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Sporadic human renal tumors display frequent allelic imbalances and novel mutations of the HRPT2 gene

Abstract

Inactivation of the HRPT2 gene encoding parafibromin was recently linked to the familial hyperparathyroidism-jaw tumor syndrome. Patients with this syndrome carry an increased risk of parathyroid and renal tumors. To determine the relevance of HRPT2 for sporadic renal tumors, clear cell, papillary and chromophobe renal cell carcinomas as well as oncocytomas and Wilms tumors were analysed for HRPT2 gene alterations. Loss of heterozygosity (LOH) of HRPT2 was found in seven of 56 (12.5%) clear cell, three of 14 (21%) papillary, six of 10 (60%) chromophobe renal cell carcinomas, three of eight (38%) oncocytomas and four of 10 (40%) Wilms tumors. In addition, two novel HRPT2 point mutations, causing K34Q and R292K changes in parafibromin, were detected in one clear cell carcinoma and one Wilms tumor, respectively. These tumors displayed LOH of the remaining wild-type allele, but interestingly no von Hippel–Lindau (VHL) mutation. Functional analysis revealed that the K34Q mutant species of parafibromin is, unlike wild-type protein, defective in suppressing cyclin D1 expression in vivo. Taken together, these results suggest that renal cancer-associated mutations in parafibromin occur in the absence of VHL mutation, which in turn may contribute to constitutively elevated cyclin D1 expression and abnormal cell proliferation.

Introduction

Renal cancer constitutes approximately 3% of all human cancers and accounts for 2.6% of all cancer deaths (Cohen and McGovern, 2005; Jemal et al., 2005). It can be classified into a number of subtypes with diverse morphologic and genetic features (Eble et al., 2004). The most common subtype is the clear cell renal cell carcinoma (RCC), which accounts for 75% of renal malignancies, followed by the papillary and the chromophobe RCC, representing 10–15% and 5% of renal epithelial neoplasms, respectively. Renal oncocytoma, which is a benign neoplasm, comprises 3–5% of renal tumors. Wilms tumor, also known as nephroblastoma, mostly affects children (Rivera and Haber, 2005). The majority of renal tumors occur sporadically, but a minority (2%) of cases are associated with hereditary renal cancer diseases, such as the von Hippel–Lindau (VHL) syndrome (Latif et al., 1993), the hereditary papillary renal cancer (HPRC) syndrome (Schmidt et al., 1997), the hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome (Tomlinson et al., 2002) and the Birt–Hogg–Dubé (BHD) syndrome (Nickerson et al., 2002).

The identification of genes responsible for hereditary renal tumor syndromes has dramatically improved our understanding of the molecular background of sporadic renal tumors, because alterations of such genes are found to be also involved in the sporadic counterparts of hereditary renal tumors (Pavlovich and Schmidt, 2004). Mutations in the VHL gene, which is the first causative gene identified for VHL syndrome, are detectable in 30–60% of sporadic clear cell RCCs (Gnarra et al., 1994; Shuin et al., 1994; Schraml et al., 2002). Furthermore, inactivation of VHL through methylation or loss of heterozygosity (LOH) occurs frequently in clear cell RCC (Herman et al., 1994; Moch et al., 1998; Brauch et al., 2000). The MET, FH and BHD genes have been identified to cause HPRC syndrome, HLRCC syndrome and BHD syndrome, respectively (Schmidt et al., 1997; Nickerson et al., 2002; Tomlinson et al., 2002) and their somatic mutations were also found in some sporadic counterparts of their hereditary tumor subtypes (Schmidt et al., 1999; Kiuru et al., 2002; Khoo et al., 2003).

Recently, a new gene, HRPT2, on chromosome 1q25–q32 has been linked to the hereditary hyperparathyroidism-jaw tumor (HPT-JT) syndrome (Carpten et al., 2002). HPT-JT is a rare autosomal dominant hereditary disease with a predisposition to parathyroid adenomas, ossifying fibromas of the jaw and various cystic and neoplastic renal lesions including papillary RCC and Wilms tumor (Jackson et al., 1990; Szabo et al., 1995; Haven et al., 2000). Germline mutations in the HRPT2 gene have been identified in more than half of the individuals from families with HPT-JT syndrome (Carpten et al., 2002). Unexpectedly, germline HRPT2 mutations were also identified in patients with apparently sporadic parathyroid cancer (Shattuck et al., 2003; Cetani et al., 2004). Somatic mutations of the HRPT2 gene have been found in up to 67% of sporadic parathyroid carcinomas, but rarely detected in parathyroid adenomas, indicating a strong association with tumor malignancy (Howell et al., 2003; Shattuck et al., 2003). LOH of the wild-type allele in the chromosomal region of 1q21–q32 with retention of the mutant allele, which was detected in individuals from some HPT-JT families and frequent biallelic inactivation of the HRPT2 gene in sporadic parathyroid tumors indicate that HRPT2 acts as a tumor suppressor gene (Carpten et al., 2002; Howell et al., 2003; Shattuck et al., 2003).

