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1.
Int J Cancer ; 122(9): 2101-5, 2008 May 01.
Article in English | MEDLINE | ID: mdl-18196582

ABSTRACT

To evaluate the associations of breast cancer risk with polymorphisms in the XPC and XPD/ERCC2 DNA nucleotide excision repair genes, a case-control study nested within a prospective cohort of 14,274 women was conducted. Genotypes were characterized for 612 incident, invasive breast cancer cases and their 1:1 matched controls. The homozygous variant of a poly(AT) insertion/deletion polymorphism in intron 9 of the XPC gene (XPC-PAT+/+), was associated with breast cancer risk [odds ratio (OR) = 1.45, 95% confidence interval: 1.07-1.97], after adjustment for other breast cancer risk factors. The breast cancer risk associated with XPC-PAT+/+ did not differ by age at diagnosis. There was an indication of an interaction (p = 0.08) between the XPC-PAT+/+ genotype and cigarette smoking. Ever smokers with the XPC-PAT+/+ genotype were at elevated risk of breast cancer (OR = 1.56, CI: 0.95-2.58), but no differences were observed among never smokers. Analyses of the ERCC2 Lys751Gln polymorphism did not show an association with breast cancer risk, either overall or at younger ages. The results suggest that breast cancer risk is related to the XPC haplotype tagged by the XPC-PAT+/+ insertion-deletion polymorphism in intron 9. Further study of the XPC haplotypes and their interactions with smoking in relation to breast cancer risk is needed.


Subject(s)
Breast Neoplasms/epidemiology , Breast Neoplasms/etiology , DNA-Binding Proteins/genetics , Polymorphism, Genetic , Smoking/adverse effects , Xeroderma Pigmentosum Group D Protein/genetics , Adult , Black or African American/genetics , Black or African American/statistics & numerical data , Alcohol Drinking/adverse effects , Breast Neoplasms/ethnology , Breast Neoplasms/genetics , Cohort Studies , Female , Genotype , Humans , Jews/genetics , Jews/statistics & numerical data , Matched-Pair Analysis , Middle Aged , Odds Ratio , Risk Assessment , Risk Factors , Surveys and Questionnaires , White People/genetics , White People/statistics & numerical data
2.
Pharmacogenet Genomics ; 17(3): 169-80, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17460545

ABSTRACT

OBJECTIVES: Retinoic acid (RA) is a critical regulator of gene expression during embryonic development and in the maintenance of adult epithelial tissues. This study was undertaken to identify genetic polymorphisms of CYP26A1 which might affect these processes. We sequenced CYP26A1 in racially diverse individuals and assessed the metabolism of retinoic acid by newly identified coding alleles of CYP26A1 in a recombinant system. METHODS: CYP26A1 was sequenced in 24 Caucasians, 24 African-Americans, 24 Asians, and 20 individuals of unknown racial origin. cDNA constructs for wild-type and coding alleles of CYP26A1 were constructed in a pcDNA3.1 expression vector and expressed in Cos-1 cells. A FLAG tag at the C-terminal end of the cDNA was used to quantitate the recombinant CYP26A1 proteins. RESULTS: A total of 13 single nucleotide polymorphisms (SNPs) were identified in CYP26A1. Three SNPs produced coding changes: R173S, F186L, and C358R. These alleles were termed as CYP26A1*2, CYP26A1*3, and CYP26A1*4, respectively, by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee at http://www.cypalleles.ki.se/. Wild type CYP26A1 protein metabolized all-trans-retinoic acid (at-RA) to 4-oxo-RA, 4-OH-RA as well as water-soluble metabolites. CYP26A1.3 (F186L) and CYP26A1.4 (C358R) allelic proteins exhibited significantly lower metabolism (40-80%) of at-RA than wild-type CYP26A1.1 protein. CONCLUSION: This is the first study to identify coding alleles of CYP26A1. Two coding alleles, CYP26A1*3 and CYP26A1*4, are predicted to be defective based on the metabolism of at-RA by the recombinant proteins. These studies suggest the need for future clinical studies of polymorphisms of CYP26A1 in embryonic development.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/physiology , Polymorphism, Single Nucleotide , Tretinoin/metabolism , Alleles , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Cytochrome P-450 Enzyme System/metabolism , DNA, Complementary , Gene Expression , Humans , Models, Molecular , Molecular Sequence Data , Recombinant Proteins/metabolism , Retinoic Acid 4-Hydroxylase , Sequence Homology, Amino Acid , Transfection
3.
Pharmacogenet Genomics ; 17(2): 145-60, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17301694

