- Email: [email protected]
ARLTS1 variants and risk of colorectal cancer
Cancer Letters 244 (2006) 172–175 www.elsevier.com/locate/canlet
ARLTS1 variants and risk of colorectal cancer Bernd Frank a,*, Kari Hemminki a,b, Hermann Brenner c, Michael Hoffmeister c, Jenny Chang-Claude d, Barbara Burwinkel a a
Division of Molecular Genetic Epidemiology, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany b Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden c Department of Epidemiology, German Centre for Research on Ageing, Heidelberg, Germany d Division of Clinical Epidemiology, German Cancer Research Center, Heidelberg, Germany Received 19 October 2005; received in revised form 30 November 2005; accepted 5 December 2005
Abstract The Cys148Arg and Trp149Stop variants in the tumour suppressor gene ARLTS1 predispose to familial breast cancer, suggesting that these variants might also contribute to colorectal carcinogenesis. As the first to evaluate the association between Cys148Arg and Trp149Stop and colorectal cancer (CRC) risk, we genotyped 611 cases with CRC (including 77 cases with a first-degree family history) and 539 controls recruited from the German DACHS study. No significant differences in the genotype frequencies of Cys148Arg and Trp149Stop were observed between cases and controls. However, we showed a non-significant increased risk of familial CRC for both variants (ORZ1.40 and 1.45), indicating a possible role of ARLTS1 in familial CRC. q 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Colorectal cancer; Tumour suppressor; Apoptosis; Polymorphism; Risk assessment
1. Introduction Colorectal cancer (CRC) is one of the most common cancers in industrialised countries [1]. Despite mainly environmental causes including diet, overweight and physical activity, genetics has a pivotal role in predisposition to CRC. About 5% of familial colorectal cancer can be ascribed to germline mutations in the high-penetrance genes APC, causing colon cancer with adenomatous polyposis coli and the mismatch repair genes MLH1 and MSH2, leading to hereditary nonpolyposis colorectal cancer (HNPCC). The remaining familial risk remains unknown, but could be due to high-penetrance mutations in yet unidentified genes or polygenic mechanisms [1–4]. Calin et al. have * Corresponding author. Tel.: C49 6221 421802; fax: C49 6221 421810. E-mail address: [email protected] (B. Frank).
identified ARLTS1 (ADP-ribosylation factor-like tumour-suppressor gene 1), a widely expressed member of the ARF-ARL (ADP-ribosylation factor/ARF-like) family, as a low-penetrance tumour suppressor gene in human cancers such as breast cancer (BC), melanoma and chronic lymphocytic leukemia (CLL) [5]. ARLTS1 expression has been shown to be dramatically decreased in lung cancer compared to normal lung cells. Moreover, transfection of wild type ARLTS1 into A549 lung cancer cells suppressed tumour formation in immunodeficient Nu/Nu mice by activating the intrinsic apoptotic pathway, while transfection of truncated ARLTS1 Stop149 had a restricted influence on apoptosis and tumour suppression [5]. While Calin et al. have revealed Trp149Stop to predispose to familial cancer, a recent study has demonstrated the association of this nonsense mutation with an increased high-risk familial and bilateral BC risk [5–7]. Additionally, ARLTS1 Cys148Arg which—according
0304-3835/$ - see front matter q 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.12.006
B. Frank et al. / Cancer Letters 244 (2006) 172–175
