GENES, CHROMOSOMES & CANCER 00:00–00 (2015)
RESEARCH ARTICLE
Metabolic Gene Variants Associated with Chromosomal Aberrations in Healthy Humans Kari Hemminki,1,2* Christoph Frank,1 Asta F€ orsti,1,2 Ludovit Musak,3 Alena Kazimirova,4 Magdalena Barancokova,4 4 5,6 Alexandra Horska, Veronika Vymetalkova, Zdenek Smerhovsky,6,7 Alessio Naccarati,5,8 Pavel Soucek,9 Ludmila Vodickova,5,6 Janka Buchancova,10 Bozena Smolkova,11 Maria Dusinska,12 and Pavel Vodicka5,6 1 Department of Molecular Genetic Epidemiology,German Cancer Research Center (DKFZ),Im Neuenheimer Feld 580, 69120, Heidelberg,Germany 2 Center for Primary Health Care Research,Lund University,Malm€ o, Sweden 3 Jessenius Faculty of Medicine in Martin,Clinic of Occupational Medicine and Toxicology,Comenius University in Bratislava and University Hospital Martin,Kollarova 2, 03601Martin, Slovakia 4 Faculty of Medicine, Slovak Medical University,Limbova12,Bratislava, Slovakia 5 Institute of Experimental Medicine, Academy of Sciences of the Czech Republic,Videnska1083,142 00 Prague,Czech Republic 6 Institute of Biology and Medical Genetics,1st Medical Faculty,Charles University, Albertov 4,128 00 Prague,Czech Republic 7 2nd Faculty of Medicine,Charles University Prague,Czech Republic 8 Human Genetics Foundation,Turin,Italy 9 National Institute of Public Health, Srobarova 48,10042 Prague10,Czech Republic 10 Department of Public Health,Jessenius Faculty of Medicine in Martin,Comenius University in Bratislava, Sklabinska 26, 03601Martin, Slovakia 11 Laboratory of Cancer Genetics,Cancer Research Institute of Slovak Academy of Sciences,Bratislava, Slovakia 12 Health Effects Laboratory MILK,NILU-Norwegian Institute for Air Research,Kjeller,Norway
Nonspecific chromosomal aberrations (CAs) are found in about 1% of lymphocytes drawn from healthy individuals. They include chromosome-type aberrations (CSAs), which are increased in exposure to ionizing radiation, and chromatid-type aberrations (CTAs) which in experimental systems are formed by DNA binding carcinogens and mutagens. The frequency of CAs is associated with the risk of cancer, but the causes of CAs in general population are unknown. Here, we want to test whether variants in metabolic genes associate with CAs in healthy volunteers. Cases were considered those whose total CA (CAtot) frequency was >2% and for CSA and CTA the limit was >1%. Controls had lower frequencies of CAs. Functional polymorphisms in seven genes were selected for analysis: cytochrome P450 1B1 (CYP1B1), epoxide hydrolase 1 (EPHX1), NAD(P)H:quinone oxidoreductase 1 (NQO1), each coding for phase 1 enzymes, and glutathione S-transferase P1 (GSTP1), glutathione S-transferases M1 (GSTM1) and T1 (GSTT1), coding for enzymes which conjugate reactive metabolites, that is, phase 2 enzymes. The number of volunteers genotyped for each gene varied from 550 to 1,500. Only EPHX1 was individually associated with CAtot; high activity genotypes decreased CAtot. A total of six significant (P < 0.01) pair-wise interactions were observed, most including a GST variant as one of the pair. In all genotype combinations with significant odds ratios for CAs a GST variant was involved. The present data provide evidence that variants in genes coding for metabolic enzymes, which individually have small effects, interact and are associated with CA frequencies in peripheral lymphoC 2015 Wiley Periodicals, Inc. V cytes of healthy volunteers.
