC2 gene. Two separate amplifications of DNA from patient 479 are shown in lanes 1 and 2. The variant bands are indicated with arrows. The normal pattern of bands, indicated with lines, is seen in DNA from two other patients in lanes 3 and 4.
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Fig. 2. Examples of DNA sequencing results. (A) Exon 9 of the TSC1 gene. Overlapping peaks at position 1112 (arrow) representing the wild-type (T) and mutant (G) bases are present in the DNA from patient 622. The wild-type sequence contains only a T at this position (arrow). (B) Exon 9 of the TSC2 gene (sequencing shown in the reverse direction). Overlapping peaks at position 880 (arrow) representing the wild-type (which is C in the reverse direction) and mutant (T) bases are present in the DNA from patient 623. DNA from the patient's father and mother contains only the wild-type C (arrows) at this position. (C) Exon 40 of the TSC2 gene. An 18-base pair deletion in one copy of TSC2 results in overlapping peaks beginning at position 5238 in DNA from patients 562 and 624. The first position affected by the deletion is indicated with an arrow. Wild-type sequence contains only a single peak at each position. (D) Exon 41 of the TSC2 gene (sequencing shown in the reverse direction). Overlapping peaks (arrows) at position 5383 are seen in DNA from patient 479, representing the wild-type (which is G in the reverse direction) and mutant (A) sequence. The wild-type sequence contains only the G at this position.
[More] [Minimize]Of the 14 patients in this study, germline mutations were identified in eight (57%). This mutation detection rate is nearly identical to that in two recent studies that used SSCP for TSC1 and TSC2 mutation detection (14, 15). Seven mutations were in the TSC2 gene and one mutation was in the TSC1 gene. No patient had a mutation detected in both the TSC1 and the TSC2 gene, consistent with all previous studies of germline TSC1 and TSC2 mutations (16). In addition, as expected, mutations were detected in one copy of either the TSC1 or TSC2 gene, consistent with the Knudson “two-hit” tumor suppressor gene model (17). Because of this, wild-type sequence was always present along with the mutant sequence, indicating the presence of the intact, wild-type copy of the gene in the peripheral blood lymphocyte DNA.
One of the eight mutations (12%) was in the TSC1 gene (Table 1). This distribution between TSC1 and TSC2 mutations is similar to that of other studies, in which approximately 80% of germline mutations are in TSC2 and 20% in TSC1 (16). The TSC1 mutation that we found in patient 622 (Figures 1A and 2A) consisted of a 1 base pair (bp) change that is predicted to change amino acid 297 from a tyrosine to a stop codon. Nearly all TSC1 mutations, including the one we identified, are predicted to cause premature protein truncation (16).
| Patient | Gene | Exon | Nucleotide Alteration | Effect of Mutation | ||||
|---|---|---|---|---|---|---|---|---|
| 622 | TSC1 | 9 | T1112G | Tyr297Stop | ||||
| 479 | TSC2 | 41 | C5383T | Arg1795Cys | ||||
| 552 | TSC2 | 30 | C3755G | Ser1252Stop | ||||
| 562 | TSC2 | 40 | Del 5238–5255 | 6 aa in-frame deletion | ||||
| 563 | TSC2 | 7 | G760T | Gln254Stop | ||||
| 590 | TSC2 | 29 | C3442T | Gln1148Stop | ||||
| 623 | TSC2 | 9 | G880A | Gly294Arg | ||||
| 624 | TSC2 | 40 | Del 5238–5255 | 6 aa in-frame deletion |
Seven mutations were identified in TSC2 (Table 1). Three of the mutations are predicted to cause premature protein truncation because of a single base pair change resulting in a premature stop codon. Two mutations are predicted to cause a six amino acid in-frame deletion in exon 40, and two mutations are predicted to cause a single amino acid “missense” change.
Patient 623 had a missense mutation in exon 9 (Figure 2B), which would be predicted to change amino acid 294 from glycine to arginine. We verified that this represents a mutation rather than a polymorphism by demonstrating that the change is not present in either of the patient's unaffected parents (Figure 2B).
Two unrelated patients (patients 562 and 624) had the same 18-bp deletion in exon 40 (Figure 2C). This was confirmed in separately collected specimens from each patient. This mutation, which would result in a six amino acid in-frame deletion, has been previously reported by three separate groups in six patients (14, 16, 18). Jones and coworkers (16) verified that this deletion is a disease-causing mutation by demonstrating that the deletion was not present in the unaffected parents of the patient. The sex of the patients previously found to have this mutation and whether they had signs or symptoms of LAM are not known.