ABSTRACT

OBJECTIVE: Cyclooxygenase-1 (COX-1, PTGS1) catalyzes the conversion of arachidonic acid to prostaglandin H2, which is subsequently metabolized to various biologically active prostaglandins. We sought to identify and characterize the functional relevance of genetic polymorphisms in PTGS1. METHODS: Sequence variations in human PTGS1 were identified by resequencing 92 healthy individuals (24 African, 24 Asian, 24 European/Caucasian, and 20 anonymous). Using site-directed mutagenesis and a baculovirus/insect cell expression system, recombinant wild-type COX-1 and the R8W, P17L, R53H, R78W, K185T, G230S, L237M, and V481I variant proteins were expressed. COX-1 metabolic activity was evaluated in vitro using an oxygen consumption assay under basal conditions and in the presence of indomethacin. RESULTS: Forty-five variants were identified, including seven nonsynonymous polymorphisms encoding amino acid substitutions in the COX-1 protein. The R53H (35+/-5%), R78W (36+/-4%), K185T (59+/-6%), G230S (57+/-4%), and L237M (51+/-3%) variant proteins had significantly lower metabolic activity relative to wild-type (100+/-7%), while no significant differences were observed with the R8W (104+/-10%), P17L (113+/-7%), and V481I (121+/-10%) variants. Inhibition studies with indomethacin demonstrated that the P17L and G230S variants had significantly lower IC50 values compared to wild-type, suggesting these variants significantly increase COX-1 sensitivity to indomethacin inhibition. Consistent with the metabolic activity data, protein modeling suggested the G230S variant may disrupt the active conformation of COX-1. CONCLUSIONS: Our findings demonstrate that several genetic variants in human COX-1 significantly alter basal COX-1-mediated arachidonic acid metabolism and indomethacin-mediated inhibition of COX-1 activity in vitro. Future studies characterizing the functional impact of these variants in vivo are warranted.


Subject(s)
Cyclooxygenase 1/genetics , Cyclooxygenase 1/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Polymorphism, Single Nucleotide , Amino Acid Sequence , Arachidonic Acid/metabolism , Cyclooxygenase 1/chemistry , Dimerization , Enzyme Inhibitors/pharmacology , Humans , Indomethacin , Inhibitory Concentration 50 , Linkage Disequilibrium , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Microsomes/enzymology , Molecular Sequence Data , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Structure, Secondary
4.
Mutat Res ; 616(1-2): 221-6, 2007 Mar 01.
Article in English | MEDLINE | ID: mdl-17161851

ABSTRACT

Systematic screens have revealed extensive DNA sequence variation existing in the human population. Studies of the role of polymorphic genetic variants in explaining the association of family history with risk of common disease have generally focused on variants predicted to disrupt protein structure and activity. Recent studies have identified genetic variation in the level of expression of many genes, variation that is potentially biologically relevant in explaining individual variation in disease risk. In a survey of data available for 108 DNA repair genes that have been systematically screened for sequence variation, an average of 3.3 SNPs per gene were found to exist at a variant allele frequency of at least 0.02 in the region 2kb upstream from the 5'-untranslated region. One-third of the genes harbored a SNP with an allele frequency of at least 0.02 within a predicted promotor element. These variants are distributed among promoter elements that average 20 elements per gene. The frequency of polymorphic SNPs in CpG islands was 0.8 per gene, while the frequency of SNPs in the 5'-UTR was 0.7 per gene. The recognition of extensive genetic variation with potential to impact levels of gene expression, and thereby exacerbate the impact of amino acid substitution variants on the activity of proteins, increases the complexity of analyses required to explain the molecular genetic basis for the familial contribution to the sporadic incidence of common disease.


Subject(s)
5' Untranslated Regions , DNA Repair , Genetic Predisposition to Disease , Genetic Variation , Polymorphism, Genetic , Databases, Genetic , Genetic Testing , Humans , Polymorphism, Single Nucleotide , Regulatory Elements, Transcriptional
5.
Mutat Res ; 616(1-2): 213-20, 2007 Mar 01.
Article in English | MEDLINE | ID: mdl-17145065

ABSTRACT

Elucidating the relationship between polymorphic sequences and risk of common disease is a challenge. For example, although it is clear that variation in DNA repair genes is associated with familial cancer, aging and neurological disease, progress toward identifying polymorphisms associated with elevated risk of sporadic disease has been slow. This is partly due to the complexity of the genetic variation, the existence of large numbers of mostly low frequency variants and the contribution of many genes to variation in susceptibility. There has been limited development of methods to find associations between genotypes having many polymorphisms and pathway function or health outcome. We have explored several statistical methods for identifying polymorphisms associated with variation in DNA repair phenotypes. The model system used was 80 cell lines that had been resequenced to identify variation; 191 single nucleotide substitution polymorphisms (SNPs) are included, of which 172 are in 31 base excision repair pathway genes, 19 in 5 anti-oxidation genes, and DNA repair phenotypes based on single strand breaks measured by the alkaline Comet assay. Univariate analyses were of limited value in identifying SNPs associated with phenotype variation. Of the multivariable model selection methods tested: the easiest that provided reduced error of prediction of phenotype was simple counting of the variant alleles predicted to encode proteins with reduced activity, which led to a genotype including 52 SNPs; the best and most parsimonious model was achieved using a two-step analysis without regard to potential functional relevance: first SNPs were ranked by importance determined by random forests regression (RFR), followed by cross-validation in a second round of RFR modeling that included ever more SNPs in declining order of importance. With this approach six SNPs were found to minimize prediction error. The results should encourage research into utilization of multivariate analytical methods for epidemiological studies of the association of genetic variation in complex genotypes with risk of common diseases.