to in silico analyses—is predicted to be functional, was associated with an increased familial BC risk [7]. Since hemizygous ARLTS1 deletions have been observed in 10% of colorectal carcinomas [5], we estimated the risk associated with the ARLTS1 Cys148Arg and Trp149Stop variants in a large colorectal cancer case– control study. 2. Material and methods 2.1. Study population Colorectal cancer cases and controls were drawn from the German DACHS (Darmkrebs: Chancen der Verhu¨tung durch Screening) study, a population-based case–control study, which is carried out in the Rhine-Neckar-Odenwald and Heilbronn regions. This analysis comprises 661 unrelated male and female case patients with incident invasive colorectal cancer diagnosed between January 2003 and March 2005 residing in the study region. Controls consist of 607 unrelated male and female individuals without a history of colorectal cancer who were randomly selected from lists of residents from the counties within the study region and matched to cases by 5-year age groups, sex, county of residence. Cases and controls were included if they were at least 30 years of age, German-speaking, and mentally and physically able to participate in a personal interview of about 1h. The study was approved by the Ethics Committees of the University of Heidelberg (Heidelberg, Germany) and the State Medical Boards of Baden-Wuerttemberg and Rhineland-Palatinate. 2.2. Data collection Details of the data collection procedures for the DACHS study are reported elsewhere [8]. Briefly, study subjects were asked to participate in an in-person interview prior to blood donation or a mouthwash when blood samples were not available. Information on demographic factors, anthropometric measures, medical history including medication and screening, familiy history of colorectal cancer, reproductive history and lifestyle factors (such as smoking, nutrition and physical activity) were collected by trained interviewers using a standardised questionnaire. 2.3. Genotyping Samples were genotyped for the ARLTS1 variants by direct sequencing as previously described [6,7,9]. Primer sequences were ARLTS1_FOR: 5 0 -GAT ATC CTC GTG TAC GTG CTG-3 0 and ARLTS1_REV: 5 0 -GAG CAA AGA TAT GCT GCT CTG T-3 0 . Standard PCR amplification was performed using 10 ng of genomic DNA, 1!PCR buffer, 1.5 mM MgCl2, 0.11 mM dNTP mixture (Invitrogen, Paisley,
173
UK), 0.15 mM each primer (MWG Biotech AG, Ebersberg, Germany) and 0.4 U PlatinumTaq DNA polymerase (Invitrogen) in an 11 ml reaction volume. Thermal cycling was carried out in GeneAmp PCR System 9700 thermocyclers (Applied Biosystems, Foster City, USA) with the following conditions: Initial denaturation for 2 min at 94 8C followed by five cycles including 30 s at 94 8C, 20 s at 64 8C, 30 s at 72 8C; four cycles including 30 s at 94 8C, 20 s at 62 8C, 30 s at 72 8C; four cycles including 30 s at 94 8C, 20 s at 60 8C, 30 s at 72 8C; six cycles including 25 s at 94 8C, 20 s at 58 8C, 30 s at 72 8C; 33 cycles consisting of 25 s at 94 8C, 20 s at 56 8C, 30 s at 72 8C and a final extension step for 4 min at 72 8C. 2.4. Statistical analysis Data of 611 case patients (33–91 years of age; median 68) including 77 patients with a first-degree family history (37–86 years of age, median 68) and 538 controls (34–94 years of age, median 67) were included in the analysis. With our present sample size, we had a power of 80% at a significance level of 0.05 to detect an OR of R1.52 for Cys148Arg and an OR of R2.47 for the rare Trp149Stop variant. HardyWeinberg equilibrium was assessed by c2 test with 1 df to compare the observed genotype frequencies with the expected genotype frequencies among the subjects using a tool offered by the Institute of Human Genetics, Technical University Munich, Munich, Germany (http://ihg.gsf.de/cgi-bin/hw/ hwa1.pl). Genotype-specific odds ratios (OR), corresponding 95% confidence intervals (95% CI) were computed by means of unconditional logistic regression using SAS version 9.1 (SAS Institute Inc., Cary, NC). P values were calculated using two-sided c2 test and P!0.05 was deemed statistically significant. Power calculation was carried out with the power and sample size calculation software PS version 2.1.31 [10] (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/ PowerSampleSize).