Functional studies on the HFPT2 gene product parafibromin revealed that it is a component of the human RNA polymerase II associated factor (PAF1) complex, which has been implicated in the transcription elongation and RNA processing pathway (Rozenblatt-Rosen et al., 2005; Yart et al., 2005). While the precise function of parafibromin within this large multiprotein complex remains to be elucidated, it is interesting to note that the segment of parafibromin required for PAF1 and RNA polymerase II (RNAP II) binding is deleted in the majority of tumor-derived mutants (Wang et al., 2005). Hence, part of parafibromin tumor suppressor function may be mediated through association with PAF1 and RNAP II. A connection between parafibromin function and components of the transcription machinery is further illustrated by the fact that the Drosophila ortholog of human parafibromin, hyrax, binds to β-catenin/Armadillo and is required for the nuclear transduction of the Wnt/Wg signal (Mosimann et al., 2006). Finally, ectopic expression of parafibromin has been shown to inhibit the expression of the cell cycle regulatory protein cyclin D1. Interestingly, a point mutant derivative of it, L64P, failed to inhibit cyclin D1 expression (Woodard et al., 2005). The latter provides an intriguing link between normal parafibromin function, cell cycle control and parathyroid cancer suppression.

To date, no data are available as to whether the HRPT2 gene is involved in sporadic renal tumors. To clarify a possible involvement of the HRPT2 gene in renal tumorigenesis, we searched the entire coding region of the HRPT2 gene for somatic mutations and allelic imbalances at the region of 1q24–q34 in a series of 117 kidney tumors. Our data demonstrate frequent LOH in different renal tumor subtypes and identify two novel HRPT2 mutations in one clear cell RCC and one Wilms tumor. Functional analysis of one of the identified mutations, K34Q, revealed a defect in the ability of this mutant to suppress cyclin D1 expression. Thus, HRPT2 gene alterations may contribute to the pathogenesis of sporadic renal tumors, possibly through deregulation of cell proliferation.

Results

Allelic imbalances of HRPT2 locus

LOH was assessed using five microsatellite markers (D1S428, D1S384, D1S412, D1S413 and D1S2622), which flank HRPT2 on its centromeric and telomeric sides and one intragenic marker, located within intron 10 of the HRPT2 gene (Figure 1a). LOH assessment was performed in 98 renal tumors including 56 clear cell, 14 papillary, 10 chromophobe RCC, eight oncocytomas and 10 Wilms tumors, for which matched normal renal tissues were available (Table 1). The intragenic marker, D1S428, D1S384, D1S412, D1S413 and D1S2622 were informative in an average of 48, 44, 53, 80, 69 and 81% of cases, respectively. Only one of 10 chromophobe and five of 14 (36%) papillary RCCs were informative for the intragenic marker. The most frequent allelic imbalances (AI) were observed in chromophobe RCC. Significant AI differences were mainly found between chromophobe and clear cell RCC, as well as between chromophobe and papillary RCC subtypes (P<0.05). LOH at the HRPT2 locus was found in seven of 56 (12.5%) clear cell, three of 14 (21%) papillary, six of 10 (60%) chromophobe RCCs, three of eight (38%) oncocytomas and four of 10 (40%) Wilms tumors (Figure 1b). LOH was detected in two of 28 (7%) pT1/2 clear cell RCCs and in five of 28 (18%) pT3 clear cell RCCs. Although LOH of the HRPT2 locus was more frequent in advanced tumor stage, this difference did not reach statistical significance. Representative examples are illustrated in Figure 2.

Figure 1
figure1

(a) Locations of microsatellite markers used in this study. These markers cover an area of about 7 cM at 1q24–q34. (b) Loss of heterozygosity (LOH) on the HRPT2 locus (chromosome 1q24–q34) in clear cell, papillary, chromophobe RCC and renal oncocytoma, R, retention of heterozygosity; L, loss of heterozygosity; -, non-informative; pos, positive; neg, negative.