Two patients in our study had a family history of TSC: patients 552 and 609. Patient 552, in whom a mutation was identified in exon 30, inherited TSC from her father and has an affected sister, who has severe LAM. Patient 609, in whom no mutation was identified, also inherited TSC from her father. She has an affected adult sister with severe mental retardation who has never had a pulmonary evaluation or CT scan.
Patient 479 had a missense mutation in exon 41 (Figures 1B and 2D), which would be predicted to change amino acid 1795 from arginine to cysteine. This nonconservative amino acid change has not been previously reported among the more than 400 patients included in the TSC Variation Database.
We tested DNA from the parents of patient 479 to determine whether the exon 41 change had arisen de novo in patient 479. The father's DNA is wild type, but the mother carries the same exon 41 change (C5383T) as patient 479. The mother has never been screened for TSC. TSC screening typically includes imaging studies of the brain and kidneys and a dermatological examination including Wood's lamp examination for hypomelanotic macules (11). Therefore, the presence of the missense change in the mother is not helpful in establishing whether the exon 41 change is a benign polymorphism, or a mutation associated with TSC.
The exon 41 missense change in patient 479 has not been previously reported as either a polymorphism or mutation among the more than 350 patients in the TSC Variation Database. Because the precise number of patients in this Database in whom exon 41 was evaluated is not known, we analyzed exon 41 in a group of 50 control DNA samples from individuals with no personal or family history of TSC. None had this change. For other studies, we have previously analyzed 35 patients with TSC or sporadic LAM in exon 41 ([19, 20] and unpublished results), none of whom had this change.
The exon 40 deletion occurs within a stretch of nine amino acids that are highly conserved between human, mouse, rat, the Japanese pufferfish Fugu rubripes, and Drosophila (Figure 3A).
Fig. 3. (A) Evolutionary conservation of exon 40. Residues that are identical between human and other species are in red, residues that are similar are in green, and residues that are different are in black. The six amino acids that are deleted in patients 562 and 624 are indicated with a box. (B) Evolutionary conservation of exon 41. The last 17 amino acids of tuberin are highly conserved between mammals and Fugu. The position of the missense change from arginine to cysteine in patient 479 is indicated with an arrow.
[More] [Minimize]Amino acid 1795 in exon 41, at which we found a nonconservative amino acid change from arginine to cysteine in patient 479, is identical in human, mouse, rat, and Fugu (but not Drosophila) (Figure 3B). This amino acid occurs within a 17 amino acid region of exon 41 (representing the last 17 amino acids of tuberin) that is highly conserved between human, mouse, rat, and Fugu (Figure 3B). These carboxy-terminal-conserved amino acids contrast sharply with the remainder of the exon, which is not conserved between mammals and Fugu.
We identified 15 variations in TSC1 and TSC2 that are likely to represent polymorphisms (Table 2). Four of these, three in TSC2 and one in TSC1, have not been previously reported by other groups. One of the previously unreported polymorphisms, in patient 622, would result in a conservative change at amino acid 84 of tuberin from alanine to valine. The other two previously unreported TSC2 polymorphisms that we found are within introns. The TSC1 polymorphism was a silent change in exon 23.
| Patient | Gene | Location | Nucleotide Alteration | Effect of Mutation | Previously Reported | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| 624 | TSC1 | Exon 10 | T1186C | Met322Thr | Yes | |||||
| 504 | TSC1 | Exon 22 | C3050T | Ala943Ala (silent) | Yes | |||||
| 603 | TSC1 | Exon22 | C3050T | Ala943Ala (silent) | Yes | |||||
| 574 | TSC1 | Exon 23 | G3416A | Thr1065Thr (silent) | No | |||||
| 502 | TSC2 | Intron 4 | T482-3C | Noncoding region | Yes | |||||
| 504 | TSC2 | Exon 14 | C1578T | Ser526Ser (silent) | Yes | |||||
| 504 | TSC2 | Intron 14 | C1600-14T | Noncoding region | Yes | |||||
| 504 | TSC2 | Intron 15 | A1716+16G | Noncoding region | No | |||||
| 504 | TSC2 | Exon 40 | T5202C | Asp1734Asp (silent) | Yes | |||||
| 504 | TSC2 | 3′UTR | Del A5424+61– | Noncoding region | Yes | |||||
| 5424+62A | ||||||||||
| 552 | TSC2 | Exon 14 | A1574G | Asn525Ser | Yes | |||||
| 552 | TSC2 | Intron 39 | C5160+38T | Noncoding region | No | |||||
| 574 | TSC2 | 3′UTR | Del A5424+61− | Noncoding region | Yes | |||||
| 5424+62A | ||||||||||
| 622 | TSC2 | Exon 3 | C251T | Ala84Val | No | |||||
| 624 | TSC2 | Exon 37 | C4959T | Serl1653Ser (silent) | Yes |
We report here the first mutational analysis of TSC patients specifically ascertained because of symptomatic LAM. Germline mutations were found in eight patients. Seven of these were in the TSC2 gene and one was in the TSC1 gene. Three of the seven TSC2 mutations were missense mutations or in-frame deletions in exons 40 and 41, which are the final two exons of the TSC2 gene.