INTRODUCTION
Chromosomal aberrations (CAs) are structural modifications of chromosomes which include nonspecific and recurrent aberrations. Recurrent CAs arise through a clonal growth of cells with specific translocations, deletions, or amplifications of chromosomal regions or whole chromosomes. CAs are a marker of cancer risk and many specific CAs are believed to be causative events in malignant transformation (Mitelman et al., 2007). These can be detected by fluorescence in situ hybridization techniques for interphase chromosomes. Nonspecific CAs include missing, fragmented or fused C 2015 Wiley Periodicals, Inc. V
chromosomal segments which are apparently not clonally expanded but may remain in lymphocytes for their lifetime (Albertini et al., 2000). They are conventionally analyzed by microscopic scoring of metaphase nuclei from cultured lymphocytes. Such microscopic techniques are not able to score Additional Supporting Information may be found in the online version of this article. *Correspondence to: Kari Hemminki. E-mail: [email protected] Received 9 October 2014; Accepted 15 December 2014 DOI 10.1002/gcc.22239 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
2
HEMMINKI ET AL.
small DNA alterations the detection of which requires sequencing techniques. Application of these to cancer cells has revealed the abundance of gene fusions and rearrangements in many cancer types. In some cancers, individual chromosomes have experienced chromothripsis, a catastrophic parsing of illegitimate chromosomal segments together (Stephens et al., 2011). Nonspecific CAs are divided into chromosometype aberrations (CSAs) and chromatid-type aberrations (CTAs). Conventionally, CSAs are thought to arise as a result of direct DNA damage by, for example, ionizing radiation which causes doublestranded breaks. A suggested alternative mechanism is replication of a damaged DNA template, resulting in CSAs or CTAs, the latter being preferentially produced by chemical carcinogens and mutagens (Albertini et al., 2000). CTAs may also be produced from preexisting DNA lesions during culture of stimulated lymphocytes through Sphase dependent clastogens. More recently it has been realized that telomere biology is intimately connected to CAs (Xu et al., 2013). Shortening of telomeres at each cell division leads ultimately to replicative crisis. Eroded telomeres may be poorly end-capped and they may be recognized by DNA repair systems as double-stranded breaks which are joined to nonhomologous chromosomes (Artandi and DePinho, 2010). CA frequency in circulating lymphocytes has been associated with a subsequent risk of many cancers and patients with many types of cancer show increased frequencies of CAs at diagnosis (Bonassi et al., 2008; Vodicka et al., 2010). Although, CAs are assumed to be random events beyond chromosome breakage syndromes our recent study on multiple myeloma showed that the recurrent t(11;14) (q13;q32) translocation was strongly associated with the cyclin D1 G870A genotype at a splice site (Weinhold et al., 2013). Furthermore, we described that the same single nucleotide polymorphism (SNP) was associated with nonspecific CA in healthy individuals (Hemminki et al., 2014). Encouraged by such findings we have now tested a number of SNPs in metabolic genes for associations with CAs in healthy volunteers. Here, we show results also on gene–gene interactions. The analyzed SNPs and deletion polymorphisms were selected based on functional data in the following genes: cytochrome P450 1B1 (CYP1B1), epoxide hydrolase 1 (EPHX1), NAD(P)H:quinone oxidoreductase 1 (NQO1), which all code for activating, that is, phase 1 enzymes, and glutathione S-transferase P1 (GSTP1), glutathione S-transferGenes, Chromosomes & Cancer DOI 10.1002/gcc
ases M1 (GSTM1) and T1 (GSTT1), coding for enzymes which conjugate reactive metabolites, that is, phase 2 enzymes. MATERIALS AND METHODS
