Affiliations of authors: Urologic Oncology Branch (CDV, YY, CPP, CATC, MMW, WML), and Laboratory of Pathology (CATC, MJM), Center for Cancer Research, National Cancer Institute, Bethesda, MD; Basic Research Program, SAICFrederick, Inc. (LSS), and Laboratory of Immunobiology, National Cancer Institute, Frederick, MD (MLN, BZ)
Correspondence to: Cathy D. Vocke, PhD, Urologic Oncology Branch, Bldg. 10, CRC, Rm. 1W-5888, National Cancer Institute, Bethesda, MD 20892 (e-mail: vockec{at}mail.nih.gov).
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ABSTRACT |
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Recently, individuals with Birt-Hogg-Dubé syndrome (BHD), a genodermatosis characterized by fibrofolliculomas (hamartomas of the hair follicle) (10) and pulmonary cysts (11,12), were found to have a seven-fold higher risk over the general population of developing kidney neoplasms (13). Unlike renal tumors in patients with other inherited kidney cancer syndromes, renal tumors from BHD patients exhibit a spectrum of histologic types, including chromophobe (34%), oncocytoma (5%), clear cell (9%), papillary (2%), and an oncocytic hybrid (50%) with features of chromophobe RCC and renal oncocytoma (1416). Germline mutations have been identified in a novel gene, BHD, in affected family members (17). BHD encodes a protein, folliculin, which is named for the hallmark dermatologic lesions found in BHD patients. All germline mutations identified to date are frameshift or nonsense mutations that are predicted to truncate folliculin, including insertions or deletions of a tract of eight cytosines (C8) in exon 11 (17,18). Mutations in this "hot spot" are found in the germline of 44% of BHD patients (17). The high frequency of germline-inactivating mutations suggests that BHD may act as a tumor suppressor gene. Identifying a somatic "second hit" in the copy of BHD without a germline mutation would support a tumor suppressor function for BHD according to the Knudson two-hit hypothesis.
To address the mutation status of the BHD gene in tumors from Birt-Hogg-Dubé patients, we analyzed a panel of 77 renal tumors by direct DNA sequence analysis. Tumor samples, as well as matched normal samples, were obtained from 12 affected members of BHD families after renal surgery at the National Cancer Institute (NCI). The protocol was approved by the institutional review board of NCI, and the patients gave written informed consent.
BHD patients were often found to have bilateral, multifocal tumors and underwent staged bilateral partial nephrectomies, providing tumor samples for this study. Out of 77 tumors, 45 frozen tumor samples were sectioned and microdissected manually to obtain both tumor and histologically normal tissue. DNA was isolated in a solution containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% Tween 20, and 0.1 mg/mL proteinase K as described (19). For the remaining 32 tumors, tissue was no longer available for microdissection, and DNA was obtained from bulk extraction of tissue under high-salt conditions (Puregene kit, Gentra Systems, Minneapolis, MN).
The entire coding region of BHD (exons 414) was sequenced in each tumor sample, following polymerase chain reaction (PCR) amplification. PCR was carried out using genomic primers for the BHD gene as previously described (17). A modified primer, SKA31, with the sequence TTCCTGCCGGTTTTGAAGGTG, was substituted for primer SKA3 because it produced more reliable amplification. Reactions were carried out in a 25 µL volume using Taq PCR Master Mix (Qiagen, Valencia, CA), 0.4 µM concentrations of each primer, and a volume of input tumor DNA that was empirically determined to result in sufficient product under standard PCR conditions. Sequencing was carried out using Big Dye Terminator chemistry and run on an ABI Prism 310 Genetic Analyzer (PE/Applied Biosystems, Foster City, CA). Each mutation was verified by repeat PCR and sequencing. To confirm a somatic BHD mutation in a tumor sample independent of the second gene copy, PCR amplicons that contained a somatic BHD mutation were cloned into the TOPO TA cloning vector or the TOPO XL cloning vector (Invitrogen, Carlsbad, CA) and characterized by sequencing as described above.
The known germline mutation from each patient was detected in every tumor. In addition, either a somatic mutation or loss of heterozygosity (LOH) was detected in at least one of the tumors in all 12 BHD patients. In total, 54 of 77 (70%) of their tumors were found to have a somatic mutation or LOH, and, at most, only a single alteration was found in each tumor (Table 1; Fig. 1). In contrast, no somatic mutations were found in any matched normal DNA. The somatic mutations that were observed in 41 of 77 (53%) tumors were distributed throughout the BHD coding region, and the majority (30 of 41, 75%) of somatic mutations were frameshift mutations within the open reading frame. Three nonsense mutations were also identified, one in exon 12 and two in exon 14. Interestingly, no missense mutations were detected in this tumor panel.