Table 1 Characteristics of patients and tumors
Figure 2
figure2

Representative examples of primary renal tumors demonstrating loss of heterozygosity (LOH) at microsatellite markers D1S2622 (left) and D1S428 (right).

HRPT2 mutation

Denaturing gradient gel electrophoresis (DGGE) was used to screen 117 renal tumors and three familial hyperparathyroidism patients for HRPT2 mutations. Of the three familial hyperparathyroidism samples, one showed mutated bands within exon 1 (Figure 3a). Sequence analysis identified a C → T substitution at nucleotide position 33 (TAC → TAT), as shown in Figure 3b. Both TAC and TAT codons encode the same amino-acid (tyrosine). This mutation does not result in an alteration in the protein sequence (Y11Y) and is considered a polymorphism. A second mutation within exon 1 was detected by DGGE in one clear cell RCC (Figure 3c). Sequence analysis revealed a nucleotide substitution at position 100 (Figure 3d), resulting in an amino-acid change from lysine to glutamine (K34Q). A third mutation, located in exon 9, was detected in one Wilms tumor (Figure 3e). This mutation showed a G → A substitution at nucleotide position 875 (AGA → AAA), leading to an amino-acid change from arginine to lysine (R292K), as shown in Figure 3f. Both K34Q and R292K have not been reported. These tumors also showed LOH of the wide-type allele at the HRPT2 locus. No VHL mutation was detected in these tumors.

Figure 3
figure3

Mutation analyses. (a) Denaturing gradient gel electrophoresis (DGGE)-based mutation analysis detected a mutation within exon 1 in a patient with familial isolated hyperparathyroidism (FIHP, lane 3). (b) Sequence analysis identified a C → T substitution at the nucleotide position 33 in this patient, which was silent (Y11Y). (c) DGGE showed a mutation within exon 1 in one clear cell RCC (lane 5). (d) Sequence analysis revealed a transversion (A → C) at position 100 in this tumor, resulting in a code change from AAG to CAG and an amino-acid change from Lysine to Glutamine (K34Q). (e) A mutation within exon 9 was detected by DGGE in one Wilms tumor (lane 5). (f) Sequencing demonstrated that this Wilms tumor harbours a G → A substitution at nucleotide position 875 (AGA → AAA), leading to an amino-acid change from arginine to lysine (R292K).

Functional analysis of the parafibromin mutant K34Q

Parafibromin is a nuclear protein and has been identified as a core component of the human Paf1 complex that interacts with RNA polymerase II and minimally contains Paf1, parafibromin, Leo1, Ctr9 and URI. To assess its subcellular localization and ability to interact with components of the Paf1 complex, we generated the K34Q parafibromin mutant, equipped it with a hemagglutinin (HA) tag. As shown in Figure 4a, when transiently expressed in HeLa cells, the parafibromin K34Q mutant species localized to the nucleus like its wild-type counterpart. Therefore, the K34Q mutation might not impair the recently described bipartite nuclear localization signal (Hahn and Marsh, 2005) and implies that the tumorigenic potential of this mutant protein may not be due to gross alterations in cellular localization. Next, we addressed whether the K34Q mutant is still able to interact with certain core components of the Paf1 complex. The K34Q mutant species was produced in parallel to wild-type parafibromin in HeLa cells and analysed for association with endogenous components of the core Paf1 complex by co-immunoprecipitation assays. As shown in Figure 4b, the K34Q mutant associated with Paf1 (upper panel), Leo1 (middle panel) and URI (bottom panel) to a similar extent as the wild-type parafibromin. Both were produced to similar levels, as verified by immunoblotting of whole-cell extracts (Figure 4b, right panels). These results suggest that the newly identified point mutant of parafibromin, K34Q, efficiently participates in complex formation with certain known components of the human Paf1 complex.