Subject(s)
DNA Repair , Genetic Variation , Polymorphism, Genetic , Polymorphism, Single Nucleotide , Cell Line , DNA Breaks, Single-Stranded , Genetic Predisposition to Disease , Genotype , Humans , Multivariate Analysis , Phenotype , Risk Assessment
6.
Cancer Causes Control ; 18(1): 91-103, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17186425

ABSTRACT

OBJECTIVE: Whether alcohol consumption influences ovarian cancer risk is unclear. Therefore, we investigated the association between alcohol intake at various ages and risk of ovarian cancer. METHODS: Among 90,371 eligible members of the California Teachers Study cohort who completed a baseline alcohol assessment in 1995-1996, 253 women were diagnosed with epithelial ovarian cancer by the end of 2003. Multivariate Cox proportional hazards regression analysis was performed to estimate relative risks (RRs) and 95% confidence intervals (CIs). RESULTS: Consumption of total alcohol, beer, or liquor in the year prior to baseline, at ages 30-35 years, or at ages 18-22 years was not associated with risk of ovarian cancer. Consumption of at least one glass per day of wine, compared to no wine, in the year before baseline was associated with increased risk of developing ovarian cancer: RR = 1.57 (95% CI 1.11-2.22), P (trend) = 0.01. The association with wine intake at baseline was particularly strong among peri-/post-menopausal women who used estrogen-only hormone therapy and women of high socioeconomic status. CONCLUSIONS: Alcohol intake does not appear to affect ovarian cancer risk. Constituents of wine other than alcohol or, more likely, unmeasured determinants of wine drinking were associated with increased risk of ovarian cancer.


Subject(s)
Alcohol Drinking , Ovarian Neoplasms/epidemiology , Wine , Adolescent , Adult , Aged , Aged, 80 and over , California/epidemiology , Cohort Studies , Female , Humans , Multivariate Analysis , Ovarian Neoplasms/etiology , Postmenopause/metabolism , Registries , Risk Factors , Socioeconomic Factors
7.
BMC Med Genet ; 7: 78, 2006 Nov 06.
Article in English | MEDLINE | ID: mdl-17087834

ABSTRACT

BACKGROUND: Although there have been studies of the genetic risk factors in the development of stroke, there have been few investigations of role of genes in the cerebral response to ischemia. The brain responds to ischemia in a series of reactions that ultimately influence the volume of a stroke that, in general, correlates with disability. We hypothesize that polymorphisms in genes encoding proteins involved in these reactions could act as modifiers of this response and impact stroke volume. One of the pathways participating in the cerebral ischemic response involves reactive oxygen species which can cause oxidative damage to nucleic acids. DNA repair mechanisms are in place to protect against such damage and imply a role for DNA repair genes in the response of the brain to ischemia and are potential candidate genes for further investigation. METHODS: We studied two common polymorphisms in the DNA repair gene, XRCC1, C26304T and G28152A, in 134 well characterized patients with non lacunar ischemic strokes. We also performed a case control association study with 113 control patients to assess whether these variants represent risk factors in the development of ischemic stroke. RESULTS: Independent of etiology, the "T" allele of the C26304T polymorphism is significantly associated with larger stroke volumes (T-test analysis, p < 0.044; multivariate regression analysis, beta = 0.23, p < 0.008). In the case control association study, we found that neither of these polymorphisms represented a risk factor for the development of stroke. CONCLUSION: Our study suggests a major gene effect of the "T" allele of the C26304T polymorphism modulating the cerebral response to ischemia in non lacunar ischemic stroke.


Subject(s)
Brain Ischemia/genetics , DNA-Binding Proteins/genetics , Polymorphism, Single Nucleotide , Stroke/genetics , Case-Control Studies , Genetic Predisposition to Disease , Humans , Risk Factors , Severity of Illness Index , X-ray Repair Cross Complementing Protein 1
8.
Mutat Res ; 602(1-2): 34-42, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-17010390

ABSTRACT

Fanconi anemia (FA) is a rare cancer predisposition disease caused by mutations in at least 12 genes encoding proteins that cooperate to maintain genomic integrity. Variants of FA genes, including FANCG, have been identified in human population screening, but their potential reduction in protein function and role in cancer susceptibility is unclear. To test for possible dysfunction, we constructed plasmids containing four FANCG polymorphisms found in the human population and introduced them in the Fancg-deficient (fancg) KO40 line derived from AA8 hamster CHO cells. Expression of wild-type human FANCG provided fancg cells with complete phenotypic correction as assessed by resistance to the DNA crosslinking agent mitomycin C (MMC), thus providing a sensitive test for detecting the degree of complementation activity for the FANCG variants. We found that all four variants conferred levels of mitomycin C resistance as well as restoration of monoubiquitination of Fancd2, a key indicator of a functional FA protein pathway, similar to those observed in wild-type transfectants. Under the same conditions, the L71P amino acid substitution mutant, identified in an FA patient, gave no complementation. Using this novel system for determining FANCG functionality, we detect no decrement in function of the human FANCG polymorphic variants examined.