Missense mutations and in-frame deletions, in contrast to mutations causing premature protein truncation, are of particular interest in tumor suppressor gene syndromes such as TSC. Most tumor suppressor gene mutations cause premature protein truncation. In TSC2, for example, approximately 80% of mutations result in premature protein truncation (16). In many cases, this truncated protein is unstable and all functions of the protein are lost. In contrast, proteins containing missense mutations and in-frame deletions can have selective functional defects that may be clinically important. Mutations that do not prematurely truncate the protein can be associated with specific clinical phenotypes (a genotype–phenotype correlation) and can help to elucidate the biological properties of the different domains of the protein.
Genotype–phenotype associations have been described for several tumor suppressor gene syndromes including neurofibromatosis type 1 (21), familial adenomatosis polyposis coli (FAP), and von Hippel Lindau disease (VHL). For example, VHL patients with germline VHL gene mutations that result in a truncated protein have a low (9%) incidence of pheochromocytoma. In contrast, VHL patients with germline missense mutations anywhere within the gene have a high (59%) incidence of pheochromocytoma, and patients with missense mutations at codon 167 have the highest (82%) incidence (22).
In FAP, which is associated with germline mutations in the APC gene, mutations in the 5′ end of the gene are associated with an attenuated form of the disease that has relatively few colonic polyps but still carries a significant risk of colon cancer (23). Desmoid tumors in FAP patients are associated with mutations in codons 1445–1578 of the APC gene, and congenital hypertrophy of the retinal epithelium is seen frequently in FAP patients with germline APC gene mutations in codons 463–1387, and rarely with germline mutations in codons 136– 302 (24).
In our study, patient 479 had a missense change in the last exon (exon 41) of the TSC2 gene. This would result in a nonconservative amino acid change (arginine to cysteine) that has not been previously identified among the more than 350 individuals included in the TSC Variation Database, nor among the more than 80 patients and controls tested in our laboratory.
In TSC as in most other tumor suppressor gene syndromes, it can be difficult to distinguish missense mutations from non– disease-associated polymorphisms. Because many TSC2 mutations arise de novo, testing of clinically unaffected parents can be useful in confirming that the DNA change is associated with the disease, and does not represent a rare polymorphism. In patient 623, for example, we were able to verify that the exon 9 TSC2 missense mutation was not present in her parents and is therefore likely to be specifically associated with TSC. In the case of patient 479, testing of family members was not helpful because the mother of patient 479 carries the change. The mother of patient 479 could have undiagnosed TSC; she has never been evaluated with imaging of the brain or kidney. It is not unusual for TSC to be diagnosed in adulthood. TSC has considerable phenotypic variability, and approximately half of affected individuals have normal intelligence.
We conclude that the exon 41 change is most likely to represent a disease-associated mutation based on two factors. First, it has not been previously identified in any patient in the Database or in the controls tested in our laboratory, suggesting that it is not a polymorphism. Second, this change would result in a nonconservative amino acid change from arginine to cysteine, within a highly conserved region of exon 41. This particular residue is identical in human, rat, mouse, and Fugu. Further studies will be required to prove that this change is disease associated and associated with abnormal function of tuberin.
Our data suggest that mutations in TSC2 exons 40 and 41 could be associated with a higher incidence of symptomatic LAM than mutations elsewhere in the gene. The TSC Variation Database includes TSC2 mutations detected in 268 different patients, 10 of which are missense mutations or in-frame deletions in exons 40 or 41 (3.1%). Of the seven TSC2 mutations we detected in TSC patients with LAM, three (43%) were in exons 40 or 41. However, no direct comparison can be made between these groups as it is not known which patients in the Database have LAM.
An association between symptomatic LAM and mutations in the carboxy-terminus of tuberin could occur if these mutations affect either the incidence and/or severity of LAM in women with TSC. Further studies will be required to address this possibility. It is important to note that we studied only TSC patients with symptomatic LAM. The incidence of undiagnosed LAM in TSC is not known. Ideally, future studies will compare the spectrum of germline TSC2 mutations in women with LAM with a control group of women with TSC, in whom careful screening has revealed no evidence of LAM.