3. Results and discussion We evaluated the impact of both ARLTS1 Cys148Arg and Trp149Stop on colorectal cancer risk. While ARLTS1 Trp149Stop has been demonstrated to affect apoptosis and tumour suppression [5], the functional relevance of Cys148Arg may be ascertained by in silico functional analyses. The cysteine to arginine exchange leads to changes in both protein secondary structure and solvent accessibility (SNP effect database) and is predicted to be ‘probably damaging’ (PolyPhen report) [11,12]. Beyond a single mutation in the yeast homologue of ARL1 (Asp151Gly), which corresponds to the human ARLTS1 Asp146, adjacent to the Cys148Arg variant, has been proven to inhibit the progression of apoptosis
174
B. Frank et al. / Cancer Letters 244 (2006) 172–175
Table 1 Genotype frequencies of ARLTS1 Cys148Arg (T442C) and Trp149Stop (G446A) in patients with colorectal cancer and control individuals, odds ratios (OR) with 95% confidence intervals (95% CI) and respective P values Males Cases (%) Cys148Arg (T442C) TT TC CC OR (95% CI), P value [CCCTC] vs. [TT]
Females Controls (%)
79 (22.0) 75 (24.5) 181 (50.4) 153 (50.0) 99 (27.6) 78 (25.5) 1.15 (0.80–1.65); 0.45
Trp149Stop (G446A) GG 352 (98.1) 300 (98.0) GA 7 (1.9) 6 (2.0) AA 0 (0) 0 (0) OR (95% CI), P 0.99 (0.33–2.99); 0.99 value [AACGA] vs. [GG]
Cases (%)
Total Controls (%)
Cases (%)
Controls (%)
56 (22.2) 60 (25.9) 133 (52.8) 103 (44.4) 63 (25.0) 69 (29.7) 1.22 (0.80–1.85); 0.35
135 (22.1) 135 (25.1) 314 (51.4) 256 (47.6) 162 (26.5) 147 (27.3) 1.17 (0.89–1.53); 0.27*
244 (96.8) 224 (96.6) 8 (3.2) 8 (3.4) 0 (0) 0 (0) 0.92 (0.34–2.49); 0.87
596 (97.5) 524 (97.4) 15 (2.5) 14 (2.6) 0 (0) 0 (0) 0.97 (0.46–2.04); 0.94*
Sex- and age-adjusted OR and P values are indicated with an asterisk (*).
due to a defect in vacuole formation [13]. Genotype frequencies of both polymorphisms analysed were in agreement with Hardy-Weinberg expectations. There was no difference in genotype frequencies between CRC cases and control individuals for either ARLTS1 Cys148Arg (ORZ1.17, 95% CIZ0.89–1.53, PZ0.27; Table 1) or Trp149Stop (ORZ0.97, 95% CIZ0.46– 2.04, PZ0.94; Table 1). Compared to alterations in the high-penetrance genes APC, MLH1 and MSH2, the contribution of both variants, especially the very rare Trp149Stop variant, to sporadic and familial CRC risk appears to be negligible, but their interactions with other variants in CRC susceptibility genes or environmental influences may modify cancer risk [4,14]. Considering the effect of ARLTS1 Cys148Arg and Trp149Stop on familial colorectal cancer risk revealed a non-significant increased risk (ORZ1.40, 95% CIZ 0.77–2.54, PZ0.27 and ORZ1.45, 95% CIZ0.40– 5.19, PZ0.23; Table 2). The risk increase is in accordance with the assessment of Cys148Arg for familial and Trp149Stop for high-risk familial BC risk (ORZ1.48 and 2.08), supporting the role of both ARLTS1 variants in familial cancers like BC, CLL and pancreatic cancer as well as melanoma and prostate carcinoma [5–7]. Much of the familial aggregation of common cancers results from inherited susceptibility [2,4,15]. Therefore, the observed elevation of CRC risk for Cys148Arg and Trp149Stop becomes evident only by investigating familial cases. In addition, cases with an extended family history appear particularly powerful
