Identification of single nucleotide polymorphisms in human DNA repair genes

Barry N. Ford1,2, Cindy C. Ruttan1, Victoria L. Kyle1, Moyra E. Brackley1 and Barry W. Glickman1,3

1 Centre for Environmental Health and the Department of Biology, University of Victoria, PO Box 3020, STN CSC, Victoria, BC, Canada V8W 3N5


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Variation in gene coding sequence represents a significant factor in predisposition to disease, including cancer. Variants of some DNA repair genes (e.g. MLH1, MSH2 and MSH6) are known to predispose to cancer. We identified single nucleotide polymorphisms (SNPs) in five DNA repair genes in 142 healthy individuals using a DNA sequencing protocol optimized for the direct detection of single nucleotide polymorphisms. This approach, called the heterozygote sequencing protocol (HSP), enables moderate-scale population surveys of SNPs. HSP uses fluorescently tagged primers and exploits the large dynamic range and low background of automated fluorescent sequencing. HSP may be used for any sequence that can be amplified by PCR. A total of 12 SNP variants in MGMT, ERCC1, CDK7, CCNH and XRCC4 were identified, 11 at polymorphic frequencies, with an average frequency of 0.22 (95% confidence interval 0.20–0.24). Among the 82 individuals for whom complete SNP profiles were available, no one person carried the GenBank reference sequence for all five genes. The extensive heterogeneity observed in these five genes is intriguing. All variants are in Hardy–Weinberg equilibrium, although the meaning of this equilibrium is unclear. Using this approach, possible associations of sequence variation, and hence of variation in DNA repair, with disease risk can be assessed.

Abbreviations: CCNH, cyclin H; CDK7, cyclin-dependent kinase 7; ERCC1, excision-repair, complementing defective, in Chinese hamster, 1; HSP, heterozygote sequencing protocol; MGMT, methylguanine-DNA methyltransferase; SNP, single nucleotide polymorphism; XRCC4, X-ray repair, complementing defective, in Chinese hamster, 4.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Current evidence supports the hypothesis that mutations are early events in carcinogenesis, so defects in DNA repair probably represent a risk factor for many types of cancer (1,2). Recent evidence that some DNA repair functions are haplo-insufficient (3,4) adds weight to the notion that sequence variants in DNA repair genes constitute part of the spectrum of defects contributing to cancer risk (5,6). DNA repair pathways are among the most critical components that mediate individual response on exposure to environmental carcinogens. Our working hypothesis is that as yet undefined DNA repair variants are involved in the etiology of many human cancers. We suggest that polymorphic variants of DNA repair genes with negative functional consequences will be differentially associated with cancer cases as compared with controls. Such variants may not cause obvious phenotypic changes. Indeed, it is possible that cancer may be the singular phenotype of DNA repair variants that do not cause other overt phenotypic effects. With few exceptions (e.g. ref. 7), however, the degree and quality of population scale variation in DNA repair genes is largely unknown.

Single nucleotide polymorphisms (SNPs) represent an important class of genetic variation, and SNPs within and outside coding sequences are under intense examination for possible associations or mechanistic links to disease (810). Recent strategies for surveying SNPs employ amplified sequences for single-strand conformational polymorphism (SSCP), oligonucleotide hybridization or DNA sequencing with computer analysis of digital trace files (9,10). Conventional analysis of coding sequences for novel variants involves cDNA preparation, followed by PCR and cloning, in order to isolate individual alleles for sequencing.

Unfortunately, the low accuracy of reverse transcriptase (RT; 11) and Taq polymerase (1214) necessitates sequencing redundant clones to filter errors. Thus, to sequence unambiguously two alleles from a single individual, it is necessary to sequence 10 or more independent clones. This is inefficient and cost-prohibitive for studies that screen large numbers of individuals, although it does have the advantage that haplotypes can be determined.