Despite the possible association between exon 40 and 41 mutations and LAM, it is clear that mutations in other regions of the gene are also associated with LAM. There are six previous reports of TSC2 mutations in women with TSC and symptomatic LAM (Table 3), none of which is in exons 40 or 41, and we found four mutations in other regions of TSC2. In VHL and other tumor suppressor gene syndromes, genotype– phenotype associations are usually not absolute (22, 23). This is likely to reflect the contribution of other genetic factors and environmental factors to disease severity (25).
| Gene | Exon | Nucleotide Alteration | Effect of Mutation | Reference | ||||
|---|---|---|---|---|---|---|---|---|
| TSC1 | 10 | C1222A | Ser334Stop | Jones and coworkers (16) | ||||
| TSC2 | 19 | T2168G | Leu717Arg | Zhang and coworkers (34) | ||||
| TSC2 | many | Large deletion | Jones and coworkers (16) | |||||
| TSC2 | 16 | C1849T | Arg611Trp | Jones and coworkers (16) | ||||
| TSC2 | 23 | A2683G | Met895Val | Niida and coworkers (14) | ||||
| TSC2 | 24 | Del 2814–2815 | Thr938fs→ 959 Stop | Beauchamp and coworkers (18) |
In all other respects, the mutations we identified appear to be similar to the general TSC population, including the distribution of mutations between TSC1 and TSC2, and the number of mutations that were identified. Our 57% rate of mutation detection (eight mutations identified out of 14 patients tested) is nearly identical to that in two recently published studies that used SSCP for TSC1 and TSC2 mutation detection (14) (15).
There are a number of possible explanations for the fact that mutations were not found in every patient. Mutations not detected by SSCP, such as mutations in the noncoding regions of the gene, large deletions, deletions of entire exons, or promotor silencing, may be present in the patients in whom we did not detect mutations. It is also possible that some of the patients in our series have mosaicism for TSC2 mutations, with the mutations present in certain tissues and not present in peripheral blood lymphocytes. Mosaicism appears to be common in tumor suppressor gene syndromes (26), including TSC (27-29).
Our data demonstrate that women with germline mutations in exons 40 and 41 of TSC2 can develop LAM. This may indicate that the carboxy-terminus of tuberin contains a domain that controls smooth muscle proliferation. The high degree of evolutionary conservation of the regions of exons 40 and 41 in which we found mutations is consistent with this hypothesis. This putative domain would be removed by premature stop codons earlier in the gene (such as those in patients 552, 563, and 590 in our study). It is possible that more proximal missense mutations (such as the exon 9 missense mutation in patient 623) also disrupt this domain through conformational changes.
The functions of tuberin and hamartin are not yet fully understood. Tuberin has a region of homology to rap1 GTPase-activating protein (GAP), encoded by exons 34 to 38 (9). Tuberin has been found to have GAP activity for both rap1 (30) and rab5 (31), and to interact with rapabtin-5, an adaptor for rab5, via the region encoded for by approximately exons 38 to 41 (31). Further studies will be required to determine whether mutations in exons 40 and 41 disrupt tuberin's GAP activity.
It is clear that LAM, which affects almost exclusively women, has a strong hormonal component. Both LAM and angiomyolipoma cells express estrogen and progesterone receptors (32), and tuberin has been shown to modulate transcription mediated by steroid hormone receptors (33). The domains of the protein responsible for this activity are not yet known. The functional impact of the mutations we identified on steroid hormone receptor-mediated transcription is an area requiring further investigation.
In summary, we screened all 41 exons of the TSC2 gene and all 21 coding exons of the TSC1 gene for mutations in 14 patients with TSC and LAM. Eight mutations were identified. Three patients had mutations in exons 40 or 41 of TSC2, which are the final two exons of this large gene. This indicates that germline alterations in the extreme carboxy-terminus of tuberin can result in LAM in women with TSC. Understanding the relationship between particular germline TSC2 mutations and smooth muscle proliferation in LAM could elucidate the underlying biology of this disease and facilitate clinical studies aimed at prevention, early diagnosis, and/or treatment of LAM.
The authors thank the patients and family members for their participation; Sue Byrnes of The LAM Foundation and Vicky Holets Whittemore of the National Tuberous Sclerosis Association for their ongoing assistance with this work; and Dr. Rebecca Raftogianis for critical review of the manuscript.
Supported by grants from the NIH (HL 60746) and The LAM Foundation (Cincinnati, OH).
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