Figure 4
figure4

Biochemical characterization of the K34Q mutation. (a) HeLa cells transfected with plasmids encoding HA-tagged parafibromin (wt), or K34Q mutant were double-stained with anti-HA mAb 12CA5 (upper panels) and DAPI (lower panels), and processed for indirect immunofluorescence microscopy. (b) HeLa cells transfected with HA-tagged parafibromin (wt) or K34Q mutant expression plasmids were lysed, and aliquots subjected to immunoprecipitation with either polyclonal anti-Paf1cp (upper panel), monoclonal anti-HA (middle panel) or monoclonal anti-URI (bottom panel) antibodies and the relevant rabbit or mouse IgG as controls. For each experiment, whole-cell extracts (WCE) were analysed in parallel for protein expression (right panels). (c) HeLa cells were transduced with pLVTHM, pLVTHM/sh-parafibromin, pLVTHM/sh-parafibromin/HA-WT, pLVTHM/sh-parafibromin/HA-K34Q or pLVTHM/sh-parafibromin/HA-R222X lentiviruses. Five days post-infection, WCE were prepared and analysed by Western blotting using anti-parafibromin, anti-HA, anti-cyclin D1 and anti-tubulin antibodies. The data shown are representative of at least three independent experiments.

Mutation in parafibromin have been recently linked to the control of cyclin D1 expression (Woodard et al., 2005). Thus, we next determined whether the K34Q parafibromin mutant is defective in this regard. To this end, we used a lentiviral-driven expression system (Wiznerowicz and Trono, 2003), that promotes efficient short hairpin RNA (shRNA)-mediated downregulation of endogenous parafibromin while at the same time allows for the expression of either wild-type or mutants of parafibromin that contain silent mutations that make them resistant to shRNA-mediated downregulation. This system is, therefore, highly suitable to analyse the effects of parafibromin mutants on cyclin D1 levels in the absence of wild-type protein. As shown in Figure 4c, transduction of HeLa cells with viruses encoding shRNA against parafibromin induced a potent downregulation of endogenous parafibromin (lane 2). Concomittantly, HA-tagged species of wild-type parafibromin (lane 3), the R222X mutant lacking the C-terminal PAF1-binding domain (lane 4) and the newly identified K34Q mutant derivative (lane 5) were efficiently produced. Consistent with previous findings demonstrating that parafibromin overexpression is correlated with decreased cyclin D1 expression (Woodard et al., 2005), we observed that downregulation of endogenous parafibromin caused an increase in cyclin D1 protein levels (compare lane 2 and 1). Re-expression of HA-tagged wild-type parafibromin reverted this effect (lane 3). Excitingly, in cells re-expressing either the R222X or the K34Q mutant, cyclin D1 levels remain elevated (lanes 4 and 5, respectively). These results demonstrate that both, the K34Q as well as the R222X mutant derivatives of parafibromin, are defective in suppressing cyclin D1 expression.

Discussion

This is the first study screening for HRPT2 alterations in a wide spectrum of kidney tumors. The results demonstrate that somatic HRPT2 mutation is likely to be rare in renal tumors, unlike its common occurrence in parathyroid carcinomas. However, frequent AIs of the HRPT2 locus are detectable in different subtypes of sporadic renal tumors.

HRPT2 is the recently identified disease-causing gene for HPT-JT syndrome (Carpten et al., 2002), which is a rare familial multiple neoplasia syndrome primarily characterized by hyperparathyroidism owing to parathyroid tumors. Patients with HPT-JT may also develop ossifying fibromas, primarily of the mandible and maxilla, and kidney lesions. To date, less than 40 families with HPT-JT have been reported in the literature (Carpten et al., 2002; Cavaco et al., 2004). The most common renal manifestation of this syndrome are cystic lesions, ranging from a few minor cysts to bilateral polycysts (Haven et al., 2000; Cavaco et al., 2001). Different renal tumors have been described to be associated with this syndrome, including mixed epithelial-stromal tumors (Teh et al., 1996), adult Wilms tumors (Kakinuma et al., 1994; Szabo et al., 1995), papillary RCC and multiple renal cell adenomas (Haven et al., 2000). This predisposition of the syndrome to renal tumors led to the speculation that the HRPT2 gene may also play a role in the pathogenesis of sporadic renal tumors.

