Urologic Oncology: Seminars and Original Investigations 31 (2013)

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1 Urologic Oncology: Seminars and Original Investigations 31 (2013) Original article HRAS T81C polymorphism modulates risk of urinary bladder cancer and predicts advanced tumors in ethnic Kashmiri population Arshad A. Pandith, Ph.D. a, Zafar A. Shah, Ph.D. a, Nighat P. Khan, Ph.D. b, Khalil M. Baba, M.D. c, M. Saleem Wani, M.D. d, Mushtaq A. Siddiqi, Ph.D. a, * a Department of Immunology and Molecular Medicine, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, India b Department of Clinical Biochemistry, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, India c Department of Pathology, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, India d Department of Urology, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, India Received 16 January 2011; received in revised form 6 March 2011; accepted 9 March 2011 Abstract Objective: Specific acquired HRAS mutations have been found to predominate in bladder cancer, and HRAS T81C polymorphism has been determined to contribute the risk of various cancers, including bladder cancer. Materials and methods: We screened the exon 1and 2 of HRAS and frequently detected polymorphism at nucleotide 81T to C (exon 1). A case-control study was conducted using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to test the genotype distribution of 140 bladder cancer patients in comparison with 160 cancer-free controls from a Kashmiri population. Results: In HRAS T81C SNP, frequencies of TT, TC, and CC genotypes among controls were 84.4%, 15.6%, and 0.0%, while in cases allele frequencies were 64.3%, 30%, and 5.7%, respectively. A significant differences was observed between the control and cases with odds ratio (OR) 3.0 and 95% confidence interval (CI) (P 0.000). Interestingly, combined TC and CC genotype abundantly presented in high grade (OR 5.4 and 95% CI ; P 0.00) and in advanced tumors (OR 3.3, 95% CI ; P 0.05). A significant association of the variant allele (TC CC) was found with male subjects ( 50) and ever smokers (P 0.001). Conclusion: It is evident from our study that HRAS T81C SNP moderately increases bladder cancer risk, and rare allele is a predictive marker of advanced bladder tumors Elsevier Inc. All rights reserved. Keywords: Polymorphism; Kashmiri; Genotype; Bladder cancer; PCR-RFLP 1. Introduction Bladder cancer accounts for approximately 5% of all cancers and is the fifth most common cancer in industrialized countries [1]. Urinary bladder cancer ranks ninth in worldwide cancer incidence. It is the seventh most common malignancy in men and the 17th most common in women [1]. The American Cancer Society estimates that 70,980 adults were diagnosed with bladder cancer in 2009, leading to 14,330 adult deaths in the United States. Bladder cancer has been associated classically with exogenous and environmental risk factors. The two best-known risk factors for bladder cancer are smoking and * Corresponding author. Tel.: , Ext: 2262; cell: ; fax: addresses: arshaajiz@gmail.com, vc.tmuck@gmail.com (M.A. Siddiqi). occupational exposure [2,3]. Compared with the general population, smokers are at two to four times greater risk of bladder cancer and heavy smokers are at five times the risk [4]. Urothelial cancers account for 5.6% of males and 1.8% of female cancers in India, with actual crude rate incidence of about 1/174 men and 1/561 women [5]. Limited data available on the incidence of bladder cancer in Kashmir ranks it as ninth in all types of cancers [6], but is reported here to be the primary malignancy of urogenital cancers. Recently, a detailed study was conducted by us here in the only tertiary care hospital between 2008 and 2010 for the bladder cancer cases registered from 2005 to This study revealed that bladder cancer ranks as the seventh leading cancer and accounts for 5.9.0% of all prevalent cancers in a Kashmiri population (data unpublished). The four cellular RAS genes encode four highly homologous 21 KDa proteins: HRAS, NRAS, KRAS4A and /$ see front matter 2013 Elsevier Inc. All rights reserved. doi: /j.urolonc