Genes for which a single PCR product may be expected, for example in the case of X-linked HPRT, are exceptions. In such cases, it is possible to sequence the PCR product directly without an intervening cloning step, because random PCR-induced errors are masked by the bulk of unmodified sequences. Such a pooling strategy has been employed in analysis of previously identified polymorphisms of human hemoglobin (15). This approach, however, has not been widely used for uncharacterized variants in autosomal genes due to technical limitations in DNA sequence data, i.e. the difficulty of discerning base substitutions or frameshifts without cloning the amplified sequences. This latter step reintroduces the problem of RT or PCR-induced sequence errors.

To begin to determine the extent of genetic variability in DNA repair genes and their possible role in cancer, we initiated a baseline survey of selected DNA repair genes (MGMT, ERCC1, CDK7, CCNH and XRCC4) in a healthy sample (n = 142) using the heterozygote sequencing protocol (HSP). Using standard redundant cloning and sequencing techniques, this survey would have required 6000–8000 individual sequencing runs, even assuming a low failure rate. Using HSP, only about 700 sequencing runs were required, making it possible to conduct reasonably large-scale population studies. Importantly, sequence changes revealed by HSP were confirmed using conventional cloning and sequencing of multiple redundant clones.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sample population
Individual samples analyzed were from healthy, anonymous volunteer participants, following a protocol approved by the University of Victoria Committee on Human Subjects Research. Results are presented for 142 individuals; of these, 82 individuals are fully characterized for all five genes. The mean age of the volunteers was 25 years, with an age range of 17–60 years. Seventy per cent of participants indicated knowledge of one or more cancer occurrences in their families. Ninety-three per cent of volunteers indicated ancestry from Europe (Britain, Western or Eastern Europe) and 5% were of Asian ancestry (Chinese, Japanese, East Indian or Korean). The remainder were from the Americas and the Middle East.

Lymphocyte collection and culture
Ten milliliters of blood was collected in heparinized Vacutainer tubes (Becton Dickinson) from each volunteer. Lymphocytes were isolated using Ficoll-Paque (Pharmacia Biotech) centrifugation following the manufacturer's instructions. Cells were then transferred to growth medium and incubated at 37°C for 7–10 days. Growth medium consisted of 40% HL-1 (BioWhittaker), 38% RPMI, 10% CBS (calf, bovine serum), 1% Fungizone (all from Gibco-BRL), 8% `4+ mixture' (containing 2535 U/ml penicillin, 2903 U/ml streptomycin sulfate, 7.3 mg/ml L-glutamine and 5.45 mg/ml pyruvic acid), 2% human serum (all from Sigma), 0.3 µg/ml phytohemagglutinin (Murex Biotech) and 5 U/ml interleukin 2 (Gibco-BRL). When sufficient growth was observed, cells were harvested by centrifuging culture for 12 min at 150xg, resuspended in 3 ml of Trizol (Gibco-BRL), aliquoted into three 1.5 ml cryovials and stored at –80°C.

RT-PCR and nested PCR reaction
Trizol samples were thawed then incubated at room temperature for 5 min before a standard phenol–chloroform (Gibco-BRL) RNA isolation was performed. RNA extracts in 20 µl DEPC (diethyl pyrocarbonate)-treated water were stored at –80°C until needed. The first-round PCR reaction was combined with the reverse transcription reaction in a single 650 µl tube. The 10x RT–PCR buffer contains 100 mM Tris–HCl (pH 9.0), 500 mM KCl and 1.0% Triton X-100 (Sigma) in sterile, deionized water. Individual primers for each gene studied and annealing temperatures are shown in Table IGo. The RT–PCR reaction mixture was 1x RT–PCR buffer, 0.25 mM each dNTP, 1.5 mM MgCl2, 2.0 µl forward outside primer, 2.0 µl reverse outside primer, 13.5 U RNAguard (Amersham Pharmacia), 1.0 µl (5 U) Taq DNA polymerase, 100 U M-MLV RT (Gibco-BRL), 2 µl RNA extract, with DEPC-treated water to a final volume of 50 µl. The RT–PCR method was: (i) 42°C for 30 min, 94°C for 4 min; (ii) 40 cycles of 94°C for 30 s, annealing temperature T1 for 60 s, 72°C for 60 s; (iii) 72°C for 5 min; (iv) 20°C for 15 min; (v) 4°C. Nested PCR conditions were 1x RT–PCR buffer, 0.25 mM each dNTP, 1.5 mM MgCl2, 2.0 or 4.0 µl forward nested primer, 2.0 µl reverse nested primer, 4.0 µl round 1 RT–PCR product, and sterile deionized water to a final volume of 100 µl. The PCR method was: (i) 94°C for 4 min; (ii) 40 cycles of 94°C for 30 s, annealing temperature T2 for 60 s, 72°C for 60 s; (iii) 72°C for 5 min; (iv) 20°C for 15 min; (v) 4°C. PCR products for MGMT were purified using Wizard PCR Preps DNA purification system (Promega). All other PCR products were purified using Qiaquick (Qiagen) PCR product purification kits. The DNA was eluted in sterile deionized water and stored at 4°C before sequencing. Longer-term storage was at –20°C.