for rare variants as Trp149Stop [16]. The analysis of variant segregation in CRC families may help to evaluate the effects of both Cys148Arg and Trp149Stop. The lack of a significant association observed, however, could also be attributed to the limited sample size. Assuming the ORs of 1.40 and 1.45 at a significance level of 0.05 for Cys148Arg and Trp149Stop, respectively, a study population of about Table 2 Genotype frequencies of ARLTS1 Cys148Arg (T442C) and Trp149Stop (G446A) in patients with a first-degree family history of colorectal cancer and control individuals, sex- and age-adjusted odds ratios (OR) with 95% confidence intervals (95% CI) and respective P values Total Familial cases (%) Cys148Arg (T442C) TT 15 (19.5) TC 43 (55.8) CC 19 (24.7) OR (95% 1.40 (0.77–2.54); 0.27 CI), P value [CCCTC] vs. [TT] Trp149Stop (G446A) GG 74 (96.1) GA 3 (3.9) AA 0 (0) OR (95% 1.45 (0.40–5.19); 0.23 CI), P value [AACGA] vs. [GG]
Controls (%)
135 (25.1) 256 (47.6) 147 (27.3)
524 (97.4) 14 (2.6) 0 (0)
B. Frank et al. / Cancer Letters 244 (2006) 172–175
850 and 4000 familial colon cancer cases and controls would be necessary to gain a power of 80% [10]. Since the proportion of familial CRC accounts for only 20% in the general population [1–3], it would be extremely difficult to recruit this number of familial cases, especially for the analysis of ARLTS1 Trp149Stop. As a result, meta-analyses would be helpful to elucidate the role of the ARLTS1 variants Cys148Arg and Trp149Stop in establishing a predisposition to familial colorectal cancer. Acknowledgements The authors wish to thank Justo Lorenzo Bermejo for statistical advice and Kerstin Wagner for critical comments on the manuscript.
References [1] A. de la Chapelle, Genetic predisposition to colorectal cancer, Nat. Rev. Cancer 4 (2004) 769–780. [2] P. Lichtenstein, N.V. Holm, P.K. Verkasalo, A. Iliadou, J. Kaprio, M. Koskenvuo, et al., Environmental and heritable factors in the causation of cancer-analyses of cohorts of twins from Sweden, Denmark, and Finland, N. Engl. J. Med. 343 (2000) 78–85. [3] K. Hemminki, B. Chen, Familial risk for colorectal cancers are mainly due to heritable causes, Cancer Epidemiol. Biomarkers Prev. 13 (2004) 1253–1256. [4] R.S. Houlston, J. Peto, The search for low-penetrance cancer susceptibility alleles, Oncogene 23 (2004) 6471–6476. [5] G.A. Calin, F. Trapasso, M. Shimizu, C.D. Dumitru, S. Yendamuri, A.K. Godwin, et al., Familial cancer associated with a polymorphism in ARLTS1, N. Engl. J. Med. 352 (2005) 1667–1676.
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[6] B. Frank, R. Klaes, B. Burwinkel, Familial cancer and ARLTS1, N. Engl. J. Med. 353 (2005) 313–314. [7] B. Frank, K. Hemminki, A. Meindl, B. Wappenschmidt, R. Klaes, R.K. Schmutzler, et al., Association of the ARLTS1 Cys148Arg variant with familial breast cancer risk, Int. J. Cancer 2005 Dec 13; [Epub ahead of print]. [8] C. Lilla, E. Verla-Tebit, A. Risch, B. Ja¨ger, M. Hoffmeister, H. Brenner, et al., Effect of NAT1 and NAT2 genetic polymorphisms on colorectal cancer risk associated with exposure to tobacco smoke and meat consumption, Cancer Epidemiol. Biomarkers Prev. 15 (2006) 99–107. [9] B. Frank, K. Hemminki, M. Wirtenberger, J.L. Bermejo, P. Bugert, R. Klaes, et al., The rare ERBB2 variant Ile654Val is associated with an increased familial breast cancer risk, Carcinogenesis 26 (2005) 643–647. [10] W.D. Dupont, W.D. Plummer Jr., Power and sample size calculations for studies involving