2 488 A.A. Pandith et al. / Urologic Oncology: Seminars and Original Investigations 31 (2013) KRAS4B. Activating Ras mutations occur in 30% of human cancers [7]. RAS genes have been elucidated as major participants in the development and progression of a series of human tumours, such as gastrointestinal cancer, lung cancer, and breast cancer [7]. It was reported that just one point mutation occurring in codon12, 13, or 61 could result in continuous stimulation of cell proliferation or, alternatively, a 5- to 50-fold amplification of the wild type gene [8]. HRAS mutations predominate in bladder cancer and vary widely from 0% to 45% between studies [7 10]. In most cases including bladder cancer, the somatic missense Ras mutations found in cancer cells introduces amino acid substitutions at positions 12, 13, and 61. These changes impair the intrinsic GTPase activity and confer resistance to GAPs, thereby causing cancer-associated mutant Ras proteins to accumulate in the active, GTP bound conformation [11]. Besides the mutation hotspots, apparently inherited polymorphisms in the HRAS sequence were described [12 14] and include a minisatellite polymorphism located 3 downstream to exon 4, the rare alleles of which were associated with bladder cancer development [15]. Rare alleles of HRAS variable numbers of tandem repeats have been associated with bladder cancer risk [15]. Another polymorphic site that could be involved in the development of cancer disease is a hexanucleotide repeat [16]. But the most important single nucleotide polymorphism identified in HRAS is at cdna position 81 T C (rs12628). It is present in codon 27 of exon 1 of HRAS, which is located in a wobble base position. HRAS SNP was shown to be associated with the risk of human cancers as well. More recently, it was demonstrated that the variant C allele of this genetic polymorphism could increase the risk of oral carcinoma, and gastric cancers [17,18]. However, no correlation between HRAS T81C SNP and colon and rectal cancers was observed in Chinese population [18]. In bladder cancer, only one study has been conducted where this SNP was reported to have a significant role in predisposition to bladder cancer [17]. These findings indicate that the distribution of HRAS T81C polymorphism seems to be genetically different in various ethnic populations. However, very few studies have been conducted to examine the HRAS T81C SNP to understand its role fully, owing to controversial results. Owing to high frequency while sequencing of HRAS gene, the HRAS 81T to C variation was considered as an informative SNP in a gene with potential relevance for carcinogenesis. Thus, we hypothesize that the HRAS T81C polymorphism may have an effect on the HRAS functional activity, which may play a role in modulating the susceptibility to bladder cancer. In order to verify our hypothesis, a population-based case-control study was conducted to investigate the association between the HRAS T81C genotypes and the risk of urinary bladder cancer in a Kashmiri population. 2. Materials and methods 2.1. Study subjects Sixty-five consecutive patients who underwent transurethral resection of bladder tumors (TURBT) in surgery department at the Sher-I-Kashmir Institute of Medical Sciences (SKIMS) between 2008 and 2010 were included in this study for sequence analysis of HRAS. All the samples were confirmed to be histologically transitional cell carcinoma (TCC) of bladder (except one adenocarcinoma). A total of 140 peripheral blood samples from urinary bladder cancer patients were collected in the urology ward and related clinics in SKIMS between March 2008 and August A pool of 160 control subjects were recruited from the same hospital and belonging to the same geographical area, ethnic background, and approximately from the similar age group. The mean age was years for the cases and years for the controls (P 0.52). The controls were healthy, free from any cancer, and were frequency-matched to the cases in terms of age ( 7 years), and gender. A written informed consent was obtained from each recruited subject and the study was approved by the local institutional ethical committee. Cases and controls went through a face-to-face interview during hospital admission using standard questionnaires DNA extraction Tissue samples (both tumor and adjacent normal tissue) collected after TURBT were snap-frozen immediately and stored at 70 C. DNA from tissues was extracted using DNA easy Tissue kit (Qiagen GmbH, Hilden, Germany) according to enclosed protocol for sequencing analysis. For HRAS T81C genotyping, the genomic DNA was extracted from peripheral blood samples (n 300, controls and cases) using standard proteinase-k digestion, phenol/chloroform extraction, and ethanol precipitation method Mutational analysis of HRAS Exon 1 and 2 of the HRAS were amplified using the previously described specific primers [19] exon1f 5 -CAG- GAGACCCTGTAGGAGGA-3 ; R 5 -TCG TCCAC AAAA TGGTT CTG-3, exon2 F 5 -TCCTGCAGGATTCCTAC- CGG-3 ; R-5 -GGTTCACCT GTAC TGGTGG A-3 ). PCR amplification was carried out in a 50 L volume containing 50 ng of genomic DNA, 1 PCR buffer containing 15 mm MgCl 2, 100 M each of datp, dgtp, dttp, dctp, 1.5 U of Taq DNA polymerase (Biotool, Madrid, Spain), and 1 M of forward and reverse primers (Genescript, Piscataway, NJ). After an initial denaturation at 95 C for 5 minutes, 35 cycles of 94 C for 1 minute, a specific annealing temperature (HRAS exon 1, 60 C; HRAS exon 2, 55 C) for 1 minute, and 72 C for 80 seconds were performed.