The HRPT2 gene is located on 1q25–q32. Previous studies have shown loss of chromosome 1 in different types of renal tumors. Loss of chromosome 1 was observed in about 90% of chromophobe RCCs (Kovacs and Kovacs, 1992; Speicher et al., 1994; Bugert et al., 1997; Nagy et al., 2004) and 25–46% of renal oncocytomas (Presti et al., 1996; Fuzesi et al., 2005). A loss of 1q was also detected in about 15% of papillary RCCs (Jiang et al., 1998; Reutzel et al., 2001) and in less than 10% of clear cell RCCs (Moch et al., 1996). In Wilms tumors, 1q gain was described to be a predominant alteration in comparative genomic hybridization analyses (Hing et al., 2001). Interestingly, LOH at loci 1q21–22 and 1q32 has been also associated with tumor adverse outcome (Law et al., 1997; Natrajan et al., 2006). Our LOH findings are consistent with these previous reports and indicate that the HRPT2 locus might be one of critical targets contributing to the development of these renal tumors.

Until now, 48 different HRPT2 mutations have been identified in familial diseases and sporadic parathyroid tumors (Wang et al., 2005). The most common mutational hot spot is exon 1, followed by exon 2, 3, 4, 5, 7 and 14. In this study, we detected one novel somatic HRPT2 mutation within exon 1 in a sporadic clear cell RCC and another novel mutation within exon 9 in a sporadic Wilms tumor. The two mutations gave rise to K34Q and R292K amino-acid change in parafibromin's primary sequence. With the exception of another missense mutation (L64P), all the described mutations arising in the HRPT2 gene cause C-terminal truncated variants of parafibromin, which lose their capability to bind to Paf1 complex components (Rozenblatt-Rosen et al., 2005; Yart et al., 2005). As the missense mutation, K34Q, gives rise to a stable gene product, it is tempting to speculate that both, K34Q and L64P, represent a specific class of mutants that share a common molecular defect.

In support of such a view, both mutant species display proper nuclear localization and retain their ability to bind to components of the Paf1 complex including Paf1, Leo1 as well as URI (this report; Rozenblatt-Rosen et al., 2005; Yart et al., 2005). Thus, it is possible that Paf1 complexes formed with either the K34Q or L64P mutant are defective in one or more aspects of the gene expression pathway. The K34Q mutant identified in renal cancer (this report), displays clear defects in normalizing cyclin D1 expression in vivo and thus behaves similar to the previously identified L64P mutant. Our data further show that also the R222X mutant is defective in cyclin D1 regulation. Notably, this mutant fails to interact with PAF1 and RNAP II. Thus, despite the fact that these mutants demonstrate distinct biochemical behavior, they each contribute to elevated cyclin D1 expression. Therefore, one might argue, that cyclin D1 overexpression may be at least one common consequence of functional inactivation of parafibromin. In this regard, deregulated cyclin D1 expression is associated with parathyroid carcinomas (Carling, 2001) and has been observed in renal cancer. Thus, cyclin D1 overexpression upon HRPT2 loss-of-function might be a key event in HRPT2-associated tumorigenesis.

Another tumor suppressor prominently mutated in clear cell RCC is the VHL gene. Over half of all sporadic clear cell RCCs carry biallelic inactivation of both VHL alleles (Foster et al., 1994; Gnarra et al., 1994; Shuin et al., 1994). However, the molecular pathway by which the VHL protein (pVHL) modulates the expression of target genes leading to tumorigenesis cannot be explained for all cases of clear cell RCC. Other mechanisms underlying the development of clear cell RCC apparently exist. In this study, we detected LOH of the HRPT2 locus in 12.5% of tumors of the clear cell subtype, with one sample exhibiting LOH of wild-type allele and retention of the mutant allele, which is consistent with Knudson's two hits theory of tumor suppressor gene inactivation. The finding that a tumor with a HRPT2 mutation showed no VHL alterations might indicate a VHL-independent, but parafibromin-dependent pathway. However, this pathway is obviously rare in clear cell RCC pathogenesis.