linear regression, Control. Clin. Trials 29 (1998) 589–601. [11] J. Reumers, J. Schymkowitz, J. Ferkinghoff-Borg, F. Stricher, L. Serrano, F. Rousseau, SNPeffect: a database mapping molecular phenotypic effects of human non-synonymous coding SNPs, Nucleic Acids Res. 33 (2005) D527–D532. [12] V. Ramensky, P. Bork, S. Sunyaev, Human non-synonymous SNPs: server and survey, Nucleic Acids Res. 30 (2002) 3894– 3900. [13] A. Abudugupur, K. Mitsui, S. Yokota, K. Tsurugi, An ARL1 mutation affected autophagic cell death in yeast, causing a defect in central vacuole formation, Cell Death Differ. 9 (2002) 158–168. [14] M.M. de Jong, I. Nolte, G.J. te Meerman, W.T. van der Graaf, J.C. Oosterwijk, J.H. Kleibeuker, et al., Genes other than BRCA1 and BRCA2 involved in breast cancer susceptibility, J. Med. Genet. 39 (2002) 225–242. [15] K. Hemminki, C. Granstrom, Familial breast cancer: scope for more susceptibility genes?, Breast Cancer Res. Treat. 82 (2003) 17–22. [16] A.C. Antoniou, D.F. Easton, Polygenic inheritance of breast cancer: implications for design of association studies, Genet. Epidemiol. 25 (2003) 190–202.
ARLTS1 variants and risk of colorectal cancer Bernd Frank a,*, Kari Hemminki a,b, Hermann Brenner c, Michael Hoffmeister c, Jenny Chang-Claude d, Barbara Burwinkel a a
Division of Molecular Genetic Epidemiology, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany b Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden c Department of Epidemiology, German Centre for Research on Ageing, Heidelberg, Germany d Division of Clinical Epidemiology, German Cancer Research Center, Heidelberg, Germany Received 19 October 2005; received in revised form 30 November 2005; accepted 5 December 2005
Abstract The Cys148Arg and Trp149Stop variants in the tumour suppressor gene ARLTS1 predispose to familial breast cancer, suggesting that these variants might also contribute to colorectal carcinogenesis. As the first to evaluate the association between Cys148Arg and Trp149Stop and colorectal cancer (CRC) risk, we genotyped 611 cases with CRC (including 77 cases with a first-degree family history) and 539 controls recruited from the German DACHS study. No significant differences in the genotype frequencies of Cys148Arg and Trp149Stop were observed between cases and controls. However, we showed a non-significant increased risk of familial CRC for both variants (ORZ1.40 and 1.45), indicating a possible role of ARLTS1 in familial CRC. q 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Colorectal cancer; Tumour suppressor; Apoptosis; Polymorphism; Risk assessment
1. Introduction Colorectal cancer (CRC) is one of the most common cancers in industrialised countries [1]. Despite mainly environmental causes including diet, overweight and physical activity, genetics has a pivotal role in predisposition to CRC. About 5% of familial colorectal cancer can be ascribed to germline mutations in the high-penetrance genes APC, causing colon cancer with adenomatous polyposis coli and the mismatch repair genes MLH1 and MSH2, leading to hereditary nonpolyposis colorectal cancer (HNPCC). The remaining familial risk remains unknown, but could be due to high-penetrance mutations in yet unidentified genes or polygenic mechanisms [1–4]. Calin et al. have * Corresponding author. Tel.: C49 6221 421802; fax: C49 6221 421810. E-mail address: [email protected] (B. Frank).