3 A.A. Pandith et al. / Urologic Oncology: Seminars and Original Investigations 31 (2013) to determine whether the polymorphisms were in Hardy- Weinberg equilibrium between cases and controls. Odds ratios (ORs) were used as estimates of the relative risk and 95% confidence intervals (CIs) were calculated to estimate the association between certain genotypes or other related risk factors of bladder cancer. 3. Results Fig. 1. Digestion of PCR product by Dra III. TT allele (186bp) shown in well 5, 6; the TC heterozygous (186 and 128 bp) in well 1, 2, 3, 7; and homozygous CC mutant allele (128 and 58 bp) in well 4 and M; 100 bp DNA ladder. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was performed for genotyping of HRAS T81C. A 186 bp DNA segment was amplified using forward primer F5 - CAGGAGACCTG- TAGGAGGA-3 and reverse primer R5 -GGCACCTG- GACGGCG GCGCTAG-3. PCR was carried out in a final volume of 25 L containing 50 ng genomic DNA template, 1 PCR buffer (Biotools, Madrid, Spain) with 2 mm MgCl2, 0.4 M of each primer (Genescript), 50 M dntps (Biotools), and 1.25 U Taq polymerase (Biotools). For PCR amplification, the standard protocol was used as follows: one initial denaturation step at 94 C for 7 minutes, followed by 35 denaturation cycles of 30 seconds at 94 C, 30 seconds of annealing at 60 C, and 30 seconds of extension at 72 C, followed by a final elongation cycle at 72 C for 5 minutes. For RFLP, the PCR products of HRAS T81C SNP were digested with DraIII (Fermentas Inc., MD) (5 U at 37 C for 16 h). For codon 81 of HRAS the CC homozygote (variant) with DraIII restriction site was cut into fragments of 128 and 58 bp, while the (TT) homozygote presented a single fragment of 186 bp and heterozygous form displayed (T/C) 186, 128, and 58 bp (Fig. 1). For quality control, each PCR reaction used distilled water instead of DNA as a negative control, and more than 20% of the samples were analyzed twice. DNA fragments were subjected to electrophoresis on a 3% agarose gel for resolution and samples were also run on 8% polyacrylamide gel to confirm results Statistical analysis The cases and controls were compared using the 2 test for categorical variables like sex and smoking status of the demographic variables. A goodness-of-fit 2 test was used Exon 1 and 2 of HRAS were screened for mutations in 65 bladder cancer cases, which revealed a mutation frequency of 13.8% (09 of 65). Mutations were observed in codon 12 and 61(preliminary report already accepted for publication). A frequent T to C substitution was identified in codon 27 of exon 1 at cdna position 81, which is located in a wobble base position. For HRAS 81 T/C SNP, a total of 140 urinary bladder cases and 160 cancer-free healthy controls were studied for polymorphic analysis. The cases included 112 males and 28 female patients (5:1) and the controls consisted of 130 males and 35 females. The cases and controls were frequency matched in terms of their age, gender, and smoking status. The mean age was years for the cases and years for the controls, and there was no significant gender-related difference. Of the total number of cases, 105 were smokers and 35 were non-smokers and almost the Table 1 Frequency distribution analysis of selected demographic and risk factors in bladder cancer cases and controls Characteristics Cases n 140 (%) Controls n 160 (%) P value Sex Male 112 (80.0) 130 (81.1) 0.79 Female 28 (20) 30 (21.9) Age 50 years 50 (35.7) 55 (34.3) years 90 (64.3) 105 (65.7) Smoking status Never smokers 35 (25.0) 42 (26.2) 0.45 Current smokers 105 (75.0) 118 (73.8) Dwelling Urban 40 (28.5) 45 (26.7) 0.93 Rural 100 (71.5) 115 (73.3) Mean age, years ( SD) 58.5 (12.3) 56.6 (11.6) 0.52 Mean no. of years smoking 28.6 (14.0) 21.0 (14.7) 0.01 ( SD) Nodal status Involved 20 (14.3) NotInvolved 120 (85.7) Histologic type GI 13 (9.3) GII 65 (46.4) GIII 62 (44.3) Clinical stage pta 25 (17.8) pt1 50 (35.7) pt2 65 (45.4)

