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Keywords:

  • renal cell cancer;
  • folliculin gene;
  • mutation;
  • allelic changes

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. Acknowledgements
  6. REFERENCES

Germline mutation of the folliculin gene (BHD) at chromosome 17p11.2 is associated with the development of multiplex hamartomas of the hair follicles, chromophobe renal cell carcinomas (RCC) and renal oncocytomas (RO). We have analyzed the folliculin gene with sequencing for mutations and the chromosome 17p11.2 with microsatellites for allelic changes in sporadic ROs and chromophobe RCCs. Allelic loss at chromosome 17 was seen in 8 of 8 chromophobe RCCs whereas none of the 8 RO showed alteration at this chromosomal region. Sequencing all exons from genomic DNA failed to disclose mutations of the folliculin gene in any of the tumors. We found a single nucleotide polymorphism (SNP) of G/A (nt 74) at the first exon in the untranslated region of the folliculin gene. We did not find a correlation between the SNP G/A or loss of the G allele and the expression level of either splice variants of the folliculin gene. Our data suggest the folliculin gene does not play a role in the tumorigenesis of sporadic chromophobe RCCs and renal oncocytomas. © 2004 Wiley-Liss, Inc.

The Birt-Hogg-Dubé (BHD) syndrome, an autosomal dominant disease, is associated with the development of multiplex hamartomas of the hair follicles, renal cell tumors and spontaneous pneumothorax.1, 2, 3 Linkage analysis of several families located the gene to chromosome 17p11.2 region.4, 5 Germline mutation of the folliculin gene (in most cases a C insertion or deletion in the hypermutable (C)8 tract at nt 1733–1740 in exon 11) has been found recently in affected family members.6, 7 A systematic histopathological survey indicated that most patients with BHD syndrome develop a particular type of renal cell tumor, the overwhelming majority of which corresponds to chromophobe RCCs (34%) or so called “hybrid” neoplasms (50%) resembling a mixture of chromophobe RCC and RO.8 Previous CGH and microsatellite studies indicated that chromosome 17 is lost in ∼80% of sporadic chromophobe RCC.9, 10 To establish the possible involvement of the folliculin gene in the tumorigenesis we have searched for mutation by sequencing the exons 1–14 in sporadic chromophobe RCC and ROs. Although microsatellite allelotyping confirmed a frequent LOH at chromosome 17p11.2 region in chromophobe RCCs, our study failed to detect mutation of the folliculin gene in the special types of sporadic tumors resembling those associated with the BHD syndrome.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. Acknowledgements
  6. REFERENCES

Tumor samples, DNA and RNA extraction

Fresh tumor and normal kidney parenchymal tissues were obtained by nephrectomy at the Departments of Urology, Hannover Medical School and the Heidelberg University, Germany between 1986–8 and 1993–7, respectively. A section of tumor was immediately snap-frozen in liquid nitrogen and stored at −80°C and the remaining tissue was fixed in 4% buffered formaldehyde for histological report. The diagnosis was established according to the Heidelberg Classification of Renal Cell Tumors.11 For DNA extraction frozen tumor sample was placed in a plastic Petri dish, covered with 2 ml TE9 buffer, and allowed to thaw. The tumor cells were then carefully scraped or pushed out to separate them from stromal tissue under an inverted microscope by a pathologist (G.K.) experienced in this technique. The stromal tissue rests were then discarded. Tumor cells were resuspended in 3–5 ml TE9 buffer with 1% SDS and 0.2 mg/ml proteinase K and were incubated for 5 hr at 55°C. DNA was precipitated with ethanol after phenol-chloroform treatment and dissolved in TE buffer. Normal control DNA was extracted from corresponding kidney parenchymal specimens by the same method. The concentration of DNA samples was adjusted to 50 ng/μl. Tumor and corresponding normal kidney tissues were homogenized in TRIzol (Invitrogen, Karlsruhe, Germany) and RNA was prepared according to the manufacturers instructions. The concentration of total RNA was measured by spectrophotometry at 260 nm. The quality of RNA was checked by electrophoresis on 1% agarose gel.