Subject(s)
Fanconi Anemia Complementation Group G Protein/genetics , Polymorphism, Genetic , Animals , CHO Cells , Cells, Cultured , Cricetinae , Dose-Response Relationship, Drug , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group D2 Protein/radiation effects , Fanconi Anemia Complementation Group G Protein/physiology , Gene Frequency , Genetic Complementation Test , Humans , Methyl Methanesulfonate/pharmacology , Mitomycin/toxicity , Ubiquitin/metabolism
9.
Carcinogenesis ; 27(3): 610-8, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16258177

ABSTRACT

Polymorphisms in six genes involved in nucleotide excision repair of DNA were examined in a large population-based case-control study of melanoma. Genotyping was conducted for 2485 patients with a single primary melanoma (controls) and 1238 patients with second or higher order primary melanomas (cases). Patients were ascertained from nine geographic regions in Australia, Canada, Italy and the United States. Positive associations were observed for XPD 312 Asn/Asn versus Asp/Asp [odds ratio (OR) = 1.5, 95% confidence interval (CI) 1.2-1.9] and XPD 751 Gln/Gln versus Lys/Lys (OR = 1.4, 95% CI 1.1-1.7) genotypes and melanoma. The combined XPD Asn (A) 312 + Gln (C) 751 haplotype was significantly more frequent in cases (32%) compared with controls (29%) (P = 0.003) and risk of melanoma increased significantly with one and two copies of the haplotype (ORs 1.2, 95% CI 1.0-1.4, and 1.6, 95% CI 1.2-2.0, trend P = 0.002). No significant associations were observed for HR23B codon 249, XPG codon 1104, XPC codon 939, XPF codon 415, XPF nt 2063, ERCC6 codon 1213 or ERCC6 codon 1230. ORs for XPD and XPC genotypes were stronger for melanoma diagnosed at an early age, but tests for interaction were not statistically significant. The results provide further evidence for a role of XPD in the etiology of melanoma.


Subject(s)
DNA Repair , Melanoma/genetics , Polymorphism, Genetic , Skin Neoplasms/genetics , Xeroderma Pigmentosum Group D Protein/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Child , Female , Genotype , Humans , Male , Melanoma/etiology , Middle Aged , Odds Ratio , Risk Factors , Skin Neoplasms/etiology
10.
J Natl Cancer Inst ; 97(11): 805-12, 2005 Jun 01.
Article in English | MEDLINE | ID: mdl-15928301

ABSTRACT

BACKGROUND: Epidemiologic studies of the association between nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and ibuprofen, and breast cancer risk have yielded inconsistent results. We investigated the association of NSAID use with risk of breast cancer in the California Teachers Study cohort, with special attention to risk of specific breast cancer subtypes and to type of NSAID used. METHODS: We analyzed data on 114 460 women in the California Teachers Study cohort who were aged 22 to 85 years and free of breast cancer at baseline in 1995 to 1996. Information on frequency and duration of NSAID use was collected through a self-administered questionnaire. A total of 2391 women were diagnosed with breast cancer during the follow-up period from 1995 to 2001. We used Cox proportional hazards regression to estimate relative risks (RR) and 95% confidence intervals (CI) of breast cancer subtypes with NSAID use. RESULTS: Neither regular use (more than once a week) of any NSAID (aspirin and ibuprofen combined) nor regular use of aspirin was associated with breast cancer risk (RR = 1.09, 95% CI = 0.97 to 1.21 for daily versus no regular use of NSAIDs and RR = 0.98, 95% CI = 0.86 to 1.13 for daily versus no regular use of aspirin). However, long-term (> or = 5 years) daily aspirin users had a non-statistically significant decreased risk of estrogen receptor and progesterone receptor (ER/PR)-positive breast cancer (RR = 0.80, 95% CI = 0.62 to 1.03). In contrast, we observed a statistically significantly increased risk of ER/PR-negative breast cancer with long-term daily use of aspirin (RR = 1.81, 95% CI = 1.12 to 2.92). In this population, 11 fewer ER/PR-positive breast cancer cases and seven excess ER/PR-negative breast cancer cases may be due to daily long-term aspirin use among 2391 breast cancer cases observed over 6 years if the association were proven to be causal. Long-term daily use of ibuprofen was also associated with an increased risk of breast cancer (RR = 1.51, 95% CI = 1.17 to 1.95), particularly of nonlocalized tumors (RR = 1.92, 95% CI = 1.24 to 2.97). If causality were subsequently proven, 16 of the observed 2391 breast cancer cases and 8 of the 713 non-localized breast cancer cases would be attributable to long-term daily use of ibuprofen. CONCLUSIONS: Long-term daily use of NSAIDs was not associated with breast cancer risk overall. Ibuprofen use was associated with an increased risk of breast cancer, and long-term daily aspirin use was associated with an increased risk of ER/PR-negative breast cancer. However, it is not clear if the observed association is causal.