4 490 A.A. Pandith et al. / Urologic Oncology: Seminars and Original Investigations 31 (2013) Table 2 Association between HRAS T81C phenotypes and clinicopathologic characteristics Cases TT TC CC Controls TT TC CC OR (95%CI) P value 64.3% 35.7% 84.4% 15.6% Overall genotype n n ( ) (%) Age group (35.7) (34.3) ( ) (64.3) (65.7) ( ) 0.00 Sex Female 28 (20) (21.9) ( ) 0.04 Male 112 (80) (81.1) ( ) 0.00 Dwelling Rural 40 (28.5) (26.7) ( ) 0.1 Urban 100 (71.5) (73.3) ( ) Smoking Never 35 (25.0) (26.2) ( ) 0.2 Ever 105 (75.0) (73.8) ( ) 0.00 Tumor a grade G-I 13 (9.2) ( ) 0.16 G-II 65 (46.4) ( ) 0.18 G-III/IV 62 (44.2) ( ) 0.00 Tumor b stage pta 25 (17.8) ( ) 0.1 pt1 50 (35.7) ( ) pt2/higher 65 (46.4) ( ) Genotype TT 90 (64.3) 135 (84.4) TC 42 (30.0) 25 (25.6) CC 08 (5.7) 00 Allele type T 222 (79.3) 295 (92.2) C 58 (20.7) 25 (7.8) Crude ORS a,b based on comparisons with the entire patient control group (n 160). same percentage was recorded in controls (Table 1); 74% of the cases were above 50 years of age and no significant age or gender-related differences were observed between the groups (P 0.05). The subjects were considered never smokers only if until the day of sample collection they had not used tobacco, and current if they are smoking presently or had quit smoking since last 6 months or less before sample collection. Table 1 shows demographic information and other parameter histories for the study subjects whereas the distribution of HRAS T81C genotypes and its allele frequency in cases and controls are shown in Table 2. Owing to the very low frequency of the CC genotype and an increased risk associated with TC and CC genotypes, TC CC was compared against TT. HRAS T81C SNP, frequencies of TT, TC, and CC genotypes among controls were 84.4%, 15.6%, and 0.0% while in cases allele frequencies were 64.3%, 30%, and 5.7%, respectively with odds ratio (OR) 3.0; 95% confidential interval (CI) ). The cases had a higher frequency of the rare allele (TC CC 35.7%) than the controls (15.6%), and this difference showed statistically significant risk with TC CC combination against TT (P 0.000) (Table 2). The frequency of mutant C allele observed in cases was 0.2 (20.7%) and 0.08 (7.8%) in controls. This observation showed a highly statistical significance of rare allele (C) between cases and controls (P 0.000). When classified further into groups, our study interestingly found higher number of rare allele (TC CC) in high grade tumors (GIII/IV) and were abundantly present as the depth of invasion increased (pt1 pt2) (P 0.00) (Table 2). Smokers were more significantly associated with combined TC and CC against TT than non-smokers (OR 3.4; 95% CI ) (P 0.001). Association of variant allele with other clinicopathologic characteristics is given in the Table 2. A meta-analysis was performed to obtain an overall assessment of the effects of the HRAS T81C polymorphism on human cancers, which was examined according to HRAS genotype in all published studies and in our patients (Table 3). The pooled odds ratio (random) was 1.7 (95% CI, ), which was significant, clearly indicating HRAS T81C as risk factor. Table 3 Meta-analysis of H-RAS T81C polymorphism for all cancers Study Type of cancer Cases Controls OR 95% CI Johne [17] Urinary bladder Castro [24] Thyroid Sathyan [21] Oral Yongjing [18] Gastrointestinal This study Urinary bladder Total (95% CI) 1,009 1,

5 A.A. Pandith et al. / Urologic Oncology: Seminars and Original Investigations 31 (2013) Discussion Several molecular alternations in RAS gene have been identified such as minisatellites and acquired mutations in various cancers. Particularly, HRAS has been predominantly studied and shown to be involved in bladder cancer [9,19]. During our study for mutational analysis of HRAS in bladder cancer [20], HRAS T81C was frequently observed and was considered to be an informative SNP. Since this polymorphism has been reported only once in bladder cancer, further evaluation was imperative, to elucidate the conformity of the results in the backdrop of different ethnic backgrounds. Thus, we conducted a case-control polymorphic study of HRAS T81C to assess the role of this SNP in bladder cancer. The distribution analysis of 3 genotypes in our study revealed allelic frequency of TT, TC, and CC as 64.3%, 30%, and 5.7% in cases and 84.4%, 15.6%, and 0.0% in controls, respectively. This pattern of distribution of allele frequency in this study was compatible with Yongjing et al. (2008) [18]. The frequency of C allele observed in cases was 20.7% compared with 7.8% in controls, and this difference was highly significant (P 0.000) and was in tune with a study conducted on oral cancer [21]. Our study revealed a 3-fold increased risk of bladder cancer in