Microsatellite analysis

Microsatellite loci D17S786 (17p13), D17S921 (flanking the BHD locus at 17p11.2) and D17S787 (17q22) were selected for our study (http://www.ncbi.nlm.nih.gov/mapview/). DNA amplification was carried out in 96-well polycarbonate plates using a PTC 200 Thermocycler (MJ Research Inc, Watertown, MA). The PCR reactions were carried out in a final volume of 10 μl with 50 ng genomic DNA, 200 μM dNTPs, 1.5 mM MgCl2, 2 pmol of each primer (the forward primer Cy5-labeled) and 0.5 U Taq-polymerase. After an initial 2 min denaturation at 94°C the samples were subjected to the following amplification program: 30 sec at 94°C, 30 sec at 55°C and 40 sec at 72°C for 28 cycles with a final elongation of 5 min at 72°C. Twenty microliters of ALF loading buffer (100% formamide with 50 mM EDTA and 5 mg/ml DextranBlue2000) were added to the completed PCR reaction. The samples were then denatured for 3 min at 94°C and 5–8 μl were loaded onto a 5% denaturing polyacrylamide gel. Electrophoresis was carried out at 600 V, 55 mA and 25 W for 200–300 min. Analysis was carried out on an automated DNA sequencer (ALFexpressII, Amersham/Pharmacia Biotech, Freiburg, Germany). The collected data were calculated by using the ALF Win Fragment Analyser 1.02 (Amersham/Pharmacia Biotech).

Sequencing

Exons 1–14 of the folliculin gene were directly sequenced on forward strand. A T7 20-mer tails (5′-TAA TAC GAC TCA CTA TAG GG-3′) have been added at the 5′ends of the sense intronic primers designed by Nickerson et al.6 PCR reactions were carried out in 25 μl of final volume by amplifying 50 ng of genomic DNA with 2.5 U of Taq DNA Polymerase (Invitrogen GmbH, Karlsruhe, Germany), 10× PCR buffer and 1.5 mM MgCl2 provided with the kit (0.2 mM of each dNTP, 5 pmol of each primer). PCR conditions were as follows: initial denaturation for 2 min at 94°C followed by 40 cycles of 45 sec at 94°C, 1 min at 64°C annealing temperature (62°C in the case of exon 9), 1 min at 72°C and a final extension step for 5 min at 72°C. Five microliters of the PCR products with 3 μl of loading buffer were analyzed on 2% agarose/EtBr gel.

Sequencing reactions were carried out using 1 μl of 1:10 dilution of the PCR products, 5 μM of sequencing primer T7 (fluorescently labeled with IR700) and the ThermoSequenase Cycle Sequencing Kit (Amersham/Pharmacia Biotech) in 4 μl of final volume. Cycling conditions were as follows: 2 min at 95°C, 30 cycles with 30 sec at 95°C, 30 sec at 55°C annealing temperature, 1 min at 72°C. The reactions were stopped by adding 4 μl of the loading buffer. Sequencing reactions were analyzed on an LI-COR Long Readir 4200 automated DNA sequencer (MWG-BIOTECH) using a 3.77% PAGE-PLUS gel (AMRESCO, Solon, OH) in 1× TBE buffer at 45°C. The raw data were collected and analyzed by using the e-Seq V2.0 analysis software and then compared to the wild-type folliculin sequences (http://www.ncbi.nlm.nih.gov).

Quantitative RT-PCR

Total RNA from paired normal and tumor RNA samples were available for 6 chromophobe RCCs and 6 ROs. Reverse transcription of 1 μg RNA was carried out in 12.5 μl reaction volume with 100 U M-MLV reverse transcriptase (RNase H Minus, Point Mutant, Promega, Mannheim, Germany), 1 μl of 50 mM oligo DT primer, 1 mM of each deoxynucleotide triphosphate and 20 U RNase inhibitor (Rnasin, Promega). The expression level was measured using a Real Time PCR Machine (Opticon, MJ Research Inc.). By using the following 4 primers we amplified cDNA fragments common in both splice variants and those occurring only in transcript variant 1 or 2 of the folliculin gene, respectively: BHD-F: 5′- CTT CTT CGG AGA TGA GCA GCA CG-3′; BHDcom-R: 5′-TTC TCC ATC TGG ACC AAG GTA TCC-3′; BHD1-R: 5′-CCT CTT CAG CCT CAG AGT TGT CC-3′; BHD2-R: 5′-GAT TCC TGC CAG GAG AGC AGA CA-3′. 1.5 μl of 1:8 diluted cDNA was amplified with 1 μM of each forward and reverse primer and 7.5 μl of the QuantiTect SYBR Green PCR Mix (Qiagen, Hilden, Germany) in 15 μl final volume. Cycling conditions were: 95°C 15 min followed by 40 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec and an additional 5 min elongation at 72°C. Samples were parallel amplified with gene specific and β-actin primers (5′-ATG GAT GAT GAT ATC GCC GCG-3′ and 5′-GTC CAT CAC GAT GCC AGT GGT AC-3′). The specificity of the product was confirmed by melting curve analysis. Standard curves were generated from a 8-step normal kidney cDNA dilution series for both gene specific and β-actin reactions. Relative quantity was calculated by dividing the gene specific expression with the appropriate β-actin expression and then multiplying with 1,000. All reactions were carried out in duplicates and the results were averaged.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. Acknowledgements
  6. REFERENCES