Subject(s)
Acetaminophen/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Breast Neoplasms/epidemiology , Breast Neoplasms/pathology , Receptors, Estrogen/analysis , Receptors, Progesterone/analysis , Acetaminophen/adverse effects , Adult , Aged , Aged, 80 and over , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Aspirin/administration & dosage , Breast Neoplasms/chemically induced , Breast Neoplasms/chemistry , California/epidemiology , Faculty/statistics & numerical data , Female , Humans , Ibuprofen/administration & dosage , Incidence , Likelihood Functions , Middle Aged , Neoplasm Staging , Proportional Hazards Models , Prospective Studies , Registries , Research Design , Risk Assessment , Risk Factors , Surveys and Questionnaires
11.
J Pharmacol Exp Ther ; 314(2): 923-31, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15914676

ABSTRACT

The human organic anion transporter hOAT1 (SLC22A6) contributes to the uptake of a range of small organic anions across the basolateral membrane of the renal proximal tubule and drives their urinary elimination. The aim of this study was to identify genetic variants of hOAT1 and to investigate potential effects on the functional properties of this transporter. Twenty single nucleotide polymorphisms (SNPs) in hOAT1 were identified in genomic DNA from 92 individuals of African, Asian, and Caucasian origin. Two SNPs encoded changes in amino acid sequence; arginine to histidine (residue 50) and lysine to isoleucine (residue 525). Significantly, these SNPs were only present in the samples of African origin. When expressed in Xenopus oocytes, wild-type R50-hOAT1 and the variants R50H-hOAT1 and K525I-hOAT1 all mediated the probenecid-sensitive uptake of the classic organic anion para-aminohippurate (PAH). Kinetic analysis indicated that the transport affinity (K(m)) for PAH was unchanged in the variants, compared with wild type. Interestingly, the K(m) for the nucleoside phosphonate analogs adefovir, cidofovir, and tenofovir seemed to be decreased in the R50H-hOAT1 variant compared with the wild type, whereas the kinetics of K525I-hOAT1 remained unchanged. In conclusion, this is the first study to identify variation of hOAT1 in a racially diverse sample and to investigate the functional properties of the resulting variants. Since hOAT1 has been suggested as the basis of nephrotoxicity induced by nucleoside phosphonate analogs, this study raises the intriguing possibility that individuals with genetic variation in hOAT1, such as R50H, may display different handling of these drugs.


Subject(s)
Organic Anion Transport Protein 1/genetics , Organic Anion Transport Protein 1/metabolism , Polymorphism, Single Nucleotide/physiology , Adenine/analogs & derivatives , Adenine/metabolism , Amino Acid Sequence , Animals , Antiviral Agents/metabolism , Cidofovir , Cytosine/analogs & derivatives , Cytosine/metabolism , DNA/genetics , Genetic Variation , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed/genetics , Oocytes/drug effects , Oocytes/metabolism , Organophosphonates/metabolism , RNA, Complementary/biosynthesis , RNA, Complementary/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tenofovir , Xenopus laevis , p-Aminohippuric Acid/metabolism
12.
Pharmacogenetics ; 14(8): 527-37, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15284535

ABSTRACT

CYP2C9 is a clinically important enzyme, responsible for the metabolism of numerous clinically important therapeutic drugs. In the present study, we discovered 38 single nucleotide polymorphisms in CYP2C9 by resequencing of genomic DNA from 92 individuals from three different racial groups. Haplotype analysis predicted that there are at least 21 alleles of CYP2C9 in this group of individuals. Six new alleles were identified that contained coding changes: L19I (CYP2C9*7), R150H (CYP2C9*8), H251R (CYP2C9*9), E272G (CYP2C9*10), R335W(CYP2C9*11) and P489S (CYP2C9*12). When expressed in a bacterial cDNA expression system, several alleles exhibited altered catalytic activity. CYP2C9*11 appeared to be a putative poor metabolizer allele, exhibiting a three-fold increase in the Km and more than a two-fold decrease in the intrinsic clearance for tolbutamide. Examination of the crystal structure of human CYP2C9 reveals that R335 is located in the turn between the J and J' helices and forms a hydrogen-bonding ion pair with D341 from the J' helix. Abolishing this interaction in CYP2C9*11 individuals could destabilize the secondary structure and alter the substrate affinity. This new putative poor metabolizer (PM) allele was found in Africans. A second potentially PM allele CYP2C9*12 found in a racially unidentified sample also exhibited a modest decrease in the Vmax and the intrinsic clearance for tolbutamide in a recombinant system. Further clinical studies are needed to determine the effect of these new polymorphisms on the metabolism of CYP2C9 substrates.


Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Ethnicity/genetics , Polymorphism, Single Nucleotide/genetics , Tolbutamide/metabolism , Alleles , Aryl Hydrocarbon Hydroxylases/metabolism , Asian People , Black People , Cytochrome P-450 CYP2C9 , Genotype , Haplotypes/genetics , Humans , Models, Molecular , Mutagenesis, Site-Directed , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transfection , White People
13.
Genomics ; 83(6): 970-9, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15177551

ABSTRACT

Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant from Tolerant (SIFT) classified 226 of 508 variants (44%) as "Intolerant." Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as "Probably or possibly damaging." Another 9-15% of the variants were classed as "Potentially intolerant or damaging." The results from the two algorithms are highly associated, with concordance in predicted impact observed for approximately 62% of the variants. Twenty-one to thirty-one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as "Tolerant" or "Benign." Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.