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For BHD to fit the model of a tumor suppressor gene, the second mutation must occur on the wild-type copy of BHD. Although mutation analysis of discrete genomic regions that are amplified by PCR is unable to distinguish between the allele with a germline mutation and the other allele except when the somatic mutation occurs in the same exon as the germline mutation, we observed germline and somatic BHD mutations occurring in the same exon in six of 41 tumors. PCR-amplified products from these exons were subcloned, permitting the identification and sequencing of both BHD alleles. We confirmed that two BHD mutations, one on each allele, had occurred in these renal tumors (Fig. 1). These data, combined with the apparent loss of the wild-type allele in 13 tumors, indicate that the first and second mutations occur on different copies of the BHD gene.
However, not all tumors exhibited LOH or somatic mutations. No LOH or somatic mutations were detected in 23 of 77 tumors (30%). In such cases, gene silencing by hypermethylation or mutations in regulatory regions of the BHD gene may play a role in its inactivation. Alternatively, normal cell contamination of tumor tissue may have interfered with detection of somatic mutations in some cases.
In our study, the frequency of somatic mutations and LOH seen differed slightly by renal tumor histology. Whereas 78% of chromophobe tumors (13 mutations plus five LOH in 23 tumors), 72% (27 mutations and seven LOH in 47 tumors) of oncocytic hybrid tumors, and 50% (three mutations and one LOH in eight tumors) of oncocytomas had second hits, no second hit was detected in either of the two clear-cell tumors. It is unclear whether these differences in mutation and LOH frequencies are due to sampling bias, association of a second hit with histologic type, or differences in the degree of normal cell contamination in these histologic types.
Our data showed that the tumors from a given BHD patient have different second hits. A schematic depiction of kidney lesions from a typical BHD patient with mutations superimposed is shown in Figure 2. Distinct somatic mutations or LOH were observed in the 15 lesions. This patient also developed several renal tumors with mixed histologic features. Again, LOH and mutation analysis were performed on tissue from the different histologic areas of three such tumors. Within each tumor, the same second hit was observed, regardless of histology. These observations strongly suggest that multiple renal tumors from some BHD patients are independent, clonal events, each arising from a separate and unique second mutation in the BHD gene. However, the three tumors with mixed histologies shared a common somatic mutation in the distinct histologic regions within each tumor. This finding suggests that in some cases, a somatic second hit precedes histologic diversification within a single tumor. The molecular mechanism that drives these events is unknown.
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In conclusion, this report is the first comprehensive evaluation of a large number of renal tumors from BHD patients with a known germline BHD mutation. Our results document the high frequency and wide spectrum of second mutations, which strongly support a tumor suppressor role for BHD. Inactivation of both copies of BHD occurred in several histologic types of renal tumors, suggesting that BHD may act at an early stage of renal oncogenesis. Further understanding of the mechanism of BHD-induced tumorigenesis awaits functional studies of the folliculin protein.
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NOTES |
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This publication has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-C0-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
We thank the BHD patients and their families for their cooperation.
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REFERENCES |
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(1) Kovacs G, Akhtar M, Beckwith BJ, Bujert P, Cooper CS, Delahunt B, et al. The Heidelberg classification of renal cell tumors. J Pathol 1997;183:1313.[CrossRef][ISI][Medline]
(2) Storkel S, Eble JN, Adlakha K, Amin M, Blute ML, Bostwick DG, et al. Classification of renal cell carcinoma: Workgroup No. 1. Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Cancer 1997;80:9879.[CrossRef][ISI][Medline]
(3) Linehan WM, Walther MM, Zbar B. The genetic basis of cancer of the kidney. J Urol 2003;170:216372.[CrossRef][ISI][Medline]
(4) Pavlovich CP, Schmidt LS, Phillips JL. The genetic basis of renal cell carcinoma. Urol Clin North Am 2003;30:43754, vii.[CrossRef][ISI][Medline]
(5) Schmidt L, Duh F-M, Chen F, Kishida T, Glenn G, Choyke P, et al. Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nat Genet 1997;16:6873.[CrossRef][ISI][Medline]
(6) Schmidt L, Junker K, Kinjerski T, Weirich G, Neumann H, Brauch H, et al. Novel mutations of the MET proto-oncogene in papillary renal carcinomas. Oncogene 1999;18:234350.[CrossRef][ISI][Medline]
(7) Launonen V, Vierimaa O, Kiuru M, Isola J, Roth S, Pukkala E, et al. Inherited susceptibility to uterine leiomyomas and renal cell cancer. Proc Natl Acad Sci U S A 2001;98:338792.