identified ARLTS1 (ADP-ribosylation factor-like tumour-suppressor gene 1), a widely expressed member of the ARF-ARL (ADP-ribosylation factor/ARF-like) family, as a low-penetrance tumour suppressor gene in human cancers such as breast cancer (BC), melanoma and chronic lymphocytic leukemia (CLL) [5]. ARLTS1 expression has been shown to be dramatically decreased in lung cancer compared to normal lung cells. Moreover, transfection of wild type ARLTS1 into A549 lung cancer cells suppressed tumour formation in immunodeficient Nu/Nu mice by activating the intrinsic apoptotic pathway, while transfection of truncated ARLTS1 Stop149 had a restricted influence on apoptosis and tumour suppression [5]. While Calin et al. have revealed Trp149Stop to predispose to familial cancer, a recent study has demonstrated the association of this nonsense mutation with an increased high-risk familial and bilateral BC risk [5–7]. Additionally, ARLTS1 Cys148Arg which—according
0304-3835/$ - see front matter q 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.12.006
B. Frank et al. / Cancer Letters 244 (2006) 172–175
to in silico analyses—is predicted to be functional, was associated with an increased familial BC risk [7]. Since hemizygous ARLTS1 deletions have been observed in 10% of colorectal carcinomas [5], we estimated the risk associated with the ARLTS1 Cys148Arg and Trp149Stop variants in a large colorectal cancer case– control study. 2. Material and methods 2.1. Study population Colorectal cancer cases and controls were drawn from the German DACHS (Darmkrebs: Chancen der Verhu¨tung durch Screening) study, a population-based case–control study, which is carried out in the Rhine-Neckar-Odenwald and Heilbronn regions. This analysis comprises 661 unrelated male and female case patients with incident invasive colorectal cancer diagnosed between January 2003 and March 2005 residing in the study region. Controls consist of 607 unrelated male and female individuals without a history of colorectal cancer who were randomly selected from lists of residents from the counties within the study region and matched to cases by 5-year age groups, sex, county of residence. Cases and controls were included if they were at least 30 years of age, German-speaking, and mentally and physically able to participate in a personal interview of about 1h. The study was approved by the Ethics Committees of the University of Heidelberg (Heidelberg, Germany) and the State Medical Boards of Baden-Wuerttemberg and Rhineland-Palatinate. 2.2. Data collection Details of the data collection procedures for the DACHS study are reported elsewhere [8]. Briefly, study subjects were asked to participate in an in-person interview prior to blood donation or a mouthwash when blood samples were not available. Information on demographic factors, anthropometric measures, medical history including medication and screening, familiy history of colorectal cancer, reproductive history and lifestyle factors (such as smoking, nutrition and physical activity) were collected by trained interviewers using a standardised questionnaire. 2.3. Genotyping Samples were genotyped for the ARLTS1 variants by direct sequencing as previously described [6,7,9]. Primer sequences were ARLTS1_FOR: 5 0 -GAT ATC CTC GTG TAC GTG CTG-3 0 and ARLTS1_REV: 5 0 -GAG CAA AGA TAT GCT GCT CTG T-3 0 . Standard PCR amplification was performed using 10 ng of genomic DNA, 1!PCR buffer, 1.5 mM MgCl2, 0.11 mM dNTP mixture (Invitrogen, Paisley,
173
UK), 0.15 mM each primer (MWG Biotech AG, Ebersberg, Germany) and 0.4 U PlatinumTaq DNA polymerase (Invitrogen) in an 11 ml reaction volume. Thermal cycling was carried out in GeneAmp PCR System 9700 thermocyclers (Applied Biosystems, Foster City, USA) with the following conditions: Initial denaturation for 2 min at 94 8C followed by five cycles including 30 s at 94 8C, 20 s at 64 8C, 30 s at 72 8C; four cycles including 30 s at 94 8C, 20 s at 62 8C, 30 s at 72 8C; four cycles including 30 s at 94 8C, 20 s at 60 8C, 30 s at 72 8C; six cycles including 25 s at 94 8C, 20 s at 58 8C, 30 s at 72 8C; 33 cycles consisting of 25 s at 94 8C, 20 s at 56 8C, 30 s at 72 8C and a final extension step for 4 min at 72 8C. 2.4. Statistical analysis Data of 611 case patients (33–91 years of age; median 68) including 77 patients with a first-degree family history (37–86 years of age, median 68) and 538 controls (34–94 years of age, median 67) were included in the analysis. With our present sample size, we had a power of 80% at a significance level of 0.05 to detect an OR of R1.52 for Cys148Arg and an OR of R2.47 for the rare Trp149Stop variant. HardyWeinberg equilibrium was assessed by c2 test with 1 df to compare the observed genotype frequencies with the expected genotype frequencies among the subjects using a tool offered by the Institute of Human Genetics, Technical University Munich, Munich, Germany (http://ihg.gsf.de/cgi-bin/hw/ hwa1.pl). Genotype-specific odds ratios (OR), corresponding 95% confidence intervals (95% CI) were computed by means of unconditional logistic regression using SAS version 9.1 (SAS Institute Inc., Cary, NC). P values were calculated using two-sided c2 test and P!0.05 was deemed statistically significant. Power calculation was carried out with the power and sample size calculation software PS version 2.1.31 [10] (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/ PowerSampleSize).