Subject(s)
Amino Acid Substitution/genetics , DNA Repair/genetics , Genetic Testing , Nuclear Proteins/genetics , Sequence Analysis, Protein/methods , Algorithms , Computational Biology/methods , Gene Frequency/genetics , Genetics, Population , Humans , Nuclear Proteins/physiology , Polymorphism, Genetic , Software
14.
Toxicol Pathol ; 32 Suppl 1: 136-45, 2004.
Article in English | MEDLINE | ID: mdl-15209414

ABSTRACT

With the vast technological and informational resources increasingly available from investments in "genomics," toxicology and much of biological science, is faced with previously undreamed of opportunities and equally daunting challenges. The ability to generate the large quantities of data becoming routinely available could not be imagined a decade ago. The complexities of data analysis are increasingly the rate-limiting element in scientific advances. The expectations that these large scientific investments will reduce the incidence of human disease and improve health are very high. An emphasis on genetic variation and Toxicogenetics is expected to yield risk estimates for specific rather than average individuals and individuals with varied lifestyles and complex patterns of exposure. Examples from studies of polymorphic variation in DNA repair genes in the healthy population and cancer risk highlight the complexity and challenges of incorporating genetic variation into quantitative estimates of risk associated with environmentally relevant exposures. Similar issues exist in selecting the animal models most appropriate for predicting human risk from environmental exposures to toxic agents.


Subject(s)
Genetic Variation , Toxicology/trends , Xenobiotics/toxicity , Animals , DNA Repair , Disease Susceptibility , Environmental Exposure , Forecasting , Genetic Testing , Humans , Neoplasms/etiology , Neoplasms/genetics , Neoplasms/prevention & control , Polymorphism, Genetic , Risk Assessment , Risk Factors , Xenobiotics/metabolism
15.
Cancer Res ; 63(23): 8536-41, 2003 Dec 01.
Article in English | MEDLINE | ID: mdl-14679022

ABSTRACT

The XRCC1 protein is involved in the base excision repair pathway through interactions with other proteins. Polymorphisms in the XRCC1 gene may lead to variation in repair proficiency and confer inherited predisposition to cancer. We prospectively assessed the associations between polymorphisms and haplotypes in XRCC1 and breast cancer risk in a nested case-control study within the Nurses' Health Study (incident cases, n = 1004; controls, n = 1385). We further investigated gene-environment interactions between the XRCC1 variations and plasma carotenoids on breast cancer risk. We genotyped four haplotype-tagging single nucleotide polymorphisms (Arg(194)Trp, C26602T, Arg(399)Gln, and Gln(632)Gln) in the XRCC1 gene. Five common haplotypes accounted for 99% of the chromosomes in the present study population of mostly Caucasian women. We observed a marginally significant reduction in the risk of breast cancer among (194)Trp carriers. As compared with no-carriers, women with at least one (194)Trp allele had a multivariate odds ratio of 0.79 (95% of the confidence interval, 0.60-1.04). The inferred haplotype harboring the (194)Trp allele was more common in controls than in cases (6.6 versus 5.3%, P = 0.07). We observed that the Arg(194)Trp modified the inverse associations of plasma alpha-carotene level (P, ordinal test for interaction = 0.02) and plasma beta-carotene level (P, ordinal test for interaction = 0.003) with breast cancer risk. No suggestion of an interaction was observed between the Arg(194)Trp and cigarette smoking. Our results suggest an inverse association between XRCC1 (194)Trp allele and breast cancer risk. The findings of the effect modification of the Arg(194)Trp on the relations of plasma alpha- and beta-carotene levels with breast cancer risk suggest a potential protective effect of carotenoids in breast carcinogenesis by preventing oxidative DNA damage.


Subject(s)
Breast Neoplasms/blood , Breast Neoplasms/genetics , Carotenoids/blood , DNA-Binding Proteins/genetics , beta Carotene/blood , Adult , Breast Neoplasms/etiology , Case-Control Studies , Female , Genetic Variation , Haplotypes , Humans , Middle Aged , Prospective Studies , Risk Factors , Smoking/adverse effects , X-ray Repair Cross Complementing Protein 1
16.
Cancer Epidemiol Biomarkers Prev ; 12(11 Pt 1): 1200-4, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14652281