We have detected the loss of one allele at all informative loci including the BHD locus at chromosome 17p11.2 region in 8 of 8 sporadic chromophobe RCCs but none in the 8 ROs. Direct sequencing of all exons of the folliculin gene failed to detect mutations in the tumors. We did not find any insertion or deletion in the poly(C) tract in exon 11, all tumors showed wild-type (C)8 sequences. We found a SNP in the first exon of untranslated region of the folliculin gene. Sequence analysis showed G/A alleles at nt 74 in 9 of 16 samples of normal kidney parenchymal cells, whereas 4 cases showed only nucleotide G and 3 cases a nucleotide A. The loss of the G allele due to loss of one allele at chromosome 17p11.2 was seen in each of the 5 chromophobe RCCs carrying the G/A polymorphism (Figs. 1, 2). We did not find a correlation between the occurrence of SNP G74A or loss of the G allele and expression of either splice variants of the folliculin gene by real time PCR (Fig. 2). Thus, we can exclude that the SNP or loss of the G allele effects the expression level of the folliculin gene. We suggest within the confines of this small sample set that the folliculin gene does not play a role in the tumorigenesis of sporadic chromophobe RCCs and renal oncocytomas. During the review process of our work, methylation of the promotor region of the folliculin gene was reported in 2 of 7 ROs, 1 of 9 chromophobe RCCs as well as in 4 of 11 papillary RCCs and 4 of 12 so-called clear cell RCC.12 Although the expression of folliculin gene has not been analyzed we suggest the role of inactivation of folliculin gene in sporadic renal cell tumors.

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Figure 1. Results of the sequence analysis in a chromophobe RCC (T) and in corresponding normal kidney parenchymal cells (N). The wild-type sequence in normal cells shows single nucleotide polymorphism with alleles G/A at nt 74, whereas G allele is deleted in tumor cells.

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Figure 2. Relative expression of the splice variants of the folliculin gene in matched normal kidney and renal oncocytoma (30-1173) as well as chromophobe RCC (204-695) samples as determined by quantitative RT-PCR. Results of sequencing at the G74A SNP within the first exon and of LOH analysis at 17p11.2 are given below. BHDcom, both splice variants; BHDvar1 and BHDvar2, splice variants 1 and 2, respectively.

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Until now, 4 genes have been identified, the germline mutation of which is associated with clinical syndromes leading to development of renal cell tumors. Germline mutation of the VHL gene results in the development of multiplex bilateral cysts of the kidneys and conventional RCCs.13 Hereditary papillary RCC is associated with the germline mutation of the MET gene and duplication of the mutated allele at chromosome 7q31 in tumor cells.14, 15 Germ-line mutation of the FH gene may lead to the development of papillary RCCs.16 The germline mutation of the folliculin gene is associated with the development of chromophobe and mixed oncocytic-chromophobe RCCs.6, 7

Somatic inactivation of the VHL gene has been found in ∼60–70% of the sporadic conventional RCCs as well.17 Despite the high frequency of allelic duplication at chromosome 7q (85%), however, only 5–8% of the sporadic papillary RCCs display an activating mutation of the MET gene and no mutation of the FH gene was detected in sporadic RCCs.14, 15, 18 Our present study indicates that the folliculin gene is not the target of mutation in sporadic chromophobe RCCs and ROs.

Clinical and detailed histopathological analyses indicate that germline mutations of the VHL, MET, FH and folliculin genes are associated with multiplex hamartomas and developmental disturbances at different sites of the body including renal cysts and embryonic rest-like structures in the kidneys. Only a few of these lesions develop clinically recognized tumors, however, suggesting that the tumorigenic process may require the inactivation/mutation of additional genes, presumably those of playing role in the development of sporadic cancers. The very low frequency of the MET mutation and lack of mutation of the FH and folliculin genes in sporadic cancers suggest this hypothesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. Acknowledgements
  6. REFERENCES