It has been described that the VHL gene is not associated with other renal tumors such as papillary, chromophobe RCC, oncocytoma as well as Wilms tumor. In papillary RCC, frequently detected genetic alterations include trisomy of chromosomes 3q, 7, 8, 12, 16, 17 and 20, and loss of the Y chromosome (Kovacs et al., 1991; Jiang et al., 1998). Although the MET proto-oncogene (7q31) is implicated in the pathogenesis of papillary RCC, only a small percentage of the cases of the sporadic papillary RCC have MET mutations (Schmidt et al., 1997). This suggests that the tumorigenic process may require aberrant alterations of additional genes. Similarly, many different genetic changes have been found in chromophobe RCC, renal oncocytoma and Wilms tumor. Chromophobe RCC is genetically characterized by widespread LOH on chromosomes 1, 2, 6, 10, 13, 17 and 21, as well as hypodiploidy (Speicher et al., 1994; Bugert et al., 1997), whereas renal oncocytoma displays frequent losses of chromosomes 1, 14 and Y (Meloni et al., 1992; Presti et al., 1996). No specific gene mutation has yet been identified as a direct contributor to their pathogenesis. Wilms tumor is known to have allelic loss at numerous loci (Grundy et al., 1994, 1998; Law et al., 1997; Klamt et al., 1998; Natrajan et al., 2006). One Wilms tumor gene (WT1) has been identified and mapped to 11p13 (Call et al., 1990; Gessler et al., 1990). The WT1 gene encoding a zinc-finger transcription factor is described to be mutated in 10–15% of sporadic Wilms tumors (Gessler et al., 1994; Varanasi et al., 1994). These same tumors often also harbour mutations in β-catenin (CTNNB1) (Maiti et al., 2000), implicating a role of Wnt pathway in the pathogenesis of Wilms tumor. Several other loci have also been linked to Wilms tumor development, including 11p15. In the present study, we detected HRPT2 mutation in 5% (1 of 19) and LOH in 40% of Wilms tumors. Our findings suggest that HRPT2 alterations may be also one of the potential contributors to the genesis of these tumor subtypes, namely papillary, chromophobe RCC, renal oncocytoma and Wilms tumors.

In conclusion, our results suggest that HRPT2 inactivation through mutation and LOH might be a critical VHL-independent event in renal tumorigenesis. As at least one corresponding mutant of parafibromin is defective in normalizing cyclin D1 expression, it is tempting to consider that deregulated expression of this cell cycle regulatory protein as a result of parafibromin mutation may contribute to renal tumorigenesis.

Materials and methods

Patients and samples

Primary renal tumor specimens were obtained from 117 patients who underwent partial or radical nephrectomy between 1993 and 2003 at the University Hospital of Zurich, Switzerland. Patient and tumor characteristics are shown in Table 1. Institutional Review Board approval for the study of human subjects was obtained from the Ethical Committee of Zurich before study initiation. Histology of all tumors was evaluated by one senior pathologist (HM) according to the World Health Organization Classification of Tumors (Eble et al., 2004). Normal renal tissues were available from all patients with the exception to four clear cell, six papillary RCC and nine Wilms tumor patients. Seventy tumor/normal tissue pairs were formalin-fixed, paraffin-embedded and 28 pairs were snap-frozen in liquid nitrogen and stored at −80°C. The 19 tumors that had no matched normal kidney specimens were frozen. Three to five tissue cylinders (0.6 mm diameter) were punched out from each paraffin block using a Beecher Microarray Instrument (Beecher Instruments Inc., Sun Prairie, WI, USA) to obtain pure tumor tissue for DNA extraction. The isolation of DNA was conducted using the Purgene kit (GentraSystems, Minneapolis, MN, USA), following the manufacturer's instructions. DNA was subjected to whole genomic amplification using WGA2 kit (Sigma, Saint Louis, MI, USA) before analyses, if the total amount of DNA was too small. Three patients with familial hyperparathyroidism served as a positive control for HRPT2 mutation analysis. Their DNA samples were isolated from peripheral blood lymphocytes.

Microsatellite analysis

PCR primer sets for specific allele loci were obtained from Microsynth (Balgach, Switzerland) and Applied Biosystems (Foster City, CA, USA). One primer of each primer pair was labelled with fluorescence dye (HEX, FAM or NED). The PCR amplification was performed in a 50 μl reaction volume with 50–100 ng of template for 40 cycles at 94°C for 30 s, 57°C for 30 s and 72°C for 30 s, followed by a 10-min final extension at 72°C. PCR product separation was performed using capillary array electrophoresis (ABI PRISM 3100 Genetic Analyzer, Applied Biosystems, Foster City, CA, USA), followed by pattern analysis using the software GeneMapper Version 3.7 (Applied Biosystems). AI value was analysed using the formula: (peak 1 height/peak 2 height in tumor DNA)/(peak 1 height/peak 2 height in normal DNA). Twenty normal DNA sample pairs, which were extracted in parallel from normal kidney tissues from 20 patients, were used as a reference for defining the LOH threshold. Based on the data from these normal reference DNA pairs, LOH was defined when one allele showed 35% reduction of peak height in tumor DNA compared with its corresponding allele in normal DNA, equating to a ratio of 0.65 or 1.54. The markers showing homozygosity, microsatellite instabilities and insufficient PCR amplification were scored as non-informative. A tumor was considered to be HRPT2 LOH-positive when LOH was found for the intragenic marker or any of four microsatellite markers. A tumor was regarded as HRPT2 LOH-negative if it demonstrated retention of the intragenic marker or retention of the two microsatellite markers (D1S384 and D1S412) that are close to HRPT2 locus. In case of doubtful LOH interpretation, AI assays were repeated to confirm the results.