3. Results and discussion We evaluated the impact of both ARLTS1 Cys148Arg and Trp149Stop on colorectal cancer risk. While ARLTS1 Trp149Stop has been demonstrated to affect apoptosis and tumour suppression [5], the functional relevance of Cys148Arg may be ascertained by in silico functional analyses. The cysteine to arginine exchange leads to changes in both protein secondary structure and solvent accessibility (SNP effect database) and is predicted to be ‘probably damaging’ (PolyPhen report) [11,12]. Beyond a single mutation in the yeast homologue of ARL1 (Asp151Gly), which corresponds to the human ARLTS1 Asp146, adjacent to the Cys148Arg variant, has been proven to inhibit the progression of apoptosis
174
B. Frank et al. / Cancer Letters 244 (2006) 172–175
Table 1 Genotype frequencies of ARLTS1 Cys148Arg (T442C) and Trp149Stop (G446A) in patients with colorectal cancer and control individuals, odds ratios (OR) with 95% confidence intervals (95% CI) and respective P values Males Cases (%) Cys148Arg (T442C) TT TC CC OR (95% CI), P value [CCCTC] vs. [TT]
Females Controls (%)
79 (22.0) 75 (24.5) 181 (50.4) 153 (50.0) 99 (27.6) 78 (25.5) 1.15 (0.80–1.65); 0.45
Trp149Stop (G446A) GG 352 (98.1) 300 (98.0) GA 7 (1.9) 6 (2.0) AA 0 (0) 0 (0) OR (95% CI), P 0.99 (0.33–2.99); 0.99 value [AACGA] vs. [GG]
Cases (%)
Total Controls (%)
Cases (%)
Controls (%)
56 (22.2) 60 (25.9) 133 (52.8) 103 (44.4) 63 (25.0) 69 (29.7) 1.22 (0.80–1.85); 0.35
135 (22.1) 135 (25.1) 314 (51.4) 256 (47.6) 162 (26.5) 147 (27.3) 1.17 (0.89–1.53); 0.27*
244 (96.8) 224 (96.6) 8 (3.2) 8 (3.4) 0 (0) 0 (0) 0.92 (0.34–2.49); 0.87
596 (97.5) 524 (97.4) 15 (2.5) 14 (2.6) 0 (0) 0 (0) 0.97 (0.46–2.04); 0.94*
Sex- and age-adjusted OR and P values are indicated with an asterisk (*).