The subjects were identified from a large cohort (over 2,000) of volunteers who have been assayed for CAs in various previous studies (Dusinska et al., 2001, 2004a, 2004b; Kazimirova et al., 2009; Vodicka et al., 2009; Horska et al., 2011; Musak et al., 2013). Many of the studies were conducted in occupational settings, including persons with defined exposures, such as asbestos and mineral fibres, small organic compounds, cytostatics, anesthetics and metals, together with unexposed controls. Several studies investigated the effect of nutrition and life factors on DNA damage, CAs and markers of oxidative stress and antioxidant protection (Dusinska et al., 2001; Kazimirova et al., 2009). Sampling of peripheral blood was carried out according to the Helsinki Declaration and the study design was approved by the local Ethical Committee of the Jessenius Medical Faculty. The participants provided an informed consent. Cytogenetic analysis was performed on cultured lymphocytes, as previously described, by microscopically analyzing (two microscopists) in a double-blind fashion coded slides of 100 mitoses per person for the frequency of total CAs, CTAs, and CSAs (Kazimirova et al., 2009; Vodicka et al., 2010; Musak et al., 2013). The CAs were categorized into a high-frequency group, “cases” (>2%) and a low-frequency group, “controls” (2%); this arbitrary cut-off point was based on our previous experience (Vodicka et al., 2010; Musak et al., 2013). For CTA and CSA, the cut-off was 1%. Genotyping methods of the present polymorphisms have been previously described (Hlavata et al., 2010; Horska et al., 2011). Briefly, in CYP1B1 two SNPs were covered (CYP1B1/432 being Leu432Val, dbSNP: rs1056836 and CYP1B1/ 453 being Asn453Ser, rs1800440) by restriction fragment length polymorphism and GSTM1 (gene deletion) and GSTT1 (gene deletion) were assayed by allele-specific multiplex PCR. Polymorphisms in GSTP1 (Ile105Val, rs1695), NQO1 (Pro187Ser, rs1800566) and EPHX1 Tyr113His (rs1051740) and His139Arg (rs2234922) were assayed by allelic discrimination using the TaqMan technology. The functional evidence appears to indicate that all the variant genotypes tested either decrease enzyme activity or completly abolish it (NQO1, GSTM1, GSTT1) (Aklillu et al., 2005; Moyer et al., 2008;
3
SNPS AND CHROMOSOMAL ABERRATIONS
TABLE 1. Numbers of Cases and Controls and Their Odds Ratios According to Backround Variables of Age, Occupational Exposure, and Smoking Status CAtot
Variable CAtot (cases/control) CTA (cases/control) CSA (cases/control) Age (min,max,mean) Occupational environment (exposed/unexposed) Sex (M/F) Smoker (S/NS)
Persons
Odds ratio
95% Confidence interval
951/1245 1042/1154 983/1213 18, 88, 43 1207/989
1.06a 2.36
1171/1025 614/1557
1.03 1.19
CSA
CTA
P-value
Odds ratio
95% Confidence interval
P-value
Odds ratio
95% Confidence interval
P-value
(1.00–1.13) (1.97–2.83)
0.07
RESEARCH ARTICLE
Metabolic Gene Variants Associated with Chromosomal Aberrations in Healthy Humans Kari Hemminki,1,2* Christoph Frank,1 Asta F€ orsti,1,2 Ludovit Musak,3 Alena Kazimirova,4 Magdalena Barancokova,4 4 5,6 Alexandra Horska, Veronika Vymetalkova, Zdenek Smerhovsky,6,7 Alessio Naccarati,5,8 Pavel Soucek,9 Ludmila Vodickova,5,6 Janka Buchancova,10 Bozena Smolkova,11 Maria Dusinska,12 and Pavel Vodicka5,6 1 Department of Molecular Genetic Epidemiology,German Cancer Research Center (DKFZ),Im Neuenheimer Feld 580, 69120, Heidelberg,Germany 2 Center for Primary Health Care Research,Lund University,Malm€ o, Sweden 3 Jessenius Faculty of Medicine in Martin,Clinic of Occupational Medicine and Toxicology,Comenius University in Bratislava and University Hospital Martin,Kollarova 2, 03601Martin, Slovakia 4 Faculty of Medicine, Slovak Medical University,Limbova12,Bratislava, Slovakia 5 Institute of Experimental Medicine, Academy of Sciences of the Czech Republic,Videnska1083,142 00 Prague,Czech Republic 6 Institute of Biology and Medical Genetics,1st Medical Faculty,Charles University, Albertov 4,128 00 Prague,Czech Republic 7 2nd Faculty of Medicine,Charles University Prague,Czech Republic 8 Human Genetics Foundation,Turin,Italy 9 National Institute of Public Health, Srobarova 48,10042 Prague10,Czech Republic 10 Department of Public Health,Jessenius Faculty of Medicine in Martin,Comenius University in Bratislava, Sklabinska 26, 03601Martin, Slovakia 11 Laboratory of Cancer Genetics,Cancer Research Institute of Slovak Academy of Sciences,Bratislava, Slovakia 12 Health Effects Laboratory MILK,NILU-Norwegian Institute for Air Research,Kjeller,Norway