ABSTRACT

Mammalian cells are constantly exposed to genotoxic agents from both endogenous and exogenous sources. Genetic variability in DNA repair contributes to deficient repair and breast cancer risk. Using samples collected in an ongoing, clinic-based, case-control study (253 cases and 268 controls), we tested whether breast cancer risk is associated with four amino acid substitution variants in three DNA repair genes, including XRCC1 Arg194Trp and XRCC1 Arg399Gln in base excision repair, XRCC3 Thr241Met in homologous recombination repair, and ERCC4/XPF Arg415Gln in nucleotide excision repair. Carriers of at least one variant allele of XRCC1 Arg194Trp [Arg/Trp and Trp/Trp versus Arg/Arg, odds ratio (OR) = 1.60, 95% confidence interval (CI) = 0.89-2.87] or two variant alleles of XRCC3 241Met/Metmay have an increased risk of breast cancer (Met/Met versus Thr/Thr and Thr/Met, OR = 1.54, 95% CI = 0.94-2.52). No association between XRCC1 Arg399Gln Dgenotype and breast cancer risk was observed. The genotype distribution of ERCC4/XPF Arg415Gln differed significantly between cases and controls (P = 0.02), and the ERCC4/XPF 415Gln/Gln genotype was found in only seven cases (3%) but not in controls. In addition, breast cancer risk was significantly associated with an increasing number of combined variant alleles of XRCC1 Arg194Trp, XRCC3 Thr241Met, and ERCC4/XPF Arg415Gln in a four-level model (P(trend) = 0.04): OR = 1.0 for those without a variant allele (referent group); OR = 1.04 (95% CI = 0.67-1.61) for those with one variant allele; OR = 1.38 (95% CI = 0.83-2.29) for those with two variant alleles; and age-adjusted OR = 2.60 (95% CI = 1.03-6.59) for those with three or more variant alleles after adjustment for age, family history, age at menarche, age at first live birth, and body mass index. We provide evidence that variants of XRCC1, XRCC3, and ERCC4/XPF genes, particularly in combination, contribute to breast cancer susceptibility.


Subject(s)
Breast Neoplasms/etiology , Breast Neoplasms/genetics , DNA Repair/genetics , Genetic Predisposition to Disease , Polymorphism, Genetic , Adult , Aged , Case-Control Studies , DNA-Binding Proteins/genetics , Female , Humans , Middle Aged , Odds Ratio , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , X-ray Repair Cross Complementing Protein 1
17.
Article in English | MEDLINE | ID: mdl-14604009

ABSTRACT

The three known mammalian CCCH tandem zinc finger proteins of the tristetraprolin (TTP) class have recently been demonstrated to be mRNA-binding proteins. The prototype, TTP, functions in normal physiology to promote the instability of the tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor mRNAs. Conversely, these mRNAs are stabilized in TTP-deficient mice, leading to an inflammatory phenotype characterized by overproduction of these cytokines. To explore sequence variations in TTP and its two related proteins, we sequenced genomic DNA encoding the TTP protein (ZFP36) and those of its two known mammalian relatives, ZFP36L1 and ZFP36L2, from 72 to 92 anonymous human subjects from various geographical and ethnic backgrounds. We also sequenced ZFP36 in genomic DNA from 92 subjects exhibiting evidence of excessive TNFalpha action. The resequencing strategy identified 13 polymorphisms in the protein-coding regions of these three genes, of which six would result in amino acid changes; other putative polymorphisms were identified by EST searches. One mutation in ZFP36L1 was a dinucleotide substitution that would prevent splicing of the single intron. This mutation was identified in only one allele of the original 144 sequenced from an adult female Aka Pygmy from the Central African Republic; a second individual with the same variant allele was found by genotyping 58 additional Aka DNA samples. Analysis of mRNA from one of these subject's lymphoblasts confirmed that ZFP36L1 mRNA levels were approximately 50% of those in a comparable sample without the mutation. The functional significance of this and the other polymorphisms identified remains to be determined by both biochemical and population linkage studies.


Subject(s)
Immediate-Early Proteins/genetics , Polymorphism, Genetic/genetics , Zinc Fingers , Amino Acid Sequence , Base Sequence , Butyrate Response Factor 1 , DNA Mutational Analysis , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Ethnicity/genetics , Female , Gene Frequency , Humans , Immediate-Early Proteins/chemistry , Male , Molecular Sequence Data , Point Mutation , Transcription Factors/chemistry , Transcription Factors/genetics , Tristetraprolin , Tumor Necrosis Factor-alpha/metabolism
18.
Pharmacogenetics ; 13(8): 461-72, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12893984