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Table I. Primers used in RT-PCR, nested PCR and sequencing reactions
 
Heterozygote sequencing protocol and data analysis
Analyzing mutations by direct sequencing similar to our approach was proposed previously (16), but has not been adapted for population-scale SNP screening. We directly sequenced PCR-amplified DNA using Li-Cor IR2 automated DNA sequencers, taking advantage of the large dynamic range and low background of the Li-Cor fluorescent dyes. Dideoxy sequencing reactions using Li-Cor fluorescently labeled primers (IRD700 forward and IRD800 reverse) were run on purified PCR products using SequiTherm EXCEL II DNA sequencing kits-LC (Epicentre Technologies). Sequencing gels were set up such that all four bases were used for the first sample, while only one base per gel was used for all other templates (e.g. ACGTAAAA...; a total of 60 samples per gel). Thus, each sample was loaded on to four separate gels. We now load 16 samples per gel, with one standard ACGT sequencing ladder, the remainder in four sets of 15 lanes each, A15C15G15 and T15, which facilitates positional alignment.

Digital image files (TIFF format) of all sequencing runs were archived and used for image analysis. Sequence variants were compared with reference sequences obtained from GenBank. Accession numbers for each cDNA are as follows: MGMT, M29971; ERCC1, M13194; CDK7, X77303; CCNH, U12685; XRCC4, 40622.

Verification/reconstruction experiments
Variant sequences were confirmed by cloning and sequencing. In the case where the same variant was observed in multiple samples, the PCR product from one heterozygous and, if available, one homozygous sample was cloned using the TOPO-TA 2.1 (Invitrogen) cloning kit. In any case where a unique variant was observed, all such samples were cloned for verification. Clones were selected on LB plates with ampicillin (50 µg/ml), and plasmid DNA was prepared for sequencing following standard methods. For each verification, 10 such clones were sequenced. The frequency of PCR-induced sequence artifacts was recorded from the cloned verification sequences.

The response range of the technique was assessed using mixtures of sequence-verified pure MGMT clones, in a reconstruction experiment of both homozygous and heterozygous genotypes. Mixtures of clone DNA representing the two alleles at one position were prepared across a range of ratios from 9:1 to 1:9. HSP and image analysis of these mixtures was performed as described above.

Statistical analysis
Individual genotypes for each variable base pair in each gene were entered into Microsoft Excel spreadsheets. These data were imported into SAS (SAS Institute Inc., 1996) to run summary statistics and preliminary tests. GENEPOP (Raymond and Rousset, 1999; http://www.cefe.cnrs-mop.fr/) was used to calculate allele frequencies and assess exact P values for Hardy–Weinberg equilibrium tests.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sequence variation in human DNA repair genes
HSP was used to analyze sequence variation in five DNA repair genes, MGMT, ERCC1, CDK7, CCNH and XRCC4, in 142 volunteers. Examples of gel images from HSP runs for ERCC1 and MGMT are shown in Figures 1 and 2GoGo, respectively. Figure 1Go clearly shows an ERCC1 C/T variant at position 354. It occurs as both heterozygous and homozygous substitutions. This C->T polymorphism is a silent mutation and has been reported previously as a variant in cell lines (17). The MGMT variant shown in Figure 2Go demonstrates an A->G transition relative to the GenBank reference sequence; this transition is also present in both heterozygous and homozygous forms. With the exception of the 225 ERCC1 and 354 ERCC1 variants (7,17), SNPs shown here have not been reported previously.