Nonspecific chromosomal aberrations (CAs) are found in about 1% of lymphocytes drawn from healthy individuals. They include chromosome-type aberrations (CSAs), which are increased in exposure to ionizing radiation, and chromatid-type aberrations (CTAs) which in experimental systems are formed by DNA binding carcinogens and mutagens. The frequency of CAs is associated with the risk of cancer, but the causes of CAs in general population are unknown. Here, we want to test whether variants in metabolic genes associate with CAs in healthy volunteers. Cases were considered those whose total CA (CAtot) frequency was >2% and for CSA and CTA the limit was >1%. Controls had lower frequencies of CAs. Functional polymorphisms in seven genes were selected for analysis: cytochrome P450 1B1 (CYP1B1), epoxide hydrolase 1 (EPHX1), NAD(P)H:quinone oxidoreductase 1 (NQO1), each coding for phase 1 enzymes, and glutathione S-transferase P1 (GSTP1), glutathione S-transferases M1 (GSTM1) and T1 (GSTT1), coding for enzymes which conjugate reactive metabolites, that is, phase 2 enzymes. The number of volunteers genotyped for each gene varied from 550 to 1,500. Only EPHX1 was individually associated with CAtot; high activity genotypes decreased CAtot. A total of six significant (P < 0.01) pair-wise interactions were observed, most including a GST variant as one of the pair. In all genotype combinations with significant odds ratios for CAs a GST variant was involved. The present data provide evidence that variants in genes coding for metabolic enzymes, which individually have small effects, interact and are associated with CA frequencies in peripheral lymphoC 2015 Wiley Periodicals, Inc. V cytes of healthy volunteers.
INTRODUCTION
Chromosomal aberrations (CAs) are structural modifications of chromosomes which include nonspecific and recurrent aberrations. Recurrent CAs arise through a clonal growth of cells with specific translocations, deletions, or amplifications of chromosomal regions or whole chromosomes. CAs are a marker of cancer risk and many specific CAs are believed to be causative events in malignant transformation (Mitelman et al., 2007). These can be detected by fluorescence in situ hybridization techniques for interphase chromosomes. Nonspecific CAs include missing, fragmented or fused C 2015 Wiley Periodicals, Inc. V
chromosomal segments which are apparently not clonally expanded but may remain in lymphocytes for their lifetime (Albertini et al., 2000). They are conventionally analyzed by microscopic scoring of metaphase nuclei from cultured lymphocytes. Such microscopic techniques are not able to score Additional Supporting Information may be found in the online version of this article. *Correspondence to: Kari Hemminki. E-mail: [email protected] Received 9 October 2014; Accepted 15 December 2014 DOI 10.1002/gcc.22239 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
2
HEMMINKI ET AL.
small DNA alterations the detection of which requires sequencing techniques. Application of these to cancer cells has revealed the abundance of gene fusions and rearrangements in many cancer types. In some cancers, individual chromosomes have experienced chromothripsis, a catastrophic parsing of illegitimate chromosomal segments together (Stephens et al., 2011). Nonspecific CAs are divided into chromosometype aberrations (CSAs) and chromatid-type aberrations (CTAs). Conventionally, CSAs are thought to arise as a result of direct DNA damage by, for example, ionizing radiation which causes doublestranded breaks. A suggested alternative mechanism is replication of a damaged DNA template, resulting in CSAs or CTAs, the latter being preferentially produced by chemical carcinogens and mutagens (Albertini et al., 2000). CTAs may also be produced from preexisting DNA lesions during culture of stimulated lymphocytes through Sphase dependent clastogens. More recently it has been realized that telomere biology is intimately connected to CAs (Xu et al., 2013). Shortening of telomeres at each cell division leads ultimately to replicative crisis. Eroded telomeres may be poorly end-capped and they may be recognized by DNA repair systems as double-stranded breaks which are joined to nonhomologous chromosomes (Artandi and DePinho, 2010). CA frequency