DGGE-based mutation analysis

All exons and their exon–intron junctions of the HRPT2 gene were PCR-amplified from tumor DNA (primer sequences are available on request). Primer sets were obtained from Microsynth (Balgach). One of each primer pair contained a 40 base pair GC-clamp at one end. Optimal DGGE conditions for each fragment were determined by computer simulation of DNA melting using the software WinMelt Version 2 (BioRad, Hercules, CA, USA). PCR amplification and DGGE were carried out, as described previously, (Perren et al., 2004) with minor modifications. Fifty nanograms of template DNA were amplified in a 50 μl mixture of 1 × polymerase chain reaction (PCR) buffer, 200 μ M dNTP (Roche, Basel, Switzerland), 1 μ M of each primer and 5 units of Taq Polymerase (Ampli Taq Gold, Applied Biosystems). Forty cycles consisted of denaturation for 60 s at 95°C, annealing for 60 s at 55–61°C (depending on the primer pair) and extension at 72°C for 60 s. After a final extension for 10 min at 72°C, heteroduplex formation was induced by initial denaturation for 15 min at 98°C, followed by incubation at 55°C for 30 min and 37°C for 30 min. For DGGE, 10 μl of the PCR product in 5 μl Ficoll-based loading buffer (4 × ) were loaded onto a 10% polyacrylamide gel with a linear increasing gradient of denaturant solution (100% denaturant=7 M urea/40% (v/v) formamide) in 1 × Tris–acetate–ethylenediaminetetraacetic acid (TAE) buffer. Electrophoreses were carried out at 60°C and 100 V for 16 h in a D Gene System (BioRad). After electrophoreses, DNA strands were visualized using a Gel Doc 2000 System with the software TDS Quantity One (BioRad). Samples showing aberrant bands were sequenced using ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).

Sequencing of involved HRPT2 exons and VHL

Samples showing aberrant bands on DGGE were sequenced using the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). To clarify a possible interaction between HRPT2 and VHL in renal tumorigenesis, all three exons of the VHL gene were also sequenced in tumors that showed a HRPT2 mutation. Primers for PCR and sequencing of involved HRPT2 exons and the VHL gene were used, as previously reported (Schraml et al., 2002; Shattuck et al., 2003). PCR was performed as described above, but without the heteroduplex formation process. PCR products were purified using the QIAquick PCR purification kit (Qiagen AG, Basel, Switzerland) following the manufacturer's protocol. Sequencing reactions were performed using the BigDye Terminator cycle sequencing kit (Applied Biosystems). Data were analysed by using the Sequencing Analysis 5.1.1 software (Applied Biosystems), and all mutations were confirmed by repeated forward and reverse sequencing of the involved exon from an independent PCR reaction. When mutations were detected in tumor DNA, the same exons in corresponding normal DNA samples were sequenced in a similar manner.

Characterization of HRPT2 mutation

The K34Q mutant of parafibromin was generated by site-directed mutagenesis, using the forward (IndexTermGGAGTTCTCCTGGCCCCAGAATGTGAAGACC) and reverse (IndexTermGGTCTTCACATTCTGGGGCCAGGAGAACTCC) primers (the modified nucleotide is underlined), and used to replace the wild-type cDNA in pcDNA3-HA-parafibromin (Yart et al., 2005). The construct was verified by sequencing.

For immunofluoresence experiments, HeLa cells plated in 35 mm dishes containing coverslips were transfected using Fugene reagent (Roche, Basel, Switzerland) with 1 μg of the indicated plasmids following manufacturer's procedure. Twenty-four hours post-transfection, cells were washed with phosphate-buffered saline (PBS), fixed for 30 min in 3.7% paraformaldehyde at 37°C, rinsed quickly 3 × with PBS, incubated for 5 min with 0.2% Triton X-100 in PBS and washed again quickly 3 × with PBS. Coverslips were incubated with 5 μg/ml anti-HA monoclonal antibody (clone 12CA5, Boehringer Mannheim, Germany) in PBS supplemented with 5% BSA and 0.2% goat serum for 1 h. After washing 3 × with PBS, fluorescein isothiocyanate-conjugated anti-mouse antibody (Jackson ImmunoResearch Europe Ltd., Cambridgeshire, UK) was applied together with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI, Sigma) for 1 h. Cells were washed as described above, mounted in Vectashield and viewed with a Zeiss fluorescence microscope.