due to a defect in vacuole formation [13]. Genotype frequencies of both polymorphisms analysed were in agreement with Hardy-Weinberg expectations. There was no difference in genotype frequencies between CRC cases and control individuals for either ARLTS1 Cys148Arg (ORZ1.17, 95% CIZ0.89–1.53, PZ0.27; Table 1) or Trp149Stop (ORZ0.97, 95% CIZ0.46– 2.04, PZ0.94; Table 1). Compared to alterations in the high-penetrance genes APC, MLH1 and MSH2, the contribution of both variants, especially the very rare Trp149Stop variant, to sporadic and familial CRC risk appears to be negligible, but their interactions with other variants in CRC susceptibility genes or environmental influences may modify cancer risk [4,14]. Considering the effect of ARLTS1 Cys148Arg and Trp149Stop on familial colorectal cancer risk revealed a non-significant increased risk (ORZ1.40, 95% CIZ 0.77–2.54, PZ0.27 and ORZ1.45, 95% CIZ0.40– 5.19, PZ0.23; Table 2). The risk increase is in accordance with the assessment of Cys148Arg for familial and Trp149Stop for high-risk familial BC risk (ORZ1.48 and 2.08), supporting the role of both ARLTS1 variants in familial cancers like BC, CLL and pancreatic cancer as well as melanoma and prostate carcinoma [5–7]. Much of the familial aggregation of common cancers results from inherited susceptibility [2,4,15]. Therefore, the observed elevation of CRC risk for Cys148Arg and Trp149Stop becomes evident only by investigating familial cases. In addition, cases with an extended family history appear particularly powerful
for rare variants as Trp149Stop [16]. The analysis of variant segregation in CRC families may help to evaluate the effects of both Cys148Arg and Trp149Stop. The lack of a significant association observed, however, could also be attributed to the limited sample size. Assuming the ORs of 1.40 and 1.45 at a significance level of 0.05 for Cys148Arg and Trp149Stop, respectively, a study population of about Table 2 Genotype frequencies of ARLTS1 Cys148Arg (T442C) and Trp149Stop (G446A) in patients with a first-degree family history of colorectal cancer and control individuals, sex- and age-adjusted odds ratios (OR) with 95% confidence intervals (95% CI) and respective P values Total Familial cases (%) Cys148Arg (T442C) TT 15 (19.5) TC 43 (55.8) CC 19 (24.7) OR (95% 1.40 (0.77–2.54); 0.27 CI), P value [CCCTC] vs. [TT] Trp149Stop (G446A) GG 74 (96.1) GA 3 (3.9) AA 0 (0) OR (95% 1.45 (0.40–5.19); 0.23 CI), P value [AACGA] vs. [GG]
Controls (%)
135 (25.1) 256 (47.6) 147 (27.3)
524 (97.4) 14 (2.6) 0 (0)
B. Frank et al. / Cancer Letters 244 (2006) 172–175
850 and 4000 familial colon cancer cases and controls would be necessary to gain a power of 80% [10]. Since the proportion of familial CRC accounts for only 20% in the general population [1–3], it would be extremely difficult to recruit this number of familial cases, especially for the analysis of ARLTS1 Trp149Stop. As a result, meta-analyses would be helpful to elucidate the role of the ARLTS1 variants Cys148Arg and Trp149Stop in establishing a predisposition to familial colorectal cancer. Acknowledgements The authors wish to thank Justo Lorenzo Bermejo for statistical advice and Kerstin Wagner for critical comments on the manuscript.
References [1] A. de la Chapelle, Genetic predisposition to colorectal cancer, Nat. Rev. Cancer 4 (2004) 769–780. [2] P. Lichtenstein, N.V. Holm, P.K. Verkasalo, A. Iliadou, J. Kaprio, M. Koskenvuo, et al., Environmental and heritable factors in the causation of cancer-analyses of cohorts of twins from Sweden, Denmark, and Finland, N. Engl. J. Med. 343 (2000) 78–85. [3] K. Hemminki, B. Chen, Familial risk for colorectal cancers are mainly due to heritable causes, Cancer Epidemiol. Biomarkers Prev. 13 (2004) 1253–1256. [4] R.S. Houlston, J. Peto, The search for low-penetrance cancer susceptibility alleles, Oncogene 23 (2004) 6471–6476. [5] G.A. Calin, F. Trapasso, M. Shimizu, C.D. Dumitru, S. Yendamuri, A.K. Godwin, et al., Familial cancer associated with a polymorphism in ARLTS1, N. Engl. J. Med. 352 (2005) 1667–1676.
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