We thank Drs. G. Staehler and W. DeRiese for providing tumor and normal kidney tissues.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. Acknowledgements
  6. REFERENCES
  • 1
    Birt AR, Hogg GR, Dubè WJ. Hereditary multiple fibrofolliculomas with trichodiscomas and acrochordons. Arch Dermatol 1977; 113: 16747.
  • 2
    Toro JR, Glenn G, Duray P, Darling T, Weirich G, Zbar B, Linehan M, Turner ML. Birt-Hogg-Dubé syndrome: a novel marker of kidney neoplasia. Arch Dermatol 1999; 135: 1195202.
  • 3
    Zbar B, Alvord WG, Glenn G, Turner M, Pavlovich CP, Schmidt L, Walther M, Choyke P, Weirich G, Hewitt SM, Duray P, Gabril F, et al. Risk of renal colonic neoplasms and spontaneous pneumothorax in the Birt-Hogg-Dubé syndrome. Cancer Epidemiol Biomarkers Prev 2002; 11: 393400.
  • 4
    Khoo SK, Bradley M, Wong FK, Hedblad MA, Nordenskjold M, Teh BT. Birt-Hogg-Dubé syndrome: mapping of a novel hereditary neoplasia gene to chromosome 17p12–q11.2. Oncogene 2001; 20: 523942.
  • 5
    Schmidt LS, Warren MB, Nickerson ML, Weirich G, Matrosova V, Toro JR, Turner ML, Duray P, Merino M, Hewitt S, Pavlovich CP, Glenn G, et al. Birt-Hogg-Dubé syndrome, a genodermatosis associated with spontaneous pneumothorax and kidney neoplasia, maps to chromosome 17p11.2. Am J Hum Genet 2001; 69: 87682.
  • 6
    Nickerson ML, Warren MB, Toro JR, Matrosova V, Glenn G, Turner ML, Duray P, Merino M, Choyke P, Pavlovich CP, Sharma N, Walther M, 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-Dubé syndrome. Cancer Cell 2002; 2: 15764.
  • 7
    Khoo SK, Giraud S, Kahnoski K, Chen J, Motorna O, Nickolov R, Binet O, Lambert D, Friedel J, Levy R, Ferlicot S, Wolkenstein P, et al. Clinical and genetic studies of Birt-Hogg-Dubé syndrome. J Med Genet 2002; 39: 90612.
  • 8
    Pavlovich CP, Walther MM, Eyler RA, Hewitt SM, Zbar B, Linehan WM, Merino MJ. Renal tumors in the Birt-Hogg-Dubé syndrome. Am J Surg Pathol 2002; 26: 154252.
  • 9
    Speicher MR, Schoell B, du Manoir S, Schrock E, Ried T, Cremer T, Storkel S, Kovacs A, Kovacs G. Loss of chromosomes 1, 2, 6, 10, 13, 17 and 21 in chromophobe renal cell carcinomas revealed by comparative genomic hybridization. Am J Pathol 1994; 145: 35664.
  • 10
    Bugert P, Gaul C, Weber K, Herbers J, Akhtar M, Ljungberg B, Kovacs G. Specific genetic changes of diagnostic importance in chromophobe renal cell carcinomas. Lab Invest 1997; 76: 2038.
  • 11
    Kovacs G, Akhtar M, Beckwith BJ, Bugert P, Cooper CS, Delahunt B, Eble JN, Fleming S, Ljungberg B, Medeiros LJ, Moch H, Reuter VE, et al. The Heidelberg classification of renal cell tumors. J Pathol 1997; 183: 1313.
  • 12
    Khoo SK, Kahnoski K, Sugimura J, Petillo D, Chen J, Shockley K, Ludlow J, Knapp R, Giraud S, Richard S, Nordenskjöld M, Teh BT. Inactivation of BHD in sporadic renal tumors. Cancer Res 2003; 63: 45837.
  • 13
    Clifford SC, Maher ER. Von Hippel-Lindau disease: clinical and molecular perspectives. Adv Cancer Res 2001; 82: 85105.
  • 14
    Schmidt L, Duh FM, Chen F, Kishida T, Glenn G, Choyke P, Scherer SW, Zhuang Z, Lubensky I, Dean M, Allikmets R, Chidambaram A, 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.
  • 15
    Fischer J, Palmedo G, von Knobloch R, Bugert P, Prayer-Galetti T, Pagano F, Kovacs G. Duplication and overexpression of the mutant allele of the MET proto-oncogene in multiple hereditary papillary renal cell tumors. Oncogene 1998; 17: 7339.
  • 16
    Tomlinson IP, Alam NA, Rowan AJ, Barclay E, Jaeger EE, Kelsell D, Leigh I, Gorman P, Lamlum H, Rahman S, Roylance RR, Olpin S, et al. Germline mutations in the fumarate hydratase gene predispose to dominantly inherited uterine fibroids, skin leiomyomata and renal cell cancer. Nat Genet 2002; 30: 40610.
  • 17
    Gnarra JR, Tory K, Weng Y, Schmidt L, Wei MH, Li H, Latif F, Liu S, Chen F, Duh FM, et al. Mutations of the VHL tumor suppressor gene in renal carcinoma. Nat Genet 1994; 7: 8590.
  • 18
    Kiuru M, Lehtonen R, Arola J, Salovaara R, Jarvinen H, Aittomaki K, Sjoberg J, Visakorpi T, Knuutila S, Isola J, Delahunt B, Herva R, et al. Few FH mutations in sporadic counterparts of tumor types observed in hereditary leiomyomatosis and renal cell cancer families. Cancer Res 2002; 62: 45547.