in circulating lymphocytes has been associated with a subsequent risk of many cancers and patients with many types of cancer show increased frequencies of CAs at diagnosis (Bonassi et al., 2008; Vodicka et al., 2010). Although, CAs are assumed to be random events beyond chromosome breakage syndromes our recent study on multiple myeloma showed that the recurrent t(11;14) (q13;q32) translocation was strongly associated with the cyclin D1 G870A genotype at a splice site (Weinhold et al., 2013). Furthermore, we described that the same single nucleotide polymorphism (SNP) was associated with nonspecific CA in healthy individuals (Hemminki et al., 2014). Encouraged by such findings we have now tested a number of SNPs in metabolic genes for associations with CAs in healthy volunteers. Here, we show results also on gene–gene interactions. The analyzed SNPs and deletion polymorphisms were selected based on functional data in the following genes: cytochrome P450 1B1 (CYP1B1), epoxide hydrolase 1 (EPHX1), NAD(P)H:quinone oxidoreductase 1 (NQO1), which all code for activating, that is, phase 1 enzymes, and glutathione S-transferase P1 (GSTP1), glutathione S-transferGenes, Chromosomes & Cancer DOI 10.1002/gcc
ases M1 (GSTM1) and T1 (GSTT1), coding for enzymes which conjugate reactive metabolites, that is, phase 2 enzymes. MATERIALS AND METHODS
The subjects were identified from a large cohort (over 2,000) of volunteers who have been assayed for CAs in various previous studies (Dusinska et al., 2001, 2004a, 2004b; Kazimirova et al., 2009; Vodicka et al., 2009; Horska et al., 2011; Musak et al., 2013). Many of the studies were conducted in occupational settings, including persons with defined exposures, such as asbestos and mineral fibres, small organic compounds, cytostatics, anesthetics and metals, together with unexposed controls. Several studies investigated the effect of nutrition and life factors on DNA damage, CAs and markers of oxidative stress and antioxidant protection (Dusinska et al., 2001; Kazimirova et al., 2009). Sampling of peripheral blood was carried out according to the Helsinki Declaration and the study design was approved by the local Ethical Committee of the Jessenius Medical Faculty. The participants provided an informed consent. Cytogenetic analysis was performed on cultured lymphocytes, as previously described, by microscopically analyzing (two microscopists) in a double-blind fashion coded slides of 100 mitoses per person for the frequency of total CAs, CTAs, and CSAs (Kazimirova et al., 2009; Vodicka et al., 2010; Musak et al., 2013). The CAs were categorized into a high-frequency group, “cases” (>2%) and a low-frequency group, “controls” (2%); this arbitrary cut-off point was based on our previous experience (Vodicka et al., 2010; Musak et al., 2013). For CTA and CSA, the cut-off was 1%. Genotyping methods of the present polymorphisms have been previously described (Hlavata et al., 2010; Horska et al., 2011). Briefly, in CYP1B1 two SNPs were covered (CYP1B1/432 being Leu432Val, dbSNP: rs1056836 and CYP1B1/ 453 being Asn453Ser, rs1800440) by restriction fragment length polymorphism and GSTM1 (gene deletion) and GSTT1 (gene deletion) were assayed by allele-specific multiplex PCR. Polymorphisms in GSTP1 (Ile105Val, rs1695), NQO1 (Pro187Ser, rs1800566) and EPHX1 Tyr113His (rs1051740) and His139Arg (rs2234922) were assayed by allelic discrimination using the TaqMan technology. The functional evidence appears to indicate that all the variant genotypes tested either decrease enzyme activity or completly abolish it (NQO1, GSTM1, GSTT1) (Aklillu et al., 2005; Moyer et al., 2008;
3
SNPS AND CHROMOSOMAL ABERRATIONS
TABLE 1. Numbers of Cases and Controls and Their Odds Ratios According to Backround Variables of Age, Occupational Exposure, and Smoking Status CAtot
Variable CAtot (cases/control) CTA (cases/control) CSA (cases/control) Age (min,max,mean) Occupational environment (exposed/unexposed) Sex (M/F) Smoker (S/NS)
Persons
Odds ratio
95% Confidence interval
951/1245 1042/1154 983/1213 18, 88, 43 1207/989
1.06a 2.36
1171/1025 614/1557
1.03 1.19
CSA
CTA
P-value
Odds ratio
95% Confidence interval
P-value
Odds ratio
95% Confidence interval
P-value
(1.00–1.13) (1.97–2.83)
0.07