For co-immunoprecipitation experiments, 50% confluent HeLa cells were transiently transfected using the calcium–phosphate method, as described previously (Krek et al., 1993), with 10 μg of plasmid DNA and harvested 24 h after removal of the precipitate. Cells were lysed in 1 ml ice-cold lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1% NP-40, 10 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 mM NaF, 1 mM Na3VO4 and 1 mM dithiothreitol (DTT). After shaking for 15 min at 4°C, whole-cell extracts were centrifuged at 4°C for 15 min at top speed in an Eppendorf microcentrifuge and supernatants were pre-incubated for 30 min at 4°C with 25 μl of a 50% (w/w) slurry of protein A-Sepharose (Pharmacia), followed by centrifugation. Supernatants were incubated with 3–5 μg of anti-HA, anti-Paf1cp (Yart et al., 2005) or anti-URI (Gstaiger et al., 2003) antibodies for 2 h at 4°C. Immunocomplexes were then collected with protein A-Sepharose for 1 h and washed 3 × in lysis buffer. Membranes were first probed with the indicated proteins to detect the co-immunoprecipitated proteins, then reprobed with the antibody used for immunoprecipitation to verify that equal amount of proteins have been pulled down. Immunoblotting was performed with standard techniques and developed by chemiluminescence.

Functional analysis of HRPT2 mutation

pLVTHM, PAX2 and pMD2G-VSVG vectors were a generous gift from Dr D Trono (Lausanne, Switzerland). To stably downregulate parafibromin expression, a shRNA-encoding primer targeting parafibromin's mRNA sequence IndexTermGGACACGAACAACTATCTTAC was cloned into pLVTHM (construct referred to as pLVTHM/sh-parafibromin). cDNA encoding HA-parafibromin-WT, HA-parafibromin-K34Q and HA-parafibromin-R222X were subcloned into pLVTHM/sh-parafibromin replacing the green fluorescent protein marker (constructs referred to as pLVTHM/sh-parafibromin/HA-WT, pLVTHM/sh-parafibromin/HA-K34Q and pLVTHM/sh-parafibromin/HA-R222X, respectively). When required, these cDNA were first made shRNA-resistant by generating silent mutations through site-directed mutagenesis using the following primers: IndexTermGTATGGAGGACGAGGACGACGATTCTACAAAGCAC and IndexTermGTGCTTTGTAGAATCGTCGTCCTCGTCCTCCATAC. Details about vectors construction are available upon request. Recombinant lentiviruses were produced by cotransfection of subconfluent 293T cells with 20 μg of plasmid vector, 15 μg of PAX2 and 5 μg of pMD2G-VSVG by calcium–phosphate precipitation. Lentiviruses-containing medium was harvested 48 h after transfection, filtered and added on target cells. Whole-cell extracts were prepared 5 days post-infection and analysed by Western blotting.

Statistics

Contingency table analysis was used for significance testing of AI frequency differences among renal tumor subtypes and between different tumor stage and grade. A P-value 0.05 was regarded as significant.

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Acknowledgements

We thank Sonja Schmidt, Silvia Behnke and Martina Storz for technical assistance and staffs of our Molecular Diagnostic Laboratory for sequencing. We are also grateful to Nobert Wey for help with photographic and computer-assisted reproduction and to Dr Adriana von Teichman for English correction. AY is the recipient of a long-term Marie Curie intra-European fellowship. The study was supported by UBS AG (made possible by an anonymous donor), the Swiss National Science Foundation and the Zurich Cancer League, Switzerland.

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Zhao, J., Yart, A., Frigerio, S. et al. Sporadic human renal tumors display frequent allelic imbalances and novel mutations of the HRPT2 gene. Oncogene 26, 3440–3449 (2007). https://doi.org/10.1038/sj.onc.1210131

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Keywords

  • LOH
  • HRPT2 mutation
  • parafibromin
  • renal tumor

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