carriers of the variant allele (TC CC) in comparison to wild-type carriers (TT) (P 0.001). The variant allele frequency (TC CC) found in cases aggregated to almost 36% compared with 15% in controls, and this difference shows a significant association with 3-fold risk of bladder cancer development (P ). Frequency of variant allele was observed mildly higher in the only study conducted by Johne et al. [17] in bladder cancer compared with our study but the fold increase in the risk was found higher in our report (47% vs. 36%; OR 2.04 vs. 3.0). This study, therefore, reveals a statistically significant increased risk for bladder cancer, particularly in the TC CC genotypes combination compared with the TT genotype, which indicates that HRAS T81C SNP is a strong susceptibility factor for the development of bladder cancer. Consistent with the tissue specificity hypothesis and various studies that had confirmed that the HRAS gene plays a more important role in bladder cancer acquired amino acid mutations in the hotspot codons 12, 13, and 61, which prolong the GTP-bound activated state of the HRAS product [22], this polymorphism HRAS T81C SNP does not lead to the alternation of ras protein structure, it affects the cancer susceptibility possibly through linkage disequilibrium with other potential functional variants of HRAS. One of the linkage candidates is a region of variable tandem repeats about 1 kb downstream exon 4, with a possible transcriptional enhancer activity [23]. Another associated polymorphic site is hexanucleotide repeat located about 80 bp upstream of the 5 -end of exon1 [16]. Yet another report has shown that HRAS T81C might be serving as a marker of other polymorphisms in intron D2 of HRAS that would act as regulators of IDX inclusion [24]. In cases, our report noticed TC and CC genotypes were frequently presented in high grade tumors (GIII/GIV) and invasive stages (pt1 and pt2) with statistically high significance (P 0.001), which is in accordance with the only study conducted on bladder cancer [17]. Homozygous CC genotypes were inclusively found in high grade and stage tumors. This indicates, presumably, that the HRAS T81C SNP results in accelerated tumor progression by conferring a growth advantage on tumor cell, but this finding needs to be evaluated in further large studies in different populations. Furthermore, ever smokers with combined TC and CC in cases had a 3.2-fold risk to bladder cancer than never smokers, which can be presumably related to HRAS gene alterations with certain environmental factors, particularly smoking [20]. In addition to the already reported clinicopathologic risk factors, we also found a significant association of the HRAS T81C SNP with age group ( 50), nodal status,, and gender identifying them as the potential risk factors for bladder cancer. 5. Conclusion The present study suggests that HRAS T81C SNP may be a risk factor for the development of bladder cancer in a Kashmiri population. We also include that variant allele is related to advanced bladder tumors and, thus, could become a useful marker to predict the bladder cancer development. However, these correlations need to be authenticated in a large sample study in the future, so as to help in the better discernment of racial differences and in determining the course of bladder cancer. Acknowledgments The authors acknowledge the technical help of Mr. Mohsin Saleem of the Department of Biochemistry. Their thanks are also due to the Head and Technical Staff, especially Mr. Nazir of the operation theater of the Department of Urological Surgery, and sister Zubaida, who helped us in procuring the tissue and blood samples. References [1] Ploeg M, Aben KK, Kiemeney L. The present and future burden of urinary bladder cancer in the world. World J Urol 2009;27: [2] Clavel J. Progress in the epidemiological understanding of gene environment interactions in major diseases: Cancer. CR Biol 2007; 330: [3] Clayson DB. Specific aromatic amines as occupational bladder carcinogens. Natl Cancer Inst Monog 1998;58:15 9. [4] Ferlay JV, Bray P, Isani DM, et al. GLOBOCAN 2002: Cancer incidence, mortality, and prevalence worldwide. IARC Cancer Base No. 5. ver IARC Press: Lyon, [5] Kamarana NM, Kamat MR, Kurkure AP. National Cancer registry project. 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