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Fig. 1. ERCC1 sequence variant, a C->T substitution at position 354. The upper panel is the C gel, the lower is the T gel, representing 60 individual samples. At this position there are 18 heterozygotes, 13 homozygous substitutions and 29 homozygous reference genotype. Note that in the lower (T) gel, three samples are unreadable. Nevertheless, their status as heterozygous or homozygous for the substitution or reference can be ascertained from the C gel image.

 


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Fig. 2. MGMT sequence variant, an A->G substitution at position 427. The upper panel is the A gel, the lower panel is the G gel. At this position there are 11 heterozygotes, two homozygous substitutions and 47 homozygous reference sequences.

 
Forty-seven per cent (45 of 96) of samples carried at least one base substitution relative to the GenBank reference in the MGMT sequence. Eighty per cent (80/100) of samples carried variant(s) in ERCC1, 73% (71/97) showed variation in CDK7 and 34% (49/142) carried variants at CCNH. Finally, all 89 readable samples carried a variant from the GenBank reference sequence for XRCC4 (Table IIGo). Five of the 12 substitutions observed are silent (159MGMT, both ERCC1 variants, 99CDK7 and 921 XRCC4). Of the remaining seven, four (250MGMT, 427MGMT, 533MGMT and 809CCNH) are conservative and three (854CDK7, 872CCNH and 401 XRCC4) non-conservative.


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Table II. Coding sequence variants by gene and base pair position
 
G:C->A:T transitions and CpG sites
It is well documented that transitions at CpG sites predominate in mammalian mutational spectra and are over-represented among variants responsible for human disease (18). This is thought to reflect the contribution of the deamination of 5-methylcytosine present when the CpG sequences are methylated (19,20). In this screening of variation in human DNA repair genes six G:C->A:T transitions are identified (Table IIGo). Of these, five occur at CpG sites. The predominance of these events at CpG sites is thus consistent with expectations.

High levels of heterozygosity in DNA repair genes
Of the 82 samples for which all five genes have been characterized, no one individual exhibits the GenBank reference genotype at all loci. If the variant allele is redefined as the least frequent allele (GenBank reference sequence compared with HSP sequence), then four of 82 individuals carry the homozygous most frequent sequence at all loci examined.

Levels of polymorphism (frequency of substitution variant) ranged between 0.02 and 0.61, with a mean frequency of substitution variant of 0.22 (95% confidence interval (CI) 0.20–0.24) (Table IIIGo). This is in close agreement with the mean variant allele frequency of 0.17 reported by Shen et al. (7) for genomic DNA of five DNA repair genes among 12 individuals. Again, if we define the least frequent allele as the substitution, then the mean least frequent allele frequency is 0.14 (95% CI 0.13–0.16), in close agreement with Mohrenweiser and Jones' (21) modified value of 0.14 (from ref. 7, with sample size increased to 36). All variants are in Hardy–Weinberg equilibrium (Table IIIGo) with the respective reference alleles, although the variant at position 354 in ERCC1 is borderline (P = 0.06).


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Table III. Allele frequency and Hardy–Weinberg calculations for each sequence variant
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An important component of differences among individuals is variation in gene coding sequence (21). Preston (22) defines two classes of genes affecting predisposition to disease, including cancer: susceptibility variants confer increased cancer risk without obvious contribution from environmental factors, whereas sensitivity variants confer increased cancer risk only in conjunction with environmental exposure. Susceptibility variants (e.g. BRCA1 and MSH2) are relatively infrequent but highly penetrant. Sensitivity variants are relatively frequent, but less penetrant than susceptibility variants. As a result, sensitivity variants are difficult to detect using standard methods for genetic epidemiology (family studies). DNA repair acts in response to exposure to environmental carcinogens, and hence repair variants are reasonable candidates for sensitivity genes.