(8) Tomlinson IP, Alam NA, Rowan AJ, Barclay E, Jaeger EE, Kelsell D, et al. Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet 2002;30:40610.[CrossRef][ISI][Medline]
(9) Toro JR, Nickerson ML, Wei MH, Warren MB, Glenn GM, Turner ML, et al. Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America. Am J Hum Genet 2003;73:95106.[CrossRef][ISI][Medline]
(10) Birt AR, Hogg GR, Dube WJ. Hereditary multiple fibrofolliculomas with trichodiscomas and acrochordons. Arch Dermatol 1977;113:167477.[Abstract]
(11) Binet O, Robin J, Vicart M, Ventura G, Beltzer-Garelly E. Fibromes perifolliculaires polypose colique familaile pneumothorax spontanes familiaux. Ann Dermatol Venereol 1986;113:92830.[ISI]
(12) Toro J, Duray PH, Glenn GM, Darling T, Zbar B, Linehan WM, et al. Birt-Hogg-Dube syndrome: a novel marker of kidney neoplasia. Arch Dermatol 1999;135:11951202.
(13) Zbar B, Alvord G, Glenn G, Turner M, Pavlovich CP, Schmidt LS, et al. Risk of renal and colon neoplasms and spontaneous pneumothorax in the Birt Hogg Dube syndrome. Cancer Epidemiol Biomarkers Prev 2002;11:393400.
(14) Pavlovich CP, Hewitt S, Walther MM, Eyler RA, Zbar B, Linehan WM, et al. Renal tumors in the Birt-Hogg-Dube syndrome. Am J Surg Pathol 2002;26:154252.[CrossRef][ISI][Medline]
(15) Tickoo SK, Reuter VE, Amin MB, Srigley JR, Epstein JI, Min KW. Renal oncocytosis: a morphologic study of fourteen cases. Am J Surg Pathol 1999;23:1094101.[CrossRef][ISI][Medline]
(16) Schmidt LS, Warren MB, Nickerson ML, Weirich G, Matrosova V, Toro JR, et al. Birt Hogg Dube syndrome, a genodermatosis associated with spontaneous pneumothorax and kidney neoplasia, maps to chromosome 17p11.2. Am J Hum Genet 2001;69:87682.[CrossRef][ISI][Medline]
(17) Nickerson ML, Warren MB, Toro JR, Matrosova V, Glenn G, Turner ML, et al. Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dube syndrome. Cancer Cell 2002;2:15764.[CrossRef][ISI][Medline]
(18) Khoo SK, Giraud S, Kahnoski K, Chen J, Motorna O, Nickolov R, et al. Clinical and genetic studies of Birt-Hogg-Dube syndrome. J Med Genet 2002;39:90612.
(19) Zhuang Z, Bertheau P, Emmert-Buck MR, Liotta LA, Gnarra JR, Linehan WM, et al. A microdissection technique for archival DNA analysis of specific cell populations in lesions <1 mm in size. Am J Pathol 1995;146:6205.[Abstract]
(20) Lamlum H, Ilyas M, Rowan A, Clark S, Johnson V, Bell J, et al. The type of somatic mutation at APC in familial adenomatous polyposis is determined by the site of the germline mutation: a new facet to Knudson's two-hit hypothesis. Nat Med 1999;5:107175.[CrossRef][ISI][Medline]
(21) Albuquerque C, Breukel C, van der Luijt R, Fidalgo P, Lage P, Slors FJ, et al. The just-right signaling model: APC somatic mutations are selected based on a specific level of activation of the beta-catenin signaling cascade. Hum Mol Genet 2002;11:154960.
(22) Crabtree M, Sieber OM, Lipton L, Hodgson SV, Lamlum H, Thomas HJ, et al. Refining the relation between first hits and second hits at the APC locus: the loose fit model and evidence for differences in somatic mutation spectra among patients. Oncogene 2003;22:425765.[CrossRef][ISI][Medline]
(23) Khoo SK, Kahnoski K, Sugimura J, Petillo D, Chen J, Shockley K, et al. Inactivation of BHD in sporadic renal tumors. Cancer Res 2003;63:45837.
(24) da Silva NF, Gentle D, Hesson LB, Morton DG, Latif F, Maher ER. Analysis of the Birt-Hogg-Dube (BHD) tumour suppressor gene in sporadic renal cell carcinoma and colorectal cancer. J Med Genet 2003;40:8204.
(25) Kahnoski K, Khoo SK, Nassif NT, Chen J, Lobo GP, Segelov E, et al. Alterations of the Birt-Hogg-Dube gene (BHD) in sporadic colorectal tumours. J Med Genet 2003;40:5115.
(26) Antonarakis SE. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat 1998;11:13.[CrossRef][ISI][Medline]
Manuscript received November 23, 2004; revised March 31, 2005; accepted April 26, 2005.
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