ABSTRACT

OBJECTIVES: Genetic polymorphisms of cytochromes P450 (CYPs) are a principal reason for inter-individual variations in the metabolism of therapeutic drugs and environmental chemicals in humans. The present study identifies 34 single nucleotide polymorphisms (SNPs) of CYP3A5 including 27 previously unidentified SNPs by direct sequencing of the exons, intron-exon junctions and 5'-upstream region of CYP3A5 from 92 racially diverse individuals (24 Caucasians, 24 Africans, 24 Asians, and 20 individuals of unknown racial origin). RESULTS: Four new CYP3A5 SNPs produced coding changes: R28C, L82R, A337T, and F446S. CYP3A5 R28C occurred in African populations (allelic frequency of 4%). CYP3A5 A337T occurred in Asians (2% allelic frequency), CYP3A5 L82R (occurred in the racially unidentified group) and CYP3A5 F446S (identified in Caucasians with a 2% allelic frequency) were on an allele containing the splice change g.6986A>G known as CYP3A5*3. The newly identified allelic proteins were constructed by site-directed mutagenesis, expressed in Escherichia coli and purified. CYP3A5 L82R was expressed only as denatured CYP420, suggesting it may be unstable. CYP3A5*1 exhibited the highest maximal clearance for testosterone followed by CYP3A5 A337T > CYP3A5 R28C >> CYP3A5 F446S. CYP3A5*1 exhibited a higher V(max) for nifedipine oxidation than CYP3A5 A337T > CYP3A5 R28C >> CYP3A5 F446S. CYP3A5 A337T and CYP3A5 R28C exhibited a 42-64% lower V(max) for nifedipine oxidation than CYP3A5*1. CYP3A5 F446S exhibited a > 95% decrease in the intrinsic clearance for both 6beta-hydroxytestosterone and nifedipine oxidation. CONCLUSION: This study identifies four new potentially defective coding alleles. CYP3A5 F446S is predicted to be more catalytically defective than the splice change alone.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Ethnicity/genetics , Polymorphism, Single Nucleotide , Alleles , Base Sequence , Cytochrome P-450 CYP3A , DNA Primers , DNA, Complementary , Escherichia coli/genetics , Humans , Mutagenesis, Site-Directed , Nifedipine/pharmacokinetics , Testosterone/pharmacokinetics
19.
Mol Pharmacol ; 64(2): 482-90, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12869654

ABSTRACT

Human soluble epoxide hydrolase (hsEH) metabolizes a variety of epoxides to the corresponding vicinal diols. Arachidonic and linoleic acid epoxides are thought to be endogenous substrates for hsEH. Enzyme activity in humans shows high interindividual variation (e.g., 500-fold in liver) suggesting the existence of regulatory and/or structural gene polymorphisms. We resequenced each of the 19 exons of the hsEH gene (EPHX2) from 72 persons representing black, Asian, and white populations. A variety of polymorphisms was found, six of which result in amino acid substitutions. Amino acid variants were localized on the crystal structure of the mouse sEH, resulting in the prediction that at least two of these (Arg287Gln and Arg103Cys) might significantly affect enzyme function. The six variants of the hsEH cDNA corresponding to each single polymorphism and one corresponding to a double polymorphism were then constructed by site-directed mutagenesis and expressed in insect cells. As predicted, Arg287Gln and the double mutant Arg287Gln/Arg103Cys showed decreased enzyme activity using trans-stilbene oxide, trans-diphenylpropene oxide, and 14,15-epoxyeicosatrienoic acid as substrates. Lys55Arg and Cys154Tyr mutants had elevated activity for all three substrates. Detailed kinetic studies revealed that the double mutant Arg287Gln/Arg103Cys showed significant differences in Km and Vmax. In addition, stability studies showed that the double mutant was less stable than wild-type protein when incubated at 37 degrees C. These results suggest that at least six hsEH variants exist in the human population and that at least four of these may influence hsEH-mediated metabolism of exogenous and endogenous epoxide substrates in vivo.


Subject(s)
Epoxide Hydrolases/genetics , Polymorphism, Genetic , Animals , Baculoviridae/genetics , Crystallization , Enzyme Stability , Epoxide Hydrolases/chemistry , Exons/genetics , Genetic Vectors/genetics , Genotype , Humans , Kinetics , Mice , Protein Conformation , Solubility , Tumor Cells, Cultured
20.
Mutat Res ; 526(1-2): 93-125, 2003 May 15.
Article in English | MEDLINE | ID: mdl-12714187

ABSTRACT

Individual risk and the population incidence of disease result from the interaction of genetic susceptibility and exposure. DNA repair is an example of a cellular process where genetic variation in families with extreme predisposition is documented to be associated with high disease likelihood, including syndromes of premature aging and cancer. Although the identification and characterization of new genes or variants in cancer families continues to be important, the focus of this paper is the current status of efforts to define the impact of polymorphic amino acid substitutions in DNA repair genes on individual and population cancer risk. There is increasing evidence that mild reductions in DNA repair capacity, assumed to be the consequence of common genetic variation, affect cancer predisposition. The extensive variation being found in the coding regions of DNA repair genes and the large number of genes in each of the major repair pathways results in complex genotypes with potential to impact cancer risk in the general population. The implications of this complexity for molecular epidemiology studies, as well as concepts that may make these challenges more manageable, are discussed. The concepts include both experimental and computational approaches that could be employed to develop predictors of disease susceptibility based on DNA repair genotype, focusing initially on studies to assess functional impact on individual proteins and pathways and then on molecular epidemiology studies to assess exposure-dependent health risk. In closing, we raise some of the non-technical challenges to the utilization of the full richness of the genetic variation to reduce disease occurrence and ultimately improve health care.


Subject(s)
Amino Acid Substitution , DNA Repair/genetics , Genetic Markers/genetics , Genetic Predisposition to Disease , Genetic Variation , Neoplasms/epidemiology , Neoplasms/genetics , Signal Transduction/genetics , Genetic Testing , Humans , Incidence , Phenotype , Risk Factors
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