Identifying coding variants by sequencing cDNA products from mRNA typically involves cloning individual copies, necessitating sequencing multiple clones in order to resolve artifacts introduced by the experimental method. Direct sequencing of pooled mRNA/cDNA, which smooths out sequence errors, has been used in the task of variant identification. However, using conventional techniques, such as autoradiography, clear identification of sequence bands present at half the intensity of the main band is difficult.

The advent of highly sensitive fluorescent sequencing technology with wide dynamic ranges for signal detection makes it possible to detect readily sequence bands that are at much lower intensity than the main band. This capability is the foundation of our technique for identifying sequence variants. Comparing multiple sequences of the same gene, single nucleotide polymorphisms are identified easily by visual inspection of digital images, where variant and reference are both visualized in a single DNA sequencing run. Additional software or computational manipulations are not required. This technique is also applicable to identifying carriers in families with suspected or known disease alleles.

All variants examined are in equilibrium. Evolutionary theory predicts that equilibrium will be obtained in the absence of drift, mutation, gene flow and selection. It is reasonable to assume that allelic association with cancer should constitute a powerful selective force and therefore tempting to argue that alleles in equilibrium do not represent likely associations with cancer risk. However, on the population level, selection occurs via genotype-associated differential fertility and/or mortality, altering parental allele frequencies in the following generation. Study samples of young, healthy individuals, such as used here, include some people who eventually will develop cancer. Alleles whose action follows the reproductive period may be in Hardy–Weinberg equilibrium and yet still be associated with disease sensitivity. Cancer is predominantly a post-reproductive event.

Further, it is only within the last hundred years, following demographic and epidemiologic transitions, that cancer has become a leading cause of death in the developed world (23). Where most deaths occur as a result of infectious diseases, and expectation of life is less than is presently the case, few individuals survive to die of cancer. Observing at the population scale the action of more frequent, less penetrant, cancer sensitivity alleles is something that could not have occurred until recently.

In summary, we used HSP to screen efficiently cDNA SNP sequence variation in the human DNA repair genes MGMT, ERCC1, CDK7, CCNH and XRCC4 in 142 healthy individuals. The magnitude and extent of variation observed reinforces the necessity of population surveys to determine variation present in the gene pool. With this baseline, distribution within and between populations, and association between specific variants and disease predisposition (susceptibility or sensitivity), may be assessed. It remains to be seen whether variants identified are associated with development of disease. In the case of ERCC1, a recent publication by Yu et al. (24) suggests that even silent substitutions may be associated with diminished mRNA and protein levels, with functional consequences in the repair of cisplatin–DNA lesions. Variants at the human XRCC1 locus have recently been associated with increased adduct levels in smokers (25). Variants at polymorphic frequencies at the XPD locus also have been associated with sub-optimal DNA repair in lymphocytes (25). Certainly both direct (11,27) and indirect (17) evidence indicates that variation in DNA repair genes may alter predisposition to disease, specifically cancer (21,27,28).


    Notes
 
2 Present address: Radiation Protection Bureau, Health Canada, 775 Brookfield Road, Ottawa, Ontario, Canada K1A 1C1 Back

3 To whom correspondence should be addressed Email: bwglick{at}uvic.ca Back


    Acknowledgments
 
We thank our volunteers for donating their blood, and S.Swanson and her colleagues for blood sample collection. J.Chen, R.Haesevoets, E.Thorleifsen and M.Russell provided technical assistance. D.Walsh, K.Sojonky and J.Kangas sequenced the cDNA. This work was supported by a grant from the Medical Research Council of Canada to Drs B.Glickman and B.Weinerman, and by a grant from the Canadian Space Agency to Dr Glickman.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 11, 2000; revised July 11, 2000; accepted July 27, 2000.